Probiotic preparations for the treatment and alleviation of metabolic and oxidative stress, inflammation and neurodegeneration

Abstract

In an optional embodiment, a probiotic preparation for treating and alleviating metabolic and oxidative stress, inflammation and neurodegeneration and a method for using them are provided, including compositions, preparations, manufactured products and kits comprising them. In an optional embodiment, a probiotic preparation as provided herein comprises or is composed of any combination of Lactobacillus fermentum NCIMB 5221, Lactobacillus plantarum NCIMB 8826 and Bifidobacteria longum spp. infantis NCIMB 702255. In an optional embodiment, a probiotic preparation as provided herein is combined with Triphala. In an optional embodiment, a probiotic preparation as provided herein is used to treat metabolic diseases such as diabetes and obesity, inflammatory diseases such as IBD, IBS, arthritis; and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. In alternative embodiments, the probiotic formulations provided herein are used to combat oxidative stress, reduce inflammation, lower blood and total glucose levels, lower triglyceride levels, and manage insulin resistance.

Description

Technical Field

[0001] The present invention relates generally to microbiology and probiotics. In an optional embodiment, probiotic preparations for treating and alleviating metabolic and oxidative stress, inflammation and neurodegeneration and methods of using them are provided, including compositions, preparations, manufactured products and kits comprising them. In an optional embodiment, the probiotic preparation as provided herein comprises or is composed of any combination of the following three probiotic strains: Lactobacillus fermentum NCIMB 5221, Lactobacillus plantarum NCIMB 8826 and Bifidobacteria longum spp. infantis NCIMB 702255. In an optional embodiment, the probiotic preparation as provided herein is combined with Triphala. In an optional embodiment, the probiotic preparation as provided herein is used to treat metabolic diseases such as diabetes and obesity, inflammatory diseases such as IBD, IBS, arthritis; and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. In alternative embodiments, probiotic formulations as provided herein are used to combat oxidative stress, reduce inflammation, lower blood and total glucose levels, lower triglyceride levels, and manage insulin resistance.

[0002] background

[0003] The gut microbiota consists of thousands of bacterial species that exist symbiotically in the gastrointestinal tract. The co-evolution of the gut microbiota with the human host has made the gut microbiota indispensable for digestive health, intestinal motility, vitamin synthesis, mineral absorption, immune and neurodevelopment, energy balance and metabolic health. Imbalances (dysbiosis) of the gut microbiota caused by poor diet, antibiotic use and normal aging contribute to chronic diseases including metabolic syndrome, depression, osteoporosis and neurodegeneration. Modulation of the microbiota with beneficial bacterial populations (probiotics) and flora-boosting indigestible carbohydrates (prebiotics) reverses dysbiosis and promotes an anti-inflammatory and antioxidant environment, while positively modulating the immune system and energy balance.

[0004] Overview

[0005] In an optional embodiment, a manufacture, composition, formulation, pharmaceutical composition or kit comprising one or more probiotics is provided, wherein the probiotics comprise or consist of any combination of Lactobacillus fermentum NCIMB 5221, Lactobacillus plantarum NCIMB 8826 and Bifidobacteria longum spp. infantis NCIMB 702255.

[0006] In alternative embodiments, the article of manufacture, composition, formulation, pharmaceutical composition or kit further comprises a prebiotic composition, wherein optionally the prebiotic composition comprises or consists of Triphala, or any of the Herbal Rosana formulas, optionally any of the Herbal Rosana formulas comprises three Terminalia spp. dry fruits, or any of the Herbal Rosana formulas consists of relatively equal portions of three Terminalia spp. dry fruits, optionally without seeds, wherein optionally the three Terminalia spp. dry fruits are Emblica officinalis, Terminalia bellirica and Terminalia chebula. chebula), and optionally the formulation or pharmaceutical composition comprising said Triphala or herbal Rosana is an aqueous or water-based formulation, and optionally about 0.5% to 50%, 1.0% to 40%, 5.0% to 30% or 10.0% to 25% of said article of manufacture, composition, formulation, pharmaceutical composition or kit is said Triphala or said herbal Rosana.

[0007] In alternative embodiments, the article of manufacture, composition, formulation, pharmaceutical composition or kit also comprises or further comprises:

[0008] - Lactobacillus fermentum NCIMB 5221,

[0009] - Lactobacillus plantarum NCIMB 8826,

[0010] - Bifidobacterium longum infantis NCIMB 702255,

[0011] - Lactobacillus fermentum NCIMB 5221 and Lactobacillus plantarum NCIMB 8826,

[0012] - Lactobacillus plantarum NCIMB 8826 and Bifidobacterium longum infantis NCIMB 702255,

[0013] - Lactobacillus fermentum NCIMB 5221 and Bifidobacterium longum infantis NCIMB 702255, or

[0014] - Lactobacillus fermentum NCIMB 5221, Lactobacillus plantarum NCIMB 8826 and Bifidobacterium longum infantis NCIMB 702255.

[0015] In an alternative embodiment, the active probiotics in the article of manufacture, composition, formulation, pharmaceutical composition or kit comprise or consist of:

[0016] - Lactobacillus fermentum NCIMB 5221,

[0017] - Lactobacillus plantarum NCIMB 8826,

[0018] - Bifidobacterium longum infantis NCIMB 702255,

[0019] - Lactobacillus fermentum NCIMB 5221 and Lactobacillus plantarum NCIMB 8826,

[0020] - Lactobacillus plantarum NCIMB 8826 and Bifidobacterium longum infantis NCIMB 702255,

[0021] - Lactobacillus fermentum NCIMB 5221 and Bifidobacterium longum infantis NCIMB 702255, or

[0022] - Lactobacillus fermentum NCIMB 5221, Lactobacillus plantarum NCIMB 8826 and Bifidobacterium longum infantis NCIMB 702255.

[0023] In an optional embodiment, one, several or all of the probiotics are alive or dead. In an optional embodiment, the probiotics are:

[0024] (a) exist in relatively equal proportions;

[0025] (b) The total number is about 10 5 CFU / ml to 10 11 CFU / ml, or about 10 5 CFU / gm to approximately 3.0x10 11 CFU / gm,

[0026] (c) The total number is about 3.0x10 9 CFU / ml or about 3.0x10 9 CFU / gm, optionally relative ratio:

[0027] - Approx. 1.0x10 9 CFU / ml of Lactobacillus fermentum NCIMB 5221 (Lf5221),

[0028] - Approx. 1.0x10 9CFU / ml Lactobacillus plantarum NCIMB 8826 (Lp8826), and

[0029] - Approx. 1.0x10 9 CFU / ml of Bifidobacterium longum infantis NCIMB 702255 (Bi702255).

[0030] In an optional embodiment, the article of manufacture, composition, formulation, pharmaceutical composition or kit is manufactured or formulated as a solution, suspension, emulsion, powder, lozenge, pill, syrup, tablet, capsule, gelatin, aerosol, suppository, chewing gum, capsule, sachet or any equivalent form thereof.

[0031] In alternative embodiments, the article of manufacture, composition, formulation, pharmaceutical composition or kit is manufactured or formulated to have a unit dosage of:

[0032] (a) A total of about 10 5 CFU / ml is about 10 11 CFU / ml, or about 10 5 CFU / gm is about 3.0x10 11 CFU / gm; or

[0033] (b) Total approximately 3.0x10 9 CFU / ml or about 3.0x10 9 CFU / gm, optionally in the following relative proportions:

[0034] - Approx. 1.0x10 9 CFU / ml of Lactobacillus fermentum NCIMB 5221 (Lf5221),

[0035] - Approx. 1.0x10 9 CFU / ml of Lactobacillus plantarum NCIMB 8826 (Lp8826), and

[0036] - Approx. 1.0x10 9 CFU / ml of Bifidobacterium longum infantis NCIMB 702255 (Bi702255).

[0037] In an alternative embodiment, a method is provided for:

[0038] (a) treating, ameliorating, preventing and / or alleviating (e.g., reducing the symptoms of) metabolic and oxidative stress, inflammation, aging and / or neurodegeneration, or slowing down the occurrence or progression of metabolic and oxidative stress, inflammation, aging and / or neurodegeneration, or prolonging lifespan in an individual in need of the method;

[0039] (b) treating, ameliorating, preventing and / or alleviating (e.g., reducing symptoms thereof) metabolic diseases such as diabetes and obesity, inflammatory diseases such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS); arthritis; neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease; oxidative stress including resistance to oxidative stress and / or inflammation including reduction of inflammation, or slowing down the occurrence or progression of metabolic diseases such as diabetes and obesity, inflammatory diseases such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS); arthritis; neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, oxidative stress including resistance to oxidative stress and / or inflammation including reduction of inflammation, in an individual in need of such method; or

[0040] (c) lowering blood and total glucose levels, lowering triglyceride levels and managing insulin resistance in an individual in need thereof,

[0041] The method comprises administering to an individual in need thereof an article of manufacture, formulation, pharmaceutical composition or kit described herein,

[0042] wherein optionally the individual is a human or an animal,

[0043] and optionally administering to said subject about 1 to 4 doses or unit doses of said article of manufacture, formulation, pharmaceutical composition or kit per day, and optionally reducing the unit dose for pediatric use, optionally by about 10% to 90%.

[0044] In an alternative embodiment, there is provided a use of an article of manufacture, formulation, pharmaceutical composition or kit as described herein for:

[0045] (a) treating, ameliorating, preventing and / or alleviating (e.g. reducing the symptoms of) metabolic and oxidative stress, inflammation, aging and / or neurodegeneration, or slowing down the occurrence or progression of metabolic and oxidative stress, inflammation, aging and / or neurodegeneration in an individual in need of such use; or prolonging lifespan;

[0046] (b) treating, ameliorating, preventing and / or alleviating (e.g., reducing symptoms thereof) metabolic diseases such as diabetes and obesity, inflammatory diseases such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS); arthritis; neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, oxidative stress including resistance to oxidative stress and / or inflammation including reduction of inflammation, or slowing down the occurrence or progression of metabolic diseases such as diabetes and obesity, inflammatory diseases such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS); arthritis; neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, oxidative stress including resistance to oxidative stress and / or inflammation including reduction of inflammation, in an individual in need of such use; or

[0047] (c) lowering blood and total glucose levels, lowering triglyceride levels and managing insulin resistance in a subject in need thereof,

[0048] It comprises administering to an individual in need of the use a manufacture, formulation, pharmaceutical composition or kit as described herein,

[0049] wherein optionally the individual is a human or an animal,

[0050] and optionally administering to said subject about 1 to 4 doses or unit doses of said article of manufacture, formulation, pharmaceutical composition or kit per day, and optionally reducing the unit dose for pediatric use, optionally by about 10% to 90%.

[0051] In alternative embodiments, there is provided an article of manufacture, formulation, pharmaceutical composition or kit as described herein for use in:

[0052] (a) treating, ameliorating, preventing and / or alleviating (e.g. reducing the symptoms of) metabolic and oxidative stress, inflammation, aging and / or neurodegeneration, or slowing down the onset or progression of metabolic and oxidative stress, inflammation, aging and / or neurodegeneration in an individual in need thereof; or prolonging lifespan;

[0053] (b) treating, ameliorating, preventing and / or alleviating (e.g., reducing the symptoms thereof) metabolic diseases such as diabetes and obesity, inflammatory diseases such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS); arthritis; neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, oxidative stress including resistance to oxidative stress and / or inflammation including reduction of inflammation, or slowing down the occurrence or progression of metabolic diseases such as diabetes and obesity, inflammatory diseases such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS); arthritis; neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, oxidative stress including resistance to oxidative stress and / or inflammation including reduction of inflammation in an individual in need thereof; or

[0054] (c) lowering blood and total glucose levels, lowering triglyceride levels and managing insulin resistance in individuals in need thereof,

[0055] It comprises administering to an individual in need thereof a manufacture, formulation, pharmaceutical composition or kit as described herein,

[0056] wherein optionally the individual is a human or an animal,

[0057] And optionally administering to said individual about 1 to 4 doses or unit doses of the article of manufacture, formulation, pharmaceutical composition or kit per day, and optionally for children, a reduced unit dose, optionally reduced by about 10% to 90%.

[0058] The details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the description that follows. Other features, objects, and advantages of the invention will become apparent from the description and drawings, and from the claims.

[0059] All publications, patents, and patent applications cited herein are expressly incorporated by reference for all purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The drawings herein are illustrative of example embodiments provided herein and are not meant to limit the scope of the invention encompassed by the claims.

[0061] Figure 1 The data showing that the exemplary probiotic formulations provided herein reduce diabetes and obesity markers in Drosophila are illustrated. Several physiological markers of diabetes phenotypes of Drosophila fed with a high sugar diet (HSD) were elevated, and the probiotic formulations effectively reduced the elevation, including (a) body weight, (b) circulating common sugar levels, and (c) total glucose levels. Several physiological markers of obesity of Drosophila fed with a high fat diet (HFD) were elevated, and the probiotic formulations effectively reduced the elevation, including (d) body weight, (e) total glucose, and (f) total triglycerides. Each group represents the percentage change of the HFD group compared with the control group of the normal diet treated with the same probiotic therapy. Each group contains samples of n=5 individuals and significance is marked as *p<0.05 and **p<0.01.

[0062] Figure 2 Graphs show data showing measurement of immune activation markers in Drosophila melanogaster treated with exemplary probiotics provided herein and exposed to acute oral infection:

[0063] Drosophila pretreated with a single or exemplary probiotic formulation provided herein were placed on media inoculated with E. coli (EC) or Staphylococcus aureus (SA). The survival rate of flies at different concentrations of E. coli (a) or S. aureus (b) was recorded as the number of days required for 50% of the flies to die. Agar diffusion tests were performed on hemolymph of flies treated with probiotics on lawns of E. coli (c) or S. aureus (d) and reported as the size of the inhibition zone. Gene expression of various inflammatory markers was shown as 5.0x10 9CFU / ml of pre-treated flies infected with E. coli (e,i,k,m) or Staphylococcus aureus (f,j,l,n). Expression of dual oxidase (Duox) (e,f), IMD (g,h), cathelicidin A (i,j), cathelicidin (k,l) and defensins (m,n) are all expressed as percentage changes between flies on the respective probiotic medium and flies infected with probiotic treatments and pathogenic strains. Each group represents a sample of n=5 individuals, with black asterisks (*) indicating the significance of differences between groups and grey asterisks (*) relative to the control group subjected to the same probiotic treatment regimen. Levels of significance are marked as *p<0.05 and **p<0.01.

[0064] Figure 3 Graphs show data measured in Drosophila melanogaster tolerability to acute oxidative stress challenge following treatment with exemplary probiotics provided herein:

[0065] Drosophila were challenged with acute oxidative stress of 3% hydrogen peroxide for 72 hours. Various biochemical assays were used to measure the levels of (a) residuals, (b) total oxidants, (c) superoxide dismutase (SOD) activity, (d) glutathione peroxidase (GPx) activity, and (e) lipid peroxidation (LPO). All values ​​are expressed as percentages compared to unchallenged controls treated with the same probiotic regimen. Grey asterisks (**) indicate significance relative to untreated controls and black asterisks (**) indicate inter-group significance, *p<0.05 and **p<0.01. Each group contained samples of n=5 individuals.

[0066] Figure 4 Data showing that exemplary probiotic formulations provided herein have preventive efficacy against several markers of Alzheimer's and Parkinson's diseases are graphically presented:

[0067] Humanized transgenic flies expressing the α-synuclein mutant A30P (Parkinson's disease; PD) or APP-BACE1 (Alzheimer's disease; AD) were placed on media containing the indicated probiotic treatments for the entire life cycle. Survival of AD (a) and PD (b) flies was recorded daily until 30 days by averaging the counts of flies alive over time. Motility was measured using a negative geotaxis test in AD (a) and PD (b) flies and expressed as the time in seconds it took 50% of the flies to reach the 10 cm mark. In AD flies, the levels of amyloid beta (Aβ) (e) were measured over time and expressed as a percentage change compared to flies on day 0. Acetylcholinesterase activity (g) was also calculated in AD flies and expressed as the number of U / min. Changes in a wide range of metabolic parameters in aging flies are expressed as total glucose in AD (h) and PD (i) models and total triglycerides in AD (j) and PD (k) models. Summary information for oxidative stress parameters in aged AD(l) and PD(m) flies is summarized as the percentage change between days 0 and 30. For inflammatory markers, the inhibition zones following the agar diffusion test are presented as the diameter of the inhibition zone for both AD(n) and PD(o) flies. Summary information for immune regulatory genes is also presented as the percentage change in gene expression between days 0 and 30 for AD(p) and PD(q) flies. Finally, mitochondrial ETC complex activity is again presented as the percentage change from day 0 to day 30 for AD(r) and PD(s) flies. In all cases, each group represents samples from n=5 individuals, where black asterisks (*) indicate significance between group comparisons and gray asterisks (*) indicate significance compared to the control group, *p<0.05 and **p<0.01.

[0068] Figure 5 Graphs show data showing that the exemplary synbiotic formulations provided herein do not interfere with the effects of the probiotic formulations in any context of gut-brain-axis communication:

[0069] Prebiotic agent - Triphala (TFLA) was combined with a probiotic formulation (synbiotic) and the activity of several physiological parameters was then compared between control, single TFLA and synbiotic formulations. Metabolic responses were measured for (i) body weight, (ii) circulating glucose and (iii) total glucose changes in fruit flies treated with (a) a high sugar diet (HSD), or (b) a high fat diet (HFD) including (i) body weight, (ii) total glucose and (iii) total triglycerides. All values ​​are expressed as a full enumeration of parameters in fruit flies on a normal diet (dark grey bars) or a diet challenged diet (light grey bars). Acute oral infection of fruit flies treated with synbiotic formulations was challenged with E. coli (c). Gene expression of (i) survival, (ii) inhibition zone diameter and (iii) dual oxidase, (iv) IMD, (v) cathelicidin A and (vi) fly cathelicidin is summarized. Acute oral infection of fruit flies treated with synbiotic formulations was also challenged with Staphylococcus aureus (d). (i) Survival, (ii) inhibition zone diameter, and gene expression of (iii) dual oxidase, (iv) IMD, (v) antimicrobial peptide A, and (vi) antimicrobial peptides are summarized. The overall level of oxidative stress (e) is shown between untreated (dark gray bars) and hydrogen peroxide challenged (light gray bars) fruit flies. (i) Survival, (ii) total oxidants, (iii) superoxide dismutase (SOD) activity, (iv) glutathione peroxidase (GPx) activity, and (v) lipid peroxidation levels are shown. Various markers of disease pathways in response to synbiotic treatment in humanized transgenic fruit fly models of Alzheimer's disease (AD) and Parkinson's disease (PD) (f) are described. Survival in AD (i) and PD (ii) fruit flies is shown as the number of flies that survived after 30 days. Motility was measured using a negative geotaxis test in AD (a) and PD (b) flies and is expressed as the time it took 50% of the flies to reach the 10 cm mark. Disease markers including (v) amyloid beta (Aβ) and acetylcholinesterase (ACh) activity are shown for AD and (vi) total dopamine levels are shown for PD flies. Metabolic markers for AD (viii) and PD (ix) flies are depicted as percentage changes including total glucose and total triglyceride levels compared to flies at day 0. Inflammatory markers for AD (x) and PD (xi) flies are shown as percentage changes in gene expression of dual oxidase, IMD, Relish, cathelicidin A, defensins, and cathelicidin between day 30 and day 0. Oxidative stress in AD (xii) and PD (xiii) flies is summarized as percentage changes between day 0 and day 30, with total oxidants, SOD, GPx, and LPO levels measured. Finally, mitochondrial complex activity in AD (xiv) and PD (xv) is shown, summarized as % change between 0 and 30 days for ETC complexes 1 through 4.In all cases, each group represents a sample of n=5 individuals, where black asterisks (*) indicate significance between group comparisons, and gray asterisks (*) indicate significance compared with the control group, *p<0.05 and **p<0.01.

[0070] Figure 6 graphically illustrates that the addition of probiotics, prebiotics or a combination thereof affects metabolic parameters in aged Drosophila melanogaster. Metabolic parameters including (a) body weight, (b) total glucose and (c) total triglycerides were measured in aged Drosophila melanogaster at days 0, 10, 20 and 30. Each group contained n=5 individuals and significance is indicated as an asterisk (*) relative to the control group at day 0, *p<0.05 and **p<0.01 and tau (τ) relative to the untreated control at the same time point, where τp<0.05.

[0071] Figure 7 illustrates the genetic markers of diabetes in aging Drosophila melanogaster supplemented with probiotic and synbiotic preparations. At days 0, 10, 20 and 30, samples of 15 flies were isolated for RNA extraction to measure the expression of several insulin signaling genes, namely (a) Drosophila insulin-like peptide (dilp) 2, (b) dilp3, (c) insulin receptor (InR), (d) dAkt, (e) dTOR and (f) dFOXO. Each group contains groups of n = 5 individuals and significance is indicated by asterisks (*) relative to the control group on day 0, *p<0.05 and **p<0.01 and tau (τ) relative to the untreated control at the same time point, where τp<0.05.

[0072] Figure 8 illustrates the genetic markers of obesity in aging Drosophila melanogaster supplemented with probiotic preparations and / or synbiotic preparations. At day 0, 10, 20 and 30, samples of 15 flies were isolated for RNA extraction to measure the expression of fat metabolism genes, namely (a) acetyl-CoA carboxylase, (b) fatty acid synthase (FAS), (c) phosphoenolpyruvate carboxylase (PEPCK), (d) sterol regulatory element binding protein (SREBP), (e) lipid storage droplet (Lsd) 2 and (f) E75. Each group contains groups of n = 5 individuals and significance is indicated as an asterisk (*) relative to the control group on day 0, *p<0.05 and **p<0.01 and tau (τ) relative to the untreated control at the same time point, where τp<0.05.

[0073] Figure 9 illustrates that age-related changes in innate immune factors in Drosophila melanogaster are ameliorated by synbiotic formulations. Drosophila melanogaster were tested for their immunological responses on media supplemented with individual probiotics, prebiotics, or probiotic and / or prebiotic combinations at day 0, 10, 20, and 30. Agar diffusion assays measuring the production of antimicrobial factors were performed on plates with (a) E. coli and (b) Staphylococcus aureus pathogenic species lawns. Gene expression of several immunological genes including (c) dual oxidase (duox), (d) immunodeficiency (IMD), (e) Relish, (f) cathelicidin A, (g) defensins, and (h) cathelicidin were also tested. Groups containing n = 5 individuals per group, significance relative to the control group at day 0 is indicated by asterisks (*), *p<0.05 and **p<0.01, relative to tau (τ) of untreated controls at the same time points, where τ p<0.05.

[0074] Figure 10 illustrates that probiotic and synbiotic formulations have a combined effect on markers of oxidative stress. Individual probiotic, prebiotic, probiotic or synbiotic formulations were supplemented to Drosophila melanogaster and the levels of (a) total oxidants, (b) superoxide dismutase activity, (c) glutathione peroxidase activity and (d) lipid peroxidation were measured at 0, 10, 20 and 30 days of life. The activities of ETC (e) complex 1 (NADH coenzyme Q reductase), (f) complex 2 (succinate dehydrogenase), (g) complex 3 (cytochrome bcl complex) and (h) complex 4 (cytochrome c oxidase) were also detected using various biochemical assays. Each group contained 25 flies with n=5 individuals, significance relative to the day 0 control group is indicated by an asterisk (*), *p<0.05 and **p<0.01, relative to tau (τ) of untreated controls at the same time point, where τ p<0.05.

[0075] Fig.11 A model describing the mechanisms of gut microbiota-host communication that influence factors of aging is described. The gut microbiota communicates with metabolic, inflammatory, and oxidative stress pathways via direct and indirect mechanisms. Because physiological changes in all three axes are cross-regulated, the simultaneous actions exerted by the gut microbiota make it a powerful intervention in aging and the development of age-related chronic diseases.

[0076] Abbreviations: Glucagon-like peptide (GLP)-1, insulin receptor (IR), insulin receptor substrate (IRS)-1, phosphatidylinositol tris-kinase (PI3K), protein kinase B (Akt), target of rapamycin (TOR), forkhead box O protein (FOXO), sterol regulatory element binding protein (SREBP), acetyl CoA carboxylase (ACC), fatty acid synthase (FAS), tight junction proteins (TJPs), lipopolysaccharide (LPS), toll-like receptor (TLR) 4, nuclear factor kappa light-chain-enhancer of activated B cells (NF-kB), activator protein (AP)-1, tumor necrosis factor (TNF)-α, interleukin (IL)-6, T helper (Th) 17, adenylate-activated protein kinase (AMPK), peroxisome proliferator-activated receptor coactivator (PGC)-1α, sirtuins (SIRT), reactive oxygen species (ROS).

[0077] The same reference numbers in different drawings represent the same elements.

[0078] Details

[0079] In an optional embodiment, a probiotic preparation is provided, including a composition, a manufactured product and a kit comprising the probiotic preparation and a method of using the same, which is used for the treatment, improvement, prevention and / or alleviation (e.g., reduction of symptoms) of metabolic and oxidative stress, inflammation and neurodegeneration. In an optional embodiment, the probiotic preparation provided herein is used for the treatment, improvement, prevention and / or alleviation (e.g., reduction of symptoms) of metabolic diseases such as diabetes and obesity, inflammatory diseases such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS); arthritis; neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, including oxidative stress to resist oxidative stress and inflammation including inflammation reduction. In an optional embodiment, the probiotic preparation provided herein is used to reduce blood and total glucose levels, reduce triglyceride levels and manage insulin resistance.

[0080] In alternative embodiments, the probiotic preparations provided herein consist of any combination of the following three probiotic strains: Lactobacillus fermentum NCIMB 5221, Lactobacillus plantarum NCIMB 8826, and Bifidobacterium longum infantis NCIMB 702255.

[0081] The probiotic formulations provided herein have been shown to be effective in combating oxidative stress, reducing inflammation, lowering blood and total glucose levels, lowering triglyceride levels, and managing insulin resistance.

[0082] In an optional embodiment, the probiotic formulation provided herein is combined with Triphala, or any herbal Rosana formulation consisting of relatively equal portions of three seedless dry fruits of the genus Terminalia: Amla (Emblica officinalis), Terminalia bellirica, and Terminalia chebula. In an optional embodiment, the use of Triphala improves the effectiveness of the probiotic formulation provided herein. This embodiment provides novel probiotic and synbiotic formulations.

[0083] In alternative embodiments, including probiotic preparations in combination with Triphala or any of the herbal Rosana formulations comprised of relatively equal portions of three seedless dried fruits of the genus Terminalia: Amla (Emblica emblica), Pili (Hairy Terminalia chebula), and Terminalia chebula (Myrobalan), the probiotic preparations provided herein provide an effective regimen for the management of age-related and diet-related chronic diseases by healing the gut flora and beneficially altering signal transduction pathways involved in gut-brain axis signaling.

[0084] In an optional embodiment, the probiotic preparation further comprises any one or several (e.g., one, two, three, four or five) of the following microorganisms and all their strains: Lactobacillus fermentum 11976, Lactobacillus leichmanni NCIMB 7854, Lactobacillus farciminis NCIMB 11717, Lactobacillus fermentum NCFB1751, Lactobacillus fermentum NCIMB 2797, Lactobacillus reuteri NCIMB 11717, Lactobacillus fermentum NCFB1751, Lactobacillus fermentum NCIMB 2797, Lactobacillus reuteri NCIMB 11717, Lactobacillus fermentum NCFB1751, Lactobacillus fermentum NCIB 2797, Lactobacillus reuteri NCIMB 11717, Lactobacillus fermentum NCIB 279 ... 11951, acid-tolerant Lactobacillus (L.acetotolerans), acid-fat Lactobacillus (L.acidifarinae), acid-fish Lactobacillus (L.acidipiscis), acidophilus Lactobacillus (L.acidophilus), agilis Lactobacillus (L.agilis), algidus Lactobacillus (L.algidus), alimentarius Lactobacillus, amylolyticus Lactobacillus, amylophilus Lactobacillus, amylotrophicus Lactobacillus, amylovorus Lactobacillus, animal Lactobacillus (L.animali), antri Lactobacillus, apodemi Lactobacillus, aviaries Lactobacillus, bifermentans Lactobacillus , Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus camelliae, Lactobacillus casei, Lactobacillus catenaformis, Lactobacillus ceti, Lactobacillus coleohominis, Lactobacillus collinoides, Lactobacillus composti, Lactobacillus concavus, Lactobacillus coryniformis, Lactobacillus crispatus, Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus delbrueckii subsp.delbrueckii subsp.lactis), Lactobacillus dextrinicus, Lactobacillus diolivorans, Lactobacillus equi, Lactobacillus equigenerosi, Lactobacillus farraginis, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus fornicalis, Lactobacillus fructivorans, Lactobacillus frumenti, Lactobacillus fuchuensis, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus gastric tricus, Lactobacillus ghanensis, Lactobacillus gramini, Lactobacillus hammesii, Lactobacillus hamster, Lactobacillus harbinensis, Lactobacillus hayakitensis, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus homoohiochii, Lactobacillus iners, Lactobacillus ingluviei, Lactobacillus intestinalis, Lactobacillus jensenii, Lactobacillus johnsonii, Kallikrein. Town Lactobacillus kalixensis, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus kimchii, Lactobacillus kitasatonis, Lactobacillus kunkeei, Lactobacillus leichmannii, Lactobacillus lindneri, Lactobacillus malefermentans, Lactobacillus mali, Lactobacillus manihotivorans, Minden Lactobacillus mindensis, Lactobacillus mucosae, Lactobacillus murinus, NeigeLactobacillus nagelii, Lactobacillus namurensis, Lactobacillus nantensis, Lactobacillus oligofermentans, Lactobacillus oris, Lactobacillus panis, Lactobacillus pantheris, Lactobacillus parabrevis, Lactobacillus parabuchneri, Lactobacillus paracasei, Lactobacillus paracollinoides, Lactobacillus parafar raginis, L. parakefiri, L. paralimentarius, L. paraplantarum, L. pentosus, L. perolens, L. plantarum, L. pontis, L. protectus, L. psittaci, L. rennini, L. reuteri, L. rhamnosus. rhamnosus, L.rimae, L.rogosa, L.rossiae, L.ruminis, L.saerimneri, L.sakei, L.salivarius, L.sanfranciscensis, L.satsumensis, L.secaliphilus, L.sharpeae, L.sili ginis), Lactobacillus spicheri, Lactobacillus suebicus, Lactobacillus thailandensis, Lactobacillus ultunensis, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus versmoldensis, Lactobacillus vini, Lactobacillus vitulinus, Lactobacillus zeae, Lactobacillus zymae, Bacillus subtilis FMCC 193, Bacillus subtilis FMCC 267, Bacillus subtilis FMCC PL-1,Bacillus subtilis FMCC 511, Bacillus subtilis NCIMB 11034, Bacillus subtilis NCIMB 3610, Bacillus pumilis ATCC 7661, Bacillus sphaericus ATCC 14577 and Bacillus licheniformis ATCC 14580, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium adolescentis, Bifidobacterium angulata, Bifidobacterium breve breve, Bifidobacteriumcatenulatum, Bifidobacterium denticolens, Bifidobacterium dentium, Bifidobacterium gallicum, Bifidobacteriuminopinatum, Bifidobacterium pseudocatenulatum, Bifidobacterium lactis, Bifidobacterium minimum, Bifidobacterium subtile, Bifidobacterium thermacidophilum, Bifidobacterium animalis, Bifidobacterium asteroids, Bifidobacterium bourn), Bifidobacterium choerinum, Bifidobacterium coryneforme, Bifidobacterium cuniculi, Bifidobacterium gallinarum, Bifidobacterium indicum,Bifidobacterium magnum, Bifidobacterium merycicum, Bifidobacterium pseudolongum subsp Pseudolongum, Bifidobacterium pseudolongum subsp Globosum, Bifidobacterium pullorum, Bifidobacterium ruminantium, Bifidobacterium saeculare, Bifidobacterium suis, Bifidobacterium thermophilum or a yeast cell selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces cerevisiae, carlsbergensis, Saccharomyces schevalieri, Accharomyces delbrueckii, Accharomyces exiguous, Saccharomyces fermentati, Saccharomyces logos, Saccharomyces mellis, Saccharomyces oviformis, Saccharomyces rosei, Saccharomyces rouxu, Saccharomyces sake, Saccharomyces uvarum, Saccharomyces willianus, Saccharomyces sp, Schizosaccharomyces octosporus, Schizosaccharomyces pombe pombe, Sporobolomyces roseus, Torulopsis Candida, Torulopsisfamta, Torulopsis globosa, Torulopsis inconspicua,Trichosporon behrendu, Trichosporon capitatum, Trichosporon cutaneum, Wickerhamia fluoresens, Candida arborea, Candida krusei, Candida lambica, Candida lipolytica, Candida parapsilosis, Candida pulcherrima, Candida rugousa, Candida tropicalis, Candida utilis, Crebrothecium ashbyu, Geotrichum candidum, Hansenula anomala, Hansenula arabitolgens, Hansenula jadinii, Hansenula saturnus, Hansenula schneggn, Hansenula subpelliculosa, Kloeckera apiculata, Lipomyces starkeyi, powder, status gnauhznePichia farinosa, Pichia membranaefaciens, Rhodospoπdium toruloides, Rhodotorula glutinis, Rhodotorula minuta, Rhodotorularubar, Rhodotorula aurantiaca, Saccharomycodes ludwigii, Saccharomycodes sinenses, Saccharomyces cerevisiae Hansen AS2375, AS2501, AS2502, AS2503, AS2504, AS2535, AS2558, AS2560, AS2561, AS2562 or IFFI1048, or Saccharomyces carlsbergensis Hansen) AS2420 and AS2444, and any equivalents or combinations thereof.

[0085] In an optional embodiment, the probiotic preparation further comprises any one or several (e.g., one, two, three, four or five) of the following probiotics or probiotic-like substances, including, for example: fructooligosaccharides, galacto-oligosaccharides, glucose, galactose, xylose, maltose, sucrose, lactose, starch, xylan, hemicellulose, inulin, rubber, ginseng (Panax ginseng, red, Korean ginseng), ginseng (white, Chinese ginseng), Rhodiola rosea (golden root), American ginseng (Panax quinquefolium, American ginseng), Eleutherococcus senticosus (Siberian ginseng), artichoke (Cynara scolymus, artichoke), Uncaria tomentosa (Cat's claw vine), Lepidium meyenii (Maca, Peruvian ginseng), Guarana (Paullinia cupana, guarana, Croton lechleri, Withania somnifera, Panax japonicus, Panax vietnamensis, Panax trifolius, Panax pseudoginseng, Panax notoginseng, Malpighia glabra, Yerba mate, Astragalus membranaceus, Stevia rebaudiana, Pfaffia paniculata, Ginkgo biloba, biloba, Tabebuia impetiginosa (pau d'or), Echinacea purpurea, Peumus boldus (Bordeaux), Gynostemma pentaphyllum (Gynostemma pentaphyllum, also known as southern ginseng or fairy grass), Sutherlandia frutescens (African ginseng), Aloe vera (Aloe vera), Cistanches alsa, Cistanche deserticola, Codonopsis pilosula ("poor man's ginseng"), Nopal opuntia (Nopal opuntia),Oranges (Citrus sinensis) and other members of the citrus family (lemons, limes, tangerines, grapefruits), tea trees (Camelia sinensis), psyllium (Plantago psyllium), amaranth (Amaranth edulis) and other amaranths (amaranthsp), Indian myrrh (Commiphora mukul), saw palmetto (Serenoa repens), saw palmetto (Serenoa serrulata), cordyceps (Cordyceps sinensis), shiitake mushrooms (Lentinula edodes), Ganoderma lucidium (Ganoderma lucidium), maitake mushrooms (Grifola rondosa), white fungus (Tremella fuciformis), Poria cocos (Poria cocos) cocos, Poria cocos), Hericium erinaceus (lion's mane), Agaricus blazei (sunshine mushroom), Phellinus linteus (Phellinus linteus), Trametes versicolor, Coriolus versicolor (turkey tail), Schizophyllum commune (white ginseng), Inonotus obliquus (scoria cone), oat bran, rice bran, flax seed, garlic, Ceratonia siliqua (locust bean gum or powdered material derived from the seeds of the carob tree), Cyanopsis tetragonoloba (guar gum, EU food additive code E412), Xanthomonas campestris (xanthan gum), and any equivalents or combinations thereof.

[0086] In alternative embodiments, the articles of manufacture, compositions, formulations, pharmaceutical compositions or kits provided herein are used prophylactically and / or therapeutically in animals (e.g., animal models) and humans suffering from type II diabetes or insulin resistance; optionally, by reducing total and circulating glucose levels, triglyceride levels, total body weight, inflammation, oxidative stress and / or any mitochondrial dysfunction.

[0087] In alternative embodiments, the articles of manufacture, compositions, formulations, pharmaceutical compositions or kits provided herein are used prophylactically and / or therapeutically in animals (e.g., animal models) and humans suffering from obesity, hyperlipidemia or overweight by reducing total body weight, triglyceride levels, glucose levels, insulin resistance, inflammation, oxidative stress and / or mitochondrial dysfunction.

[0088] In alternative embodiments, the articles of manufacture, compositions, formulations, pharmaceutical compositions or kits provided herein are used prophylactically and / or therapeutically in animals (e.g., animal models) and humans suffering from inflammatory diseases including symptoms of pathogen infection, arthritis, inflammatory bowel disease, inflammatory bowel syndrome, Crohn's disease, ulcerative colitis and inflammatory aging by reducing circulating inflammatory markers (cytokines, chemokines, etc.).

[0089] In alternative embodiments, the articles of manufacture, compositions, formulations, pharmaceutical compositions or kits provided herein are used prophylactically and / or therapeutically in animals (e.g., animal models) and humans suffering from acute and / or chronic oxidative stress by reducing total oxidant counts, lipid peroxidation and increasing the activity of superoxide dismutase and glutathione peroxidase.

[0090] In alternative embodiments, the articles of manufacture, compositions, formulations, pharmaceutical compositions or kits provided herein are used prophylactically and / or therapeutically in animals (e.g., animal models) and humans suffering from neurodegeneration including Alzheimer's disease and Parkinson's disease by reducing metabolic diseases (e.g., high sugar, high triglycerides, obesity, insulin resistance), inflammation, oxidative stress and by affecting the activity of the electron chain complexes of mitochondria.

[0091] In alternative embodiments, the probiotic formulations provided herein are combined with any other probiotic or prebiotic substances, optionally providing a potentiating effect, and optionally without any negative effects on the efficacy of the formulation with respect to the aforementioned parameters.

[0092] The present invention will be further described with reference to the examples described herein; however, it will be understood that the present invention is not limited to such examples. Example

[0093] The Drosophila melanogaster model is an effective and predictable model for evaluating various treatments in humans, such as treating diet-induced diabetes / insulin resistance and / or obesity / hyperlipidemia; treating or improving inflammation; or treating or improving acute stress; treating or improving, or slowing down the progression or occurrence of neurodegenerative diseases including Alzheimer's disease (AD) and Parkinson's disease (PD). Although Drosophila melanogaster may be small, they retain a large number of human homology of cells and molecules, including regulation of gene expression, membrane trafficking, neuronal connectivity, cell signaling, synaptogenesis, and cell death. In addition, Drosophila is highly genetically amenable, making it more feasible to model genetic diseases than higher organisms. One of the most common expression transgenic systems in Drosophila is the UAS (upstream activation system)-Gal4 system, which allows driving genes downstream of UAS elements through specific cell expression of Gal4 driven by a selected promoter. In fact, 75% of human genes implicated in disease share functional homologs with flies with 80-90% conservation of functional domains (Reiter, Potocki, Chien, Gribskov, & Bier, 2001). Drosophila also have an organized brain, albeit one with fewer cells and a different neuroanatomical structure. The simplicity of the fruit fly may be its most significant strength, as the functional redundancy found in mammals is not widely found in flies, allowing for the deciphering of molecular pathways. Finally, its short life cycle and fully sequenced genome are another experimental advantage the organism possesses (Sang & Jackson, 2005); see also: Reiter et al. (2001) Systematic analysis of human disease-associated gene sequences in Drosophila melanogaster. Genome Research, 11(6), 1114-1125. and Sang, T.-K. et al. (2005) Drosophila models of neurodegenerative diseases. Neuroscience Reviews: American Association for Experimental Neurotherapeutics, 2(3), 438-446.

[0094] Example 1: Example compositions for treating diabetes and obesity

[0095] The following examples demonstrate the effectiveness of exemplary probiotic formulations for treating or ameliorating diabetes and obesity.

[0096] We have outlined several parameters of diabetes and obesity that are positively impacted by probiotic formulations, supported by mechanistic studies outlining how probiotics communicate with host physiology, which support the robust nature of probiotic treatments. We evaluated the ability of the exemplary probiotic formulations provided herein to impact the physiological attributes of diet-induced diabetes / insulin resistance or obesity / hyperlipidemia in an art-accepted model of Drosophila melanogaster.

[0097] method

[0098] Bacterial culture

[0099] Bacterial cultures (Lp8826, Lf5221, Bi702255) from frozen glycerol stocks were grown in Man-Rogosa-Sharpe (MRS) medium obtained from Sigma Aldrich (Oakville, ON, Canada) at 37°C on MRS-agar plates or in liquid culture. After one round of liquid culture, several bacterial stocks were prepared in MRS containing 20% ​​(v / v) glycerol and stored at -80°C as working stocks. Because all experiments required continuous culture to be performed, the bacterial stocks were refreshed from frozen stocks every two weeks to maintain the purity of the cultures. For each individual experiment, a 1% (v / v) inoculum was used for subculture, inoculated at 37°C for 18 hours and removed immediately before use. To prepare individual fruit fly treatment bottles, overnight cultures were centrifuged at 4000 rpm for 10 minutes at 4°C. The pellet was washed once and resuspended in 0.85% (w / v) sterile saline. Total colony counts were determined spectrophotometrically by comparison with a standard curve of colony forming units (CFU) on MRS plates.

[0100] Fruit fly feeding

[0101] Wild-type Drosophila melanogaster (Oregon R) was obtained from the Bloomington Drosophila Stock Center (Indiana University, Bloomington, Indiana). The flies were fed on standard oatmeal-sucrose-yeast medium with inactive yeast culture, prepared by boiling oatmeal (83 g), sucrose (50 g) and yeast extract (30 g) in distilled water for 30 minutes. The flies were kept under controlled conditions of 25° C. 12 h:12 h light-dark cycle. The inoculation medium was prepared by dividing the concentrated bacterial culture into a cooled but still liquid medium. It is believed that bacteria can survive up to 2 weeks in the fruit fly medium before the concentration loss can be detected by daily CFU counts. The survival rate of the strain inoculated in the GIT of the flies can also be verified by real-time PCR quantification. Nevertheless, flies were transferred to freshly inoculated bottles every 3 to 4 days during the experiment to ensure continuous inoculation of pure probiotic bacteria.

[0102] In the control medium, or containing 1.0x10 9 Freshly mounted flies were adapted for 7 days on a probiotic preparation containing 1.0 × 10 CFU / ml of each bacterium, and a control medium containing 3.0 × 10 9CFU / ml of a single bacteria (Lp8826, Lf5221, Bi702255) inoculated medium. According to the experimental procedure, each group consists of 5 independent samples, each sample has 20 to 50 flies. After environmental adaptation and the correct construction of the intestinal flora, the flies are transferred to similarly treated medium supplemented with a high sugar diet (HSD) or a high fat diet (HFD), wherein the high sugar diet contains 1M sucrose, and the high fat diet contains 30% coconut oil in addition to the normal medium. The flies are allowed to feed on the newly treated medium for 10 days, and then they are anesthetized, weighed, rinsed in 10% bleach solution and rinsed three times in water before treatment.

[0103] Physiological metabolic parameters

[0104] Physiological parameters of freshly anesthetized flies were assessed. Body weight was assessed by weighing 10 flies (repeated 5 times). Glucose measurements were taken from the hemolymph (circulating glucose) and whole body homogenates of fruit flies. Before measurement, the homogenates were heated at 70°C for 20 minutes to remove any complexes. Glucose levels were measured in 2 μl hemolymph or 5 μl whole body homogenates using a glucose (HK) assay kit (Sigma, Oakvilla, ON, Canada) according to the manufacturer's instructions. Whole body triglycerides were determined in 10 μl homogenates using a triglyceride assay kit (endpoint method) (Stanbio, TX, USA) according to the manufacturer's instructions. The measured values ​​in each sample were standardized to protein content and statistical differences were calculated using a two-way ANOVA with Turkey post hoc analysis.

[0105] Gene expression analysis

[0106] According to the manufacturer's instructions, RNA was extracted from whole flies using Trizol (ThermoFisher, MA, USA). According to the manufacturer's instructions, cDNA was synthesized from 1 μg RNA measured with a ND-2000 spectrophotometer (FisherScientific, Ottawa, ON, Canada) using a high-capacity cDNA synthesis kit (ThermoFisher, MA, USA). Real-time PCR was performed on an Eco real-time PCR system (Illumina, CA, USA) using the SybrGreen detection method (Diamed, Mississauga, ON, Canada) and according to the 2-ddCT method, Rp49 was used as a reference standard for quantification. Primer pairs and annealing temperatures are listed in Table 1:

[0107] Table 1: Primer pairs and annealing temperatures

[0108]

[0109]

[0110] statistics

[0111] All statistics were performed using R statistical software. Multivariate two-way ANOVA with Turkey post hoc analysis was performed for all groups. p<0.05 was considered significant.

[0112] Results and Discussion

[0113] Physiological parameters measured to assess the effects of HSD (mimicking diet-induced diabetes) or HFD (mimicking obesity) on flies included 1) total body weight, 2) circulating glucose, 3) total glucose, and 4) total triglycerides. In HSD flies, total body weight of control flies increased by 32.4% ( Figure 1 a). Lp8826, Lf5221, and Bi702255 significantly reduced this increase to 9.8%, 14.3%, and 3.4%, respectively, while the probiotic preparation reduced the total body weight gain by 18.1%. As expected for the diabetic phenotype, circulating glucose levels in the control flies increased dramatically by 78.7% ( Figure 1 b). This increase was significantly greater than that of the Bi702255 and probiotic preparation groups, which showed a 2.2% and 5.7% decrease in circulating glucose levels, respectively, compared to the untreated control group. For the measurement of circulating glucose, the probiotic preparation was more effective in reducing glucose levels than the Lp8826 and Lf5221 single probiotic treatments. Similarly, total glucose levels were significantly increased by 151.4% in the HSD control group ( Figure 1 c). This increase was significantly higher than the Lf5221, Bi702255 and probiotic preparation groups, which showed increases of 41.0%, 71.0% and 58.7%, respectively, compared to the control group. In summary, the probiotic preparations consistently demonstrated the ability to reduce the physiological parameters of diet-induced diabetes, although not to a greater extent than the individual probiotics. Although some individual probiotics showed the same degree of reduction in the physiological parameters of diabetes as the probiotic preparations, no probiotic consistently reduced all parameters to the same level as the probiotic preparations, indicating that the preparations are able to effectively treat all aspects of diet-induced diabetes.

[0114] In D. melanogaster fed HFD, total body weight of the control group increased significantly by 41.0% ( Figure 1d). This increase was significantly higher than that of the Lp8826, Bi702255 and probiotic preparation groups, which showed 20.4%, 11.9% and 9.1% weight increases, respectively, compared to the control group. The total glucose level of the control group flies increased by 148% ( Figure 1 e), which was significantly higher than any single probiotic or probiotic preparation. Notably, the probiotic preparation showed significantly lower total glucose levels compared to the Lp8826 and Bi702255 groups (31.0% higher than the untreated control group). Finally, total triglycerides in the HFD-fed control group were significantly increased by 187% ( Figure 1 f). This increase was significantly greater than that of flies treated with Lf5221, Bi702255, and the probiotic formulation, which showed a 119.0%, 91.2%, and 32.3% increase in total triglycerides, respectively. Notably, the probiotic formulation showed the smallest increase in total triglycerides compared to any of the individual probiotic treatment groups, suggesting a cumulative effect. Again, although some of the individual probiotics showed similar abilities to the probiotic formulations to reduce physiological parameters of diet-induced obesity, none of the probiotics reduced all physiological parameters of obesity as powerfully as the probiotic formulations.

[0115] Various genetic markers of insulin signaling, fatty acid metabolism, and their potential signaling pathways were evaluated by real-time PCR. All reported values ​​are expressed as percentage changes compared to control flies (same probiotic treatment but normal diet). Of particular importance, the expression of the insulin receptor (InR) gene in HSD control flies increased by 148.1%, which was significantly higher than that in flies treated with Lp8826 (7.0% increase) or flies treated with the probiotic formulation (6.5% increase). Similarly, the insulin effector molecule (Drosophila insulin-like peptide; dilp) was significantly increased by 174.7% (dilp2) and 118.2% (dilp3) in HSD-fed control flies. The increase in dilp2 in the control flies was significantly higher than that in Lp8826 (13.0% increase), Lf5221 (24.3% increase), or the probiotic formulation, which actually showed a 58.3% decrease in dilp2 expression, significantly lower than any other single probiotic. Similarly, the probiotic preparation significantly reduced dilp3 expression by 37.2%, which was significantly lower than any of the individual probiotic treatment groups. This suggests that the probiotic preparation is much more effective than individual probiotics in reducing HSD-induced genetic variation that leads to insulin resistance, which had only mild and inconsistent effects on key markers of diabetes.

[0116] Analyzing the results more closely, the probiotic preparation also significantly affected the downstream of insulin signaling pathways, such as the canonical Akt-TOR-FOXO pathway. dAkt was significantly increased by 241.0% in the HSD control flies, which was significantly higher than each of the individual probiotic treatment groups Lp8826 (33.3% increase), Lf5221 (83.6% increase), and Bi702255 (130.4% increase). The probiotic preparation completely reduced the increase in dAkt signaling in HSD-fed flies compared to the normal control diet, which was significantly lower than each of the individual probiotics. dTOR was also significantly increased (164.1%) in the control group of fruit flies fed the HSD diet. As with the probiotic preparations that completely rescued the increase in dTOR expression, each of the individual probiotic preparations Lp8826 (1.5% increase), Lf5221 (3.0% decrease) and Bi702255 (68.3% increase) significantly reduced this increase. A similar phenomenon was noted for the transcription factor dFOXO, which was significantly reduced by the HSD diet by 62.5%, a reduction that was significantly lower than that of Lf5221 (4.7%) and in particular the probiotic preparations, which again completely rescued the reduction in dFOXO expression.

[0117] Similar trends were observed in the regulation of fatty acid oxidation genes in HFD-fed flies. Several genetic markers of fatty acid synthesis (SREBP, ACC, FAS), gluconeogenesis (PEPCK), and regulation of triglyceride content (E78 and LSD) were evaluated by real-time PCR. Acetyl-CoA carboxylase (ACC) increased by 79.9% in the control group of HFD-fed flies, which was significantly higher than flies treated with Lf5221 (0.8% increase) or Bi702255 (27.8% increase). The probiotic formulation rescued the expression of ACC, which was significantly lower than each individual probiotic. Fatty acid synthase (FAS) was also significantly increased by 82.1% in the control group of HSD-fed flies. This increase was significantly higher than flies treated with Lf5221 (26.7% decrease) or flies treated with the probiotic formulation (which rescued the expression of FAS in HFD-fed flies). Phosphoenolpyruvate carboxykinase (PEPCK) was significantly upregulated by 239.8% in the HFD-fed control group of flies. This increase was significantly higher than any of the individual probiotics Lp8826 (36.8% increase), Lf5221 (49.9% increase), and Bi702255 (165.8% increase); however, PEPCK expression was completely rescued by the probiotic preparation, which was significantly lower than any of the individual probiotics.

[0118] Looking at upstream regulators, sterol regulatory element binding protein (SREBP), which regulates the expression of all the aforementioned fatty acid oxidation genes, was elevated by 165.0% in the control group of flies fed with HFD. Each of the individual probiotics Lf5221 (109.1% increase), Lp8826 (51.7% increase), and Bi702255 (42.1% decrease) significantly reduced this elevation, as did the probiotic preparation (114.2% increase). dTOR was also significantly upregulated in flies in the control group (93.7%), which was significantly higher than each of the individual probiotics Lp8826 (52.0% increase), Lf5221 (5.9% decrease), and Bi702255 (29.3% increase) and was completely rescued by the probiotic preparation. Ecdysin-induced protein 78 (E78) in Drosophila is a nuclear receptor that, like Rev-erbα and PPARγ, is known to be involved in energy regulation in Drosophila. E78 was elevated by 253.6% in the control HFD-treated flies, which was significantly reduced by Lf5221 (53.7% increase) and Bi702255 (84.1% increase) and rescued by Lp8826 and the probiotic formulation. Finally, Drosophila lipid storage droplet (LSD) 2 controls the storage of triglycerides in the fly's fat body. LSD2 was elevated by 169.8% in the control HFD flies, which was only reduced in the probiotic-treated flies (7.5% increase). When genetic markers of diabetes and obesity were examined, similar phenomena were found as for the physiological markers, as the probiotic formulation more strongly and consistently rescued disease markers, whereas individual probiotics had only inconsistent effects on gene expression.

[0119] in conclusion

[0120] The individual probiotics tested (Lp8826, Lf5221 and Bi702255) all had some efficacy on various markers of diet-induced diabetes in Drosophila; however, their effects were inconsistent in all parameters tested and were limited to one or two of the underlying genetic pathways. Insulin signaling is a dynamic, multi-pathway system with natural robustness, so the greatest benefit comes from consistently reducing the classical signaling pathway (InR-Akt-TOR-FOXO) while controlling the expression of insulin and insulin effectors (dilps). Similarly, for fatty acid oxidation, there are several levels of regulation to control the accumulation of fatty acids in adipose tissue, which is most effectively controlled by simultaneous targeting through probiotic preparations. In this case, probiotic preparations provide more complete, consistent and reliable effects on physiological and genetic parameters of diabetes and obesity, indicating that the exemplary Lp8826, Lf5221 and Bi702255 preparations provided herein are better than single probiotics in treating the entire disease pathology.

[0121] Example 2: Example compositions for treating inflammation

[0122] The following examples illustrate the effectiveness of exemplary probiotic formulations for treating or ameliorating inflammation.

[0123] To test the inflammatory resistance of D. melanogaster, they were treated with probiotic preparations and challenged with oral bacterial infection with either Gram-negative bacteria (Escherichia coli) or Gram-positive bacteria (Staphylococcus aureus). Basic immune parameters including survival and production of antimicrobial agents as well as genetic markers of immune activation were evaluated.

[0124] method

[0125] strain

[0126] Two pathogenic strains, Escherichia coli ATCC 8739 (Gram-negative pathogen) and Staphylococcus aureus ATCC10832 (Gram-positive pathogen) were maintained in 20% glycerol stocks at -80°C until streaked onto nutrient agar plates containing 3 g / L beef extract and 5 g / L peptone, adjusted to pH 7.0 (nutrient broth; NB). Colonies were inoculated into NB and grown for 18 hours at 37°C with shaking (250 rpm). Overnight colonies were inoculated into NB with 1% inoculum and bacterial suspensions were isolated for incorporation into Drosophila medium or smearing onto nutrient agar plates.

[0127] Oral infection

[0128] Immediately after eclosion, flies were transferred to culture medium supplemented with probiotics (Lp8826, Lf5221, Bi702255 or probiotic preparations). Ten days later, flies were treated with 0, 1.0x10 9 , 5.0x10 9 or 1.0x10 10 CFU / ml, the flies were transferred to the medium containing the probiotic therapy and E. coli or S. aureus. The survival rate of 10 flies isolated from the medium inoculated with S. aureus or E. coli was measured (three times), and the time taken for 50% of the flies to die was recorded. Otherwise, the flies were kept on the medium for 5 days (the time required for approximately 50% mortality in the control group), and then the flies were removed and rinsed in 10% bleach and 3x distilled water before being placed at -80° for further processing.

[0129] Agar disk diffusion assay

[0130] To evaluate the ability of flies to directly inhibit the growth of E. coli and S. aureus, hemolymph from flies fed on media containing probiotic therapy for 10 days was placed on a lawn of S. aureus or E. coli on TSC-agar plates. To isolate hemolymph, 50 flies were pierced in the thoracic region with a fine needle and placed in a small microcentrifuge tube with several holes, which was then placed in a larger microcentrifuge tube. The pierced flies were centrifuged at 3000xg for 20 minutes at 4°C to remove enough hemolymph for analysis. Hemolymph (20 μl) was diluted to 50 μl with sterile saline and pipetted onto a 5 mm sterile filter paper loop on a nutrient agar plate that had just been coated with 100 μl of an overnight solution of S. aureus or E. coli. The inoculated plates with hemolymph saturated filter paper were placed at 37°C for 24 hours and then the inhibition zone was measured. Each treatment was replicated three times on a separate nutrient agar plate.

[0131] Real-time PCR

[0132] mRNA isolation, cDNA synthesis and real-time PCR were performed as described in the metabolism section. Specific primers for analysis of inflammatory markers are summarized in Table 2:

[0133] Table 2: Primers used for analysis of inflammatory markers

[0134]

[0135]

[0136] result

[0137] Survival rate

[0138] As expected, there was a dose-dependent decrease in survival in the control group fed with E. coli or S. aureus. 9 , 5.0x10 9 Increased to 1.0x10 10 CFU / ml, the 50% survival rate in the control group decreased from 7.8 days, 3.1 days to 2.5 days ( Figure 2 a; p<0.01). In all treatment groups, there was a dose-dependent decrease in survival, although the extent varied between groups. 1.0x10 9 There was little difference in CFU / ml of E. coli between the different treatments, however, at 5.0x10 9 In the E. coli group, there was a significant increase in survival rate for the Bi702255 and probiotic preparation groups ( Figure 2 a; p<0.01). 10CFU / ml groups, only the triple formulation group had a significant increase in survival rate ( Figure 2 a, p < 0.01), indicating a synergistic immune effect against E. coli challenge.

[0139] The survival rate of flies orally infected with Staphylococcus aureus was similar to that of flies infected with Escherichia coli. 9 , 5.0x10 9 Increased to 1.0x10 10 CFU / ml, 50% survival rate decreased from 6.5 days, 2.8 days to 2.4 days ( Figure 2 b; p<0.01), although at 5.0x10 9 and 1.0x10 10 There was no significant difference between the CFU / ml groups. Similar to E. coli, 1.0x10 9 There was no significant difference in survival rate between treatment groups at 5.0x10 9 CFU / ml group, when treated with Bi702255 or probiotic preparation, there was a significantly higher survival rate ( Figure 2 b, p<0.01). 10 There was also a significant increase in survival for all treatment groups, with the probiotic preparation being slightly more effective than the single probiotic treatment groups.

[0140] Flies pretreated with each probiotic therapy were subjected to an agar plate diffusion assay, where they were exposed to plates containing either E. coli or a lawn containing S. aureus. Hemolymph was isolated from the flies and spotted onto small filter paper disks placed on the inoculated plates. On the E. coli plates, there was a significant increase in inhibition zones ( ) in the Lp8826, Bi702255, and probiotic formulation groups compared to the untreated control. Figure 2 c, p<0.01). In addition, the inhibition zone created by the probiotic preparation was larger than that of each individual probiotic group, indicating a synergistic effect. Similarly, for the agar plates inoculated with S. aureus, there was a significant increase in inhibition zones in all treatment groups ( Figure 2 d; p < 0.05), and the probiotic preparation group had a larger inhibition zone compared with the Lp8826 and Lf5221 groups. In conclusion, the probiotic preparations were more synergistically effective against E. coli and S. aureus challenges than the individual probiotic species, indicating their potential in inhibiting both Gram-negative and Gram-positive bacterial challenges.

[0141] To evaluate immune activity at the molecular level, the expression of several immune activation genes was assessed by real-time PCR. Based on existing survival data, the optimal acute inoculum concentration for E. coli and S. aureus was selected to be 5.0x10 9 CFU / ml, and only genetic analyses at this concentration are shown. To investigate the upstream and first response elements of the Drosophila immune response, the expression of Duox, IMD, and Relish was examined. Dual oxidase (Duox) is a member of the intestinal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family that is involved in several aspects of gut microbiota interactions, such as clearance of microorganisms, intestinal epithelial cell renewal, and redox regulation of signaling pathways (e.g., see Kim & Lee, 2014). In response to invading pathogens and signals from the Drosophila gut microbiota, Duox produces ROS particles in any branch of the innate immune system to clear those pathogens. Typically, duox expression is elevated in conjunction with immune insults and is elevated in flies challenged with E. coli (197.1% increase; Figure 2 e) and S. aureus -challenged flies (814% increase; Figure 2 This result was precisely observed in the control group of Lf5221. In E. coli treated flies, all treatment groups had significantly reduced Duox expression levels, with the probiotic formulation group having significantly lower Duox expression than the Lf5221 group ( Figure 2 e; p<0.01). Similarly, in the S. aureus group, all treatment groups significantly reduced Duox expression ( Figure 2 f; p<0.01).

[0142] The immune deficiency (IMD) factor in Drosophila is strongly activated downstream of Gram-negative bacterial infection and works to elicit the expression of effector molecules including the cytokine-like Relish and the AMPs antimicrobial peptide A and dipterin. As expected, there was a strong upregulation of IMD expression in the E. coli-treated control group (165.4% increase; Figure 2 g; p<0.01). IMD expression was significantly downregulated in all treatment groups, with the probiotic preparation having a significantly greater effect than the Lp8826 and Lf5221 groups. After S. aureus treatment, there was a significant increase in IMD expression of 41.1% in the control flies, which was significantly higher than that in the Lp8826, Bi702255 and probiotic preparation groups ( Figure 2 h; p < 0.05). Notably, the probiotic preparation reduced IMD expression to a greater extent than Lf5221 and Bi702255.

[0143] Antimicrobial peptides (AMPs) are effector molecules of the Drosophila immune system and have several modes of action due to their amphipathic nature, but generally target specific subunits of invading pathogens. For example, defensins target Gram-positive bacteria, while antimicrobial peptides and dipterans target Gram-negative bacteria. In flies challenged with E. coli, as expected, there was a strong increase in antimicrobial peptide A expression in the control group for Gram-negative bacteria (232.7% increase) ( Figure 2 i, p<0.01). This increase was significantly higher than in any treatment group. There was also a significant increase in antimicrobial peptide A expression in the control group treated with S. aureus (183.9% increase; Figure 2 j; p<0.01), which was significantly higher than any of the treatment groups. As expected, dipterin expression in the E. coli control group was strongly elevated (251.2% increase, Figure 2 k; p<0.01) and this height was significantly higher than any treatment group. It is worth noting that the expression of dipterin in the probiotic preparation group was significantly lower than that in the Lp8826 treatment group. In the S. aureus control group, there was also a significant increase in dipterin expression (115.1% increase), which was greater than that in Bi702255 and the probiotic preparation ( Figure 2 l; p < 0.01). Finally, defensin expression was evaluated and found to be modestly upregulated in the E. coli-inoculated control group (41.0%; Figure 2 m). This was significantly higher than in the Lp8826, Lf5221 and probiotic groups, which actually reduced defensin expression to lower levels than the control group. In the S. aureus control group, as expected for a Gram-positive challenge, there was a significant increase in defensin expression (329.9%; Figure 2 n). This increase was significantly higher than that in any treatment group, with the lowest defensin expression in the Bi702255 group, which was at or close to baseline levels.

[0144] in conclusion

[0145] The increased survival of flies treated with the exemplary probiotic formulations provided herein, coupled with the reduced expression of immune effector genes, suggests that the intestinal flora of the treated group was favorably altered to an environment that is less permissive for the growth of pathogenic microorganisms. This may include mechanisms such as reduced intestinal permeability or competition between good bacteria and pathogenic strains for growth resources.

[0146] Example 3: Example compositions for treating acute stress

[0147] The following examples illustrate the effectiveness of exemplary probiotic formulations for treating or ameliorating acute stress.

[0148] method :

[0149] Drosophila and oxidative challenge

[0150] Wild-type Drosophila melanogaster (OregenR) were raised on a regular oatmeal-yeast-sucrose medium as described in the previous section and immediately after eclosion, the flies were transferred to a 3.0×10 9 CFU / ml Lp2389, Lf5221, Bi702255 or triple formulations. After 10 days, flies were either transferred to control medium or to medium containing 3.0% hydrogen peroxide to mimic acute oral oxidative stress. After 3 days, when approximately 50% mortality was observed in the control-hydrogen peroxide group, flies were anesthetized and immediately homogenized in Tris-EDTA-Triton X-100 buffer to release intracellular antioxidant enzymes. Several enzymes were tested:

[0151] Total oxidants

[0152] Total oxidants were determined using 2'-7'-dichlorofluorescein acetoacetate (DCFA) (Sigma, Oakville, ON) as outlined by Hosamani et al. (Archives Insect Biochem). Briefly, 20 μl of fly homogenate was mixed with 170 μL of Locke buffer. Next, 10 μl of 1 mM DCFA solution was added to each well and 3 minutes after inoculation, fluorescence was read at 474 nm excitation and 530 nm emission.

[0153] Superoxide dismutase (SOD) assay

[0154] SOD was determined using xanthine-xanthine oxidase to generate superoxide radicals, and nitrotetrazolyl blue (NBT) reduction was used as an indicator of superoxide generation (Mockett 2002, Methods Enzymol). Briefly, the working solution consisted of 110 μl of potassium phosphate buffer with 20 mg / ml BSA, 6.25 μl catalase (40 U / ml), 6.25 μl of NBT and 50 μl xanthine (1.8 mM). 7.5 μl of sample and 20 μl of xanthine oxidase (XOD, 5 U / ml) were added to the working solution and incubated in the dark at 37°C with stirring for 20 minutes to allow color development. The absorbance of the reduced NBT at 570 nm was read and compared with the standard curve of SOD. Glutathione peroxidase (GPx) activity

[0155] GPx activity was assessed based on the oxidation of glutathione (GSSG), which was continuously supplied by excess glutathione reductase (GR). To measure GPx activity, the subsequent reduction of the co-substrate NADPH was monitored at 340nm as outlined in (Weydert & Cullen 2010 Nature Protocols). Briefly, a GPx buffer containing 0.5M phosphate (pH 7.2), 100mM EDTA and 1.1mM sodium azide was prepared. The GPx assay buffer consisted of 1.33mM GSSH and 1.33U / ml GR of GPx buffer. The assay solution consisted of 160μl GPx assay solution, 10μl NADPH (5mM), 20μl hydrogen peroxide (0.5%) and 15μl homogenate. The absorbance at 340nm was monitored for 3 minutes and the linear portion of the curve was evaluated for GPx activity.

[0156] Lipid peroxidation

[0157] Adapted from Gerard-Monnier, Chem. Red Toxicol. 1998, lipid peroxidation was assessed as bound propylene glycol (MDA) hydrolyzed in the presence of butylated hydroxytoluene (BHT). The reaction solution contained 10 mM 1-methyl-2-phenylindole in a mixture of acetonitrile:methanol in a ratio of 3:1. 20 μl of sample and 40 μl of 37% HCl were added to 120 μl of this solution and allowed to react at 100°C for 60 minutes. The absorbance of the solution thus obtained was measured at 550 nm and compared with an MDA standard solution.

[0158] result

[0159] After transfer to medium containing 3% hydrogen peroxide, flies adapted to the untreated medium showed 50% mortality after 3.4 days, which is represented by a 33.3% change compared to the untreated control group ( Figure 3 a; p < 0.01). This reduction in survival was significantly enhanced by Lf5221 and probiotic treatment (p < 0.01). Notably, the probiotic was more efficient than Lp8826 and Bi702255 in improving the survival of hydrogen peroxide-treated flies. All treatment groups significantly reduced measures of total oxidants, with no significant improvement with the probiotic compared to the individual probiotic treatments ( Figure 3 b; p<0.01).

[0160] The activity of antioxidant enzymes reflects not only the ability of the flies to cope with oxidative challenges, but also the level of inherent protection due to probiotic treatment. The moderately elevated SOD activity (132.3%) in the hydrogen peroxide-treated control group was not affected by any of the probiotic treatments. In contrast, the significantly elevated glutathione peroxidase activity (192.9%) in the hydrogen peroxide-treated control group was significantly reduced by both Lp8826 and the probiotic preparation treatment, with the probiotic preparation having a greater effect than any of the individual probiotic groups, including Lp8826 ( Figure 3 d). Finally, the significantly elevated LPO levels in the hydrogen peroxide-treated control group (278.4%) were significantly reduced by Lp8826, Lf5221 and the probiotic formulation, and the probiotic formulation completely rescued the levels of lipid peroxidation ( Figure 3 e).

[0161] in conclusion

[0162] Acute oxidative stress challenge reduces the survival rate of fruit flies and the activity of two key antioxidant enzymes SOD and GPx, and increases the level of total oxidants and lipid peroxidation. In terms of increasing the survival rate to oxidative stress and reducing the level of total oxidants and lipid peroxidation, the exemplary probiotic preparations provided herein are the most effective; however, the exemplary probiotic preparations also show a beneficial effect on the activity of the increased SOD and GPx antioxidant enzymes.

[0163] Example 4: Example compositions for treating neurodegenerative diseases

[0164] The following examples illustrate the effectiveness of exemplary probiotic preparations for treating or ameliorating neurodegenerative diseases including Alzheimer's disease (AD) and Parkinson's disease (PD), or slowing the progression of neurodegenerative diseases including Alzheimer's disease (AD) and Parkinson's disease (PD), or slowing the occurrence of neurodegenerative diseases including Alzheimer's disease (AD) and Parkinson's disease (PD).

[0165] We show that modifying the gut microbiota with probiotics can mitigate the three main environmental causes of neurodegeneration (inflammation, oxidative stress, and faulty metabolism) and thus improve molecular markers of the disease. This unique construct exploits the gut-brain-axis mechanism to treat neurodegenerative pathology at its source in a sustainable and safe manner.

[0166] method

[0167] Drosophila genetic models of Parkinson's and Alzheimer's diseases

[0168] Genetic models of PD and AD are established in Drosophila using the UAS (upstream activating sequence)-GAL4 system. For PD, a neuron-specific elav-GAL4 driver (Indiana University, Bloomington, IN) is used to mate female A30P flies with male elav-GAL4 flies, and highly aggregated human α-synuclein mutants (UAS-A30P are kindly donated by Dr. Mel Feany of Harvard University, Massachusetts) will be expressed in Drosophila neurons. After 30 days, the expression of A30P-α-synuclein induces PD phenotypes, for example, see Feany & Bender, 2000. UAS-(β-secretase) BACE1-APP gene (Indiana University, Bloomington, IN) is expressed in neurons using elav-GAL4 to establish an AD model. Drosophila have an endogenous APP ortholog and the γ-secretase machinery but Drosophila β-secretase has low activity and Drosophila APP lacks homology in the region corresponding to the deleterious Aβ peptide, see, e.g., Luo, Tully, & White, 1992. However, expression of human BACE1 produces Aβ-like particles within 3-4 weeks that stimulate AD pathogenesis.

[0169] After eclosion, flies were placed on medium supplemented with control, Lf5221, TFLA, or probiotic formulations. Bottles were changed every 3 to 4 days to prevent accumulation of waste and mold.

[0170] Physiological tests

[0171] The entire lifespan and motility of flies with PD and AD were evaluated to detect the impact of their age on overall health. The lifespan of fruit flies was calculated by first dividing 10 flies into separate vials and then counting the number of live flies in each treatment group every day. Each test was performed with 5 individual vials of 10 flies. Motility was assessed using a negative geotaxis test. Briefly, 10 flies were lightly anesthetized and transferred to an empty bottle 15 cm high. After acclimating to the environment for 45 minutes, the flies were gently tapped to the bottom of the bottle, and the time it took for 50% of the flies to reach the 10 cm mark on the bottle was recorded. Each test was repeated with three independent samples of 10 flies.

[0172] Metabolic testing

[0173] To assess metabolic activity in AD and PD flies, total glucose and total triglyceride assays were performed as described above.

[0174] Acetylcholinesterase activity assay

[0175] ACh activity in fly head homogenates was assessed using a modified Ellman's method, see, e.g., Benabent, Vilanova, Sogorb, & Estevez, 2014. Homogenize 20 fly heads with 200 μL of ice-cold Tris-EDTA-Triton X-100 in the presence of protease inhibitors and centrifuge at 10,000 rpm for 2 minutes to remove body debris. Add 5 μL of the homogenate to 100 μL of 10 mM thioacetylcholine and incubate at 37°C for 10 minutes for the enzyme reaction. Stop the reaction with 50 μL of 6 mM dibenzothiazole disulfide (2-nitrobenzoic acid). After dilution with 50 μL of buffer, measure the absorbance at 410 nm and normalize the total acetylcholine activity to the total protein of the sample.

[0176] Total amyloid content

[0177] Total amyloid content was assessed using the Thioflavin T (Tht) assay protocol, see, e.g., Khurana et al., 2005. Similar to the ACh activity assay, 20 Drosophila heads were homogenized in Tris-EDTA-Triton X-100 buffer containing protease inhibitors and centrifuged to remove cell debris. A stock solution was prepared by diluting 8 mg of Tht into 10 mL of PBS, pH 7.0. For the working solution, the Tht stock solution was diluted 1:5 into PBS and 2 μL of the sample homogenate was added to 198 μL of the working Tht solution. Fluorescence was measured at 440 nm excitation / 482 nm emission and the total quantification of amyloid was normalized to the total protein in the sample.

[0178] Dopamine quantification

[0179] Dopamine quantification will be performed using a commercial rapid dopamine ELISA kit (LDN; Nordhom Germany) with some modifications. A homogenate of 40 fly heads was prepared in PBS with assay buffer (1 M HCl) before addition to the elution plate. The rest of the procedure was performed according to the manufacturer's instructions.

[0180] result

[0181] AD and PD pathology were measured by a wide range of physiological parameters (lifespan and motility) as well as specific neural markers of disease. In AD flies, control flies had only 40% survival at day 30, while flies treated with Lf5221 and the probiotic formulation had no significant decrease in survival (30% at day 30), while the probiotic formulation significantly increased survival to 60% ( Figure 4a; p < 0.01). A similar trend was observed in PD flies. The survival rate of control flies was 40%, while there was no change in survival after Lf5221 treatment, and the probiotic preparation increased the survival rate to 60% ( Figure 4 b; p < 0.01). Motility is a good indicator of the overall physical fitness of aging flies. Over time, the negative geotaxis test time of AD control flies increased from 6.4 s, 7.5 s, 8.2 s, 12 s on days 0, 10, 20, and 30 ( Figure 4 c; p<0.01). Although the negative geotaxis time of both Lf5221 and probiotic preparation groups increased significantly from day 20, the negative geotaxis time of the probiotic preparation group was significantly reduced compared with the Lf5221 control group on day 30 (p<0.01). In PD flies, the time to complete the negative geotaxis test in the control group also increased steadily from 5.7s, 8.1s, 10.1s, and 14.3s on days 0, 10, 20, and 30 ( Figure 4 d; p<0.01). The negative geotaxis time of the Lf5221 group and the probiotic preparation group was significantly higher on the 30th day (p<0.01) and on the 20th day (p<0.01). On the 10th, 20th, and 30th days, the negative geotaxis time of the Lf5221 group and the probiotic preparation group was significantly lower than that of the control group (p<0.01).

[0182] To measure the specific disease pathology of AD, we measured the levels of Aβ plaques and ACh activity in Drosophila head homogenates over time. As expected, the number of Aβ plaques in the control group increased by 30% on day 30 ( Figure 4 e; p<0.01). On the 30th day, the ACh activity of the Lf5221 group (26%) and the probiotic preparation group (21%) increased significantly, the Lf5221 group was significantly lower than the control group (p<0.05), and the probiotic preparation was lower than the Lf5221 group and the control group (p<0.01). As expected, the ACh activity of the control group decreased significantly on the 20th and 30th days ( Figure 4 g; p<0.01). Compared with Lf5221 and control groups, the Lf5221-treated group significantly increased ACh activity on days 20 and 30, while the probiotic preparation significantly increased ACh activity on days 20 and 30 (p<0.01).

[0183] To measure the progression of PD, we measured dopamine levels in head homogenates. As expected, total dopamine levels in the control group steadily decreased from 0.67, 0.61, 0.55 to 0.52 μg / mg protein on days 0, 10, 20, and 30, respectively ( Figure 4 f; p<0.01). By day 30, dopamine levels in both Lf5221 and probiotic preparation treatment groups were significantly increased (p<0.01).

[0184] We evaluated changes in basic metabolic parameters in aged AD and PD flies. In AD flies, total glucose levels in control flies rose to 1.24 (g / mg protein on day 30, a 60% increase compared to day 0 ( Figure 4 h; p<0.01). Although the Lf5221 and probiotic preparation groups also increased significantly on day 30, the increase in the probiotic preparation group was significantly lower than that in the control group and Lf5221 group (p<0.05). On day 30, the total glucose of PD flies also increased significantly to 1.11 μg / mg protein, an increase of 27% compared with day 0 ( Figure 4 i; p<0.01). The blood glucose level of the control group was significantly higher than that of the Lf5221 group and the probiotic preparation treatment group (p<0.05). The total triglyceride level of the AD control group fruit flies increased by 20% on the 30th day ( Figure 4 j; p>0.05). However, the probiotic preparation significantly reduced total triglyceride levels on days 20 and 30 compared with the control and Lf5221 groups (p<0.05). In flies in the PD control group, total triglyceride increased significantly on days 10, 20, and 30, increasing by 88% on day 30 ( Figure 4 k, p<0.01). On day 30, Lf5221 and probiotic preparations reduced total triglyceride levels significantly less than the control group, and the probiotic preparation was more effective than Lf5221 in reducing total triglyceride levels (p<0.01).

[0185] Oxidative stress levels were measured by measuring total oxidants, peroxidation, and antioxidant enzyme activity. In AD flies, total oxidants increased by 30% from day 0 to day 30 compared with control flies ( Figure 4 l; p<0.01). At day 30, total oxidant levels were reduced by 5% in Lf5221-treated AD flies, while the probiotic formulation reduced them by 11%: both reductions were significantly less than the increase in control flies. Superoxide dismutase activity in control AD ​​flies was significantly reduced by 36%, while it remained low at day 30 in the Lf5221 (41% reduction) and probiotic formulation groups (38% reduction). In contrast, glutathione peroxidase activity was relatively unchanged at day 30 in control flies (increased by 9%); however, GPx activity was slightly increased (34%) in the Lf5221-treated group, while it was greatly increased (257%) by the probiotic formulation at day 30 ( Figure 4 l; p<0.01). Finally, LPO levels were significantly increased by 38% in the control flies. LPO levels were also significantly increased in the Lf5221-treated group (62%); however, the probiotic formulation significantly reduced LPO levels (41% reduction) compared to both the control and lf5221-treated flies ( Figure 4 l; p<0.01).

[0186] Similar effects were found in PD flies. In the control group, total oxidants increased by 24% by day 30, which was rescued by Lf5221 treatment and reduced by 20% by the probiotic preparation ( Figure 4 m; p<0.01). Again, in aged PD flies, SOD levels were slightly reduced in all groups. On day 30, GPx activity was significantly reduced in control flies (15%), but Lf5221 activity was increased (34%), and the probiotic preparation increased it by 252%, which was very effective ( Figure 4 m, p<0.01). Finally, on day 30, the LPO level in the control group increased significantly to 37%, which was rescued by the probiotic preparation ( Figure 4 m; p<0.01).

[0187] The production of antimicrobial agents was directly measured by agar diffusion test to determine the activity of the immune system. The diameter of the inhibition zone was recorded as the inhibition zone. In the AD control fruit flies, the inhibition zone of the aged fruit flies decreased significantly from the 10th day, indicating that the fruit flies produced less antimicrobial agents ( Figure 4 n; p < 0.01). In contrast, reduced inhibition zones were observed only on day 30 in flies treated with Lf5221 while no reduction in inhibition zones was observed in flies treated with the probiotic formulation, suggesting that probiotic treatment significantly enhances the production of antimicrobial agents and thus immune tolerance in aged AD flies ( Figure 4 n; p<0.01). Similarly, in PD flies, the inhibition zone of the control flies decreased significantly from the 10th day ( Figure 4 o); p<0.01). Treatment with Lf5221 or probiotic preparations delayed the reduction of the inhibition zone to day 30 ( Figure 4 o; p<0.01). Several immunological gene expressions were also examined to determine the specific immune response of aged AD and PD flies. In the control group AD flies, Duox expression was significantly decreased, and the expression of IMD and AMPs antimicrobial peptide A, defensin, and dipterin was significantly increased ( Figure 4 p; p<0.01). The probiotic preparation significantly increased the expression of Duox, while both Lf5221 and the probiotic preparation decreased the expression of IMD ( Figure 4 p; p<0.05). Although the probiotic preparation significantly reduced the expression of defensins, the probiotic treatment had little effect on the expression of AMPs. In the control PD flies, the expression of Duox and IMD was significantly reduced, while the expression of AMPs was also increased ( Figure 4 q; p<0.01). Lf5221 treatment only reduced the expression of antimicrobial peptides, while the probiotic preparation significantly reduced the expression of IMD, defensins, and antimicrobial peptides ( Figure 4q; p<0.01). With age, the fly immune system is known to become less efficient, leading to an increased pathogenic load. This could explain the decrease in arbitrary immune genes (Duox and IMD) and the increase in pathogen-specific factors (AMPs).

[0188] Finally, the activity of mitochondrial ETC complexes was assessed in individual aged AD and PD flies. In control AD ​​flies, the activity of complexes 1, 2, and 3 was significantly decreased ( Figure 4 r; p<0.01). Lf5221 treatment significantly increased the activity of complex 3 on day 30 (p<0.05), while the probiotic preparation increased the activity of all complexes ( Figure 4 r; p<0.05). In the control PD flies, all mitochondrial ETC complexes 1 to 4 were significantly reduced. Both Lf5221 and the probiotic preparation significantly increased the activities of mitochondrial complexes 1, 2, and 4, while the probiotic preparation was more effective in increasing the activities of ETC complexes 2 and 3 ( Figure 4 s; p<0.05).

[0189] in conclusion

[0190] Many typical physiological changes in AD and PD, including disease markers, metabolic changes, immune stress, oxidative stress, and mitochondrial complex activity, are positively affected by administration of the exemplary probiotic formulations provided herein, which provide more beneficial effects than a single probiotic alone.

[0191] Example 5: Example Compositions in Combination with Triphala L. (TFLA)

[0192] The following examples illustrate the efficacy of exemplary probiotic formulations in combination with Triphala lanolin (TFLA).

[0193] In an optional embodiment, the article of manufacture, composition, formulation, pharmaceutical composition or kit further comprises Triphala Lalala (TFLA), which is a multi-herbal formula known in the traditional Indian medical practice (Ayurveda) that has a positive effect on digestive discomfort. Traditionally, TFLA is taken as a cold infusion, where its powdered formulation is mixed in cold water and taken on an empty stomach to increase gastrointestinal transit, relieve bloating and increase nutrient absorption. TFLA literally means "three fruits" and the formula contains equal amounts of Emblica officinalis (Amla), Terminalia chebula (Myrobalan) and Terminalia chebula (Herba Chebula). TFLA is an antioxidant-rich herbal formula and a rasayana (rejuvenating agent) with adaptogenic properties. Recently, the ethnomedicine claims of TFLA have been confirmed by researchers, and these activities include antioxidant, anti-inflammatory, maintenance of serum cholesterol levels, analgesia, wound healing, homeopathy, hypoglycemia and chemopreventive effects. Furthermore, the traditional use of TFLA as a gastrointestinal tonic includes relief of gastrointestinal tension, constipation, relief of diarrhea, symptoms of IBD and IBS, and increased gastrointestinal motility, all of which suggest that TFLA may have prebiotic activity and that it produces some of its beneficial effects by modifying the intestinal flora.

[0194] Based on the wide range of physiological effects of TFLA and its untapped potential as a prebiotic preparation, the TFLA-water extract was combined with the exemplary probiotic preparation provided herein to create a novel synbiotic preparation (a product containing both probiotics and prebiotics). This exemplary preparation was tested to determine that the addition of the prebiotic agent did not affect the in vivo activity of the probiotic preparation.

[0195] We tested whether a novel synbiotic formulation prepared by adding TFLA of a plant formula to the exemplary probiotic formulation provided herein would have any effect on the efficacy of the probiotic formulation: the results showed that this synbiotic formulation did not affect the efficacy of the original exemplary probiotic formulation, or enhanced its efficacy.

[0196] method

[0197] Triphala and synbiotic preparations

[0198] The dry ingredients of Triphala (Emblica officinalis, Terminalia chebula and Terminalia chebula) were obtained from the Ayurvedic Pharmacy, Banaras Hindu University, Varanasi, India. Each ingredient was weighed individually and combined in equal proportions (by weight) before being crushed and ground into a fine powder manually using a mortar and pestle. The fine powder thus obtained was used for all extractions. The extraction method was to gently stir 5 g of Triphala powder in 100 mL of double distilled water at room temperature for 72 h. After extraction, the solvent phase was filtered and rotary evaporated under vacuum pressure at 40 °C to achieve drying. The dry mass was recorded, reconstituted in 100 mL of double distilled water and stored at 4 °C until further processing.

[0199] To prepare TFLA and synbiotic media for Drosophila, the TFLA extract described above was processed into a 10X preparation and added to the cooled liquid culture medium along with the probiotic preparation described above. All physiological tests were performed as described in the above section.

[0200] result

[0201] TFLA and synbiotic preparations were tested on the same metabolic, inflammatory, and oxidative stress combinations in AD and PD neurodegeneration models. In the diabetic HSD metabolic model, total body weight was reduced by 30% and 22% in the TFLA and synbiotic groups, respectively ( Figure 5 ai, p<0.01). In addition, TFLA and synbiotic preparations rescued circulating glucose levels ( Figure 4 aii, p<0.05) and total glucose levels ( Figure 4 iii; p<0.01 in HSD-treated flies). Similarly, in the HFD metabolic model of obesity, weight gain was significantly reduced in flies treated with HFD using either TFLA (8.5%) or synbiotic formulations (20%) ( Figure 5 bi; p<0.01). TFLA and synbiotic preparations also rescued the increase in total glucose in HFD-treated flies ( Figure 5 bii, p<0.01) and total triglyceride levels ( Figure 5 biii; p<0.01). These results suggest that TFLA is highly effective in reducing metabolic stress markers and that its combination with a probiotic formulation does not alter its efficacy against metabolic shock and may even enhance its effectiveness as a synbiotic formulation.

[0202] Also received TFLA or synbiotic preparations pretreatment, followed by 1.0x10 10 Inflammatory markers were assessed in flies acutely oral challenged with either CFU / ml E. coli or S. aureus. In response to E. coli stress, the nearly 70% reduction in survival in the control group was significantly increased by both TFLA (25% reduction) and the synbiotic formulation (20% reduction). Figure 5 ci; p<0.01). In the agar diffusion assay, the inhibition zones of the TFLA and synbiotic preparations also increased significantly, with the synbiotic preparation having a larger inhibition zone compared to the TFLA group alone ( Figure 5cii; p<0.01). In terms of typical inflammatory gene expression, Duox expression in the control group increased significantly after E. coli infection, while TFLA and synbiotic preparations significantly reduced this effect (Figure ciii; p<0.01). Similar to the expression of IMD, the strong increase in IMD in the control group was significantly reduced by TFLA and synbiotic preparations (Figure civ; p<0.01). Antimicrobial peptide A and Gram-negative sensitive AMP were also significantly reduced by TFLA and synbiotic preparations ( Figure 5 Finally, in the control E. coli treatment group, the expression of Gram-negative sensitive AMP fly antimicrobial peptide was also extremely high, but both were significantly reduced by TFLA and synbiotic treatment ( Figure 5 c vi; p<0.01). In general, after oral infection with E. coli, TFLA treatment had a strong anti-inflammatory effect and did not affect the effect of the probiotic preparation in the synbiotic preparation, and even enhanced the effect of the probiotic preparation.

[0203] Acute oral infection with the Gram-positive pathogen Staphylococcus aureus was also performed. The survival rate of flies exposed to S. aureus was significantly reduced by 70% in the control group, 35% after TFLA treatment, and 20% with the synbiotic formulation (data not shown; p<0.01). Similarly, TFLA and synbiotic formulations increased the clearance diameter by 72% and 98%, respectively, by agar diffusion assay (data not shown, p<0.01). In terms of gene expression, duox expression was significantly increased in S. aureus treated control flies and significantly decreased under synbiotic formulation (data not shown; p<0.01). The expression of IMD was moderately increased by S. aureus and further increased by TFLA and synbiotic formulations (data not shown, p<0.01). In control flies, expression of AMP antimicrobial peptide A was elevated by 180%, whereas both TFLA treatment (43% increase) and the synbiotic formulation (7% increase) significantly reduced this expression (data not shown, p < 0.01). Finally, Gram-positive responses to AMP defensins were significantly elevated by nearly 300% following treatment with S. aureus, whereas TFLA and synbiotic formulation treatment completely rescued this response (data not shown, p < 0.01). Similar to oral infection with E. coli, S. aureus infection produced a potent immune response, but treatment with TFLA and synbiotic formulations attenuated this response; similar to the probiotic formulation, this suggests that prebiotic supplementation does not affect the efficacy of the probiotic formulation.

[0204] Acute oxidative stress was introduced by exposing flies to hydrogen peroxide after pretreatment with either TFLA or synbiotics. After exposure to hydrogen peroxide, the survival of control flies was greatly reduced by nearly 70%, while the survival of the TFLA-treated group was reduced by 30%, and the survival of the synbiotic group was reduced by 25% (data not shown; p<0.01). The increase in total oxidant levels in the control group was also significantly reduced by TFLA treatment and rescued by synbiotic treatment (data not shown; p<0.01). Superoxide dismutase activity was increased in the control group under hydrogen peroxide stress, which was significantly reduced by TFLA pretreatment and rescued by synbiotic pretreatment (data not shown; p<0.01), indicating that pretreatment can protect flies from a significant antioxidant response. Glutathione peroxidase was also significantly increased in the control group treated with hydrogen peroxide, while it was significantly reduced by synbiotic alone (data not shown; p<0.01). Finally, lipid peroxidation levels were significantly increased by nearly 300% in the control group, an increase that was completely rescued by TFLA pretreatment and the synbiotic preparation (data not shown; p<0.01). The reduction in total oxidants and enzyme induction by antioxidants suggests that, similar to the probiotic preparation, TFLA and synbiotics together have a higher intrinsic antioxidant defense and that the combination of TFLA with the probiotic preparation does not hinder the efficacy of the probiotic preparation against oxidative stress.

[0205] The effects of TFLA and synbiotics were also tested in flies with AD and PD. In AD flies, both TFLA and synbiotics increased the survival of aging flies, and synbiotics were more effective than TFLA (data not shown; p<0.01). Similarly, in PD flies, both TFLA and synbiotics significantly increased the survival of flies, and synbiotics were more effective than TFLA alone (data not shown; p<0.05). Measuring motility, aging AD flies gradually lost motility, most significantly at day 30. TFLA and synbiotics significantly increased the motility of 12% and 39% of AD flies, respectively (data not shown, p<0.05). The decrease in motility of PD flies in the control group became increasingly evident at day 10 and continued to worsen. Both TFLA and synbiotics delayed the onset of motor impairment until day 20, and significantly increased motility at days 20 and 30 compared with the control group (data not shown; p<0.01).

[0206] As expected, Aβ levels increased over time in control AD ​​flies. Although there was some variation, both TFLA and synbiotic preparations reduced total Aβ accumulation by day 30, almost to the level of flies at day 0 (data not shown; p < 0.01). Similarly, for acetylcholinesterase activity, as predicted, ACh activity decreased over time in control AD ​​flies, while TFLA and synbiotic preparations significantly increased ACh activity at day 30 (data not shown; p < 0.01). Dopamine levels in control PD flies decreased significantly starting at day 10 and continued to decrease until day 30. Both synbiotic preparations delayed the decrease in dopamine levels until day 30 compared to the control group, and both TFLA and synbiotic preparations induced an increase in dopamine levels at day 30 (data not shown; p < 0.05).

[0207] Finally, to assess the mechanisms by which TFLA and synbiotics affect the gut-brain axis in the context of AD and PD, we assessed the same physiological markers. In control AD ​​flies, total glucose levels increased significantly by 157%, and this level was significantly reduced by the synbiotics (data not shown; p<0.01). Similarly, total triglyceride levels increased by 120% in control AD ​​flies, and this level was significantly reduced by synbiotics treatment (data not shown; p<0.01). In PD flies, total glucose levels increased significantly by 127%, and were unchanged by either TFLA or synbiotics (data not shown; p>0.05). However, in PD flies, total triglycerides increased significantly by 187%, and were significantly reduced by TFLA and synbiotics, by 143% and 119%, respectively (data not shown; p<0.01), with the synbiotics being more effective compared to TFLA treatment.

[0208] The same panel of inflammatory markers in aged flies was assessed in AD and PD flies treated with TFLA or synbiotics. In PD flies, Duox expression was significantly reduced by 50% at day 30 (data not shown; p < 0.01), whereas synbiotic treatment significantly increased Duox expression. IMD was elevated in aged PD flies, although neither TFLA nor synbiotics significantly affected its expression. Relish was not significantly altered over time in controls, despite a significant decrease in its expression following TFLA treatment. AMP expression was significantly elevated in aged PD flies in controls. The 180% increase in anthelmin A was not affected by TFLA or synbiotics (data not shown; p > 0.05). However, TFLA and synbiotics completely rescued a similar increase in defensins (p < 0.01). Finally, TFLA did not affect the nearly 200% increase in anthelmin expression, although synbiotics significantly reduced it.

[0209] In PD flies, Duox expression was reduced in aged control flies and was not affected by TFLA or synbiotics (data not shown; p>0.05). In aged control flies, IMD expression was reduced by approximately 20%, while TFLA and synbiotics significantly increased it (data not shown; p<0.01). Similar to the AD group, Relish expression did not change significantly with age or treatment. AMP expression was increased in PD flies in aged controls. The almost 200% increase in antimicrobial peptide A was significantly reduced by TFLA and synbiotics treatment (data not shown; p<0.01). Similarly, the increase in defensin expression levels was only reduced by synbiotics, but not by TFLA (p<0.01). Finally, both TFLA and synbiotics significantly reduced the nearly 300% increase in antimicrobial peptide expression in flies (p<0.01). In general, TFLA and synbiotic preparations had positive effects on affected immune parameters in Drosophila disease models of AD and PD, and the combination of TFLA and probiotic preparations did not negatively affect their efficacy and even improved it in some cases.

[0210] Considering markers of oxidative stress, total oxidants increased by 30% in control flies in the aged AD model. Both TFLA and synbiotic preparations significantly reduced this increase, with synbiotic preparations being more effective than TFLA treatment alone (data not shown; p<0.01). In aged control flies, superoxide dismutase activity was relatively unchanged and had no effect on either TFLA or synbiotic treatment. Glutathione peroxidase activity increased by nearly 10% in aged control AD ​​flies, which was significantly reduced by TFLA treatment (data not shown; p<0.01). Finally, lipid peroxidation levels increased by 38% in aged control AD ​​flies, which was significantly reduced by both TFLA and synbiotic preparations, with synbiotic preparations being slightly better than TFLA preparations in reducing LPO levels (data not shown; p<0.01). In aged PD flies, 24% of the increase in total oxidants in control flies was reduced only by synbiotic preparations (data not shown; p<0.01). Similar to AD fruit flies, there was no significant difference in SOD activity in the aging flies of the control group, and neither TFLA treatment nor synbiotic preparation treatment had much effect on SOD activity.

[0211] GPx activity was slightly reduced in control aged PD flies, but was significantly increased by both TFLA and synbiotic treatments, with TFLA treatment increasing GPx activity significantly more than synbiotic treatment (data not shown; p < 0.01). Finally, the 37% increase in lipid peroxidation in control aged PD flies was significantly reduced only by synbiotic treatment, which actually completely reduced LPO levels to levels seen in young controls (data not shown; p < 0.01). TFLA prebiotics significantly affected several markers of oxidative stress, and the combination with the probiotic did not affect the efficacy of the preparation and even enhanced some parameters.

[0212] Mitochondrial ETC complex activity in AD and PD was also affected by treatment with TFLA and synbiotics. The activity of ETC complex 1 in aged AD flies in the control group was reduced by 57% after 30 days, whereas complex 1 activity was significantly increased with TFLA synbiotics but not TFLA treatment (data not shown; p<0.05). The activity of ETC complex 2 was also reduced in aged AD flies, unaffected by TFLA treatment, and completely rescued by synbiotics at day 30 (data not shown; p<0.01). The activity of ETC complex 3 was reduced by nearly 50% in aged AD flies in the control group, whereas TFLA and synbiotics increased the activity of ETC complex 3 to the same extent (data not shown; p<0.01). Finally, ETC complex 4 in AD flies was not affected by TFLA and synbiotics over time (data not shown; p>0.05). In aged PD flies, the activity of ETC complex 1 was significantly reduced by 70%, and this reduction was significantly increased after TFLA and synbiotic treatment (data not shown; p<0.01). In control PD flies, the activity of ETC complex 2 was reduced by more than 50%, and TFLA and synbiotic treatment further increased the activity of ETC complex 2 (data not shown; p<0.01). There was a very strong 80% reduction in the activity of ETC complex 3 in aged PD flies, which was only increased by synbiotic treatment (data not shown; p<0.05). Finally, in aged PD flies, the activity of ETC complex 4 was reduced by nearly 60%, which was only increased by synbiotic treatment (data not shown; p<0.05). In summary, the reduction in the activity of ETC complexes in both AD and PD fly models was increased by TFLA treatment and the combination of TFLA and probiotic treatment, such that the synbiotic treatment did not affect the efficacy of the treatment and increased the efficacy of the treatment.

[0213] in conclusion

[0214] The overall combination of the prebiotic plant product TFLA with the probiotic formulation did not have any effect on the efficacy of the exemplary synbiotic formulations and even improved their efficacy in some cases.

[0215] Example 6: Exemplary methods for enhancing lifespan: Drosophila melanogaster extends lifespan via gut-brain-axis communication

[0216] The following examples describe data showing the efficacy of exemplary compositions and methods provided herein for enhancing (increasing) lifespan and improving aging.

[0217] In this study, novel probiotic and synbiotic formulations were shown to combinatorially extend lifespan in Drosophila melanogaster through gut-brain-axis communication mechanisms relevant to chronic disease management. Both probiotic and synbiotic formulations rescued metabolic stress markers by managing insulin resistance and energy regulatory pathways. Aging and the development of chronic diseases are multifactorial processes involving the cumulative effects of metabolic disturbances, inflammation, oxidative stress, and mitochondrial dynamics, and changes in the gut microbiota are associated with age-related phenotypes.

[0218] Both formulations also improved inflammation, elevated oxidative stress, and loss of mitochondrial complex integrity. In nearly all pathways measured, the synbiotic formulation had more powerful effects than its individual components, indicating a combined effect. The concomitant effects of the gut microbiota on each of the key risk factors for aging make it a powerful therapeutic tool to combat neurodegeneration, diabetes, obesity, cardiovascular disease, and other age-related chronic diseases.

[0219] This study describes how novel probiotic and synbiotic preparations affect lifespan in Drosophila melanogaster via a GBA mechanism. The current probiotic and synbiotic preparations exhibit combined effects in reducing physiological stress markers, oxidative stress, inflammation, and mitochondrial ETC complex integrity, thus targeting most of the major mechanisms of aging. This effect will not only benefit longevity, but will also prevent many age-related chronic diseases associated with the above-mentioned states.

[0220] result

[0221] Probiotic and synbiotic preparations have beneficial effects on physiological and metabolic markers of aging in Drosophila melanogaster

[0222] Starting from the first day after eclosion, aged flies were exposed to probiotics, prebiotics, probiotics, or synbiotics, and physiological metabolic parameters were measured every 10 days for 30 days. As expected, total weight of untreated control flies increased in a time-dependent manner (Figure 6a; F(3,18) = 3.24; p < 0.05). This trend was observed in all groups; however, at day 30, total weight was significantly reduced in the probiotic and synbiotic groups compared with the control, individual probiotic, and TFLA-treated groups (Figure 6a). Similarly, total glucose measurements in aged flies were also time-dependent, with total glucose peaking at day 30 in control flies, increasing 2.5-fold (Figure 6b; F(3,18) = 4.13, p < 0.05). Each individual probiotic and TFLA prebiotic showed similar time-dependent increases in total glucose levels, but at day 30, total glucose levels were reduced in all groups. Notably, the probiotic and synbiotic preparations completely rescued the increase in total glucose at day 30, which was significantly lower than that of the Lp8826, Bi702255, and TFLA treatment groups (p < 0.01). Finally, total triglyceride levels in the untreated control group (F(3,18) = 5.34, p < 0.05) increased 1.7-fold at day 30 (Fig. 6c, p < 0.01). Total triglyceride levels decreased in the Lf5221, Bi702255, probiotic, and synbiotic preparation groups by day 30, with total triglyceride levels in the Lf5221 and synbiotic preparation groups completely decreasing to the levels of day 0 flies. Overall, the probiotic preparations were the most effective in reducing all physiological markers of metabolic stress in Drosophila, while the synbiotic preparations showed almost the same effects.

[0223] Insulin-like signaling in aged Drosophila melanogaster is positively affected by probiotic and / or prebiotic treatment

[0224] To evaluate the potential genetic regulatory mechanisms underlying the metabolic effects of prebiotic or probiotic supplementation, the expression of a panel of insulin signaling genes was assessed using real-time PCR. Insulin-like peptide (dilp) 2 expression in control flies (F(3,18) = 3.47, p < 0.05) was elevated at days 20 and 30 (Fig. 7a; p < 0.01). Only the TFLA-supplemented group reduced dilp2 expression at day 20, while Bi702255 and the synbiotic formulation reduced dilp2 expression by 33% and 57%, respectively, at day 30 (p < 0.01). Notably, only the synbiotic formulation was able to rescue dilp2 expression, which was significantly lower than that of any other treatment group. Similarly, dilp3 expression in the control group (F(3,18) = 4.87, p < 0.05) was elevated at day 30 (Fig. 7b; p < 0.01), with significant decreases in all groups. Lp8826 and TFLA supplementation restored dilp3 expression to the level of the control on day 0, while probiotic and synbiotic preparations further reduced dilp3 expression; it was significantly lower than Lf5221 and Bi702255 on day 30. The insulin receptor (InR) of the control flies (F(3,18=4.53, p<0.05) was also increased on days 20 and 30 (Fig. 7c; p<0.01). On day 20, only the probiotic preparation significantly reduced the expression of InR, while on day 30, Lp8826, Lf5221, probiotics and synbiotic preparations all significantly reduced the expression of InR (p<0.01). It is worth noting that on day 30, the level of InR reduced by the probiotic preparation was significantly higher than all other groups, while the synbiotic preparation was lower than the groups without probiotic preparation.

[0225] We obtained similar but interesting results by examining the insulin signaling pathway downstream of the insulin receptor. Expression of dAkt in aged control flies (F(3,18)=4.82, p<0.05) increased from day 10 to day 30 (Fig. 7d; p<0.01). At day 30, Lp8826, TFLA, and synbiotic supplementation all significantly reduced dAkt expression (p<0.05). Notably, TFLA, probiotics, and synbiotics completely rescued dAkt levels at day 30, and the synbiotics reduced dAkt expression more than Lf5221 and Bi702255. A direct downstream of dAkt is dTOR (F(3,18)=4.91, p<0.01), whose expression in control flies was also increased from day 10 to day 30 (Fig. 7e; p<0.01). At day 30, only the Lp8826 group did not reduce dTOR levels, while TFLA, probiotics, and synbiotics all completely rescued expression. Finally, the transcription factor dFOXO (F(3,18)=4.12, p<0.01), which is downregulated by dAkt, was downregulated from day 10 to day 30 in control aged flies (Figure 7f; p<0.01). Only probiotics and synbiotics increased the expression level of dFOXO, and only the synbiotics completely rescued its expression to the level of the day 0 control group. Overall, individual probiotics and TFLA had different but positive effects on the insulin signaling pathway in aged flies; however, probiotics and synbiotics demonstrated a combined effect to rescue potential insulin signaling dysregulation in aged flies.

[0226] Fatty acid metabolism in aged Drosophila melanogaster benefits from supplementation with probiotics and synbiotic preparations

[0227] To determine the mechanism of elevated total triglycerides in aged flies and how probiotics mitigate this effect, we assessed the expression of several adipogenic and lipogenic regulators. Acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) are both lipogenic factors activated by the transcription factor sterol regulatory element binding protein (SREBP). ACC (F(3,18) = 4.12, p < 0.05) (Figure 8a) and FAS (F(3,18) = 3.91, p < 0.05) ( Figure 3b) were significantly increased from day 10 to day 30 (p<0.01), indicating that lipogenesis was increased in aged control flies. On day 30, Lf5221, TFLA, probiotics, and synbiotics reduced the expression of ACC, with the synbiotics having a greater effect on ACC expression than the probiotics (p<0.05). On day 30, only TFLA, probiotics, and synbiotics reduced the expression of FAS, with TFLA and synbiotics rescuing the expression of FAS to the level of the control group on day 0. The expression of SREBP (F(3,18)=3.2, p<0.05) also showed the same trend (Fig. 8c). Lp826 and Lf5221 reduced the increase on days 20 and 30 (p<0.01), while probiotics and synbiotics rescued the increase on day 30 (p<0.01). Phosphoenolpyruvate carboxykinase (PEPCK), a master regulator of gluconeogenesis, increased in control flies at days 20 and 30, as did lipogenic genes (F(3,18)=4.63, p<0.05) (Fig. 8d; p<0.01). TFLA, probiotics, and synbiotics all reduced PEPCK expression at day 30, and TFLA and synbiotics rescued PEPCK expression to day 0 control levels (p<0.01). Lipid storage droplet (LSD) 2 is expressed in the fat body and controls triglyceride accumulation. LSD2 expression increased at day 30 in control flies (F(3,18)=3.28, p<0.05) (Fig. 8e); and Lp8826, TFLA, probiotics, and synbiotics rescued LSD2 expression (p<0.01). Finally, E75 is a transcriptional target of the Drosophila PPARγ homologue responsible for adipogenesis and insulin sensitivity. E75 expression in the aged control group (F(18) = 5.09, p < 0.05) was significantly decreased between days 10 and 30 (p < 0.01), and on day 30, it was decreased in the Bi702255 group, and only increased in the probiotic and synbiotic groups, and the effect of the synbiotic was greater than that of the probiotic (p < 0.05).

[0228] Inflammatory markers that increase with age are reduced by combined prebiotic and probiotic treatment

[0229] The agar diffusion assay measured the amount of secretable immunogenic factors and their immunogenicity against pathogenic bacterial lawns on nutrient agar plates. The control flies showed a significant reduction in immunogenicity against E. coli (F(3,18) = 3.98, p < 0.05), with a 47% decrease in clearance diameter from day 0 to day 30 (Fig. 9a; p < 0.01). At day 20, TFLA, probiotics, and synbiotic preparations all increased clearance diameter (p < 0.01), while at day 30, all supplementation groups had a positive effect.

[0230] The synbiotic formulation was most effective in increasing clearance diameter at days 20 and 30, supporting its combined effect. A similar trend was seen for clearance diameter on S. aureus plates (F(3,18) = 4.12, p < 0.05), with control flies experiencing a 56% decrease in clearance diameter from day 0 to day 30 (Fig. 9b; p < 0.01). All supplementation groups had a positive effect at days 20 and 30; however, either the probiotic or the synbiotic formulation had a greater effect than either the probiotic alone or TFLA alone.

[0231] In terms of regulation of immune genetic markers, expression of dual oxidase (Duox), a major innate immune response that stimulates ROS release after pathogenic insult, was reduced in untreated flies from day 10 to day 30 (F(3,18)=3.78, p<0.05) (Fig. 9c; p<0.01). Treatment with probiotics and synbiotics rescued Duox expression compared to day 0 controls at day 30, but only at day 30 did synbiotics increase Duox expression compared to untreated controls. Immunodeficiency (IMD) is another innate immune response, but is slightly more sensitive to Gram-negative bacteria. From day 10 to day 30, IMD expression in the control flies (F(3,18)=4.71, p<0.05) decreased (Fig. 9d; p<0.01), and only the synbiotic preparation increased IMD expression on day 30. However, due to differences in baseline expression, Lp8826, TFLA, probiotics, and the synbiotic preparation all effectively rescued IMD expression on day 30 (p>0.05). Relish is a cytokine-like factor downstream of the IMD pathway (F(3,18)=2.54, p>0.05), and its expression in the control flies was upregulated on day 30 (Fig. Figure 4 e; p<0.01). The effects of probiotics were limited and varied, although probiotic and synbiotic preparations abolished any variation in Relish expression in aged flies.

[0232] Antimicrobial peptides (AMPs) are effector molecules downstream of the innate immune system-induced IMD and toll pathways. Both Gram-positive and Gram-negative bacterial insults activated AMP A transcription (F(3,18)=3.73, p<0.05), and in this model, AMP A transcription was significantly increased from days 10 to 30 in control aged flies (Fig. 9f; p<0.01). Bi702255, TFLA, and synbiotic preparations reduced AMP A expression on day 30, and synbiotic preparations reduced AMP A expression to day 0 control levels (p<0.01). Defensins are activated downstream of the toll pathway and Gram-positive bacterial insults. Like AMP A, defensin expression was increased in control aged flies (F(3,18)=4.13, p<0.05) (Fig. 9g); except for Lp8826, it was decreased in all supplemented groups on day 30. Finally, antimicrobial peptides activated downstream of IMD by Gram-negative challenged flies (F(3,18)=2.31, p>0.05) were also elevated in control aged flies from days 10 to 30 (Fig. 9h; p<0.01), although any probiotic and / or prebiotic treatment had little effect.

[0233] Age-related increases in oxidative stress may be mitigated by probiotic and / or prebiotic treatment

[0234] The gradual accumulation of oxidative stress is a major factor contributing to aging, and in this model, we observed an increase in oxidants and a corresponding decrease in antioxidant enzyme activity. Total oxidant levels in control flies (F(3,18) = 3.78, p < 0.05) were significantly elevated at days 20 and 30, peaking at day 30 with a 50% increase (Fig. 10a; p < 0.01). At day 30, only the probiotic or synbiotic preparation supplementation groups reduced total oxidative load, and both groups were significantly greater than the effects of the probiotic or TFLA alone (p < 0.05). Notably, Lf5221, Bi702255, and TFLA all caused a beneficial reduction in total oxidants at day 20. Superoxide dismutase (SOD) is required for the conversion of superoxide anions from less harmful hydrogen peroxide. From day 0 to day 30, SOD activity in flies in the control group (F(3,18)=3.92, p<0.05) decreased by 50% (Fig. 10b; p<0.01), and probiotic or synbiotic preparations had significant effects on SOD activity only on days 10, 20, and 30 (p<0.05). TFLA preparations also increased SOD activity on days 10 and 20; however, the extent of the increase was less than that of probiotic and synbiotic preparations. Similarly, glutathione peroxidase (GPx) activity, which converts hydrogen peroxide to water, decreased by 42% from day 0 to day 30 in aged flies in the control group (F(3,18)=4.08, p<0.05) (Fig. 10c; p<0.01). On day 30, Lf5221, probiotic or synbiotic preparations had a beneficial effect, with the synbiotic preparation having a more significant effect than any other supplementation group. Finally, lipid peroxidation (LPO) levels in control flies (F(3,18) = 5.31, p < 0.01) increased significantly by 26% from day 0 to day 30 (Fig. 10d; p < 0.01); at day 30, Lf5221, TFLA, probiotics, and the synbiotic formulation all reduced lipid peroxidation (LPO) levels. Once again, the synbiotic formulation had a significantly greater effect than the probiotic formulation and any of the individual treatments, being the only group to rescue LPO levels compared to the day 0 control group (p < 0.01). Clearly, this synbiotic formulation had the strongest favorable and consistent effects on oxidative stress markers, suggesting its combinatorial effect.

[0235] The increase in oxidative stress is a result of increased production of ROS particles due to mitochondrial dysfunction and reduced antioxidant capacity. To test mitochondrial function, we tested the activity of each ETC complex in aged flies before and after supplementation with probiotics and / or prebiotics. NADH coenzyme Q reductase (ETC complex I) accepts electrons from the Krebs cycle from the electron carrier NADH and transfers them to complex II. In control flies, the activity of ETC complex I (F(3,18)=4.31, p<0.05) decreased at days 10, 20, and 30, and decreased by 62% by day 30 (Figure 10e; p<0.01). TFLA, probiotic, and synbiotic supplementation increased the activity of complex I at day 30, and the probiotic and synbiotic preparations completely rescued the activity of complex I (p<0.01). Complex II (succinate dehydrogenase) activity (F(3,18) = 2.53, p > 0.05) was also reduced on days 20 and 30 (Figure 10f; p < 0.05), although no treatment improved Complex II activity on day 30. However, the activity of ETC Complex II was reduced to the level of the control group on day 0 in the TFLA and synbiotic preparation groups (p > 0.05). Complex III (cytochrome bc1 complex) activity (F(3,18) = 3.98, p < 0.05) was also reduced by 76% in the control group on day 30 compared with day 0 (Figure 10g; p < 0.01). On day 30, each probiotic and / or prebiotic group had a beneficial effect on Complex III activity; however, the probiotic and synbiotic preparations rescued Complex III expression to the level of the control group on day 0 (p > 0.05). The effect of the probiotic preparation was significantly greater than that of the individual probiotics, and the effect of the synbiotic preparation was greater than that of Bi702255. Finally, at day 30, the activity of complex IV (cytochrome c oxidase) (F(3,18) = 4.01, p < 0.05) was also reduced by 67% in the control group (p < 0.01), and all probiotic and / or prebiotic treatments enhanced this effect ( Fig.11 d; p < 0.05). Lf5221, Bi702255, and TFLA treatment rescued complex IV activity to the level of the control group at day 0, however, at day 30, the synbiotic preparations had a significantly greater effect on complex IV expression than Lp8826 or Lf5221

[0236] Modifying the gut microbiota to treat neurological diseases

[0237] We designed novel probiotic and synbiotic formulations to optimize changes in the intestinal flora and elicit the greatest potential impact on neurological diseases. The probiotic formulation contains three probiotic species, Lactobacillus plantarum NCIMB 8826, Lactobacillus fermentum NCIMB 5221, and Bifidobacterium longum infantis NCIMB 702255, each dosed at 1.0x10 9 CFU / ml, total dose is 3.0x10 9 CFU / ml of Drosophila medium.

[0238] To design a synbiotic formulation, a recently characterized polyphenol-rich aqueous extract of Triphala, a polyherbal preparation, was combined with a probiotic preparation. The prebiotic activity of Triphala was characterized using in vitro batch cultures of isolated Lactobacillus and Bifidobacterium strains, an in vivo Drosophila model, and a humanized gastrointestinal tract in vitro model, demonstrating in each model an increase in the population of beneficial bacteria while reducing the population of pathogenic bacterial species. At the same time, Triphala at a concentration of 0.5% w / v has been shown to be non-toxic to Drosophila, does not induce an immune response, and even has a beneficial effect on Drosophila lifespan and motility, suggesting a metabolically beneficial effect.

[0239] Finally, in these physiological indices (lifespan and motility) measured, the probiotic and synbiotic preparations showed a combined effect compared to either individual probiotics or Triphala alone.

[0240] discuss

[0241] The gut microbiota of Drosophila melanogaster changes significantly with age, with strong expansion of most bacterial groups, and pathogenic Gammaproteobacteria spp. and Enterococcus spp. 28 This is a known phenomenon in Drosophila that reflects age-related immunosenescence and the accumulation of intestinal barrier dysfunction from proinflammatory pathogens. 29 These changes are similar to age-related changes observed in aging humans. 14,19 Treatment with probiotics and / or prebiotics, as used in this study, has previously been shown to reduce bacterial load in aged flies. 28 , has immunomodulatory effects 30 , regulate metabolism 31 , enhance lifespan 28 , suggesting the potential of probiotic-based therapeutics for treating age-related diseases.

[0242] Compared with flies fed a regular diet, the synbiotic formulation extended lifespan by 60%, while the probiotic formulation extended lifespan by 55%. 28There are other studies showing the life-extending potential of probiotic supplementation. 32-35 , however, described herein is the first study that demonstrates the simultaneous and multifaceted effects of the novel synbiotic formulation described herein on several markers of aging due to its lifespan-extending effects.

[0243] Inhibition of insulin-like growth factor (IGF)-1 and insulin signaling is thought to be the primary mechanism by which calorie restriction leads to lifespan extension 36 .

[0244] In this study, all levels of metabolic impairment in aged flies in the control group were rescued by the synbiotic preparation, including total weight, glucose and triglyceride levels. Previous studies have shown that downregulation of dilps and insulin receptors can extend the lifespan of flies. 37 , which are regulated by diet 38 .

[0245] In terms of the insulin signaling pathway, synbiotic treatment rescued the increase in dAkt and dTOR and the decrease in dFOXO, and the effects of individual probiotics and TFLA were beneficial but different. Inhibition of TOR signaling with its natural inhibitor rapamycin has been shown to extend yeast survival by mimicking the effects of caloric restriction. 39 , Nematodes 40 , fruit flies 41 and mice 42 Downstream of TOR signaling is the FOXO family of transcription factors. Activation of AMPK by calorie restriction is also associated with increased FOXO expression and pro-longevity effects. 43 , including simultaneous inhibition of ROS production 44 , NF-κB induction 45 ,senescence 46 and prevention of apoptosis 47 , and promotes mitophagy and the protective mechanism of autophagy 48 Some studies have linked polymorphisms in human FOXO3 to increased longevity. 5,49 , while in Drosophila 50 and mice 51 Interestingly, some studies have shown that the intake of polyphenols, including green tea gallic acid peptide, curcumin, and resveratrol, can reduce NF-κB, TNF-α, ROS, inflammation, and oxidative stress by increasing the expression of SOD, GPx, and sirtuin 1. 52,53 , stimulating FOXO expression, thereby extending lifespan. Similarly, in the study, FOXO expression was upregulated by synbiotic preparations.

[0246] Similar to glucose regulatory factors, many potential adipogenic factors were also positively affected by the probiotic and synbiotic preparations. There was an improvement in adipogenic dysregulation, as indicated by the restored expression of FAS, SREBP, and LSD2 in flies supplemented with the synbiotic preparations. In aged flies in the control group, the intrinsic increase in adipogenic ACC and FAS genes was most significantly downregulated by the probiotic and synbiotic preparations, as was the gluconeogenic PEPCK factor. These changes may be related to the transcriptional regulation of SREBP, whose expression levels were reduced to the levels of young flies under the action of probiotics and synbiotic preparations. Importantly, SREBP regulation is controlled by the ILS pathway, which is activated by both Akt and TOR signaling. 54 Regulation of fatty acid lipogenesis is an important consideration for many aspects of age and age-related diseases, especially inflammation, as obesity is intrinsically linked to a pro-inflammatory state. 55 , a condition known to be preventable by adequate probiotic and prebiotic treatment 56,57 .

[0247] Previous studies have shown that PPARγ-mediated responses are central to the effects of synergism in the Drosophila melanogaster metabolic stress model 31 PPARγ is highly expressed in adipose tissue and is a key regulator of adipogenesis and lipogenesis 58 , also the main insulin sensitizer 59 It is also believed that PPARγ expression in other tissues is involved in regulating their metabolic and inflammatory responses. 60 This makes it central to many hypotheses about aging. 61 In this study, the PPARY target E75 was significantly downregulated in aged flies in the control group, an effect that was only ameliorated in the probiotic and synbiotic groups, and PPARY expression increased over time in the synbiotic group. This effect would explain the beneficial effects on adipogenic factors and insulin sensitivity, supporting the idea that synbiotic treatment modulates metabolic stress at a high level in aged flies and explains the broad metabolic effects.

[0248] Aging is associated with immunosenescence, which is caused by the exhaustion of stem cells, reduced regenerative capacity of the immune system, accumulation of antigens, and thymic atrophy 62 Dysfunctional immune cells prevent the body from mounting an appropriate immune response, leading to the accumulation of damaged cells and the release of pro-inflammatory cytokines. Chronic low-grade inflammation (inflammatory aging) is associated with many age-related diseases such as neurodegeneration, cardiovascular disease, insulin resistance, diabetes, osteoporosis, cognitive decline, dementia, frailty, cancer, and importantly, death ( 63 in ).

[0249] The gut microbiota of the elderly reflects a pro-inflammatory constitution enriched in Proteobacteria and lacking in butyrate-producing bacteria2. With aging, the integrity of the epithelial lining of the gastrointestinal tract (GIT) is compromised, allowing bacteria and bacterial products to leak into the host's bloodstream, thereby contributing to inflammatory aging. 64 All of these factors are related to the intestinal flora 65 Age-related changes in the microbiota can reduce the integrity of the intestinal epithelium, leading to atypical intestinal hyperplasia. 66 The pro-inflammatory environment also promotes the activation of NF-KB through LPS-TLR4 interaction and promotes the differentiation of naive T cells into pro-inflammatory Th17 cells. 67 The gradual accumulation of chronic low-grade inflammatory aging may be the source of many age-related chronic diseases because inflammation is comorbid with elevated ROS generation, mitochondrial dysfunction, and metabolic abnormalities.

[0250] As previously reported, there is upregulation of pro-inflammatory pathogenic organisms in aged flies, which is downregulated by synergistic agents 28 Indeed, aged flies are more susceptible to infection and have impaired immune functions, such as phagocytosis and melanization. 68 A general increase in immune-related genes, increased bacterial load, and more persistent AMP activation following infection can also be observed in flies, similar to the chronic inflammatory state observed in humans. 69 In the present study, flies in the control group showed an age-related decline in innate immune function. In particular, active immune responses to both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) challenges were reduced, an effect that was significantly affected by all probiotic treatments but was greatest by the probiotic and synbiotic preparations. This may reflect a reduced ability of aged flies to mount an immune attack against invading pathogens, as previously noted. 70 Compared to other studies that observed increases in immune gene expression 68 The reduction in Duox and IMD expression observed in control aged flies suggests a weakened innate immune response and supports the reduced immunity to pathogenic insults observed in the agar diffusion assay. Duox and IMD are at the first line of defense of the Drosophila innate immune system and are directly regulated by antigen recognition by invading pathogens. Therefore, it is possible that the responses of core immune regulatory cells in the fat body are compromised by age-affected Duox and IMD production. This may involve the Janus kinase / signal transducer activator of transcription (JNK / STAT) pathway, which has been shown to have either competitive or cooperative effects on the systemic immune response in Drosophila. 71 .

[0251] Although IMD expression decreased in aged flies, an increase in AMP expression was observed, particularly cathelicidin A, defensins, and cathelicidin, with significant benefit from supplementation with individual probiotics, prebiotics, and probiotic and synbiotic preparations. 72 , and is directly linked to gut barrier dysfunction, which is associated with lifespan in Drosophila 73 The reason for the difference between IMD signaling and AMP expression may be related to the different levels of AMP expression regulation. Increased AMP expression in aged flies is associated with increased oxidative stress 74 Some AMPs, including cathelicidin A, can be co-regulated by inflammatory factors such as AP-1 and NF-KB proteins and HDAC activity. 72 In addition, AMPs are affected by hormonal signals, namely 20-hydroxyecdysone and juvenile hormone 75, which are affected differently by aging. IlS also affects the expression of AMPs 68 , which could explain the significant effect of probiotic treatment on AMP expression in aged flies. Indeed, dFOXO has been shown to regulate AMP expression, especially in the presence of defects in the Toll and IMD pathways. 76 .

[0252] As we age 77 Mitochondria gradually lose their energy capacity due to changes in morphology, decrease in number, loss of protein and mutation of mtDNA. 78 Mitochondrial autophagy and dysregulation of the mitochondrial fission-fusion cycle can also impair mitochondrial integrity, leading to elevated ROS generation and continuous mitochondrial damage. 79 .

[0253] The study confirmed a decline in mitochondrial function, as the activity of each ETC complex decreased over time in control flies. Mitochondria play a key role in the aging process, and fruit flies have been established as a powerful model for studying mitochondrial activity in aging. 80 Similar phenomena were observed in various tissues of mice and rats, but only the activity of complexes I and IV was decreased. 81 , while the activities of complexes II and III remained relatively unchanged 82 Compared with the control group, TFLA, probiotics, and synbiotic preparations were able to increase the activities of ETC complexes 1, 3, and 4 at day 30, which is important to illustrate how probiotic treatment affects the integrity of mitochondrial complexes and, in turn, the production of ROS particles.

[0254] One of the key regulators of mitochondrial biogenesis is the PGC-1 family of transcriptional coactivators, whose expression declines with age 83Loss of PGC-1α is associated with reduced mitochondrial biogenesis, decreased fission-fusion cycles, and dysfunctional mitophagy 84 Nutrient deprivation is a key modulator of mitochondrial dynamics, as caloric restriction decreases AMP / ATP and NADH / NAD+ ratios, ultimately stimulating mitochondrial biogenesis and activity via PGC-1a and SIRT1, respectively. Results in Drosophila showed that overexpression of a PGC-1a homologue (dPGC-1 / Asparagus) was sufficient to increase mitochondrial activity, and that tissue-specific expression of dPGC-1 in the digestive tract extended lifespan. 83 In support of this, E75, a Drosophila PPARγ target, was shown to decline with age in this study, likely representing a decline in the PGC-1α equivalent. Individual probiotic treatments had little effect on E75 expression; however, E75 expression was elevated at day 30 in both probiotic and synbiotic treatments. This suggests that PPARγ may be one of the key mechanisms by which the gut microbiota manages lifespan through mitochondrial complex integrity.

[0255] ROS production is an essential component of health, but too much ROS can drive disease. Immunosenescence exacerbates redox stress and vice versa, stimulating a positive feedback loop between ROS production and inflammation. 85 . In the current aging model, ROS levels were significantly increased, and the control flies had increased levels of total oxidants and LPO, while SOD and GPx activities were significantly reduced. Probiotics and synbiotic preparations had little effect on antioxidant enzyme activities, but significantly reduced the levels of total oxidants and lipid peroxide (LPO), among which the synbiotic preparation had a greater effect on reducing the levels of lipid peroxide (LPO). This is very important in the context of neurodegeneration, because there are high levels of PUFA in neuronal cell membranes, and in Alzheimer's disease, the level of LPO is associated with the degree of cognitive impairment. 86 .

[0256] In this study, an optimized probiotic formulation containing three bioactive probiotics, such as the exemplary formulation provided by the present invention, was combined with the novel polyphenol-rich prebiotic Triphala to create a novel lifespan-extending synbiotic formulation. This synbiotic formulation has a combined effect on various aging markers, including essential signaling pathways that govern the cross-regulation of these markers. By understanding how the gut microbiota and probiotic treatments interact with these key aging pathways, specific formulations can be formulated early in life to prevent the onset of chronic diseases including cardiovascular disease, diabetes, obesity, cancer, and even neurodegeneration.

[0257] Materials and methods

[0258] Cultivation of probiotics

[0259] Three probiotic strains - Lactobacillus plantarum NCIMB 8826 (Lp8826), Lactobacillus fermentum NCIMB 5221 (Lf5221) and Bifidobacterium longum infantis NCIMB 702255 (Bi702255) were obtained from the NCIMB Culture Collection (Aberdeen, Scotland, UK). Cells were cultured on Man-Rogosa-Sharpe (MRS) medium obtained from Sigma Aldrich (Oakville, ON, Canada) on MRS-agar plates or in liquid culture at 37°C. After one round of liquid culture, several bacterial stocks were prepared in MRS medium containing 20% ​​(v / v) glycerol and stored at -80°C. Since all experiments required continuous cultivation, frozen stocks were renewed every two weeks to maintain the purity of the culture. To prepare for each individual experiment, a 1% (v / v) inoculum was used for subculture, incubated at 37°C for 18 hours, and removed immediately before use.

[0260] Probiotics and synbiotic preparations

[0261] The dried ingredients of Triphala (TFLA; Phyllanthus emblica, Terminalia chebula, and Terminalia chebula) were obtained from the Ayurvedic Pharmacy, Banaras Hindu University, Varanasi, India. Each ingredient was weighed individually and combined in equal proportions (by weight) before being manually crushed and ground into a fine powder using a mortar and pestle. During boiling, 5 g of TFLA powder was mixed with 1 L of Drosophila medium to a final TFLA concentration of 0.5% in the entire medium. The total amount of probiotics contained in the probiotic preparation was 3.0 x 10 9 CFU / ml, evenly distributed among Lp8826 (1.0x109 CFU / ml), Lf5221 (1.0x109 CFU / ml) and Bi702255 (1.0x109 CFU / ml). The synbiotic preparation contained the probiotic preparation and added 0.5% TFLA powder.

[0262] Fruit fly feeding

[0263] Wild-type Drosophila melanogaster (Oregon R) was purchased from the Bloomington Drosophila Stock Center (Indiana University, Bloomington Indiana). Flies were raised on standard oatmeal-sucrose-yeast medium without activated yeast culture, which was prepared by boiling oatmeal (83 g), sucrose (50 g) and yeast extract (30 g) in distilled water for 30 minutes. The flies were kept under controlled conditions of 25°C with a 12h:12h light-dark cycle. To prepare fruit fly bottles inoculated with probiotics and / or pathogens, the culture was left overnight and centrifuged at 3000 x g for 10 minutes at 4°C. The pellet was washed once and resuspended in 0.85% (w / v) saline. The total number of colonies was determined spectrophotometrically and compared to a standard curve prepared by colony forming units (CFUs) on MRS plates. The inoculation medium was prepared by splitting the concentrated bacterial culture into cooled liquid medium to a specified final concentration of CFU / ml medium. This is a validated method for oral inoculation of Drosophila, as bacterial cells survive in Drosophila culture medium for up to two weeks with a decrease in concentration detectable by daily CFU counts. However, during the experiment, flies were transferred to new inoculated vials every 3 to 4 days.

[0264] Metabolic measurements in Drosophila

[0265] Body weight was assessed by weighing 10 flies under anesthesia, with five replicates. Glucose was measured in hemolymph and whole body homogenates of flies, representing circulating and total glucose levels. Hemolymph was extracted by puncturing anesthetized flies with a fine tungsten needle, placed in a small tube with several perforations in a large tube, and centrifuged at 4000 rpm for 10 min. For whole body homogenates, total protein content was first determined using the Bradford method, and the resulting quantification of metabolic markers was normalized to total protein content to account for variations in fly mass. Subsequently, the homogenates were heat treated at 70 °C for 20 min to remove any complexes. Glucose levels were measured in 2 μl of hemolymph or 5 μl of whole body homogenates using a glucose (HK) assay kit (Sigma, Oakvilla, ON, Canada) according to the manufacturer's instructions. Whole body triglycerides were measured in 10 μl of homogenates using a triglyceride liquid staining test (Stanbio, TX, USA) according to the manufacturer's instructions.

[0266] Genetic variation in metabolic markers

[0267] RNA was extracted from 25 whole flies using Trizol (ThermoFisher, MA, USA) according to the manufacturer's instructions. cDNA synthesized from 1 μg of RNA was measured using a high-capacity cDNA synthesis kit (ThermoFisher, MA, USA) according to the manufacturer's instructions using an ND-2000 spectrophotometer (Fisher Scientific, Ottawa, ON, Canada). Expression of various immune factor genes was performed using SybrGreen (EvaGreen qPCR Mastermix, Diamed, Mississauga, Canada) real-time quantitative PCR (Eco Real-Time PCR System, Illumina, CA, USA). Primers, their sequences, and annealing temperatures are listed in Table 3. 2 ddCT Methods The final gene expression levels were calculated.

[0268] Table 3: Primer sequences for Drosophila melanogaster metabolic markers

[0269]

[0270]

[0271] Oxidative stress markers

[0272] Oxidative stress markers were detected in fresh Drosophila homogenates prepared from 25 flies in Tris-EDTA-Triton X-100 buffer, pH 7.4, filtered through fine cloth to remove solid particles. 2'-7'-Dichlorofluorescein diacetate (DCFA) (Sigma, Oakville, ON) was used as described previously. 92 Determination of total oxidants. Briefly, 20 μl of fly homogenate was mixed with 170 μl of Locke buffer. Then, 10 μl of 1 mM DCFA solution was added to each well and after 3 min of incubation, fluorescence was read at 474 nm excitation and 530 nm emission. Quantification was normalized to the amount of protein in each sample.

[0273] Superoxide radicals were generated by xanthine-xanthine oxidase reaction, and the reduction of nitro blue tetrazolium (NBT) was used as an indicator of superoxide generation. 93, SOD activity was determined. Briefly, the working solution consisted of 110 μl of potassium phosphate buffer with 20 mg / ml BSA, 6.25 μl of catalase (40 U / ml), 6.25 μl of NBT, and 50 μl of xanthine (1.8 mM). 7.5 μl of homogenate and 20 μl of xanthine oxidase (XOD, 5 U / ml) were added to the working solution and stirred in the dark at 37°C for 20 minutes to allow the color to develop. The absorbance of reduced NBT was recorded at 570 nm, compared with the standard curve of SOD, and standardized to the protein content in the sample.

[0274] The assessment of GPx activity is based on the oxidation of glutathione (GSSG), which is continuously supplied by excess glutathione reductase (GR). To measure the activity of GPx, the consequent decrease in the co-substrate NADPH was monitored at 340 nm, as previously shown 94 . Briefly, the GPx buffer contained 0.5 M sodium phosphate (pH 7.2), 100 mM EDTA, and 1.1 mM sodium azide. The GPx assay buffer consisted of 1.33 mM GSSH and 1.33 U / ml GPx buffer. The assay solution consisted of 160 μl of GPx assay solution, 10 μl of NADPH (5 mM), 20 μl of hydrogen peroxide (0.5%), and 15 μl of homogenate. The absorbance was monitored at 340 nm for 3 minutes, and the linear portion of the curve was evaluated for GPx activity, and the quantification was normalized to the amount of protein in the sample.

[0275] Lipid peroxidation (LPO) levels were assessed as adapted from 95 The bound malondialdehyde (MDA) was hydrolyzed in the presence of butylated hydroxytoluene (BHT). The reaction solution contained 10 mM 1-methyl-2-phenylindole in a 3:1 mixture of acetonitrile:methanol. To 120 μl of this solution, 20 μl of sample and 40 μl of 37% HCl were added and reacted at 100°C for 60 minutes. The absorbance of the resulting solution was measured at 550 nm, compared with an MDA standard solution and normalized to the content of total protein in the sample.

[0276] Agar disk diffusion assay

[0277] To evaluate the ability of Drosophila to directly inhibit the growth of E. coli and S. aureus, hemolymph obtained from flies fed on media containing probiotic and / or prebiotic therapy for 10 days was placed on lawns of S. aureus or E. coli on tryptic sulfite cycloserine (TSC)-agar (Sigma, Oakville, ON) plates. 50 flies were pierced in the thoracic region with a fine needle and placed in a small microcentrifuge tube with several holes, which was then placed in a larger microcentrifuge tube. The pierced flies were centrifuged at 3000 x g for 20 minutes at 4°C to remove enough hemolymph for analysis. Hemolymph (20 μl) was diluted to 50 μl with sterile saline and pipetted onto a 5 mm sterile filter paper loop on a nutrient agar plate that had been freshly coated with 100 μl of an overnight solution of S. aureus or E. coli. The inoculated plates with hemolymph-saturated filter paper were placed at 37°C for 24 hours before measuring the inhibition zone. Each treatment was replicated three times on separate nutrient agar plates.

[0278] Inflammatory markers

[0279] We assessed the expression of genetic inflammatory markers as described in the metabolic markers section, except that the primer sequences listed in Table 4 were used.

[0280] Table 4: Primer sequences for Drosophila melanogaster inflammatory markers

[0281]

[0282]

[0283] Mitochondrial electron transport chain (ETC) complex activity

[0284] Mitochondria were isolated from Drosophila by homogenizing 40 anesthetized flies in 4 mL of chilled hypotonic buffer (25 mM K2HPO4 and 5 mM MgCl2) on ice using a pre-cooled smooth pestle grinder (Corning, NY, USA). Large pieces were filtered out using a fine nylon mesh, and the remaining isolate was quickly frozen on dry ice before storage at -80°C. Before use, the homogenate was subjected to 3 freeze-thaw cycles to open the mitochondrial membranes.

[0285] As mentioned before 75The activity of ETC complex I (NADH-ubiquinone oxidoreductase) was evaluated by measuring the electron transfer from NADH to decylubiquinone. The assay buffer contained potassium phosphate buffer (50 mM, pH 7.2), bovine serum albumin BSA (50 mg / mL), potassium cyanide (KCN; 10 mM), NADH ((10 mM) and rotenone (1 mM). To start the reaction, 25 μL of the homogenate and 10 μM decylubiquinone were added to 150 μL of assay buffer. The change in absorbance at 340 nm was monitored over two minutes, and the slope of NADH reduction was recorded and normalized to the total protein in the sample.

[0286] As mentioned before 75 , ETC complex II (succinate-ubiquinone oxidoreductase) was determined as the reduction rate of 2,6-bisphenol hydroxyindoxyphenol (DCPIP). The assay buffer contained potassium phosphate buffer (50 mM, pH 7.6), BSA (50 mg / mL), potassium cyanide (KCN, 10 mM), succinate (400 mM) and DCPIP (0.015% w / v). To start the reaction, 10 μL of the homogenate and 65 mM phenazine methyl sulfate were added to 160 μL of assay buffer. The reduction rate at 600 nm was determined over two minutes, and the activity of ETC complex II was measured as the linear portion of the DCPIP reduction slope and normalized to the amount of protein in the sample.

[0287] The activity of ETC complex III (NADH-cytochrome c oxidoreductase) was determined as the antimycin-dependent reduction rate of cytochrome c. The assay buffer contained potassium phosphate buffer (50 mm, pH 7.2), as well as oxidized cytochrome c (1 mM), KCN (10 mm), EDTA (5 mM), Tween-20 (2.5%) and antimycin A (1 mg / mL). To start the reaction, 20 μL of sample homogenate and 10 μL of decylubiquinone (10 mM) were added to the assay buffer. Cytochrome c reduction was monitored at 550 nm in the presence and absence of antimycin A over two minutes and the difference in reaction rate was taken as the antimycin A-sensitive complex activity and normalized to the amount of protein in the sample.

[0288] Finally, the activity of complex IV (cytochrome c oxidase) was determined by monitoring the rate of cytochrome c oxidation. The assay buffer contained potassium phosphate buffer (50 mM, pH 8.0) supplemented with reduced cytochrome c (1 mM). Cytochrome c was reduced by adding a few grains of sodium dithiocyanate to the oxidized cytochrome c solution until the red color changed to orange and the absorbance was greater than 6 at 550 nm to 565 nm. 25 μl of the homogenate was added at the beginning of the reaction and the reduction rate was recorded at 550 nm within 3 minutes. The complex rate was normalized to the amount of total protein in the sample.

[0289] statistics

[0290] All statistical analyses were performed using [R] software. All experiments were performed with five independent trials. Changes in body weight and motility over the probiotic dose curve were assessed by one-way ANOVA with Tukey's post hoc comparison method. The significance of metabolic markers and gene expression was assessed by two-way ANOVA with Tukey's post hoc comparison method. Significance was considered significant if p < 0.05 and was evaluated at p < 0.01.

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[0388] Many embodiments of the present invention have been described. However, it should be understood that various modifications can be made without departing from the spirit and scope of the present invention. Therefore, other embodiments are also within the scope of the following claims.

Claims

1. A probiotic preparation, which consists of three strains: Lactobacillus fermentum NCIMB 5221, Lactobacillus plantarum NCIMB 8826 and Bifidobacteria longum spp. infantis NCIMB 702255.

2. A synbiotic preparation, which consists of the probiotic preparation according to claim 1 and a prebiotic composition, wherein the prebiotic composition consists of Triphala.

3. The probiotic preparation according to claim 1, wherein the strain: (a) are present in equal proportions; or (b) The total number of 5 CFU / ml to 10 11 CFU / ml or 10 5 CFU / gm to 3.0x10 11 CFU / gm present; or (c) Total 3.0 x 10 9 CFU / ml or 3.0x 10 9 CFU / gm present.

4. The probiotic preparation according to claim 1, wherein the strains have the following relative proportions: 1.0x 10 9 CFU / ml of Lactobacillus fermentum NCIMB 5221, 1.0x 10 9 CFU / ml of Lactobacillus plantarum NCIMB 8826, and 1.0x 10 9 CFU / ml of Bifidobacterium longum infantis NCIMB 702255.

5. The probiotic preparation according to claim 1, which is manufactured or formulated in the form of a solution, suspension, emulsion, powder, lozenge, pill, syrup, tablet, capsule, aerosol, suppository, or chewing gum.

6. Use of the probiotic preparation according to claim 1 in the preparation of a medicament for preventing diabetes, obesity, Staphylococcus aureus or Escherichia coli infection.

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