WO2023066973A1 - Probiotic strains for reducing blood cholesterol and/or treating dyslipidemia, and methods for using and producing the same - Google Patents

Probiotic strains for reducing blood cholesterol and/or treating dyslipidemia, and methods for using and producing the same Download PDF

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WO2023066973A1
WO2023066973A1 PCT/EP2022/079057 EP2022079057W WO2023066973A1 WO 2023066973 A1 WO2023066973 A1 WO 2023066973A1 EP 2022079057 W EP2022079057 W EP 2022079057W WO 2023066973 A1 WO2023066973 A1 WO 2023066973A1
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lactobacillus
strains
activity
lactobacillus acidophilus
composition
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PCT/EP2022/079057
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French (fr)
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Mathiew MILLETTE
Martin FRAPPIER
Julie AUCLAIR
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Kerry Group Services International Limited
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/113—Acidophilus
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/169—Plantarum

Definitions

  • the present disclosure relates generally to bacterial strains from the family Lactobacillaceae that exhibit one or more of a bile salt hydrolase activity, feruloyl esterase activity, cholesterol assimilation activity, gut microbiome modulatory activity, and endocannabinoidome modulatory activity.
  • the present disclosure further relates to compositions containing the bacterial strain(s), and methods for reducing blood cholesterol and/or treating dyslipidemia in a subject, by administering one or more of the bacterial strains.
  • the present disclosure even further relates to the production of bacterial compositions for reducing blood cholesterol and/or treating dyslipidemia in a subject.
  • Cardiovascular disease is currently the leading causes of death worldwide. See, e.g., Collaborators, G.B.D.C.o.D ("Global, regional, and national age-sex- specific mortality for 282 causes of death in 195 countries and territories, 1980-2017: a systematic analysis for the Global Burden of Disease Study 2017,” Lancet, 2018, 392(10159): 1736-1788).
  • dyslipidemia abnormal levels of blood lipids, especially low-density lipoprotein cholesterol (LDL-C)
  • LDL-C low-density lipoprotein cholesterol
  • MRFIT Multiple Risk Factor Intervention Trial
  • Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. See, e.g., Hill et al. ("Expert consensus document. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic,” Nat. Rev. Gastroenterol. Hepatol., 2014, 11(8): 506-14).
  • Probiotic strains with cholesterol- lowering activity have been identified, see, e.g., Miremadi etal. (“Cholesterol reduction mechanisms and fatty acid composition of cellular membranes of probiotic Lactobacilli and Bifidobacteria, "Journal of Functional Foods, 2014, 9: 295-305); Miremadi etal.
  • lactobacillus spp. The most frequently used probiotic species are Lactobacillus spp., Bifidobaterium spp. and Saccharomyces spp.
  • Several mechanisms of action have been proposed to explain their effects, including, for example, the production of antimicrobial substances, competition for gastro-intestinal colonization and available nutrients, immunomodulation, and the promotion of lactose digestion.
  • probiotic strains, and probiotic combinations and compositions that can effectively reduce blood cholesterol and/or treat dyslipidemia, while also effectively resisting gastrointestinal conditions in vivo, such as pH and bile salts, so as to survive passage through the gastrointestinal tract.
  • the present disclosure provides bacterial strains from the family Lactobacillaceae that exhibit one or more of a bile salt hydrolase activity, feruloyl esterase activity, cholesterol assimilation activity, gut microbiome modulatory activity, and endocannabinoidome modulatory activity.
  • These bacterial strains in certain embodiments, exhibit resistance to gastrointestinal conditions in vivo, such as pH and bile salts, and can therefore be used, singly or in combination, to reduce blood cholesterol and/or treating dyslipidemia in a subject.
  • Non-limiting embodiments of the disclosure include as follows.
  • a composition for reducing cholesterol and/or treating dyslipidemia comprising one or more strains of Lactobacillaceae, and wherein at least one of said one or more strains exhibits bile salt hydrolase (BSH) activity, and wherein at least one of said one or more strains exhibits feruloyl esterase (EF) activity.
  • BSH bile salt hydrolase
  • EF feruloyl esterase
  • composition according to [1] wherein said composition comprises
  • Lactiplantibacillus plantarum CHOL-200 Lactiplantibacillus plantarum CHOL-200.
  • composition according to [1] wherein said composition comprises
  • Lactobacillus acidophilus CL1285 Lactobacillus acidophilus CL1285.
  • composition according to [1] wherein said composition comprises
  • composition according to [8], wherein said composition comprises one or more additional probiotic strains.
  • composition according to [9], wherein at least one of said one or more additional probiotic strains is selected from Lactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacillus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus, Lentilactobacillus, Convivina, Frucctobacillus, , Oenococcus, Weis
  • composition according to [10], wherein at least one of said one or more additional probiotic strains is selected from Lacticaseibacillus casei LBC80R, Lacticaseibacillus casei ATCC 393, Lacticaseibacillus rhamnosus ATCC 53103, Lactiplantibacillus pentosus BK1034, Lactiplantibacillus plantarum BK300, Lactiplantibacillus plantarum BK303, Lactiplantibacillus plantarum BK324, Lactiplantibacillus plantarum BK1135, Lactiplantibacillus plantarum BK1039, Lactiplantibacillus plantarum BK1040, Lactobacillus acidophilus BK1197, Lactobacillus acidophilus ATCC 314, Lactobacillus acidophilus ATCC 4355, Lactobacillus acidophilus ATCC 4356, Lactobacillus acidophilus
  • composition according to [12], wherein said composition comprises at least one pharmaceutically-acceptable carrier or additive.
  • composition according to [12] wherein said composition is a feed, foodstuff, or food supplement.
  • composition according to any one of [1] -[15], wherein said composition further comprises at least one additional agent for reducing cholesterol or treating dyslipidemia.
  • composition according to [16], wherein said additional agent is selected from the group consisting of Atorvastatin, Fluvastatin, Lovastatin, Pitavastatin, Pravastatin, Rosuvastatin, Simvastatin, Ezetimibe, Bempedoic acid, Cholestyramine, Colesevelam, Colestipol, Amlodipine, Fenofibrate, Gemfibrozil, niacin, omega-3 fatty acids, fibers, soluble fibers, beta-glucans, and oat beta glucan.
  • said additional agent is selected from the group consisting of Atorvastatin, Fluvastatin, Lovastatin, Pitavastatin, Pravastatin, Rosuvastatin, Simvastatin, Ezetimibe, Bempedoic acid, Cholestyramine, Colesevelam, Colestipol, Amlodipine, Fenofibrate, Gemfibrozil, niacin, omega-3 fatty acids, fibers, soluble fibers
  • a method for reducing cholesterol and/or treating dyslipidemia comprising administering, to a subject in need thereof, one or more strains of Lactobacillaceae, wherein at least one of said one or more strains exhibits bile salt hydrolase (BSH) activity, and wherein at least one of said one or more strains exhibits feruloyl esterase (EF) activity.
  • BSH bile salt hydrolase
  • EF feruloyl esterase
  • Lactiplantibacillus plantarum CHOL-200 Lactiplantibacillus plantarum CHOL-200.
  • Lactobacillus acidophilus CL1285 Lactobacillus acidophilus CL1285.
  • CL1285 and Lactiplantibacillus plantarum CHOL-200 are administered to the subject at the same or different times.
  • At least one of said one or more additional probiotic strains is selected from Lactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacillus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus, Lentilactobacillus, Convivina, Frucctobacillus, , Oenococcus, Weis
  • FIG. 1 depicts a visual representation of BSH and EF-positive activities of L. acidophilus CL1285 and L. plantarum CHOL-200. Strains were grown in the presence oftaurocholic acid (TCA) and glycocholic acid (GCA) for 72 h under an anaerobic atmosphere at 37°C, or in the presence of ethyl ferulate (EF) for up to 6 days under an anaerobic atmosphere at 37°C. No EF activity was detected for L. plantarum CHOL-200.
  • FIG. 2 depicts the percentage of cholesterol assimilation by L. plantarum CHOL-200 ("1”), L. acidophilus CL1285 (“2”), and L. acidophilus AT CC 43121 (3”). Numbers bearing a different letter are significantly different (p ⁇ 0.05).
  • FIG. 3 depicts survival of L. acidophilus CL1285 and L. plantarum CHOL- 200 when exposed to a Simulated Gastric Fluid at pH 1.5 to 2.5 for 30 and 60 min.
  • Black bar L. acidophilus CL1285
  • grey bar L. plantarum CHOL-200.
  • FIGS. 4A-4I depict metabolic parameters in hypercholesterolemic and hypertriglyceridemic hamsters after probiotic interventions. Following the 30 -day high- fat diet run-in period (Week 0), hamsters were gavaged with probiotic formulas for 6 weeks (Week 6). Body weight, food intake and liver weight at week 6 are illustrated in FIGS. 4A-4C, respectively. Metabolic parameters including fasting glucose, insulin, triglycerides, cholesterol, non-HDL cholesterol and HDL cholesterol to total cholesterol ratio for Week 0 and Week 6 are illustrated in FIGS. 4D-4I, respectively, with mean ⁇ SEM. One-way ANOVA with a Tukey post-hoc. * p ⁇ 0.05 vs low fat of the run-in period, # p ⁇ 0.05 vs compared to LF-Milk and p ⁇ 0.05 vs HF-Milk post-intervention.
  • FIGS. 5A-5B depict the relative abundance of specific bacterial families in the microbiota of the ileum and the caecum after diet and probiotic interventions.
  • FIG. 6 depicts the association between gut microbiota and metabolic parameters in hypercholesterolemic and hypertriglyceridemic hamsters. Heatmap detailing significant Spearman rho coefficients (p ⁇ 0.05) between microbiota families in the ileum and caecum (top and bottom halves, respectively) and metabolic parameters. Family names in red identify those significantly modified by the probiotic interventions in one or both intestinal segments.
  • FIGS. 7A-7B depicts the reversal of diet-induced alterations in circulating levels of endocannabinoidome by probiotic intervention.
  • FIG. 7A depicts relative changes of NAEs and 2 -MAGs in the plasma, ileum, inguinal adipose tissue (SAT) and epididymal adipose tissue (VAT) compared to LF-Milk hamsters. *0ne-way ANOVA and Tukey post-hoc p ⁇ 0.05 vs. HF-Milk.
  • FIG. 7B depicts a heatmap detailing significant Spearman rho coefficients (p ⁇ 0.05) between plasma endocannabinoidome mediators and metabolic parameters.
  • FIG. 8 depicts changes in gut microbiota composition associated with changes in NAEs and 2 -MAGs. Heatmap illustrating significant Spearman rho correlation coefficients (p ⁇ 0.05) between the relative abundance of selected bacterial families in the ileum and NAE or 2 -MAG concentrations in the plasma, ileum, inguinal adipose tissue (SAT) and epididymal adipose tissue (VAT).
  • a composition for reducing blood cholesterol and/or treating dyslipidemia contains a single Lactobacillaceae strain. In other embodiments, a composition for reducing blood cholesterol and/or treating dyslipidemia contains a plurality of Lactobacillaceae strains.
  • the composition may demonstrate, by virtue of the presence of the strain or strains therein, one or more of a bile salt hydrolase activity, feruloyl esterase activity, cholesterol assimilation activity, gut microbiome modulatory activity, and endocannabinoidome modulatory activity. In some embodiments, the composition may demonstrate, by virtue of the presence of the strain or strains therein, at least 1, at least
  • composition may demonstrate, by virtue of the presence of the strain or strains therein, all of these activities.
  • the present inventors identified Lactobacillaceae strains having desirable activities for reducing blood cholesterol, including, e.g., bile salt hydrolase activity, feruloyl esterase activity, cholesterol assimilation activity, gut microbiome modulatory activity, and endocannabinoidome modulatory activity.
  • the present inventors further identified the ability of particular Lactobacillaceae strains having these beneficial properties to effectively resist gastrointestinal conditions in vivo, such as pH and bile salts, so as to survive passage through the gastrointestinal tract.
  • isolated when referring to a probiotic strain, such as a strain of Lactobacillaceae for example, that the strain is found in an environment, composition, or mixture, distinct from that which it occurs in nature.
  • the term “purified” as used herein preferably means at least 75% by weight, more preferably at least 85% by weight, more preferably still at least 95% by weight, and most preferably at least 98% by weight, of the same molecule is present.
  • engineered preferably means at least 75% by weight, more preferably at least 85% by weight, more preferably still at least 95% by weight, and most preferably at least 98% by weight, of the same molecule is present.
  • the terms “engineered,” “genetically engineered,” “genetically modified,” “recombinant,” “modified,” “non-naturally occurring,” and “non-native” indicate intentional human manipulation of the genome of an organism or cell.
  • the terms encompass methods of genomic modification that include genomic editing, as well as techniques that alter gene expression or inactivation, enzyme engineering, directed evolution, knowledge-based design, random mutagenesis methods, gene shuffling, codon optimization, and the like. Methods for genetic engineering are known in the art. Genetically modified probiotic strains are within the scope
  • expression refers to transcription of a polynucleotide from a D NA template, resulting in, for example, a messenger RNA [mRNA], The term further refers to the process through which transcribed mRNA is translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides maybe referred to collectively as "gene products.”
  • a "coding sequence” or a sequence that "encodes” a selected polypeptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when under the control of appropriate regulatory sequences.
  • module refers to a change in the quantity, degree or amount of a function.
  • the term "between” is inclusive of end values in a given range e.g., between about 1 and about 50 nucleotides in length includes 1 nucleotide and 50 nucleotides.
  • amino acid refers to natural and synthetic (unnatural) amino acids, including amino acid analogs, modified amino acids, peptidomimetics, glycine, and D or L optical isomers.
  • polypeptide As used herein, the terms “peptide,” “polypeptide,” and “protein” are interchangeable and refer to polymers of amino acids.
  • a polypeptide may be of any length. It may be branched or linear, it may be interrupted by non-amino acids, and it may comprise modified amino acids.
  • the terms also refer to an amino acid polymer that has been modified through, for example, acetylation, disulfide bond formation, glycosylation, lipidation, phosphorylation, pegylation, biotinylation, cross -linking, and/or conjugation (e.g., with a labeling component or ligand).
  • the terms "subject,” “individual,” or “patient” are used interchangeably herein and refer to any member of the phylum Chordata, including, without limitation, humans and other primates, including non-human primates, such as rhesus macaques, chimpanzees, and other monkey and ape species; farm animals, such as cattle, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals, including rabbits, mice, rats, and guinea pigs; birds, including domestic, wild, and game birds, such as chickens, turkeys, and other gallinaceous birds, ducks, and geese; and the like.
  • the term does not denote a particular age or gender. Thus, the term includes adult, young, and newborn individuals as well as males and females.
  • the terms "effective amount” or “therapeutically effective amount” of a composition or agent refer to a sufficient amount of the composition or agent to provide the desired response. Preferably, the effective amount will prevent, avoid, or eliminate one or more harmful side-effects.
  • the exact treatment amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, and the particular treatment used, mode of administration, and the like. An appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
  • 'Treatment” or "treating” a particular disease includes: (1) preventing the disease, for example, preventing the development of the disease or causing the disease to occur with less intensity in a subject that may be predisposed to the disease, but does not yet experience or display symptoms of the disease; (2) inhibiting the disease, for example, reducing the rate of development, arresting the development or reversing the disease state; and/or (3) relieving symptoms of the disease, for example, decreasing the number of symptoms experienced by the subject.
  • pharmaceutically acceptable refers to the suitability of, for example, a substance, carrier, composition, or vehicle, etc., for administration in vivo, such as to a mammal, including a human.
  • the term "nutritionally acceptable,” as used herein, refers to the suitability of, for example, a substance, composition, or vehicle, etc., for ingestion in vivo, such as to a mammal, including a human.
  • strain refers to a genetic variant, or subtype, of a microorganism species (such as a subtype of a bacterial species that is useful as a probiotic).
  • probiotic strain typically refers to a live strain of a microorganism, including a strain capable of future replication and/or metabolism (e.g., strains in spore form, lyophilized form, spray-dried form, etc.).
  • cholesterol-assimilation activity refers to the ability of a strain, such as a probiotic strain, to capture cholesterol and incorporate it into its bacterial cellular structure.
  • the assimilation of cholesterol by a particular strain can be assessed, for example, by using fluorescently-labeled cholesterol (or fluorescent analogues of cholesterol), and measuring incorporation of the labeled cholesterol, or cholesterol analog, into the strain by fluorometry or fluorocytometry, for example.
  • viability refers to the ability of a cell, such as a bacterial cell, to be capable of future replication, cell division, and/or cellular metabolism.
  • viable bacterial cells may be in a metabolically-active state, a dividing state, a vegetative state, and/or in a dormant state e.g., strains in spore form, lyophilized form, spray-dried form, etc.').
  • a bacterial cell that is non-viable indicates that the cell is not capable of cellular metabolic activity, or replication, at present or in the future. It will be appreciated that non-viable cells include dead cells. Viability can be determined by assays known in the art. For example, cytolysis or membrane leakage assays, such as lactate dehydrogenase assays, propidium iodide assays, trypan blue assays, and 7 -aminoactinomycin D assays, as well as genomic and proteomic assays.
  • active growth phase refers to the stage of bacterial growth characterized by an increase in total biomass or cell numbers.
  • Bacterial growth in culture can typically be characterized into different phases, including lag phase, log (or exponential) phase, and stationary phase.
  • lag phase bacteria adapt their growth conditions, and the rate of increase in cell number/biomass is minimal.
  • log phase the growth rate increases to reach a constant and maximal growth rate under the particular growth conditions.
  • stationary phase the growth rate declines, typically to a point where growth is absent or negligible.
  • cells may become non-viable (e.g., dead), which is sometimes referred to as the death phase.
  • Bacterial growth may be measured, for example, by measuring bacterial cell number (e.g., by measuring optical density of the culture), biomass, and/or the metabolism, catabolism, or production of various substances.
  • substantially pure culture refers to a culture or composition e.g., of bacteria) in which a particular bacterial strain or strains, depending on the embodiment, represent at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, of the total bacterial strains in the culture or composition.
  • culture refers to a population of microorganisms within a defined environment, for instance, within an in vitro vessel, container, fermenter, vat, or flask. Where the organism is a bacteria, the culture may be known as a "bacterial culture.”
  • a culture may comprise one or more additional elements, including, for example, culture medium and/or additives for promoting the growth of the desired microorganism(s).
  • Culture medium maybe in a liquid, solid, or semi-solid state (including gelatinous media such as agar, agarose, gelatin and collagen, for example).
  • agents may be added to suppress the growth of undesired microorganisms e.g., antibiotics and/or anti-fungal agents).
  • lipids and/or lipid proteins refers to an abnormal level and/or profile of lipids and/or lipid proteins in the blood. For example, an amount of one or more lipids may be increased or decreased with respect to the levels thereof in a healthy subject.
  • lipids and lipid proteins may include, for example, cholesterol, glycerides (such as triglycerides), and lipoproteins (such as low-density lipoprotein (LDL) and high-density lipoprotein (HDL).
  • LDL low-density lipoprotein
  • HDL high-density lipoprotein
  • the present disclosure provides bacterial strains from the family Lactobacillaceae that exhibit one or more of a bile salt hydrolase activity, feruloyl esterase activity, cholesterol assimilation activity, gut microbiome modulatory activity, and endocannabinoidome modulatory activity.
  • these bacterial strains exhibit resistance to gastrointestinal conditions in vivo, such as pH and bile salts, and can therefore be used, singly or in combination, to reduce blood cholesterol and/or treat dyslipidemia in a subject.
  • Bacterial strains from the family Lactobacillaceae include, without limitation, homofermentative and heterofermentative organisms, such as members of the genus Lactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacillus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus, Lentilactobacillus, Convivina, Frucctobac
  • the bacterial strain or strains exhibits bile salt hydrolase (BSH) activity, typically through bacterial production and secretion of BSH.
  • BSH breaks the peptide linkage between primary bile acid, and either glycine or taurine, thereby liberating the amino acid from the sterol core.
  • the resultant unconjugated bile acids are less soluble than the conjugated form from which they were liberated, and accordingly, are retained in the intestinal lumen (where they mix with the surrounding intestinal content).
  • BSH-producing bacteria may be identified, for example, by incubating bacteria in the presence of sodium taurocholic acid hydrate and/or or sodium glycocholate hydrate, and assessing colony morphology and/or precipitate, as described in Pereira etal. ("An in vitro study of the probiotic potential of a bile-salt-hydrolyzing Lactobacillus fermentum strain, and determination of its cholesterol-lowering properties,” Appl. Environ. Microbiol., 2003, 69(8): 4743-52). Other techniques known in the art for detecting BSH expression and/or activity may also be used.
  • the bacterial strain or strains exhibits feruloyl esterase (EF) activity, typically through bacterial production and secretion of EF.
  • EF is naturally involved in the liberation of phenolic compounds, such as ferulic acid (FA), from plant cell walls.
  • FA ferulic acid
  • EF can also act as a competitive inhibitor against one of the main enzymes responsible for de novo synthesis of cholesterol, namely hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase), which is the same enzyme targeted by statins.
  • HMG-CoA reductase hydroxymethylglutaryl-CoA reductase
  • EF can also activate peroxisome proliferator-activated receptor alpha (PPAR-a), a nuclear transcription factor implicated in the synthesis of two-cholesterol transporters (HDL and LDL).
  • PPAR-a activation has been associated with an increase in HDL and a decrease in LDL. See, e.g., Dominguez-Avila etal. ("Modulation of PPAR Expression and Activity in Response to Polyphenolic Compounds in High Fat Diets,” Int. J. Mol. Sci., 2016,
  • EF-producing bacteria may be identified, for example, by incubating bacteria in the presence of ethyl ferulate, and assessing colony morphology and/or clearing around colonies, as generally described in Donaghy etal. ("Detection of ferulic acid esterase production by Bacillus spp. and lactobacilli,’’ Appl. Microbiol. Biotechnol, 1998, 50(2): 257-60). Other techniques known in the art for detecting EF expression and/or activity may also be used.
  • the bacterial strain or strains exhibits cholesterol assimilation activity, such as by incorporating cholesterol into the bacterial cell membrane.
  • This cholesterol assimilation activity thereby has an effect of sequestering cholesterol from the host, and may lead to the indirect excretion of cholesterol by the host via excretion of the bacteria in feces, thereby decreasing the overall pool of circulating blood cholesterol.
  • Pereira etal. (“Effects of consumption of probiotics and prebiotics on serum lipid levels in humans,” Crit. Rev. Biochem. Mol. Biol., 2002, 37(4): 259-81); Gilliland etal. ("Assimilation of cholesterol by Lactobacillus acidophilus," Appl. Environ.
  • bacteria having cholesterol assimilation activity can be identified, for example, by measuring the amount of cholesterol incorporated into the bacteria, as described by, e.g., Gilliland etal. ("Assimilation of cholesterol by Lactobacillus acidophilus,” Appl. Environ. Microbiol., 1985, 49(2): 377-81) and Rudel etal. ("Determination of cholesterol using o- phthalaldehyde,”/. Lipid Res., 1973, 14(3): 364-6). Other techniques known in the art for detecting incorporation and/or assimilation of cholesterol into bacterial cells may also be used.
  • the bacterial strain or strains exhibits one or both of gut microbiome modulatory activity and endocannabinoidome modulatory activity.
  • Endocannabinoids (2-arachidonoyl-glycerol; 2 -AG and /V-arachidonoylethanolamine; AEA) are endogenously produced bioactive lipids that act on the receptor target for the principle active component of cannabis (delta9- tetrahydrocannabinol; THC), cannabinoid receptor 1 and 2 (CBiand2) and other targets.
  • endocannabinoids show some redundancy at the level of receptor targets with endocannabinoids, but also act on a host of receptors, such as G protein-coupled receptors, peroxisome proliferator-activated receptors (PPARs) and transient receptor potential cation channels (TRPs), which often have opposite physiological effects to CBi.
  • G protein-coupled receptors such as G protein-coupled receptors, peroxisome proliferator-activated receptors (PPARs) and transient receptor potential cation channels (TRPs), which often have opposite physiological effects to CBi.
  • PPARs peroxisome proliferator-activated receptors
  • TRPs transient receptor potential cation channels
  • the endocannabinoid system links gut microbiota to adipogenesis,” Mol. Syst. Biol., 2010, 6, Doi: 10.1038/msb.2010.46); Everard eta/. (“Intestinal epithelial N- acylphosphatidylethanolamine phospholipase D links dietary fat to metabolic adaptations in obesity and steatosis,” Nat. Commun., 2019, Doi: 10.1038/s41467-018- 08051-7); and Manca etal. (“Germ-free mice exhibit profound gut microbiota-dependent alterations of intestinal endocannabinoidome signaling,” J. Lipid Res., 2020, Doi: 10.1194/jlr.RA119000424). Additionally, it is suggested that the gut microbiota- endocannabinoidome axis forms a triangular relationship with metabolic health.
  • the bacterial strain or strains upon administration to a subject, are able to modulate the gut microbiota composition e.g., in the small intestine and/or caecum) and/or alter the plasma and/or tissue levels of one or more endocannabinoidome mediators e.g., members of the /V-acylethanolamine (NAE) and 2- monoacylglycerol (2 -MAG) endocannabinoidome mediator families).
  • endocannabinoidome mediators e.g., members of the /V-acylethanolamine (NAE) and 2- monoacylglycerol (2 -MAG) endocannabinoidome mediator families.
  • the bacterial strain or strains are one or more selected from Lacticaseibacillus casei LBC80R, Lacticaseibacillus casei ATCC 393, Lacticaseibacillus rhamnosus ATCC 53103, Lactiplantibacillus pentosus BK1034, Lactiplantibacillus plantarum BK300, Lactiplantibacillus plantarum BK303, Lactiplantibacillus plantarum BK324, Lactiplantibacillus plantarum BK1135, Lactiplantibacillus plantarum BK1039, Lactiplantibacillus plantarum BK1040,
  • Lactiplantibacillus plantarum CHOL-200 Lactobacillus acidophilus BK1197, Lactobacillus acidophilus ATCC 314, Lactobacillus acidophilus ATCC 4355, Lactobacillus acidophilus ATCC 4356, Lactobacillus acidophilus AT CC 4796, Lactobacillus acidophilus AT CC 53544, Lactobacillus acidophilus ATCC 53671, Lactobacillus acidophilus ATCC 9224, Lactobacillus acidophilus LMG 11466, Lactobacillus acidophilus CL1285, Lactobacillus acidophilus FERM BP-4980, Lactobacillus acidophilus FERM BP-4981, Lactobacillus crispatus BK343, Lactobacillus crispatus BK482, Lactobacillus crispatus ATCC 55221, Lactobacillus gallinarum 9435, Lactobacillus gasseri 33323, Levilactobacillus (Lacto
  • the bacterial strain is selected from Lactobacillus acidophilus CL1285 (deposited November 15, 1994, as Accession Number LALC 1285, under the terms of the Budapest Treaty, with the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25-28 rue du Dondel Roux, 75724 Paris Cedex 15, France) and Lactiplantibacillus plantarum CHOL-200 (deposited August 25, 2021, as Accession Number CNCM 1-5742, under the terms of the Budapest Treaty, with the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25-
  • Lactobacillus acidophilus CL1285 is a bacterium of genus Lactobacillus and species Lactobacillus acidophilus.
  • Lactiplantibacillus plantarum CHOL-200 is a bacterium of genus Lactiplantibacillus and species Lactiplantibacillus plantarum. Relevant information on the characteristics of Lactobacillus acidophilus CL1285 and Lactiplantibacillus plantarum CHOL-200 can be found throughout the present application, in particular the description and drawings.
  • suitable media for propagation of Lactobacillus acidophilus CL1285 and Lactiplantibacillus plantarum CHOL-200 are described in the examples, including but not limited to paragraphs [00144], [00145], [00152], and [00159],
  • Lactobacillus acidophilus CL1285 and Lactiplantibacillus plantarum CHOL-200 are used in combination, such as, for example, by being combined within a single composition; or being co-administered to a subject.
  • the present disclosure also encompasses variants of the strains listed above, such as, for example, variants of the Lactobacillus acidophilus CL1285 and Lactiplantibacillus plantarum CHOL-200 strains.
  • Such variant strains may or may not have the same identifying biological characteristics of the specific strains listed herein, provided they share similar advantageous properties in terms of, for example, one or more of bile salt hydrolase activity, feruloyl esterase activity, cholesterol assimilation activity, gut microbiome modulatory activity, and endocannabinoidome modulatory activity.
  • the 16S rRNA genes of a variant strain as contemplated herein may share at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, with a strain disclosed herein.
  • the degree of relatedness between a variant and a parent strain may be defined according to the average nucleotide identity (ANI), which reflects DNA conservation of the core genome. See, e.g., Konstantinidis et al. Proc. Natl. Acad. Sci. USA, 2005, 102: 2567-2592).
  • ANI average nucleotide identity
  • the ANI between a variant and a parent strain is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.99%, at least 99.999%, at least 99.9999%, at least 99.99999%, or at least 99.999999%.
  • the degree of relatedness between a variant and a parent strain may be defined according to the degree of similarity obtained when analyzing the genomes of the parent and variant strains by Pulsed-Field Gel Electrophoresis (PFGE), using one or more restriction endonucleases.
  • the degree of similarity obtained by PFGE can be measured by the Dice similarity coefficient.
  • the Dice similarity coefficient between the variant and the parent strain is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.99%, at least 99.999%, at least 99.9999%, at least 99.99999%, or at least 99.999999%.
  • the degree of relatedness between a variant and a parent strain may be defined according to ribotype, as obtained using any of the methods known in the art.
  • the degree of relatedness between a variant and a parent strain may be defined according to the Pearson correlation coefficient obtained by comparing the genetic profiles of both strains obtained by repetitive extragenic palindromic element-based PCR (REP-PCR).
  • the Pearson correlation coefficient obtained by comparing the REP-PCR profiles of the variant and the parent strain is at least 0.99, at least 0.999, at least 0.9999, at least 0.99999, at least 0.999999, or at least 0.999999.
  • the degree of relatedness between a variant and a parent strain may be defined according to the linkage distance obtained by comparing the genetic profiles of both strains obtained by Multilocus sequence typing (MLST).
  • the linkage distance obtained by MLST of the variant and the parent strain is at least 0.99, at least 0.999, at least 0.9999, at least 0.99999, at least 0.999999, or at least 0.999999.
  • the probiotic strain or strains are preferably in the form of viable cells.
  • the viable cells are in the form of living cells in a metabolically-active state, a dividing state, a vegetative state, and/or in a dormant state e.g., strains in spore form, lyophilized form, spray-dried form, etc.'), in some embodiments, the probiotic strain or strains in a culture or composition have a viability of at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, in terms of the total number of cells of the probiotic strain(s).
  • the probiotic strain or strains are present in a substantially pure culture.
  • the probiotic strain or strains in the substantially pure culture represent at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, of the total bacterial strains in the culture.
  • compositions containing one or more of the probiotic strains disclosed herein.
  • at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 probiotic strains as contemplated herein are included in a composition.
  • one or more probiotic strains of the family Lactobacillaceae are present in a composition together with one or more non-Lactobacillaceae microorganisms, such as, for example, one or more microorganisms from the genera Saccharomyces, Streptococcus, Bacteroides, Pediococcus, Enterococcus, Leuconostoc, and/or Bifidobacterium.
  • non-Lactobacillaceae microorganisms such as, for example, one or more microorganisms from the genera Saccharomyces, Streptococcus, Bacteroides, Pediococcus, Enterococcus, Leuconostoc, and/or Bifidobacterium.
  • a single probiotic strain may constitute up to 1%, up to 5%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, or up to 99%, of the total probiotic cells in a composition.
  • At least one of the probiotic strains in the composition is Lactobacillus acidophilus CL1285. In some embodiments, at least one of the probiotic strains in the composition is Lactiplantibacillus plantarum CHOL-200. In some embodiments, the composition contains both of Lactobacillus acidophilus CL1285 and Lactiplantibacillus plantarum CHOL-200. In some embodiments, Lactobacillus acidophilus CL1285 and Lactiplantibacillus plantarum CHOL-200 are the only probiotic strains in the composition.
  • Lactobacillus acidophilus CL1285 and Lactiplantibacillus plantarum CHOL-200 are the only Lactobacillaceae in the composition.
  • multiple compositions containing different probiotic strains are provided.
  • a first composition may contain Lactobacillus acidophilus CL1285
  • a second composition may contain Lactiplantibacillus plantarum CHOL-200.
  • These compositions maybe administered, for example, to a subject at the same or different times, and/or, at different frequencies.
  • Such multiple compositions may be packaged together, for example, within a kit, together with instructions for their use.
  • compositions and probiotic strain(s) in the manufacture of a medicament to reduce cholesterol and/or treat dyslipidemia.
  • Probiotic-containing compositions of the present disclosure include, for example, compositions in a solid, liquid, or semi-solid form, including, without limitation, gels, granules, powders, capsules, bars, blocks, jellies, etc. Such compositions can be formulated as, for example, pharmaceutical, nutraceutical, feed, foodstuff, food supplement, or beverage, compositions.
  • the pharmaceutical product or pharmaceutical product may be formulated as, for example, a powder, granule, pill, soft capsule, hard capsule, tablet, chewable tablet, quick-disintegrating tablet, syrup, liquid, suspension, suppository, injection, or the like.
  • the probiotic-containing composition is formulated for delivery to the oral, gastric and/or to the intestinal mucosal surface.
  • compositions formulated for alternate delivery routes such as nasopharyngeal, respiratory, reproductive or glandular mucosa, and it may be administered to a subject by an oral, nasal, ocular, rectal, topical and/or vaginal route.
  • compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablet), pills, granules, powders, capsules (including soft and hard capsules), syrups, emulsions, suspensions, and the like.
  • compositions may be produced using methods known to persons of skill in the art, and may also contain additives or carriers generally used in the pharmaceutical field, such as excipients, binders, disintegrants, lubricants, for instance.
  • excipients include animal and plant oils, polyvalent alcohols (such as polyethylene glycol, propylene glycol, glycerol, sorbitol), surfactants (such as sorbitan fatty acid ester, sucrose fatty acid ester, glycerin fatty acid ester, polyglycerol fatty acid ester), purified water, lactose, starch, crystalline cellulose, D -mannitol, lecithin, gum arable, sorbitol solution, carbohydrate solution, for example.
  • polyvalent alcohols such as polyethylene glycol, propylene glycol, glycerol, sorbitol
  • surfactants such as sorbitan fatty acid ester, sucrose fatty acid ester, glycerin fatty acid
  • binders include hydroxypropylmethylcellulose, hydroxypropylcellulose, gelatin, pregelatinized starch, and polyvinylpyrrolidone, polyvinylalcohol.
  • disintegrants include carmellose calcium, carmellose sodium, croscarmellose sodium, crospovidone, low-substituted hydroxypropylcellulose, and cornstarch.
  • lubricants include talc, hydrogenated vegetable oil, waxes, stearic acid, magnesium stearate, calcium stearate, and aluminum stearate.
  • compositions of the present disclosure may also contain one or more of a sweetener, a colorant, a pH adjuster, a flavor, various amino acids, etc.
  • compositions of the presently claimed invention may include, or be used in combination with, other drugs, treatments, and/or medicaments, for reducing cholesterol and/or treating dyslipidemia.
  • compositions of the presently claimed invention may include, or be used in combination with: a statin e.g., Atorvastatin, Fluvastatin, Lovastatin, Pitavastatin, Pravastatin, Rosuvastatin, Simvastatin, etc.); Ezetimibe; Alirocumab; Evolocumab; Bempedoic acid; Cholestyramine; Colesevelam; Colestipol; Amlodipine; Fenofibrate; Gemfibrozil; niacin; omega-3 fatty acids, fibers, soluble fibers, beta-glucans, and oat beta glucan.
  • a statin e.g., Atorvastatin, Fluvastatin, Lovastatin, Pitavastatin, Pravastatin, Rosuvastatin, Simvastatin, etc.
  • Ezetimibe e.g., Atorvastatin, Fluvastatin, Lovastatin, Pitavastatin, Pravastatin, Ros
  • the food (or feed) or food (or feed) containing the additive is not particularly limited as long as it permits oral ingestion, such as a solution, suspension, powder, solid formed particles, granules, and bars, for example.
  • supplements include supplements (powders, granules, soft capsules, hard capsules, tablets, chewable tablets, quick-disintegrating tablets, syrups, liquids, etc.); drinks (carbonated drinks, milk drinks, sport drinks, fruit juice drinks, vegetable drinks, soymilk drink, coffee drinks, tea drinks, powder drinks, concentrated drinks, nutrition drinks, alcohol drinks etc.); dairy products (yogurt, butter, cheese, ice cream etc.); and confectionery (gummies, jellies, gums, chocolates, cookies, candies, caramels, etc.), and carbohydrate-based products (bread, pasta, noodles, cake mixes, breads, etc.).
  • the above-mentioned foods can contain, in some embodiments, one or more nutrients, vitamins e.g., vitamin A, vitamin Bl, vitamin B2, vitamin B6, vitamin C, vitamin D, vitamin E, vitamin K, etc.), minerals ⁇ e.g., magnesium, zinc, iron, sodium, potassium, selenium, etc.), dietary fiber, a dispersing agent, a stabilizer, an emulsifier, a sweetener, and/or a flavoring acid ⁇ e.g., citric acid, malic acid, etc.).
  • vitamins e.g., vitamin A, vitamin Bl, vitamin B2, vitamin B6, vitamin C, vitamin D, vitamin E, vitamin K, etc.
  • minerals ⁇ e.g., magnesium, zinc, iron, sodium, potassium, selenium, etc.
  • dietary fiber e.g., a dispersing agent, a stabilizer, an emulsifier, a sweetener, and/or a flavoring acid ⁇ e.g., citric acid, mal
  • compositions of the present disclosure may be formulated to contain, or so as to provide as a daily dose, at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , or at least 10 12 , viable cells of a probiotic strain or strains.
  • Therapeutic Uses at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , or at least 10 12 , viable cells of a probiotic strain or strains.
  • the present disclosure further provides, in some embodiments, methods for treating elevated cholesterol; methods for reducing cholesterol, including blood cholesterol; and methods for preventing cholesterol elevation.
  • the present disclosure thereby provides, in some embodiments, therapeutic and preventative compositions and methods against additional diseases, disorders, and ailments, caused by, associated with, or resulting from, elevated cholesterol levels.
  • the present disclosure further provides, in some embodiments, methods for treating or preventing dyslipidemia, including, for instance, modulating the level of one or more of cholesterol, glycerides (such as triglycerides), and lipoproteins (such as low-density lipoprotein (LDL) and high-density lipoprotein (HDL).
  • the present disclosure thereby provides, in some embodiments, therapeutic and preventative compositions and methods useful as a treatment, or adjunct therapy, for additional diseases, disorders, and ailments that are caused by, associated with, or resulting from, dyslipidemia (such as, for example, insulin resistance, metabolic syndrome, atherosclerosis, heart failure, or diabetes).
  • the dyslipidemia is hypercholesterolemia e.g., where blood cholesterol levels are elevated above clinically and/or medically recommended levels).
  • hypercholesterolemia may be determined by measuring the levels of one or more of: HDL, LDL, and triglycerides. In some embodiments, hypercholesterolemia may be determined by measuring all of HDL, LDL, and triglycerides.
  • a subject may be judged or diagnosed to have hypercholesterolemia, and/or be suitable for cholesterol management, if the measured LDL levels are, for example, above 70 mg/dl, above 80 mg/dl, above 90 mg/dl, above 100 mg/dl, above 110 mg/dl, above 120 mg/dl, above 130 mg/dl, above 140 mg/dl, above 150 mg/dl, above 160 mg/dl, above 170 mg/dl, above 180 mg/dl, or above 200 mg/dl.
  • the measured LDL levels are, for example, above 70 mg/dl, above 80 mg/dl, above 90 mg/dl, above 100 mg/dl, above 110 mg/dl, above 120 mg/dl, above 130 mg/dl, above 140 mg/dl, above 150 mg/dl, above 160 mg/dl, above 170 mg/dl, above 180 mg/dl, or above 200 mg/dl.
  • there may be differing degrees of severity of hypercholesterolemia depending on
  • a subject may be judged or diagnosed to have hypercholesterolemia, and/or be suitable for cholesterol management, if the measured HDL levels are, for example, below 60 mg/dl, below 55 mg/dl, below 50 mg/dl, below 45 mg/dl, below 40 mg/dl, below 35 mg/dl, below 30 mg/dl, below 25 mg/dl, or below 20 mg/dl.
  • the measured HDL levels are, for example, below 60 mg/dl, below 55 mg/dl, below 50 mg/dl, below 45 mg/dl, below 40 mg/dl, below 35 mg/dl, below 30 mg/dl, below 25 mg/dl, or below 20 mg/dl.
  • a subject may be judged or diagnosed to have hypercholesterolemia, and/or be suitable for cholesterol management, if the measured triglyceride levels are, for example, above 140 mg/dl, above 145 mg/dl, above 150 mg/dl, above 155 mg/dl, above 160 mg/dl, above 170 mg/dl, above 180 mg/dl, above 190 mg/dl, above 200 mg/dl, above 300 mg/dl, above 400 mg/dl, or above 500 mg/dl.
  • the measured triglyceride levels are, for example, above 140 mg/dl, above 145 mg/dl, above 150 mg/dl, above 155 mg/dl, above 160 mg/dl, above 170 mg/dl, above 180 mg/dl, above 190 mg/dl, above 200 mg/dl, above 300 mg/dl, above 400 mg/dl, or above 500 mg/dl.
  • there may be differing degrees of severity of hypercholesterolemia depending on
  • cholesterol may be measured using free plasma cholesterol.
  • Hypercholesterolemia may be judged or diagnosed in a subject if, for example, the measured free cholesterol levels are above 200 mg/dl, above 210 mg/dl, or above 220 mg/dl.
  • one or more probiotic strains disclosed herein may be administered.
  • at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 probiotic strains as contemplated herein are administered to a subject.
  • one or more probiotic strains of the family Lactobacillaceae are administered in conjunction e.g., before, together with, or after) with one or more non-Lactobacillaceae microorganisms, such as, for example, one or more microorganisms from the genera Saccharomyces, Streptococcus, Bacteroides,
  • a single probiotic strain may constitute up to 1%, up to 5%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, or up to 99%, of the total probiotic cells administered.
  • At least one of the probiotic strains administered is Lactobacillus acidophilus CL1285. In some embodiments, at least one of the probiotic strains administered is Lactiplantibacillus plantarum CHOL-200. In some embodiments, both of Lactobacillus acidophilus CL1285 and Lactiplantibacillus plantarum CHOL-200 are administered. In some embodiments, Lactobacillus acidophilus CL1285 and Lactiplantibacillus plantarum CHOL-200 are the only probiotic strains administered to a subject. In some embodiments, Lactobacillus acidophilus CL1285 and Lactiplantibacillus plantarum CHOL-200 are the only Lactobacillaceae administered to a subject.
  • compositions containing different probiotic strains are administered to a subject.
  • a first composition may contain Lactobacillus acidophilus CL1285
  • a second composition may contain Lactiplantibacillus plantarum CHOL-200.
  • These compositions maybe administered, for example, to a subject at the same or different times, and/or, at different frequencies.
  • probiotic strains of the present disclosure may be administered in conjunction e.g., before, together with, or after) with one or more of: a statin ⁇ e.g., Atorvastatin, Fluvastatin, Lovastatin, Pitavastatin, Pravastatin, Rosuvastatin, Simvastatin, etc.); Ezetimibe; Alirocumab; Evolocumab; Bempedoic acid;
  • a statin e.g., Atorvastatin, Fluvastatin, Lovastatin, Pitavastatin, Pravastatin, Rosuvastatin, Simvastatin, etc.
  • Ezetimibe e.g., Atorvastatin, Fluvastatin, Lovastatin, Pitavastatin, Pravastatin, Rosuvastatin, Simvastatin, etc.
  • Ezetimibe e.g., Atorvastatin, Fluvastatin, Lovastatin, Pitavastatin, Pravastatin, Rosuva
  • a subject is administered a daily dose of at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , or at least 10 12 , viable cells of a probiotic strain or strains.
  • the subject may be administered the probiotic strain or strains at varying frequencies, depending on the nature of the disorder or the proposed use of the composition, and/or the characteristics of the subject involved.
  • therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures, e.g., with experimental animals, such as by calculating the ED, (the dose therapeutically effective in 50% of the population) or LD (the dose lethal to 50% of the population) statistics.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD/ED ratio.
  • the effect may be dose-dependent ⁇ e.g., the higher the dose, the higher the protective and/or therapeutic activity).
  • the dosage may also vary depending upon the dosage form employed, the sensitivity of the patient, and the route of administration. The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment.
  • Dosage and administration are adjusted to provide sufficient levels or to maintain the desired effect.
  • Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy.
  • Probiotic strains may be administered several times per day ⁇ e.g., 1, 2, 3, 4, or 5 times per day), every day, every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days, or monthly depending on, e.g., the clearance rate, tolerance by the subject, and response characteristics of the subject.
  • the probiotic strain or strains are administered by delivery to the oral, gastric and/or to the intestinal mucosal surface.
  • alternate delivery routes such as nasopharyngeal, respiratory, reproductive or glandular mucosa, and it may be administered to a subject by an oral, nasal, ocular, rectal, topical and/or vaginal route.
  • Subjects may be administered probiotic strains according to the various compositions and administration routes described herein.
  • the administered probiotic cells are in the form of living cells in a metabolically-active state, a dividing state, a vegetative state, and/or in a dormant state (e.g., strains in spore form, lyophilized form, spray-dried form, etc.).
  • Non-limiting embodiments of the present invention are illustrated in the following Examples. Efforts have been made to ensure accuracy with respect to numbers used e.g., amounts, concentrations, percent changes, and the like), but some experimental errors and deviations should be accounted for.
  • BSH activity was evaluated as described in Pereira et al. ("An in vitro study of the probiotic potential of a bile -salt-hydrolyzing Lactobacillus fermentum strain, and determination of its cholesterol-lowering properties,” Appl. Environ. Microbiol., 2003, 69(8): 4743-52.
  • EF activity was evaluated using a semi-quantitative method described by Donaghy etal. ("Detection of ferulic acid esterase production by Bacillus spp. and lactobacilli.,’’ Appl. Microbiol. Biotechnol., 1998, 50(2): 257-60), with some modifications. Specifically, a modified MRS medium with agar (1.5% w/v) was prepared using all the same ingredients for the MRS medium broth, but without glucose. The medium was sterilized at 121°C for 20 min and cooled at 50-55°C.
  • ethyl 4-hydroxy-3 -methoxy cinnamate (ethyl ferulate) (10% w/v; Sigma- Aldrich) dissolved in N, N-Dimethylformamide (Sigma-Aldrich) was added to 500 mL of medium and gently mixed. The medium was then poured into sterile Petri dishes and solidified at room temperature. A loop of fresh bacterial culture was streaked on the surface of each Petri dish and the plates were incubated under an anaerobic atmosphere at 37°C for 3 to 6 days.
  • EF activity was determined by comparing the growth of each bacteria and the disappearance of the fogginess around each bacterial colony on an agar plate with and without ethyl ferulate. The results are depicted in Table 1 below ("EF” columns). Plus “+” symbols were used to describe EF activity, and the number of symbols is proportional to the EF activity. A negative result is indicated by a negative symbol, meaning the bacteria could grow on the medium, but there was no activity detected when compared with the same medium without ethyl ferulate.
  • BSH Bile salt hydrolase
  • FAE feruloyl esterase
  • BSH activity (10 exhibiting selectivity towards the taurine-conjugated form of bile acids; 5 exhibiting selectivity towards glycine-conjugated bile acids; and 3 strains (corresponding to Lactobacillus acidophilus, Lacticaseibacillus casei and Lactiplantibacillus plantarum) showing the capacity to deconjugate bile acid with either taurine or glycine).
  • Table 1 also shows that 26 of the 70 tested strains tested positive for EF activity (7 from the genus Lactiplantibacillus, 14 from the genus Lactobacillus, 1 from the genus Levilactobacillus, and 4 from the genus Limosilactobacillus) .
  • Eight of the EF-positive strains were isolated from a plant source (olive oil), which is consistent with the reported literature that EF breaks the bonds that maintain the cell wall of plants, and releases different phenolic compounds including FA. See, e.g., Mukdsi et al. ("Administration of Lactobacillus fermentum CRL1446 increases intestinal feruloyl esterase activity in mice,” Lett. Appl. Microbiol., 2012, 54(1): 18-25.
  • the other EF-positive bacteria were isolated from human feces or vaginal mucosa.
  • L. acidophilus CL1285 exhibited particularly strong EF activity. Representation of BSH and EF-positive activities are shown in FIG. 1, using L. acidophilus CL1285 and L. plantarum CHOL-200 results as reference.
  • Example 2
  • L. plantarum CHOL-200 and L. acidophilus CL1285 were selected for further characterization, and in particular, their capacity to assimilate cholesterol
  • Cholesterol micelles were prepared according to the method developed by Razin etal. ("Phospholipid and cholesterol uptake by Mycoplasma cells and membranes,” Biochim. Biophys. Acta, 1980, 598(3): 628-40), with some modifications.
  • Sonication was performed on ice using a Sonic Dismembrator 500 (Fisher Scientific) at maximum amplitude with a power of between 100 and 120 watts for three periods of 15 min, renewing ice between each period.
  • the solution was then centrifuged (Avanti J-26S XPI, Beckman Coulter, Brea, CA, USA) in high-speed centrifuge tubes for 30 min at 30,000 x g, to eliminate metal particles from the sonicator probe.
  • the blank consisted of 4 mL of o- phthalaldehyde solution and 2 mL of concentrated sulfuric acid.
  • the standard curve was generated using a cholesterol solution of 0, 25, 50, 75, 100, 200, and 400 pg/rnL.
  • TC was measured from the thioglycolate- and bile-supplemented MRS media containing cholesterol micelles from which value of thioglycolate- and bile-supplemented MRS medium without cholesterol was subtracted. All standards and samples were subjected to identical procedures. [00154]
  • the assimilation percentage was calculated using the following formula: > 100
  • strains CHOL-200 and CL1285 showed the ability to assimilate cholesterol present in the culture media.
  • a cholesterol removal rate of 76.3 % was observed for L. plantarum CHOL-200, and a cholesterol removal rate of 61.5 % was observed for L. acidophilus CL1285.
  • probiotic bacteria have to resist the harsh acidic and bilious environments of the stomach and the small intestine, respectively.
  • SGF simulated gastric fluid
  • SGFs of pH 1.5, 2 and 2.5 were prepared as follows. 2 g of sodium chloride (Fisher Scientific) and 3.2 g of pepsin (Sigma-Aldrich) were dissolved in 900 mL of osmosis filtered water. The pH was adjusted with 5 M hydrochloric acid (Fisher Scientific) and measured using an Orion 3
  • L. acidophilus CL1285 was unaffected (difference from the initial bacterial count ⁇ 1 Logio CFU/mL) up to 60 min in an SGF of pH 2 or 2.5. L. acidophilus CL1285 also resisted a pH of 1.5 for 30 min. However, its viability was altered after 60 min, as shown by a marked decrease of 3.07 Logio CFU/mL. L. plantarum CHOL-200 survived up to 60 min in an SGF of pH 2.5, and for 30 min in an SGF of pH 2. However, its viability decreased by nearly 1.9 Logio CFU/mL after 60 min at pH 2. The CHOL-200 strain was less resistant in the SGF of pH 1.5 for 30 or 60 min, as shown by a reduction of 2.84 and 5.43 Logio CFU/mL, respectively.
  • Bile bovine consisting of 70% bile salts, 22% phospholipids, 4% cholesterol, 3% proteins and 0.3% bilirubin (source: Sigma Aldrich).
  • Bile acids (as cholic acid) corresponding to > 45% of dry matter (source: Organotechnie).
  • the hamsters were housed in individual cages, and fed ad libitum with a standard laboratory chow diet for a 5 to 7-day acclimatization period.
  • Nine (9) hamsters were randomly assigned to the low-fat (LF) purified diet (D 14042701 Research Diet) for 30 days, while 45 hamsters were assigned to the high-fat (HF) purified diet (D12079B Research Diet) for the same period.
  • LF low-fat
  • HF high-fat
  • Such HF-feeding run-in is a recognized means of promoting weight gain and inducing a dysmetabolic state of hypercholesterolemia and hypertriglyceridemia in hamsters.
  • Both diets had comparable fiber content and were composed mainly of butter (rich in saturated and oleic fatty acids), corn oil (rich in oleic and linoleic acids), sucrose and corn starch.
  • Table 3 The study design is shown in Table 3 below.
  • HF-fed hamsters were then randomly assigned to a 6- week intervention period with 1) pasteurised milk (HF-Milk), 2) L. acidophilus CL1285 rehydrated in pasteurized milk (HF-La), 3) L. plantarum CHOL-200 in pasteurized milk (HF-Lp), 4) L. acidophilus and L. plantarum in pasteurized milk (HF- LaLp) or 5) Cholestyramine (HF-CHY; 300 mg/kg/day) (Table 1).
  • the fermented milk was stored at 4°C and comprised at least 50 x 10 9 colony forming units (CFU) /100 g of three proprietary strains (L. acidophilus CL1285, L.
  • L. acidophilus CL1285 and L. plantarum CHOL-200 were supplied as freeze-dried pure culture and stored at -20°C until utilization.
  • Probiotic and pharmacologic interventions were administered by daily intragastric gavage (2 mb).
  • Body weight and food intake were monitored every two days and every week, respectively. Sixteen (16) to 18-hour fasted hamsters were sacrificed at the end of the intervention period by cardiac puncture.
  • FIGS. 4A-4I Metabolic response to interventions
  • FIGS. 4A-4I Weight gain and metabolic measures of the hamsters following the run-in and the intervention period are illustrated in FIGS. 4A-4I.
  • HF-fed hamsters had a higher body weight than LF-fed hamsters (130.6 ⁇ 12.00 gvs. 90.2 ⁇ 14.3 g).
  • HF-feeding elevated fasting insulin levels (FIG. 4E), as well as fasting levels of triglycerides (FIG. 4F), total cholesterol (FIG. 4G), HDL cholesterol and non-HDL cholesterol (FIG. 4H), and reduced HDL/total cholesterol ratio compared to LF-fed hamsters (FIG.
  • the HF diet used was not chosen to induce hepatic fat deposition, steatosis or fibrosis - often requiring more than 40% of total calories from fat - thus greater liver weights observed do not necessarily correlate with such phenotypes and ensuing dysmetabolisms.
  • L. acidophilus CL1285 either alone or in combination with L plantarum CHOL-200, was distinct from that of the HF-Milk hamsters.
  • the intervention with L. acidophilus CL1285 tends to bring gut microbiota composition of HF-fed hamsters to a state closer to that observed in LF-fed hamsters.
  • Shannon Alpha-diversity index a marker of taxa richness and evenness, was not significantly influenced by the probiotic interventions, except for an increased diversity in the caecum when L. acidophilus CL1285 and L. plantarum CHOL-200 were combined (HF-Milk: 3.1 [2.6-3.5] vs. HF-LaLp: 3.8 [3.7-3.9], p ⁇ 0.05).
  • Table 3 Bacterial families significantly modified by probiotic interventions in the caecum.
  • HF feeding also decreased the relative abundance of the Deferribacteraceae family in the ileum (FIG. 5A), which was negatively associated with circulating levels of insulin, cholesterol, non-HDL cholesterol and triglycerides (FIG. 6).
  • Supplementation with both L. acidophilus CL1285 containing probiotics HF-La and HF-LaLp) induced an elevation of the relative abundance of Lactobacillaceae and of its genus Lactobacillus spp. in the ileum (FIG. 5A). Lactococcus genus, were positively associated with elevated levels of insulin, triglycerides, total cholesterol and non-HDL cholesterol across the intervention groups.
  • Plasma, ileum, caecum, inguinal and epididymal adipose tissues were extracted to measure /V-acetylethanolamines (NAEs) and 2 -monoacylglycerols (2-MAGs) by HPLC-MS/MS. Briefly, samples (40 pl of samples or 5-10 mg of tissue) were brought to 500 pl in 50 mM Tris (pH 7) then mixed/crushed in 500 pl methanol containing 0.01% acetic acid and 5 ng of deuterated standards. Lipids were extracted 3 times by adding 1 ml chloroform, vortexing for 1 minute and centrifuging (3,500 x g, 10 min).
  • the method can differentiate monoacylglycerol isomers at positions 1 and 2 but signals from both isomers of unsaturated fatty acids were summed - and identified as 2-MAGs - prior to analysis in order to account for their rapid interconversion.
  • NAEs were quantitated: /V-palmitoylethanolamine (PEA), /V-oleoylethanolamine (OEA), /V-linoleoylethanolamine (LEA), /V-arachidonoylethanolamine (AEA), /V-docosahexaenoylethanolamine (DHEA); and the following 2-MAGs: 2-palmitoyl-glycerol (2-PG), 2-oleoyl-glycerol (2-OG), 2-linoleoyl- glycerol (2 -LG), 2-arachidonoyl-glycerol (2 -AG), 2-eicosapenaenoyl-glycerol (2 -EPG), 2-docosapentaenoyl(n-3)-glycerol (2-DPG), and 2-docosaheaenoyl-glycerol (2-DHG).
  • Endocannabinoidome mediator profiling was not performed for HF-CHY hamsters, as plasma and tissue biopsies were not collected. All 2 -MAG congeners were increased in the plasma following HF feeding (FIG. 7A). These elevations were exacerbated by some probiotic formulations. Greater 2-AG, 2-DHG and 2-DPG levels were observed in the plasma following the supplementation with L. acidophilus CL1285 but not by its combinations with L. plantarum CHOL-200. In contrast, HF feeding decreased plasma levels of NAEs, especially AEA, OEA and LEA, and the supplementations with L. acidophilus CL1285, alone or in combination with L. plantarum CHOL-200, prevented such decreases in LEA levels.
  • NAE congeners but not 2 -MAGs, were significantly influenced by the interventions in the inguinal adipose tissue (FIG. 7 A). Namely, AEA levels tended to be higher in HF-Milkthan in LF-Milk hamsters and were increased by the intervention with both L. acidophilus CL1285 and L. plantarum CHOL-200.
  • the OEA decreases in the subcutaneous adipose tissue of HF-fed hamsters were also attenuated by the combination of L. acidophilus CL1285 and L. plantarum CHOL-200, and instead somewhat enhanced in the subcutaneous adipose tissue when L. plantarum was administered alone.
  • DHEA levels in this visceral adipose tissue depot were completely restored by the supplementation with L. acidophilus CL1285 alone (FIG. 7A).
  • OEA levels in the epididymal adipose tissue were decreased by the HF diet and were partially restored closer to those of the LF-Milk hamsters by L. acidophilus CL1285 and L. plantarum CHOL- 200 interventions, alone or in combination.
  • L. acidophilus CL1285 alone or in combination with L. plantarum CHOL-200, is effective at normalizing the HF diet-induced metabolic alterations, and at producing variations in the gut microbiota composition and the plasma/adipose tissue NAE levels. Therefore, if a sequential relationship exists among probiotic-induced changes in gut microbiota composition, endocannabinoidome signaling, and beneficial metabolic effects, this is likely mediated by NAEs. Additionally, the magnitude of the cholesterol-lowering effect of both L. acidophilus CL1285-based interventions i.e., HF-La and HF-LaLp) was comparable to that observed in cholestyramine-treated hamsters.

Abstract

The present disclosure relates generally to bacterial strains from the family Lactobacillaceae that exhibit one or more of a bile salt hydrolase activity, feruloyl esterase activity, cholesterol assimilation activity, gut microbiome modulatory activity, and endocannabinoidome modulatory activity. The present disclosure further relates to compositions and methods for reducing blood cholesterol and/or treating dyslipidemia in a subject, by administering one or more of the bacterial strains. The present disclosure even further relates to the production of bacterial compositions for reducing blood cholesterol and/or treating dyslipidemia in a subject.

Description

PROBIOTIC STRAINS FOR REDUCING BLOOD CHOLESTEROL AND/OR TREATING DYSLIPIDEMIA, AND METHODS FOR USING AND PRODUCING THE SAME
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0001] Not applicable.
TECHNICAL FIELD
[0002] The present disclosure relates generally to bacterial strains from the family Lactobacillaceae that exhibit one or more of a bile salt hydrolase activity, feruloyl esterase activity, cholesterol assimilation activity, gut microbiome modulatory activity, and endocannabinoidome modulatory activity. The present disclosure further relates to compositions containing the bacterial strain(s), and methods for reducing blood cholesterol and/or treating dyslipidemia in a subject, by administering one or more of the bacterial strains. The present disclosure even further relates to the production of bacterial compositions for reducing blood cholesterol and/or treating dyslipidemia in a subject.
Figure imgf000002_0001
BACKGROUND
[0003] Cardiovascular disease (CVD) is currently the leading causes of death worldwide. See, e.g., Collaborators, G.B.D.C.o.D ("Global, regional, and national age-sex- specific mortality for 282 causes of death in 195 countries and territories, 1980-2017: a systematic analysis for the Global Burden of Disease Study 2017,” Lancet, 2018, 392(10159): 1736-1788). In this connection, dyslipidemia (abnormal levels of blood lipids, especially low-density lipoprotein cholesterol (LDL-C)) is a major risk factor for CVD. See, e.g., Imamura etal. ("LDL cholesterol and the development of stroke subtypes and coronary heart disease in a general Japanese population: the Hisayama study,” Stroke, 2009, 40(2): 382-8; and Stamler etal. ("The Multiple Risk Factor Intervention Trial (MRFIT) - importance then and now," JAMA, 2008, 300(11): 1343-5).
[0004] Although numerous small molecular weight compounds are used clinically for normalization of serum lipids in hypercholesterolemic patients, the use of the vast majority of them is associated with adverse events such as muscular pain, diabetes, a decrease in renal function, and an increase in depression-like symptoms. See, e.g., Ramkumar etal. ("Statin Therapy: Review of Safety and Potential Side Effects,” Acta Cardiol Sin., 2016, 32(6): 631-639). For instance, over 30% of all patients receiving statins experience unwanted side effects such as muscle pain. See, e.g., Davies etal.
Figure imgf000003_0001
("Current and Emerging Uses of Statins in Clinical Therapeutics: A Review,” Lipid Insights, 2016, 9: LPI.S37450).
[0005] As an alternative, many individuals seek natural or alternative medicine products to improve lipid metabolism, such as various foods or supplements e.g., green tea, soluble fiber, or garlic). See, e.g., Cicero etal. ("Lipid lowering nutraceuticals in clinical practice: position paper from an International Lipid Expert Panel,” Arch. Med. Sci., 2017, 13(5): 965-1005); and Yeh et al. ("Use of complementary therapies in patients with cardiovascular disease,” Am. J. Cardiol., 2006, 98(5): 673-80). Probiotics are another. See, e.g., Hunter et al. ("Functional foods and dietary supplements for the management of dyslipidaemia,” Nature Reviews Endocrinology, 2017, 13(5): 278-288). [0006] Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. See, e.g., Hill et al. ("Expert consensus document. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic,” Nat. Rev. Gastroenterol. Hepatol., 2014, 11(8): 506-14). Probiotic strains with cholesterol- lowering activity have been identified, see, e.g., Miremadi etal. ("Cholesterol reduction mechanisms and fatty acid composition of cellular membranes of probiotic Lactobacilli and Bifidobacteria, "Journal of Functional Foods, 2014, 9: 295-305); Miremadi etal.
("Hypocholesterolaemic effect and anti-hypertensive properties of probiotics and
Figure imgf000004_0001
prebiotics: A review," Journal of Functional Foods, 2016, 25: 497-510); Wu etal. ("Effect of probiotic Lactobacillus on lipid profile: A systematic review and meta-analysis of randomized, controlled trials,” PLoS One, 2017, 12(6): e0178868); and Sun etal. ("Effects of probiotics consumption on lowering lipids and CVD risk factors: a systematic review and meta-analysis of randomized controlled trials, ’’Ann. Med., 2015, 47(6): 430- 40).
[0007] The most frequently used probiotic species are Lactobacillus spp., Bifidobaterium spp. and Saccharomyces spp. Several mechanisms of action have been proposed to explain their effects, including, for example, the production of antimicrobial substances, competition for gastro-intestinal colonization and available nutrients, immunomodulation, and the promotion of lactose digestion.
[0008] However, there remains a need for probiotic strains, and probiotic combinations and compositions, that can effectively reduce blood cholesterol and/or treat dyslipidemia, while also effectively resisting gastrointestinal conditions in vivo, such as pH and bile salts, so as to survive passage through the gastrointestinal tract.
SUMMARY OF THE INVENTION
[0009] The present disclosure provides bacterial strains from the family Lactobacillaceae that exhibit one or more of a bile salt hydrolase activity, feruloyl
Figure imgf000005_0001
esterase activity, cholesterol assimilation activity, gut microbiome modulatory activity, and endocannabinoidome modulatory activity. These bacterial strains, in certain embodiments, exhibit resistance to gastrointestinal conditions in vivo, such as pH and bile salts, and can therefore be used, singly or in combination, to reduce blood cholesterol and/or treating dyslipidemia in a subject.
[0010] Non-limiting embodiments of the disclosure include as follows.
[0011] [1] A composition for reducing cholesterol and/or treating dyslipidemia, wherein said composition comprises one or more strains of Lactobacillaceae, and wherein at least one of said one or more strains exhibits bile salt hydrolase (BSH) activity, and wherein at least one of said one or more strains exhibits feruloyl esterase (EF) activity. [0012] [2] The composition according to [1], wherein at least one of said one or more strains exhibits BSH activity and EF activity.
[0013] [3] The composition according to [1], wherein at least two of said one or more strains exhibits BSH activity.
[0014] [4] The composition according to any one of [1] -[3], wherein at least one of said one or more strains exhibits cholesterol assimilation activity.
[0015] [5] The composition according to any one of [1] -[4], wherein at least one of said one or more strains exhibits gut microbiome modulatory activity and/or endocannabinoidome modulatory activity.
Figure imgf000006_0001
[0016] [6] The composition according to [1], wherein said composition comprises
Lactiplantibacillus plantarum CHOL-200.
[0017] [7] The composition according to [1], wherein said composition comprises
Lactobacillus acidophilus CL1285.
[0018] [8] The composition according to [1], wherein said composition comprises
Lactobacillus acidophilus CL1285 and Lactiplantibacillus plantarum CHOL-200.
[0019] [9] The composition according to [8], wherein said composition comprises one or more additional probiotic strains.
[0020] [10] The composition according to [9], wherein at least one of said one or more additional probiotic strains is selected from Lactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacillus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus, Lentilactobacillus, Convivina, Frucctobacillus, , Oenococcus, Weissella, Saccharomyces, Streptococcus, Bacteroides, Pediococcus, Enterococcus, Leuconostoc, and/or
Bifidobacterium.
Figure imgf000007_0001
[0021] [11] The composition according to [10], wherein at least one of said one or more additional probiotic strains is selected from Lacticaseibacillus casei LBC80R, Lacticaseibacillus casei ATCC 393, Lacticaseibacillus rhamnosus ATCC 53103, Lactiplantibacillus pentosus BK1034, Lactiplantibacillus plantarum BK300, Lactiplantibacillus plantarum BK303, Lactiplantibacillus plantarum BK324, Lactiplantibacillus plantarum BK1135, Lactiplantibacillus plantarum BK1039, Lactiplantibacillus plantarum BK1040, Lactobacillus acidophilus BK1197, Lactobacillus acidophilus ATCC 314, Lactobacillus acidophilus ATCC 4355, Lactobacillus acidophilus ATCC 4356, Lactobacillus acidophilus AT CC 4796, Lactobacillus acidophilus AT CC 53544, Lactobacillus acidophilus ATCC 53671, Lactobacillus acidophilus ATCC 9224, Lactobacillus acidophilus LMG 11466, Lactobacillus acidophilus FERM BP-4980, Lactobacillus acidophilus FERM BP-4981, Lactobacillus crispatus BK343, Lactobacillus crispatus BK482, Lactobacillus crispatus A CC 55221, Lactobacillus gallinarum 9435, Lactobacillus asseri 33323, Levilactobacillus (Lactobacillus') namurensis BK214, Limosilactobacillus (Lactobacillus) vaginalis BK337, Limosilactobacillus (Lactobacillus) vaginalis BK372, Limosilactobacillus (Lactobacillus) vaginalis BK373, Limosilactobacillus (Lactobacillus) vaginalis BK349, Limosilactobacillus (Lactobacillus) vaginalis BK488, and Limosilactobacillus (Lactobacillus) vaginalis BK384.
Figure imgf000008_0001
[0022] [12] The composition according to any one of [1] -[11], wherein said composition is a pharmaceutical, nutraceutical, feed, foodstuff, food supplement, or beverage, composition.
[0023] [13] The composition according to [12], wherein said composition comprises at least one pharmaceutically-acceptable carrier or additive.
[0024] [14] The composition according to [12], wherein said composition is a feed, foodstuff, or food supplement.
[0025] [15] The composition according to any one of [1]-[14], wherein at least one of said one or more strains is in a lyophilized or spray-dried form.
[0026] [16] The composition according to any one of [1] -[15], wherein said composition further comprises at least one additional agent for reducing cholesterol or treating dyslipidemia.
[0027] [17] The composition according to [16], wherein said additional agent is selected from the group consisting of Atorvastatin, Fluvastatin, Lovastatin, Pitavastatin, Pravastatin, Rosuvastatin, Simvastatin, Ezetimibe, Bempedoic acid, Cholestyramine, Colesevelam, Colestipol, Amlodipine, Fenofibrate, Gemfibrozil, niacin, omega-3 fatty acids, fibers, soluble fibers, beta-glucans, and oat beta glucan.
[0028] [18] A method for reducing cholesterol and/or treating dyslipidemia, wherein said method comprises administering, to a subject in need thereof, one or more
Figure imgf000009_0001
strains of Lactobacillaceae, wherein at least one of said one or more strains exhibits bile salt hydrolase (BSH) activity, and wherein at least one of said one or more strains exhibits feruloyl esterase (EF) activity.
[0029] [19] The method according to [18], wherein at least one of said one or more administered strains exhibits BSH activity and EF activity.
[0030] [20] The method according to [18], wherein at least two of said one or more administered strains exhibits BSH activity.
[0031] [21] The method according to any one of [18] -[20], wherein at least one of said one or more administered strains exhibits cholesterol assimilation activity.
[0032] [22] The method according to any one of [18] -[21], wherein at least one of said one or more administered strains exhibits gut microbiome modulatory activity and/or endocannabinoidome modulatory activity.
[0033] [23] The method according to [18], wherein said subject is administered
Lactiplantibacillus plantarum CHOL-200.
[0034] [24] The method according to [18], wherein said subject is administered
Lactobacillus acidophilus CL1285.
[0035] [25] The method according to [18], wherein said subject is administered
Lactobacillus acidophilus CL1285 and Lactiplantibacillus plantarum CHOL-200.
Figure imgf000010_0001
[0036] [26] The method according to [25], wherein Lactobacillus acidophilus
CL1285 and Lactiplantibacillus plantarum CHOL-200 are administered to the subject at the same or different times.
[0037] [27] The method according to [25], wherein said subject is administered one or more additional probiotic strains.
[0038] [28] The method according to [27], wherein at least one of said one or more additional probiotic strains is selected from Lactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacillus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus, Lentilactobacillus, Convivina, Frucctobacillus, , Oenococcus, Weissella, Saccharomyces, Streptococcus, Bacteroides, Pediococcus, Enterococcus, Leuconostoc, and/or Bifidobacterium.
[0039] [29] The method according to claim 28, wherein at least one of said one or more additional probiotic strains is selected from Lacticaseibacillus casei LBC80R, Lacticaseibacillus casei ATCC 393, Lacticaseibacillus rhamnosus ATCC 53103,
Lactiplantibacillus pentosus BK1034, Lactiplantibacillus plantarum BK300,
Figure imgf000011_0001
Lactiplantibacillus plantarum BK303, Lactiplantibacillus plantarum BK324, Lactiplantibacillus plantarum BK1135, Lactiplantibacillus plantarum BK1039, Lactiplantibacillus plantarum BK1040, Lactobacillus acidophilus BK1197, Lactobacillus acidophilus ATCC 314, Lactobacillus acidophilus ATCC 4355, Lactobacillus acidophilus ATCC 4356, Lactobacillus acidophilus AT CC 4796, Lactobacillus acidophilus AT CC 53544, Lactobacillus acidophilus ATCC 53671, Lactobacillus acidophilus ATCC 9224, Lactobacillus acidophilus LMG 11466, Lactobacillus acidophilus FERM BP-4980, Lactobacillus acidophilus FERM BP-4981, Lactobacillus crispatus BK343, Lactobacillus crispatus BK482, Lactobacillus crispatus A CC 55221, Lactobacillus gallinarum 9435, Lactobacillus asseri 33323, Levilactobacillus (Lactobacillus') namurensis BK214, Limosilactobacillus (Lactobacillus) vaginalis BK337, Limosilactobacillus (Lactobacillus) vaginalis BK372, Limosilactobacillus (Lactobacillus) vaginalis BK373, Limosilactobacillus (Lactobacillus) vaginalis BK349, Limosilactobacillus (Lactobacillus) vaginalis BK488, and Limosilactobacillus (Lactobacillus) vaginalis BK384.
[0040] [30] The method according to any one of [18]-[29], wherein said one or more strains of Lactobacillaceae are administered to said subject in a pharmaceutical, nutraceutical, feed, foodstuff, food supplement, or beverage, composition.
[0041] [31] The method according to any one of [18] -[30], wherein said administration is oral administration.
Figure imgf000012_0001
[0042] [32] The method according to [30], wherein said administered composition comprises at least one pharmaceutically-acceptable carrier or additive.
[0043] [33] The method according to [30], wherein said administered composition is a feed, foodstuff, or food supplement.
[0044] [34] The method according to any one of [18] -[33], wherein at least one of said one or more administered strains is in a lyophilized or spray-dried form.
[0045] [35] The method according to any one of [18] -[34], wherein said subject has, or is, receiving at least one additional agent for reducing cholesterol or treating dyslipidemia.
[0046] [36] The method according to any one of [18]-[34], wherein said method further comprises administering at least one additional agent for reducing cholesterol or treating dyslipidemia.
[0047] [37] The method according to [35] or [36], wherein said additional agent is selected from the group consisting of Atorvastatin, Fluvastatin, Lovastatin, Pitavastatin, Pravastatin, Rosuvastatin, Simvastatin, Ezetimibe, Bempedoic acid, Cholestyramine, Colesevelam, Colestipol, Amlodipine, Fenofibrate, Gemfibrozil, niacin, omega-3 fatty acids, fibers, soluble fibers, beta-glucans, and oat beta glucan.
Figure imgf000013_0001
INCORPORATION BY REFERENCE
[0048] All patents, publications, and patent applications cited in the present specification are herein incorporated by reference as if each individual patent, publication, or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.
BRIEF DESCRIPTION OF THE FIGURES
[0049] The features of the invention are set forth with particularity in the appended claims. Abetter understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying figures.
[0050] FIG. 1 depicts a visual representation of BSH and EF-positive activities of L. acidophilus CL1285 and L. plantarum CHOL-200. Strains were grown in the presence oftaurocholic acid (TCA) and glycocholic acid (GCA) for 72 h under an anaerobic atmosphere at 37°C, or in the presence of ethyl ferulate (EF) for up to 6 days under an anaerobic atmosphere at 37°C. No EF activity was detected for L. plantarum CHOL-200.
Figure imgf000014_0001
[0051] FIG. 2 depicts the percentage of cholesterol assimilation by L. plantarum CHOL-200 ("1”), L. acidophilus CL1285 ("2”), and L. acidophilus AT CC 43121 (3”). Numbers bearing a different letter are significantly different (p < 0.05).
[0052] FIG. 3 depicts survival of L. acidophilus CL1285 and L. plantarum CHOL- 200 when exposed to a Simulated Gastric Fluid at pH 1.5 to 2.5 for 30 and 60 min. Black bar: L. acidophilus CL1285, grey bar: L. plantarum CHOL-200.
[0053] FIGS. 4A-4I depict metabolic parameters in hypercholesterolemic and hypertriglyceridemic hamsters after probiotic interventions. Following the 30 -day high- fat diet run-in period (Week 0), hamsters were gavaged with probiotic formulas for 6 weeks (Week 6). Body weight, food intake and liver weight at week 6 are illustrated in FIGS. 4A-4C, respectively. Metabolic parameters including fasting glucose, insulin, triglycerides, cholesterol, non-HDL cholesterol and HDL cholesterol to total cholesterol ratio for Week 0 and Week 6 are illustrated in FIGS. 4D-4I, respectively, with mean ± SEM. One-way ANOVA with a Tukey post-hoc. * p< 0.05 vs low fat of the run-in period, # p< 0.05 vs compared to LF-Milk and p< 0.05 vs HF-Milk post-intervention.
[0054] FIGS. 5A-5B depict the relative abundance of specific bacterial families in the microbiota of the ileum and the caecum after diet and probiotic interventions.
Relative abundances of selected gut bacterial families of interest in response to interventions in the ileum (FIG. 5A) and the caecum (FIG. 5B). Plots detail the log CSS-
Figure imgf000015_0001
normalised bacterial counts for each intervention groups. Boxes extend from first to third quartile, horizontal lines indicate median and whiskers extend to the maximal and minimal values within 1.5 times the interquartile range (IQR) from QI and Q3. * denote Tukey post-hoc p < 0.1 vs HF-Milk.
[0055] FIG. 6 depicts the association between gut microbiota and metabolic parameters in hypercholesterolemic and hypertriglyceridemic hamsters. Heatmap detailing significant Spearman rho coefficients (p < 0.05) between microbiota families in the ileum and caecum (top and bottom halves, respectively) and metabolic parameters. Family names in red identify those significantly modified by the probiotic interventions in one or both intestinal segments.
[0056] FIGS. 7A-7B depicts the reversal of diet-induced alterations in circulating levels of endocannabinoidome by probiotic intervention. FIG. 7A depicts relative changes of NAEs and 2 -MAGs in the plasma, ileum, inguinal adipose tissue (SAT) and epididymal adipose tissue (VAT) compared to LF-Milk hamsters. *0ne-way ANOVA and Tukey post-hoc p < 0.05 vs. HF-Milk. FIG. 7B depicts a heatmap detailing significant Spearman rho coefficients (p < 0.05) between plasma endocannabinoidome mediators and metabolic parameters. Names in red identify mediators that were significantly modified by the interventions.
Figure imgf000016_0001
[0057] FIG. 8 depicts changes in gut microbiota composition associated with changes in NAEs and 2 -MAGs. Heatmap illustrating significant Spearman rho correlation coefficients (p < 0.05) between the relative abundance of selected bacterial families in the ileum and NAE or 2 -MAG concentrations in the plasma, ileum, inguinal adipose tissue (SAT) and epididymal adipose tissue (VAT).
DETAILED DESCRIPTION OF THE INVENTION
[0058] The present inventors conceived of using bacterial strains from the family Lactobacillaceae, that exhibit one or more of a bile salt hydrolase (BSH) activity, feruloyl esterase (EF) activity, cholesterol assimilation activity, gut microbiome modulatory activity, and endocannabinoidome modulatory activity, to effectively reduce blood cholesterol and/or treat dyslipidemia in a subject. In some embodiments, a composition for reducing blood cholesterol and/or treating dyslipidemia contains a single Lactobacillaceae strain. In other embodiments, a composition for reducing blood cholesterol and/or treating dyslipidemia contains a plurality of Lactobacillaceae strains. The composition may demonstrate, by virtue of the presence of the strain or strains therein, one or more of a bile salt hydrolase activity, feruloyl esterase activity, cholesterol assimilation activity, gut microbiome modulatory activity, and endocannabinoidome modulatory activity. In some embodiments, the composition may
Figure imgf000017_0001
demonstrate, by virtue of the presence of the strain or strains therein, at least 1, at least
2, at least 3, or at least 4, of these activities. In some embodiments, the composition may demonstrate, by virtue of the presence of the strain or strains therein, all of these activities.
[0059] As discussed herein, the present inventors identified Lactobacillaceae strains having desirable activities for reducing blood cholesterol, including, e.g., bile salt hydrolase activity, feruloyl esterase activity, cholesterol assimilation activity, gut microbiome modulatory activity, and endocannabinoidome modulatory activity. The present inventors further identified the ability of particular Lactobacillaceae strains having these beneficial properties to effectively resist gastrointestinal conditions in vivo, such as pH and bile salts, so as to survive passage through the gastrointestinal tract.
[0060] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the present specification and the appended claims, the singular forms "a,” "an,” and "the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide” includes one or more polynucleotides, and reference to "a vector” includes one or more vectors.
[0061] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to
Figure imgf000018_0001
which the invention pertains. Although other methods and materials similar, or equivalent, to those described herein can be useful in the present invention, preferred materials and methods are described herein.
[0062] By "isolated” is meant, when referring to a probiotic strain, such as a strain of Lactobacillaceae for example, that the strain is found in an environment, composition, or mixture, distinct from that which it occurs in nature.
[0063] The term "purified” as used herein preferably means at least 75% by weight, more preferably at least 85% by weight, more preferably still at least 95% by weight, and most preferably at least 98% by weight, of the same molecule is present. [0064] The terms "engineered,” "genetically engineered,” "genetically modified,” "recombinant,” "modified,” "non-naturally occurring,” and "non-native” indicate intentional human manipulation of the genome of an organism or cell. The terms encompass methods of genomic modification that include genomic editing, as well as techniques that alter gene expression or inactivation, enzyme engineering, directed evolution, knowledge-based design, random mutagenesis methods, gene shuffling, codon optimization, and the like. Methods for genetic engineering are known in the art. Genetically modified probiotic strains are within the scope of the present disclosure.
[0065] As used herein, "expression” refers to transcription of a polynucleotide from a D NA template, resulting in, for example, a messenger RNA [mRNA], The term
Figure imgf000019_0001
further refers to the process through which transcribed mRNA is translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides maybe referred to collectively as "gene products.” A "coding sequence” or a sequence that "encodes” a selected polypeptide, is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when under the control of appropriate regulatory sequences.
[0066] As used herein, the term "modulate” refers to a change in the quantity, degree or amount of a function.
[0067] As used herein, the term "between” is inclusive of end values in a given range e.g., between about 1 and about 50 nucleotides in length includes 1 nucleotide and 50 nucleotides.
[0068] As used herein, the term "amino acid” refers to natural and synthetic (unnatural) amino acids, including amino acid analogs, modified amino acids, peptidomimetics, glycine, and D or L optical isomers.
[0069] As used herein, the terms "peptide,” "polypeptide,” and "protein” are interchangeable and refer to polymers of amino acids. A polypeptide may be of any length. It may be branched or linear, it may be interrupted by non-amino acids, and it may comprise modified amino acids. The terms also refer to an amino acid polymer that has been modified through, for example, acetylation, disulfide bond formation,
Figure imgf000020_0001
glycosylation, lipidation, phosphorylation, pegylation, biotinylation, cross -linking, and/or conjugation (e.g., with a labeling component or ligand).
[0070] The terms "subject,” "individual,” or "patient” are used interchangeably herein and refer to any member of the phylum Chordata, including, without limitation, humans and other primates, including non-human primates, such as rhesus macaques, chimpanzees, and other monkey and ape species; farm animals, such as cattle, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals, including rabbits, mice, rats, and guinea pigs; birds, including domestic, wild, and game birds, such as chickens, turkeys, and other gallinaceous birds, ducks, and geese; and the like. The term does not denote a particular age or gender. Thus, the term includes adult, young, and newborn individuals as well as males and females.
[0071] The terms "effective amount” or "therapeutically effective amount” of a composition or agent, refer to a sufficient amount of the composition or agent to provide the desired response. Preferably, the effective amount will prevent, avoid, or eliminate one or more harmful side-effects. The exact treatment amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, and the particular treatment used, mode of administration, and the like. An appropriate "effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
Figure imgf000021_0001
[0072] 'Treatment” or "treating” a particular disease, includes: (1) preventing the disease, for example, preventing the development of the disease or causing the disease to occur with less intensity in a subject that may be predisposed to the disease, but does not yet experience or display symptoms of the disease; (2) inhibiting the disease, for example, reducing the rate of development, arresting the development or reversing the disease state; and/or (3) relieving symptoms of the disease, for example, decreasing the number of symptoms experienced by the subject.
[0073] The term "pharmaceutically acceptable,” as used herein, refers to the suitability of, for example, a substance, carrier, composition, or vehicle, etc., for administration in vivo, such as to a mammal, including a human.
[0074] The term "nutritionally acceptable,” as used herein, refers to the suitability of, for example, a substance, composition, or vehicle, etc., for ingestion in vivo, such as to a mammal, including a human.
[0075] The term "strain,” as used herein, refers to a genetic variant, or subtype, of a microorganism species (such as a subtype of a bacterial species that is useful as a probiotic). The term "probiotic strain,” as used herein, typically refers to a live strain of a microorganism, including a strain capable of future replication and/or metabolism (e.g., strains in spore form, lyophilized form, spray-dried form, etc.).
Figure imgf000022_0001
[0076] The term "cholesterol-assimilation activity,” as used herein, refers to the ability of a strain, such as a probiotic strain, to capture cholesterol and incorporate it into its bacterial cellular structure. The assimilation of cholesterol by a particular strain can be assessed, for example, by using fluorescently-labeled cholesterol (or fluorescent analogues of cholesterol), and measuring incorporation of the labeled cholesterol, or cholesterol analog, into the strain by fluorometry or fluorocytometry, for example. [0077] The term "viability,” as used herein, refers to the ability of a cell, such as a bacterial cell, to be capable of future replication, cell division, and/or cellular metabolism. For instance, viable bacterial cells may be in a metabolically-active state, a dividing state, a vegetative state, and/or in a dormant state e.g., strains in spore form, lyophilized form, spray-dried form, etc.'). Conversely, a bacterial cell that is non-viable indicates that the cell is not capable of cellular metabolic activity, or replication, at present or in the future. It will be appreciated that non-viable cells include dead cells. Viability can be determined by assays known in the art. For example, cytolysis or membrane leakage assays, such as lactate dehydrogenase assays, propidium iodide assays, trypan blue assays, and 7 -aminoactinomycin D assays, as well as genomic and proteomic assays.
[0078] The term "active growth phase,” as used herein, refers to the stage of bacterial growth characterized by an increase in total biomass or cell numbers.
Figure imgf000023_0001
Bacterial growth in culture can typically be characterized into different phases, including lag phase, log (or exponential) phase, and stationary phase. During lag phase, bacteria adapt their growth conditions, and the rate of increase in cell number/biomass is minimal. In log phase, however, the growth rate increases to reach a constant and maximal growth rate under the particular growth conditions. During stationary phase, the growth rate declines, typically to a point where growth is absent or negligible. After stationary phase, cells may become non-viable (e.g., dead), which is sometimes referred to as the death phase. Bacterial growth may be measured, for example, by measuring bacterial cell number (e.g., by measuring optical density of the culture), biomass, and/or the metabolism, catabolism, or production of various substances.
[0079] The term "substantially pure culture,” as used herein, refers to a culture or composition e.g., of bacteria) in which a particular bacterial strain or strains, depending on the embodiment, represent at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, of the total bacterial strains in the culture or composition.
[0080] The term "culture,” as used herein, refers to a population of microorganisms within a defined environment, for instance, within an in vitro vessel, container, fermenter, vat, or flask. Where the organism is a bacteria, the culture may be known as a "bacterial culture.” A culture may comprise one or more additional
Figure imgf000024_0001
elements, including, for example, culture medium and/or additives for promoting the growth of the desired microorganism(s). Culture medium maybe in a liquid, solid, or semi-solid state (including gelatinous media such as agar, agarose, gelatin and collagen, for example). Conversely, agents may be added to suppress the growth of undesired microorganisms e.g., antibiotics and/or anti-fungal agents).
[0081] The term "dyslipidemia,” as used herein, refers to an abnormal level and/or profile of lipids and/or lipid proteins in the blood. For example, an amount of one or more lipids may be increased or decreased with respect to the levels thereof in a healthy subject. Such lipids and lipid proteins may include, for example, cholesterol, glycerides (such as triglycerides), and lipoproteins (such as low-density lipoprotein (LDL) and high-density lipoprotein (HDL).
Probiotic Strains
[0082] The present disclosure provides bacterial strains from the family Lactobacillaceae that exhibit one or more of a bile salt hydrolase activity, feruloyl esterase activity, cholesterol assimilation activity, gut microbiome modulatory activity, and endocannabinoidome modulatory activity.
[0083] In embodiments herein, these bacterial strains exhibit resistance to gastrointestinal conditions in vivo, such as pH and bile salts, and can therefore be used,
Figure imgf000025_0001
singly or in combination, to reduce blood cholesterol and/or treat dyslipidemia in a subject.
[0084] Bacterial strains from the family Lactobacillaceae include, without limitation, homofermentative and heterofermentative organisms, such as members of the genus Lactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacillus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus, Lentilactobacillus, Convivina, Frucctobacillus, Leuconosto, Oenococcus and Weissella. In some embodiments, the bacterial strain or strains is at least one selected from the group consisting of Lactobacillus and Lactiplantibacillus.
[0085] In some embodiments, the bacterial strain or strains exhibits bile salt hydrolase (BSH) activity, typically through bacterial production and secretion of BSH. BSH breaks the peptide linkage between primary bile acid, and either glycine or taurine, thereby liberating the amino acid from the sterol core. The resultant unconjugated bile acids are less soluble than the conjugated form from which they were liberated, and accordingly, are retained in the intestinal lumen (where they mix with the surrounding
Figure imgf000026_0001
intestinal content). This ultimately results in excretion of the unconjugated bile acids from the host in feces, necessitating the synthesis of new bile acid molecules de novo using cholesterol present in the host, thereby reducing the amount of circulating blood cholesterol in the host. See, e.g., Liong et al. ("Bile salt deconjugation ability, bile salt hydrolase activity and cholesterol co-precipitation ability of lactobacilli strains,” International Dairy Journal, 2005, 15(4): 391-398; and Pereira etal. ("Effects of consumption of probiotics and prebiotics on serum lipid levels in humans,” Crit. Rev. Biochem. Mol. Biol., 2002, 37(4): 259-81).
[0086] As demonstrated by the present inventors herein, BSH-producing bacteria may be identified, for example, by incubating bacteria in the presence of sodium taurocholic acid hydrate and/or or sodium glycocholate hydrate, and assessing colony morphology and/or precipitate, as described in Pereira etal. ("An in vitro study of the probiotic potential of a bile-salt-hydrolyzing Lactobacillus fermentum strain, and determination of its cholesterol-lowering properties,” Appl. Environ. Microbiol., 2003, 69(8): 4743-52). Other techniques known in the art for detecting BSH expression and/or activity may also be used.
[0087] In some embodiments, the bacterial strain or strains exhibits feruloyl esterase (EF) activity, typically through bacterial production and secretion of EF. EF is naturally involved in the liberation of phenolic compounds, such as ferulic acid (FA),
Figure imgf000027_0001
from plant cell walls. See, e.g., Benoit eta/. ("Biotechnological applications and potential of fungal feruloyl esterases based on prevalence, classification and biochemical diversity,” Biotechnol. Lett., 2008, 30(3): 387-96); Esteban-Torres etal.
("Characterization of a feruloyl esterase from Lactobacillus plantarum,’’ Appl. Environ. Microbiol., 2013, 79(17): 5130-6); and Tomaro-Duchesneau etal. ("Lactobacillus fermentum NCIMB 5221 has a greater ferulic acid production compared to other ferulic acid esterase producing Lactobacilli," International Journal of Probiotics and Prebiotics, 2012, 7(1): 23-32). However, EF can also act as a competitive inhibitor against one of the main enzymes responsible for de novo synthesis of cholesterol, namely hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase), which is the same enzyme targeted by statins. See, e.g., Kim etal. ("Lipid-lowering efficacy of hesperetin metabolites in high-cholesterol fed rats,” Clin. Chim. Acta, 2003, 327(1-2): 129-37). EF can also activate peroxisome proliferator-activated receptor alpha (PPAR-a), a nuclear transcription factor implicated in the synthesis of two-cholesterol transporters (HDL and LDL). PPAR-a activation has been associated with an increase in HDL and a decrease in LDL. See, e.g., Dominguez-Avila etal. ("Modulation of PPAR Expression and Activity in Response to Polyphenolic Compounds in High Fat Diets,” Int. J. Mol. Sci., 2016,
17(7)).
Figure imgf000028_0001
[0088] As demonstrated by the present inventors herein, EF-producing bacteria may be identified, for example, by incubating bacteria in the presence of ethyl ferulate, and assessing colony morphology and/or clearing around colonies, as generally described in Donaghy etal. ("Detection of ferulic acid esterase production by Bacillus spp. and lactobacilli,’’ Appl. Microbiol. Biotechnol, 1998, 50(2): 257-60). Other techniques known in the art for detecting EF expression and/or activity may also be used.
[0089] In some embodiments, the bacterial strain or strains exhibits cholesterol assimilation activity, such as by incorporating cholesterol into the bacterial cell membrane. This cholesterol assimilation activity thereby has an effect of sequestering cholesterol from the host, and may lead to the indirect excretion of cholesterol by the host via excretion of the bacteria in feces, thereby decreasing the overall pool of circulating blood cholesterol. See, e.g., Pereira etal. ("Effects of consumption of probiotics and prebiotics on serum lipid levels in humans,” Crit. Rev. Biochem. Mol. Biol., 2002, 37(4): 259-81); Gilliland etal. ("Assimilation of cholesterol by Lactobacillus acidophilus," Appl. Environ. Microbiol., 1985, 49(2): 377-81); Lye etal. ("Removal of cholesterol by lactobacilli via incorporation and conversion to coprostanol,” Journal of Dairy Science, 2010, 93: 1383-92); and Noh etal. ("Incorporation of Cholesterol into the Cellular Membrane of Lactobacillus acidophilus ATCC 431211, "Journal of Dairy Science,
1997, 80(12): 3107-3113).
Figure imgf000029_0001
[0090] As demonstrated by the present inventors herein, bacteria having cholesterol assimilation activity can be identified, for example, by measuring the amount of cholesterol incorporated into the bacteria, as described by, e.g., Gilliland etal. ("Assimilation of cholesterol by Lactobacillus acidophilus,” Appl. Environ. Microbiol., 1985, 49(2): 377-81) and Rudel etal. ("Determination of cholesterol using o- phthalaldehyde,”/. Lipid Res., 1973, 14(3): 364-6). Other techniques known in the art for detecting incorporation and/or assimilation of cholesterol into bacterial cells may also be used.
[0091] In some embodiments, the bacterial strain or strains exhibits one or both of gut microbiome modulatory activity and endocannabinoidome modulatory activity. [0092] Endocannabinoids (2-arachidonoyl-glycerol; 2 -AG and /V-arachidonoylethanolamine; AEA) are endogenously produced bioactive lipids that act on the receptor target for the principle active component of cannabis (delta9- tetrahydrocannabinol; THC), cannabinoid receptor 1 and 2 (CBiand2) and other targets. A host of endocannabinoid congeners belonging to the 2 -monoacylglycerol (2-MAG) and /V-acylethanolamine (NAE) families have been identified and are rapidly produced/degraded by a redundant series of enzymes that also act on classical endocannabinoids. See, e.g., Di Marzo etal. ("New approaches and challenges to targeting the endocannabinoid system,” Nat. Rev. Drug Discov., 2018, Doi:
Figure imgf000030_0001
10.1038/nrd.2018.115). These related molecules show some redundancy at the level of receptor targets with endocannabinoids, but also act on a host of receptors, such as G protein-coupled receptors, peroxisome proliferator-activated receptors (PPARs) and transient receptor potential cation channels (TRPs), which often have opposite physiological effects to CBi. This collection of related lipid-derived mediators, their regulatory enzymes and receptor targets are collectively referred to as the endocannabinoidome. See, e.g., Di Marzo etal. ("New approaches and challenges to targeting the endocannabinoid system,” Nat. Rev. Drug Discov., 2018, Doi: 10.1038/nrd.2018.115). This system was shown to intervene in regulating several aspects of energy homeostasis, food intake, inflammation and immune response, among other biological functions. See, e.g., Silvestri et al. ("The endocannabinoid system in energy homeostasis and the etiopathology of metabolic disorders,” Cell Metab., 2013, 475-90. Doi: 10.1016/j.cmet.2013.03.001); and Di Marzo etal. ("Lifestyle and metabolic syndrome: Contribution of the endocannabinoidome,” Nutrients, 2019, Doi: 10.3390/null081956).
[0093] Evidence suggests that manipulation of the gut microbiome modulates the activity of the endocannabinoidome, while the reverse relationship has also been shown in mice with blunted endocannabinoidome metabolic pathways. See, e.g., Muccioli etal.
("The endocannabinoid system links gut microbiota to adipogenesis,” Mol. Syst. Biol.,
Figure imgf000031_0001
2010, 6, Doi: 10.1038/msb.2010.46); Everard eta/. ("Intestinal epithelial N- acylphosphatidylethanolamine phospholipase D links dietary fat to metabolic adaptations in obesity and steatosis,” Nat. Commun., 2019, Doi: 10.1038/s41467-018- 08051-7); and Manca etal. ("Germ-free mice exhibit profound gut microbiota-dependent alterations of intestinal endocannabinoidome signaling,” J. Lipid Res., 2020, Doi: 10.1194/jlr.RA119000424). Additionally, it is suggested that the gut microbiota- endocannabinoidome axis forms a triangular relationship with metabolic health.
[0094] In some embodiments, the bacterial strain or strains, upon administration to a subject, are able to modulate the gut microbiota composition e.g., in the small intestine and/or caecum) and/or alter the plasma and/or tissue levels of one or more endocannabinoidome mediators e.g., members of the /V-acylethanolamine (NAE) and 2- monoacylglycerol (2 -MAG) endocannabinoidome mediator families).
[0095] In some embodiments, the bacterial strain or strains are one or more selected from Lacticaseibacillus casei LBC80R, Lacticaseibacillus casei ATCC 393, Lacticaseibacillus rhamnosus ATCC 53103, Lactiplantibacillus pentosus BK1034, Lactiplantibacillus plantarum BK300, Lactiplantibacillus plantarum BK303, Lactiplantibacillus plantarum BK324, Lactiplantibacillus plantarum BK1135, Lactiplantibacillus plantarum BK1039, Lactiplantibacillus plantarum BK1040,
Lactiplantibacillus plantarum CHOL-200, Lactobacillus acidophilus BK1197, Lactobacillus
Figure imgf000032_0001
acidophilus ATCC 314, Lactobacillus acidophilus ATCC 4355, Lactobacillus acidophilus ATCC 4356, Lactobacillus acidophilus AT CC 4796, Lactobacillus acidophilus AT CC 53544, Lactobacillus acidophilus ATCC 53671, Lactobacillus acidophilus ATCC 9224, Lactobacillus acidophilus LMG 11466, Lactobacillus acidophilus CL1285, Lactobacillus acidophilus FERM BP-4980, Lactobacillus acidophilus FERM BP-4981, Lactobacillus crispatus BK343, Lactobacillus crispatus BK482, Lactobacillus crispatus ATCC 55221, Lactobacillus gallinarum 9435, Lactobacillus gasseri 33323, Levilactobacillus (Lactobacillus') namurensis BK214, Limosilactobacillus (Lactobacillus) vaginalis BK337, Limosilactobacillus (Lactobacillus) vaginalis BK372, Limosilactobacillus (Lactobacillus) vaginalis BK373, Limosilactobacillus (Lactobacillus) vaginalis BK349, Limosilactobacillus (Lactobacillus) vaginalis BK488, and Limosilactobacillus (Lactobacillus) vaginalis BK384. [0096] In some embodiments, the bacterial strain is selected from Lactobacillus acidophilus CL1285 (deposited November 15, 1994, as Accession Number LALC 1285, under the terms of the Budapest Treaty, with the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris Cedex 15, France) and Lactiplantibacillus plantarum CHOL-200 (deposited August 25, 2021, as Accession Number CNCM 1-5742, under the terms of the Budapest Treaty, with the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25-
28 rue du Docteur Roux, 75724 Paris Cedex 15, France). Lactobacillus acidophilus
Figure imgf000033_0001
CL1285 is a bacterium of genus Lactobacillus and species Lactobacillus acidophilus. Lactiplantibacillus plantarum CHOL-200 is a bacterium of genus Lactiplantibacillus and species Lactiplantibacillus plantarum. Relevant information on the characteristics of Lactobacillus acidophilus CL1285 and Lactiplantibacillus plantarum CHOL-200 can be found throughout the present application, in particular the description and drawings. For example, suitable media for propagation of Lactobacillus acidophilus CL1285 and Lactiplantibacillus plantarum CHOL-200 are described in the examples, including but not limited to paragraphs [00144], [00145], [00152], and [00159],
[0097] In some embodiments, Lactobacillus acidophilus CL1285 and Lactiplantibacillus plantarum CHOL-200 are used in combination, such as, for example, by being combined within a single composition; or being co-administered to a subject. [0098] The present disclosure also encompasses variants of the strains listed above, such as, for example, variants of the Lactobacillus acidophilus CL1285 and Lactiplantibacillus plantarum CHOL-200 strains. Such variant strains may or may not have the same identifying biological characteristics of the specific strains listed herein, provided they share similar advantageous properties in terms of, for example, one or more of bile salt hydrolase activity, feruloyl esterase activity, cholesterol assimilation activity, gut microbiome modulatory activity, and endocannabinoidome modulatory activity.
Figure imgf000034_0001
[0099] For example, the 16S rRNA genes of a variant strain as contemplated herein may share at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, with a strain disclosed herein.
[00100] In other embodiments, the degree of relatedness between a variant and a parent strain may be defined according to the average nucleotide identity (ANI), which reflects DNA conservation of the core genome. See, e.g., Konstantinidis et al. Proc. Natl. Acad. Sci. USA, 2005, 102: 2567-2592). In some embodiments, the ANI between a variant and a parent strain is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.99%, at least 99.999%, at least 99.9999%, at least 99.99999%, or at least 99.999999%.
[00101] In other embodiments, the degree of relatedness between a variant and a parent strain may be defined according to the degree of similarity obtained when analyzing the genomes of the parent and variant strains by Pulsed-Field Gel Electrophoresis (PFGE), using one or more restriction endonucleases. The degree of similarity obtained by PFGE can be measured by the Dice similarity coefficient. In some embodiments, the Dice similarity coefficient between the variant and the parent strain is
Figure imgf000035_0001
at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.99%, at least 99.999%, at least 99.9999%, at least 99.99999%, or at least 99.999999%.
[00102] In other embodiments, the degree of relatedness between a variant and a parent strain may be defined according to ribotype, as obtained using any of the methods known in the art.
[00103] In other embodiments, the degree of relatedness between a variant and a parent strain may be defined according to the Pearson correlation coefficient obtained by comparing the genetic profiles of both strains obtained by repetitive extragenic palindromic element-based PCR (REP-PCR). In some embodiments, the Pearson correlation coefficient obtained by comparing the REP-PCR profiles of the variant and the parent strain is at least 0.99, at least 0.999, at least 0.9999, at least 0.99999, at least 0.999999, or at least 0.999999.
[00104] In other embodiments, the degree of relatedness between a variant and a parent strain may be defined according to the linkage distance obtained by comparing the genetic profiles of both strains obtained by Multilocus sequence typing (MLST). In some embodiments, the linkage distance obtained by MLST of the variant and the parent
Figure imgf000036_0001
strain is at least 0.99, at least 0.999, at least 0.9999, at least 0.99999, at least 0.999999, or at least 0.999999.
[00105] In embodiments herein, the probiotic strain or strains are preferably in the form of viable cells. In some embodiments, the viable cells are in the form of living cells in a metabolically-active state, a dividing state, a vegetative state, and/or in a dormant state e.g., strains in spore form, lyophilized form, spray-dried form, etc.'), in some embodiments, the probiotic strain or strains in a culture or composition have a viability of at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, in terms of the total number of cells of the probiotic strain(s). [00106] In some embodiments, the probiotic strain or strains are present in a substantially pure culture. In some embodiments, the probiotic strain or strains in the substantially pure culture represent at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, of the total bacterial strains in the culture.
Compositions, Foods, and Nutritional Products
Figure imgf000037_0001
[00107] The present disclosure provides compositions containing one or more of the probiotic strains disclosed herein. In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 probiotic strains as contemplated herein are included in a composition. In some embodiments, one or more probiotic strains of the family Lactobacillaceae are present in a composition together with one or more non-Lactobacillaceae microorganisms, such as, for example, one or more microorganisms from the genera Saccharomyces, Streptococcus, Bacteroides, Pediococcus, Enterococcus, Leuconostoc, and/or Bifidobacterium.
[00108] A single probiotic strain may constitute up to 1%, up to 5%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, or up to 99%, of the total probiotic cells in a composition.
[00109] In some embodiments, at least one of the probiotic strains in the composition is Lactobacillus acidophilus CL1285. In some embodiments, at least one of the probiotic strains in the composition is Lactiplantibacillus plantarum CHOL-200. In some embodiments, the composition contains both of Lactobacillus acidophilus CL1285 and Lactiplantibacillus plantarum CHOL-200. In some embodiments, Lactobacillus acidophilus CL1285 and Lactiplantibacillus plantarum CHOL-200 are the only probiotic
Figure imgf000038_0001
strains in the composition. In some embodiments, Lactobacillus acidophilus CL1285 and Lactiplantibacillus plantarum CHOL-200 are the only Lactobacillaceae in the composition. In some embodiments, multiple compositions containing different probiotic strains are provided. For instance, in some embodiments, a first composition may contain Lactobacillus acidophilus CL1285, and a second composition may contain Lactiplantibacillus plantarum CHOL-200. These compositions maybe administered, for example, to a subject at the same or different times, and/or, at different frequencies. Such multiple compositions may be packaged together, for example, within a kit, together with instructions for their use.
[00110] Also provided herein are uses of the compositions and probiotic strain(s) in the manufacture of a medicament to reduce cholesterol and/or treat dyslipidemia. [00111] Probiotic-containing compositions of the present disclosure include, for example, compositions in a solid, liquid, or semi-solid form, including, without limitation, gels, granules, powders, capsules, bars, blocks, jellies, etc. Such compositions can be formulated as, for example, pharmaceutical, nutraceutical, feed, foodstuff, food supplement, or beverage, compositions.
[00112] When the probiotic strain or strains of the present disclosure is provided as a pharmaceutical product or pharmaceutical additive, the pharmaceutical product or pharmaceutical product may be formulated as, for example, a powder, granule, pill, soft
Figure imgf000039_0001
capsule, hard capsule, tablet, chewable tablet, quick-disintegrating tablet, syrup, liquid, suspension, suppository, injection, or the like.
[00113] Preferably the probiotic-containing composition is formulated for delivery to the oral, gastric and/or to the intestinal mucosal surface. However, also contemplated herein are compositions formulated for alternate delivery routes, such as nasopharyngeal, respiratory, reproductive or glandular mucosa, and it may be administered to a subject by an oral, nasal, ocular, rectal, topical and/or vaginal route. [00114] Examples of compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablet), pills, granules, powders, capsules (including soft and hard capsules), syrups, emulsions, suspensions, and the like.
[00115] Such compositions may be produced using methods known to persons of skill in the art, and may also contain additives or carriers generally used in the pharmaceutical field, such as excipients, binders, disintegrants, lubricants, for instance. Examples of excipients include animal and plant oils, polyvalent alcohols (such as polyethylene glycol, propylene glycol, glycerol, sorbitol), surfactants (such as sorbitan fatty acid ester, sucrose fatty acid ester, glycerin fatty acid ester, polyglycerol fatty acid ester), purified water, lactose, starch, crystalline cellulose, D -mannitol, lecithin, gum arable, sorbitol solution, carbohydrate solution, for example.
Figure imgf000040_0001
[00116] Examples of binders include hydroxypropylmethylcellulose, hydroxypropylcellulose, gelatin, pregelatinized starch, and polyvinylpyrrolidone, polyvinylalcohol. Examples of disintegrants include carmellose calcium, carmellose sodium, croscarmellose sodium, crospovidone, low-substituted hydroxypropylcellulose, and cornstarch. Examples of lubricants include talc, hydrogenated vegetable oil, waxes, stearic acid, magnesium stearate, calcium stearate, and aluminum stearate. In some embodiments, compositions of the present disclosure may also contain one or more of a sweetener, a colorant, a pH adjuster, a flavor, various amino acids, etc.
[00117] In some embodiments, compositions of the presently claimed invention may include, or be used in combination with, other drugs, treatments, and/or medicaments, for reducing cholesterol and/or treating dyslipidemia.
[00118] For instance, compositions of the presently claimed invention may include, or be used in combination with: a statin e.g., Atorvastatin, Fluvastatin, Lovastatin, Pitavastatin, Pravastatin, Rosuvastatin, Simvastatin, etc.); Ezetimibe; Alirocumab; Evolocumab; Bempedoic acid; Cholestyramine; Colesevelam; Colestipol; Amlodipine; Fenofibrate; Gemfibrozil; niacin; omega-3 fatty acids, fibers, soluble fibers, beta-glucans, and oat beta glucan.
[00119] When a probiotic strain or strains is provided in the form of a food (or feed) or food additive (or feed additive), the food (or feed) or food (or feed) containing
Figure imgf000041_0001
the additive is not particularly limited as long as it permits oral ingestion, such as a solution, suspension, powder, solid formed particles, granules, and bars, for example. [00120] Specific examples include supplements (powders, granules, soft capsules, hard capsules, tablets, chewable tablets, quick-disintegrating tablets, syrups, liquids, etc.); drinks (carbonated drinks, milk drinks, sport drinks, fruit juice drinks, vegetable drinks, soymilk drink, coffee drinks, tea drinks, powder drinks, concentrated drinks, nutrition drinks, alcohol drinks etc.); dairy products (yogurt, butter, cheese, ice cream etc.); and confectionery (gummies, jellies, gums, chocolates, cookies, candies, caramels, etc.), and carbohydrate-based products (bread, pasta, noodles, cake mixes, breads, etc.). [00121] The above-mentioned foods (or feed) can contain, in some embodiments, one or more nutrients, vitamins e.g., vitamin A, vitamin Bl, vitamin B2, vitamin B6, vitamin C, vitamin D, vitamin E, vitamin K, etc.), minerals {e.g., magnesium, zinc, iron, sodium, potassium, selenium, etc.), dietary fiber, a dispersing agent, a stabilizer, an emulsifier, a sweetener, and/or a flavoring acid {e.g., citric acid, malic acid, etc.). [00122] In some embodiments, compositions of the present disclosure may be formulated to contain, or so as to provide as a daily dose, at least 104, at least 105, at least 106, at least 107, at least 108, at least 109, at least 1010, at least 1011, or at least 1012, viable cells of a probiotic strain or strains.
Figure imgf000042_0001
Therapeutic Uses
[00123] The present disclosure further provides, in some embodiments, methods for treating elevated cholesterol; methods for reducing cholesterol, including blood cholesterol; and methods for preventing cholesterol elevation. The present disclosure thereby provides, in some embodiments, therapeutic and preventative compositions and methods against additional diseases, disorders, and ailments, caused by, associated with, or resulting from, elevated cholesterol levels.
[00124] The present disclosure further provides, in some embodiments, methods for treating or preventing dyslipidemia, including, for instance, modulating the level of one or more of cholesterol, glycerides (such as triglycerides), and lipoproteins (such as low-density lipoprotein (LDL) and high-density lipoprotein (HDL). The present disclosure thereby provides, in some embodiments, therapeutic and preventative compositions and methods useful as a treatment, or adjunct therapy, for additional diseases, disorders, and ailments that are caused by, associated with, or resulting from, dyslipidemia (such as, for example, insulin resistance, metabolic syndrome, atherosclerosis, heart failure, or diabetes).
[00125] In some embodiments, the dyslipidemia is hypercholesterolemia e.g., where blood cholesterol levels are elevated above clinically and/or medically recommended levels). In some embodiments, hypercholesterolemia may be determined
Figure imgf000043_0001
by measuring the levels of one or more of: HDL, LDL, and triglycerides. In some embodiments, hypercholesterolemia may be determined by measuring all of HDL, LDL, and triglycerides.
[00126] In some embodiments, a subject may be judged or diagnosed to have hypercholesterolemia, and/or be suitable for cholesterol management, if the measured LDL levels are, for example, above 70 mg/dl, above 80 mg/dl, above 90 mg/dl, above 100 mg/dl, above 110 mg/dl, above 120 mg/dl, above 130 mg/dl, above 140 mg/dl, above 150 mg/dl, above 160 mg/dl, above 170 mg/dl, above 180 mg/dl, or above 200 mg/dl. As will be appreciated by persons of skill in the art, there may be differing degrees of severity of hypercholesterolemia, depending on the measured LDL value. [00127] In some embodiments, a subject may be judged or diagnosed to have hypercholesterolemia, and/or be suitable for cholesterol management, if the measured HDL levels are, for example, below 60 mg/dl, below 55 mg/dl, below 50 mg/dl, below 45 mg/dl, below 40 mg/dl, below 35 mg/dl, below 30 mg/dl, below 25 mg/dl, or below 20 mg/dl. As will be appreciated by persons of skill in the art, there may be differing degrees of severity of hypercholesterolemia, depending on the measured HDL value.
[00128] In some embodiments, a subject may be judged or diagnosed to have hypercholesterolemia, and/or be suitable for cholesterol management, if the measured triglyceride levels are, for example, above 140 mg/dl, above 145 mg/dl, above 150
Figure imgf000044_0001
mg/dl, above 155 mg/dl, above 160 mg/dl, above 170 mg/dl, above 180 mg/dl, above 190 mg/dl, above 200 mg/dl, above 300 mg/dl, above 400 mg/dl, or above 500 mg/dl. As will be appreciated by persons of skill in the art, there may be differing degrees of severity of hypercholesterolemia, depending on the measured triglycerides value.
[00129] Alternatively, or additionally, cholesterol may be measured using free plasma cholesterol. Hypercholesterolemia may be judged or diagnosed in a subject if, for example, the measured free cholesterol levels are above 200 mg/dl, above 210 mg/dl, or above 220 mg/dl.
[00130] In some embodiments of the therapeutic uses and methods disclosed herein, one or more probiotic strains disclosed herein may be administered. In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 probiotic strains as contemplated herein are administered to a subject.
[00131] In some embodiments, one or more probiotic strains of the family Lactobacillaceae are administered in conjunction e.g., before, together with, or after) with one or more non-Lactobacillaceae microorganisms, such as, for example, one or more microorganisms from the genera Saccharomyces, Streptococcus, Bacteroides,
Pediococcus, Enterococcus, Leuconostoc, and/or Bifidobacterium.
Figure imgf000045_0001
[00132] A single probiotic strain may constitute up to 1%, up to 5%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, or up to 99%, of the total probiotic cells administered.
[00133] In some embodiments, at least one of the probiotic strains administered is Lactobacillus acidophilus CL1285. In some embodiments, at least one of the probiotic strains administered is Lactiplantibacillus plantarum CHOL-200. In some embodiments, both of Lactobacillus acidophilus CL1285 and Lactiplantibacillus plantarum CHOL-200 are administered. In some embodiments, Lactobacillus acidophilus CL1285 and Lactiplantibacillus plantarum CHOL-200 are the only probiotic strains administered to a subject. In some embodiments, Lactobacillus acidophilus CL1285 and Lactiplantibacillus plantarum CHOL-200 are the only Lactobacillaceae administered to a subject.
[00134] In some embodiments, multiple compositions containing different probiotic strains are administered to a subject. For instance, in some embodiments, a first composition may contain Lactobacillus acidophilus CL1285, and a second composition may contain Lactiplantibacillus plantarum CHOL-200. These compositions maybe administered, for example, to a subject at the same or different times, and/or, at different frequencies.
Figure imgf000046_0001
[00135] Additionally, probiotic strains of the present disclosure may be administered in conjunction e.g., before, together with, or after) with one or more of: a statin {e.g., Atorvastatin, Fluvastatin, Lovastatin, Pitavastatin, Pravastatin, Rosuvastatin, Simvastatin, etc.); Ezetimibe; Alirocumab; Evolocumab; Bempedoic acid;
Cholestyramine; Colesevelam; Colestipol; Amlodipine; Fenofibrate; Gemfibrozil; niacin; omega-3 fatty acids; fibers; soluble fiber; beta-glucans; and oat beta glucan.
[00136] In some embodiments, a subject is administered a daily dose of at least 104, at least 105, at least 106, at least 107, at least 108, at least 109, at least 1010, at least 1011, or at least 1012, viable cells of a probiotic strain or strains. The subject may be administered the probiotic strain or strains at varying frequencies, depending on the nature of the disorder or the proposed use of the composition, and/or the characteristics of the subject involved.
[00137] For instance, therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures, e.g., with experimental animals, such as by calculating the ED, (the dose therapeutically effective in 50% of the population) or LD (the dose lethal to 50% of the population) statistics. The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD/ED ratio. In some embodiments, the effect may be dose-dependent {e.g., the higher the dose, the higher the protective and/or therapeutic activity).
Figure imgf000047_0001
[00138] The dosage may also vary depending upon the dosage form employed, the sensitivity of the patient, and the route of administration. The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Probiotic strains may be administered several times per day {e.g., 1, 2, 3, 4, or 5 times per day), every day, every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days, or monthly depending on, e.g., the clearance rate, tolerance by the subject, and response characteristics of the subject.
[00139] Preferably, the probiotic strain or strains are administered by delivery to the oral, gastric and/or to the intestinal mucosal surface. However, also contemplated herein are alternate delivery routes, such as nasopharyngeal, respiratory, reproductive or glandular mucosa, and it may be administered to a subject by an oral, nasal, ocular, rectal, topical and/or vaginal route.
[00140] Subjects may be administered probiotic strains according to the various compositions and administration routes described herein. In some embodiments, the administered probiotic cells are in the form of living cells in a metabolically-active state,
Figure imgf000048_0001
a dividing state, a vegetative state, and/or in a dormant state (e.g., strains in spore form, lyophilized form, spray-dried form, etc.).
Experimental
[00141] Non-limiting embodiments of the present invention are illustrated in the following Examples. Efforts have been made to ensure accuracy with respect to numbers used e.g., amounts, concentrations, percent changes, and the like), but some experimental errors and deviations should be accounted for.
[00142] Unless indicated otherwise, temperature is in degrees Centigrade and pressure is at or near atmospheric. It should be understood that these Examples are given by way of illustration only and are not intended to limit the scope of what the inventor regards as various embodiments of the present invention. Not all of the following steps set forth in each Example are required nor must the order of the steps in each Example be as presented.
Example 1
Identification of Lactobacillaceae Exhibiting BSH and/or EF Activity
[00143] Seventy Lactobacillaceae strains, isolated from human gut, oral, vaginal and milk samples, foods, or purchased from commercial suppliers, were analyzed for
Figure imgf000049_0001
BSH and EF activity. Of these strains, 15 were of the genus Lacticaseibacillus, 14 were of the genus Lactiplantibacillus, 17 were of the genus Lactobacillus, 7 were of the genus Lentilactobacillus, 10 were of the genus Levilactobacillus, and 7 were of the genus Limosilactobacillus.
[00144] BSH activity was evaluated as described in Pereira et al. ("An in vitro study of the probiotic potential of a bile -salt-hydrolyzing Lactobacillus fermentum strain, and determination of its cholesterol-lowering properties,” Appl. Environ. Microbiol., 2003, 69(8): 4743-52. Specifically, for each strain tested, a loop of fresh bacterial culture was streaked on the surface of MRS agar (1.5% w/v) supplemented with either sodium taurocholic acid hydrate (TCA) (0.5% w/v; Sigma-Aldrich; St- Louis, MO, USA) or sodium glycocholate hydrate (GCA) (0.5% w/v; Sigma-Aldrich), and calcium chloride anhydrous (0.37 g/L; Labo MAT, Montreal, Canada). Each plate was incubated in an anaerobic atmosphere at 37°C for 72 h. BSH activity was then visually determined by observing the presence of a precipitate around the colony, a rough aspect of the colony, or both. The results of the analysis are shown in Table 1 below ("BSH” columns). Semi-quantitative data are expressed by the "+” symbol comparing the growth and/or precipitation zone of each bacteria on an agar plate with and without bile salts; the number of "+” symbols is proportional to the intensity of the activity. A negative result is indicated by a "-” symbol, meaning the
Figure imgf000050_0001
bacteria could grow on the medium but there was no activity detected when compared with the same medium without bile salts.
[00145] EF activity was evaluated using a semi-quantitative method described by Donaghy etal. ("Detection of ferulic acid esterase production by Bacillus spp. and lactobacilli.,’’ Appl. Microbiol. Biotechnol., 1998, 50(2): 257-60), with some modifications. Specifically, a modified MRS medium with agar (1.5% w/v) was prepared using all the same ingredients for the MRS medium broth, but without glucose. The medium was sterilized at 121°C for 20 min and cooled at 50-55°C. Six mL of a solution containing ethyl 4-hydroxy-3 -methoxy cinnamate (ethyl ferulate) (10% w/v; Sigma- Aldrich) dissolved in N, N-Dimethylformamide (Sigma-Aldrich) was added to 500 mL of medium and gently mixed. The medium was then poured into sterile Petri dishes and solidified at room temperature. A loop of fresh bacterial culture was streaked on the surface of each Petri dish and the plates were incubated under an anaerobic atmosphere at 37°C for 3 to 6 days. EF activity was determined by comparing the growth of each bacteria and the disappearance of the fogginess around each bacterial colony on an agar plate with and without ethyl ferulate. The results are depicted in Table 1 below ("EF” columns). Plus "+” symbols were used to describe EF activity, and the number of symbols is proportional to the EF activity. A negative result is indicated by a negative
Figure imgf000051_0001
symbol, meaning the bacteria could grow on the medium, but there was no activity detected when compared with the same medium without ethyl ferulate.
Table 1: Bile salt hydrolase (BSH) and feruloyl esterase (FAE) activities negative, +: positive, ++: strongly positive, GCA: glycine-conjugated bile acid, TCA: taurine- conjugated bile acid).
Figure imgf000052_0002
Figure imgf000052_0001
Figure imgf000053_0002
Figure imgf000053_0001
Figure imgf000054_0002
Figure imgf000054_0001
Figure imgf000055_0002
[00146] As shown in Table 1 above, of these 70 bacterial isolates, 18 exhibited
BSH activity (10 exhibiting selectivity towards the taurine-conjugated form of bile acids; 5 exhibiting selectivity towards glycine-conjugated bile acids; and 3 strains (corresponding to Lactobacillus acidophilus, Lacticaseibacillus casei and
Figure imgf000055_0001
Lactiplantibacillus plantarum) showing the capacity to deconjugate bile acid with either taurine or glycine).
[00147] Among these 3 strains, two exhibited stronger BSH activity than the others: L. acidophilus CL1285 and L. plantarum CHOL-200 exhibited, in the presence of either GCA or TCA, robust BSH activity compared to all other Lactobacillaceae tested.
[00148] Additionally, Table 1 also shows that 26 of the 70 tested strains tested positive for EF activity (7 from the genus Lactiplantibacillus, 14 from the genus Lactobacillus, 1 from the genus Levilactobacillus, and 4 from the genus Limosilactobacillus) . Eight of the EF-positive strains were isolated from a plant source (olive oil), which is consistent with the reported literature that EF breaks the bonds that maintain the cell wall of plants, and releases different phenolic compounds including FA. See, e.g., Mukdsi et al. ("Administration of Lactobacillus fermentum CRL1446 increases intestinal feruloyl esterase activity in mice,” Lett. Appl. Microbiol., 2012, 54(1): 18-25. The other EF-positive bacteria were isolated from human feces or vaginal mucosa.
[00149] L. acidophilus CL1285 exhibited particularly strong EF activity. Representation of BSH and EF-positive activities are shown in FIG. 1, using L. acidophilus CL1285 and L. plantarum CHOL-200 results as reference.
Figure imgf000056_0001
Example 2
Characterization of Cholesterol Assimilation Activity
[00150] L. plantarum CHOL-200 and L. acidophilus CL1285 were selected for further characterization, and in particular, their capacity to assimilate cholesterol
(which, together with BSH and EF activities, may decrease cholesterol levels in vivo) was assessed. Cholesterol assimilation was also measured for L. acidophilus AT CC 43121, as a positive control. See, e.g., Gilliland etal. ("Assimilation of cholesterol by Lactobacillus acidophilus," Appl. Environ. Microbiol., 1985, 49(2): 377-81); and Noh etal.
("Incorporation of Cholesterol into the Cellular Membrane of Lactobacillus acidophilus ATCC 431211,” Journal of Dairy Science, 1997, 80(12): 3107-3113).
[00151] Cholesterol micelles were prepared according to the method developed by Razin etal. ("Phospholipid and cholesterol uptake by Mycoplasma cells and membranes,” Biochim. Biophys. Acta, 1980, 598(3): 628-40), with some modifications.
Briefly, 100 mg of cholesterol (Sigma- Aldrich) and 220 mg of lecithin soybean (a mixture of phosphatidylcholine and lysolecithin (American Lecithin Company, Oxford, CT, USA)) were solubilized in 10 mL of chloroform (Fisher Scientific, Ottawa, Canada). Chloroform was evaporated using nitrogen gas (Praxair Canada Inc., Laval, Quebec). Next, 30 mL of a 0.4 M sucrose solution (Fisher Scientific) was added and the cholesterol/lecithin
Figure imgf000057_0001
mixture was sonicated, to produce cholesterol micelles. Sonication was performed on ice using a Sonic Dismembrator 500 (Fisher Scientific) at maximum amplitude with a power of between 100 and 120 watts for three periods of 15 min, renewing ice between each period. The solution was then centrifuged (Avanti J-26S XPI, Beckman Coulter, Brea, CA, USA) in high-speed centrifuge tubes for 30 min at 30,000 x g, to eliminate metal particles from the sonicator probe.
[00152] After two successive overnight anaerobic incubations in MRS broth, each bacteria was washed using saline water (NaCl 0.85% w/v) and the pellet was suspended in sterile water. Then, 100 pL of the pellet suspension and 1 mL of cholesterol micelles were mixed with pre-reduced MRS medium supplemented with sodium thioglycolate (0.2% w/v) and bovine bile (0.6% w/v). Subsequently, all tubes were incubated under an anaerobic atmosphere at 37°C for 48 h.
[00153] Quantification of the cholesterol assimilation by bacteria was measured using the method described by Rudel etal. ("Determination of cholesterol using o- phthalaldehyde,”/. Lipid Res., 1973, 14(3): 364-6), with some modifications. Briefly, the content of each tube was centrifuged at 3,700 x g for 15 min and the pellets, containing the assimilated cholesterol, were suspended with the same quantity of sterile water.
One mL of each sample was transferred in a borosilicate tube containing 3 mL of ethanol (95% v/v; Les Alcools de Commerce, Montreal, Canada) and 2 mL potassium hydroxide
Figure imgf000058_0001
solution (50% w/v; Acros Organics; Fair Lawn, NJ, USA). Each tube was vortexed 20 s and then heated in a water bath at 60°C for 10 min. Then, the tubes were cooled at room temperature for 10 min. A volume of 5 mL of n-hexane (Acros Organics) and 3 mL of sterile distilled water were added to the tubes and vortexed for 30 s. The tubes were kept at room temperature for 15 min for phase separation. After that, 2.5 mL of the superior phase was transferred in another borosilicate tube. The tube was warmed in hot water and the hexane was completely evaporated using nitrogen gas. The pellet was suspended with 4 mL of a fresh solution of o-phthalaldehyde (Sigma- Aldrich) at a concentration of 0.5 mg/mL previously dissolved in glacial acetic acid (Fisher Scientific). The tubes were kept at room temperature for 10 min. Then, 2 mL of concentrated sulfuric acid (Fisher Scientific) was added, mixed for 20 s by vortexing, and kept for 10 min at room temperature. The OD550nm of each sample was measured using a BioPhotometer plus (Eppendorf, Hamburg, Germany). The blank consisted of 4 mL of o- phthalaldehyde solution and 2 mL of concentrated sulfuric acid. The standard curve was generated using a cholesterol solution of 0, 25, 50, 75, 100, 200, and 400 pg/rnL. TC was measured from the thioglycolate- and bile-supplemented MRS media containing cholesterol micelles from which value of thioglycolate- and bile-supplemented MRS medium without cholesterol was subtracted. All standards and samples were subjected to identical procedures.
Figure imgf000059_0001
[00154] The assimilation percentage was calculated using the following formula: > 100
Figure imgf000060_0001
[00155] For each bacteria, the percentage of cholesterol assimilation was evaluated from three independent samples processed in duplicate. Values are expressed as means ± standard deviation. Data were analyzed by one-way analysis of variance (ANOVA), using SPSS Statistics for Windows, version 19.0 software (IBM Corporation, Armonk, NY, USA). Differences among the groups were analyzed with a Post Hoc Duncan’s multiple range test. Differences between means were considered significant at
P < 0.05.
Figure imgf000060_0002
[00156] As shown in FIG. 2, strains CHOL-200 and CL1285 showed the ability to assimilate cholesterol present in the culture media. A cholesterol removal rate of 76.3 % was observed for L. plantarum CHOL-200, and a cholesterol removal rate of 61.5 % was observed for L. acidophilus CL1285. These were comparable to the cholesterol removal rate obtained with the control strain L. acidophilus AT CC 43121.
Example 3
Characterization of Gastric Survival
[00157] To exert their beneficial properties, probiotic bacteria have to resist the harsh acidic and bilious environments of the stomach and the small intestine, respectively. To characterize further the probiotic properties of L. acidophilus CL1285 and L. plantarum CHOL-200, both strains were challenged in a simulated gastric fluid (SGF), simulating the acidic and enzymatic stomach conditions, of pH 1.5 to 2.5 for 30 and 60 minutes.
[00158] Briefly, the day of the experiment, SGFs of pH 1.5, 2 and 2.5 were prepared as follows. 2 g of sodium chloride (Fisher Scientific) and 3.2 g of pepsin (Sigma-Aldrich) were dissolved in 900 mL of osmosis filtered water. The pH was adjusted with 5 M hydrochloric acid (Fisher Scientific) and measured using an Orion 3
Star pH BenchTop (Thermo Scientific) equipped with a Ross Ultra pH/ATC Triode
Figure imgf000061_0001
(Thermo Scientific). Following pH adjustment, the SGF volume was completed to 1 L using sterile osmosis water. Then, a volume of 19 mL of SGF was transferred in 50 mL conical tubes. Before the experiment, each tube was placed in an incubator at 37°C for one hour to warm the solution.
[00159] One mL of an overnight culture of L. acidophilus CL1285 and L. plantarum CHOL-200 was added to each SGF, and each tube was immediately placed into a MaxQ 4450 Incubator-shaker (Thermo Scientific) previously warmed at 37°C and agitated at 250 rpm for 30 and 60 min. After incubation, 1 mL of each SGF-bacterial mixture was neutralized in 9 mL of a sterile solution of 0.1 M sodium phosphate buffer (Fisher Scientific). Bacterial enumeration was then obtained by tenfold serial dilution in peptone water (1 g/L peptone and 0.5 g/L sodium chloride). Appropriate dilutions were plated on MRS agar (1.5% w/v) and incubated under aerobic atmosphere at 37°C for
48h.
Figure imgf000062_0001
[00160] As shown in FIG. 3, L. acidophilus CL1285 was unaffected (difference from the initial bacterial count < 1 Logio CFU/mL) up to 60 min in an SGF of pH 2 or 2.5. L. acidophilus CL1285 also resisted a pH of 1.5 for 30 min. However, its viability was altered after 60 min, as shown by a marked decrease of 3.07 Logio CFU/mL. L. plantarum CHOL-200 survived up to 60 min in an SGF of pH 2.5, and for 30 min in an SGF of pH 2. However, its viability decreased by nearly 1.9 Logio CFU/mL after 60 min at pH 2. The CHOL-200 strain was less resistant in the SGF of pH 1.5 for 30 or 60 min, as shown by a reduction of 2.84 and 5.43 Logio CFU/mL, respectively.
Example 4
Characterization of Bile Salt Tolerance
[00161] It has been reported that probiotic bacteria should be able to grow in the presence of 0.15% to 0.3% of bile salts. See, e.g., Goldin etal. ("Probiotics for humans," in Probiotics, D. Springer, Editor, 1992). L. acidophilus CL1285 and L. plantarum CHOL-200 were thus evaluated for their tolerance to 0.15% and 0.3% of three different commercial mixtures of bile acids. Bile salt tolerance was performed on L. acidophilus CL1285 and L. plantarum CHOL-200 using three different types of bile. Tolerance to Ox gall powder (Sigma-Aldrich), Bile Salts (Sigma Aldrich) and Bacteriological Bile (OrganoTechnie, La Courneuve, France) was performed by inoculating both bacteria on media containing
Figure imgf000063_0001
0.15% and 0.3% (w/v) of bile. Each bacteria was streaked on the surface of a bile salt supplemented MRS and incubated under an anaerobic atmosphere at 37°C for 48h. Growth on MRS agar without bile salts was used as a positive control to determine the capacity of each strain to grow on each supplemented MRS agar.
[00162] As shown in Table 2 below, the CL1285 strain was able to grow in the presence of Ox gall powder, bacteriological bile, and 0.15% of bile salts. However, an inhibitory effect was observed at 0.3% of bile salts. The CHOL-200 strain grew, to different extents, on MRS supplemented with all three bile salts, regardless of the concentration tested. Of the three products tested, both strains showed a greater resistance in the presence of Ox gall powder. In this connection, the tested Ox gall powder has been reported to be more similar to human bile, based on bile acid ratios, indicating that the tested Ox gall powder may be the most appropriate for evaluating the bile resistance of probiotic bacteria.
Table 2. Growth of L. acidophilus CL1285 and L. plantarum CHOL-200, after 48h under anaerobic atmosphere, on MRS supplemented with 0.15% and 0.3% of bile salts. Bile concentrations are expressed in % (w/ ).
Figure imgf000064_0001
Bacteria MRS Ox gall powder1 Bile salts2 Bacteriological bile3
0.15% 0.3% 0.15% 0.3% 0.15% 0.3%
CHOL-200 ++ ++ ++ ++ + ++ ++
CL1285 ++ ++ ++ + - + +
1. Bile bovine consisting of 70% bile salts, 22% phospholipids, 4% cholesterol, 3% proteins and 0.3% bilirubin (source: Sigma Aldrich).
2. Mixture of-50% cholic acid and-50% deoxycholic acid (source: Sigma Aldrich).
3. Bile acids (as cholic acid) corresponding to > 45% of dry matter (source: Organotechnie).
Example 5
Effects on metabolic health, gut microbiota composition, and endocannabinoidome mediators
[00163] The effect of L. acidophilus CL1285 and L. plantarum CHOL-200 on metabolic health, gut microbiota composition, and endocannabinoidome mediators, in an animal model of hypercholesterolemia, was determined. Briefly, five groups of nine Syrian hamsters (n=45) were fed either a low- fat or high-fat (HF) diet to induce hypercholesterolemia and gavaged for a period of 6 weeks with either Lactobacillus acidophilus CL1285, Lactiplantibacillus plantarum CHOL-200, or a combination of the two. The hamsters were housed in individual cages, and fed ad libitum with a standard laboratory chow diet for a 5 to 7-day acclimatization period. Nine (9) hamsters were
Figure imgf000065_0001
randomly assigned to the low-fat (LF) purified diet (D 14042701 Research Diet) for 30 days, while 45 hamsters were assigned to the high-fat (HF) purified diet (D12079B Research Diet) for the same period. Such HF-feeding run-in is a recognized means of promoting weight gain and inducing a dysmetabolic state of hypercholesterolemia and hypertriglyceridemia in hamsters. Both diets had comparable fiber content and were composed mainly of butter (rich in saturated and oleic fatty acids), corn oil (rich in oleic and linoleic acids), sucrose and corn starch. The study design is shown in Table 3 below.
Table 3
Groups N Diet Intervention Daily probiotic dose
LF-Milk 9 Low fat Pasteurised milk
HF-Milk 9 High fat Pasteurised milk
HF-La 9 High fat L. acidophilus CL1285 2 x 109 cfu
HF-Lp 9 High fat L. plantarum CHOL-200 5.4 x l09 cfu
H HFF-L LaLLnp 99 H Hiigghh t faatt L a p c^ia°nPta^r^u u m s C CLH1O2L8-52 a0n0d 44, 7/ x ll0U9 cftu
HF-CHY 9 High fat Cholestyramine 300 mg/kg
At the end of the run-in period, HF-fed hamsters were then randomly assigned to a 6- week intervention period with 1) pasteurised milk (HF-Milk), 2) L. acidophilus CL1285 rehydrated in pasteurized milk (HF-La), 3) L. plantarum CHOL-200 in pasteurized milk
Figure imgf000066_0001
(HF-Lp), 4) L. acidophilus and L. plantarum in pasteurized milk (HF- LaLp) or 5) Cholestyramine (HF-CHY; 300 mg/kg/day) (Table 1). The fermented milk was stored at 4°C and comprised at least 50 x 109 colony forming units (CFU) /100 g of three proprietary strains (L. acidophilus CL1285, L. casei LBC80R and L. rhamnosus CLR2). L. acidophilus CL1285 and L. plantarum CHOL-200 were supplied as freeze-dried pure culture and stored at -20°C until utilization. Probiotic and pharmacologic interventions were administered by daily intragastric gavage (2 mb). Cholestyramine (CHY), a bile acid sequestrant, was selected as a positive control for response to a cholesterol- lowering treatment. Body weight and food intake were monitored every two days and every week, respectively. Sixteen (16) to 18-hour fasted hamsters were sacrificed at the end of the intervention period by cardiac puncture. One aliquot was centrifuged (1,780 x g, 10 min) to obtain plasma for the analyses described below as another aliquot was allowed to clot for 30-45 minutes prior to being centrifuged. Ileum (1 cm proximal to the ileocecal junction) and caecum were dissected, and their luminal contents were collected in PBS by gentle scraping. Liver, subcutaneous (inguinal) and visceral (epididymal) adipose tissues were rapidly collected, and all samples were stored at - 80°C until batch analysis.
[00164] Metabolic response to interventions
Figure imgf000067_0001
[00165] Weight gain and metabolic measures of the hamsters following the run-in and the intervention period are illustrated in FIGS. 4A-4I. At the end ofthe 30-day run- in period (Week 0), HF-fed hamsters had a higher body weight than LF-fed hamsters (130.6 ± 12.00 gvs. 90.2 ± 14.3 g). HF-feeding elevated fasting insulin levels (FIG. 4E), as well as fasting levels of triglycerides (FIG. 4F), total cholesterol (FIG. 4G), HDL cholesterol and non-HDL cholesterol (FIG. 4H), and reduced HDL/total cholesterol ratio compared to LF-fed hamsters (FIG. 41). Altogether, these HF-induced metabolic adaptations were suggestive of the development of insulin resistance and dyslipidemia. [00166] At the end ofthe 6-week intervention period, HF-fed hamsters still had greater body weights than LF-fed hamsters (90.2 ± 14.3 gvs. 130.6 ± 12.0 g), and mean body weights between all HF-fed groups were not significantly different from one another (FIG. 4A). All probiotic supplemented groups had lower food consumption than those without probiotics (FIG. 4B). Moreover, hamsters supplemented with L. acidophilus CL1285, either alone or combined with L. plantarum CHOL-200, had the sharpest decline in food intake during the intervention compared to the run-in period (- 52.4% and -49.9%, respectively). Between-group variations in food intake cannot explain variations in weight gain or other metabolic parameters, as groups fed L. acidophilus CL1285, either alone or combined with L. plantarum CHOL-200, showed the lowest food intakes and greater weight gains, for example. HF-fed hamsters had
Figure imgf000068_0001
increased liver weight compared to the LF-Milk hamsters (FIG. 4C). The HF diet used was not chosen to induce hepatic fat deposition, steatosis or fibrosis - often requiring more than 40% of total calories from fat - thus greater liver weights observed do not necessarily correlate with such phenotypes and ensuing dysmetabolisms.
[00167] Fasting glucose levels remained relatively unaffected by the diets following the run-in and the intervention periods. HF-Lp and HF-CHY hamsters had slightly reduced fasting glycemia than HF-Milk hamsters (FIG. 4D). On the other hand, fasting insulin was increased in all HF-fed hamsters with the exception of hamsters supplemented with L. acidophilus CL1285 alone, in which insulin levels remained similar to the LF-Milk hamsters (FIG. 4E). The HF diet induced hypertriglyceridemic and hypercholesterolemic states following the run-in period, and this effect was maintained during the interventions (FIGS. 4F-4G). Intervention with L. plantarum CHOL-200 increased circulating levels of triglycerides, total cholesterol and non-HDL cholesterol levels (FIGS. 4F-4H). Interestingly, hamsters on either L. acidophilus CL1285 interventions (HF-La and HF-LaLp) showed similar triglyceride levels than HF-Milk hamsters and the combination of both strains (HF-LaLp) induced lower non-HDL cholesterol levels (FIG. 4F and FIG. 4H). These interventions beneficially raised the HDL/total cholesterol ratio compared to the HF-Milk hamsters (FIG. 41). The intervention with cholestyramine successfully reduced total cholesterol, non-HDL
Figure imgf000069_0001
cholesterol levels and HDL/total cholesterol ratio but did not alter triglyceride levels (FIGS. 4F-4I). The magnitude of the cholesterol-lowering effect observed in both L. acidophilus CL1285 supplemented groups (HF-La and HF-LaLp) was comparable to that of the hamsters receiving cholestyramine.
[00168] Gut microbiota response to the probiotic interventions
[00169] Global bacterial community composition assessed by 16S rRNA gene sequencing revealed significant differences between the ileum and the caecum in principal component analysis (PCA). Comparison of the Bray-Curtis dissimilarity indexes revealed significant differences in beta-diversity between both intestinal segments (PERMANOVA p<0.001) and supports the notion that the interindividual variation within a given intestinal section is lower than regional variation within an individual. Moreover, subsequent analyses revealed that the microbiota composition within each intestinal segment was modified differently by the diets and the probiotic interventions. Pairwise PERMANOVA analyses using Bray-Curtis distances between gut microbiota confirmed that gut bacterial composition of all HF-fed hamsters is different from that of the LF-Milk hamsters in both intestinal segments. These analyses also revealed that ileal and caecal microbiota of the HF-Lp hamsters remain similar to that of the HF-Milk hamsters.
Figure imgf000070_0001
[00170] In contrast, the gut microbiota of hamsters supplemented with
L. acidophilus CL1285, either alone or in combination with L plantarum CHOL-200, was distinct from that of the HF-Milk hamsters. The intervention with L. acidophilus CL1285 tends to bring gut microbiota composition of HF-fed hamsters to a state closer to that observed in LF-fed hamsters. Shannon Alpha-diversity index, a marker of taxa richness and evenness, was not significantly influenced by the probiotic interventions, except for an increased diversity in the caecum when L. acidophilus CL1285 and L. plantarum CHOL-200 were combined (HF-Milk: 3.1 [2.6-3.5] vs. HF-LaLp: 3.8 [3.7-3.9], p < 0.05). The Firmicutes to Bacteroidetes ratio, which is usually increased in diet-induced obesity in mice, was lower in the caecum of HF-fed animals (LF-Milk: 1.05 [1.03-1.06] vs. HF- Milk: 1.00 [1.00-1.01], p < 0.05). This unexpected result could be explained by the choice of a LF control diet with similar fiber content as the HF diet, which is in contrast with the chow diet used as a control diets in several studies.
[00171] Analysis of variance identified several families whose relative abundance in both segments were altered by the interventions, i.e. Peptostreptococcaceae, Eubacteriaceae, and Helicobacteraceae. This analysis also identified bacterial families whose relative abundance was altered only in the ileum, i.e. Lactobacillaceae, Deferribacteraceae, Streptococcaceae and Erysipelotrichaceae, or only in the caecum, i.e. Coriobacteriaceae Bifidobacteriaceae and Clostridiaceae (Tables 4 and 5, and FIG. 5).
Figure imgf000071_0001
Table 2: Bacterial families significantly modified by probiotic interventions in the ileum.
LF-Milkvs HF-Lavs HF-Lp vs HF-LaLp vs
KW HF-Milk HF-Milk HF-Milk HF-Milk
Eubacteriaceae < 0.001 0.002 0.02 - 0.02
Lactobacillaceae 0.003 - 0.03 - 0.004
Deferribacteraceae 0.02 0.02
Streptococcaceae 0.02 _ _ _ _
Peptostreptococcaceae 0.09 0.002 0.04 0.03 0.03
Erysipelotrichaceae 0.10 - 0.02 0.08 0.06
Helicobacteraceae 0.10
Only taxa with Kruskal-Wallis p < 0.1 (KW) are included in the table; - : NS
Table 3: Bacterial families significantly modified by probiotic interventions in the caecum.
LF-Milkvs HF-Lavs HF-Lp vs HF-LaLp vs
KW HF-Milk HF-Milk HF-Milk HF-Milk
Eubacteriaceae < 0.001 < 0.001 0.05 0.07 0.03
Coriobacteriaceae 0.02 0.003 _ _ _
Helicobacteraceae 0.05 0.10 0.08
Peptostreptococcaceae 0.05 0.004 0.005 0.04 0.005
Bifidobacteriaceae 0.05 - 0.07 0.01 0.03
Clostridiaceae_l 0.10 0.01 0.03 0.06 0.003
Only taxa with Kruskal-Wallis p < 0.1 (KW) are included in the table; - : NS
Figure imgf000072_0001
[00172] Interestingly, the relative abundance of Eubacteriaceae, and of one of its key genera Pseudoramibacter), was decreased in both segments in response to the HF diet and was restored to the level of the LF-fed hamsters following the intervention with L. acidophilus CL1285 alone or in combination with L. plantarum CHOL-200 (FIGS. 5A-5B). The relative abundance of Eubacteriaceae across intervention groups was negatively correlated with the levels of insulin, total cholesterol, non-HDL cholesterol and triglycerides (FIG. 6). HF feeding also decreased the relative abundance of the Deferribacteraceae family in the ileum (FIG. 5A), which was negatively associated with circulating levels of insulin, cholesterol, non-HDL cholesterol and triglycerides (FIG. 6). [00173] Supplementation with both L. acidophilus CL1285 containing probiotics (HF-La and HF-LaLp) induced an elevation of the relative abundance of Lactobacillaceae and of its genus Lactobacillus spp. in the ileum (FIG. 5A). Lactococcus genus, were positively associated with elevated levels of insulin, triglycerides, total cholesterol and non-HDL cholesterol across the intervention groups. Finally, the elevation in the relative abundance of Helicobacteraceae observed in the caecum of HF-fed hamsters was prevented by the addition of L. acidophilus CL1285 when administered alone, with a similar but non-significant reduction when co-administered with L. plantarum CHOL- 200. This family was positively associated with circulating levels of triglycerides, total cholesterol and non-HDL cholesterol (FIG. 5B and FIG. 6).
Figure imgf000073_0001
[00174] Endocannabinoidome response to the probiotic interventions
[00175] Circulating and tissue levels of 2-MAGs and NAEs following the probiotic interventions were then assessed.
[00176] Plasma, ileum, caecum, inguinal and epididymal adipose tissues were extracted to measure /V-acetylethanolamines (NAEs) and 2 -monoacylglycerols (2-MAGs) by HPLC-MS/MS. Briefly, samples (40 pl of samples or 5-10 mg of tissue) were brought to 500 pl in 50 mM Tris (pH 7) then mixed/crushed in 500 pl methanol containing 0.01% acetic acid and 5 ng of deuterated standards. Lipids were extracted 3 times by adding 1 ml chloroform, vortexing for 1 minute and centrifuging (3,500 x g, 10 min). Pooled organic phases were evaporated under nitrogen and resuspended in 50 pl of our mobile phase (50% B). Samples were injected (40 pl) onto an HPLC column (Kinetex C8, 150x2.1 mm, 2.6 pm, Phenomenex) and eluted at a flow rate of 400 pl/min using a discontinuous gradient solvent A (1 mM ammonium acetate + 0,05% acetic acid) and solvent B (acetonitrile/water; 95/5 + 1 mM ammonium acetate + 0.05% acetic acid). The HPLC system was interfaced with the electrospray source of a Shimadzu 8050 triple quadrupole mass spectrometer and mass spectrometric analysis was done in the positive ion mode using multiple reaction monitoring.
[00177] The method can differentiate monoacylglycerol isomers at positions 1 and 2 but signals from both isomers of unsaturated fatty acids were summed - and identified
Figure imgf000074_0001
as 2-MAGs - prior to analysis in order to account for their rapid interconversion. The following NAEs were quantitated: /V-palmitoylethanolamine (PEA), /V-oleoylethanolamine (OEA), /V-linoleoylethanolamine (LEA), /V-arachidonoylethanolamine (AEA), /V-docosahexaenoylethanolamine (DHEA); and the following 2-MAGs: 2-palmitoyl-glycerol (2-PG), 2-oleoyl-glycerol (2-OG), 2-linoleoyl- glycerol (2 -LG), 2-arachidonoyl-glycerol (2 -AG), 2-eicosapenaenoyl-glycerol (2 -EPG), 2-docosapentaenoyl(n-3)-glycerol (2-DPG), and 2-docosaheaenoyl-glycerol (2-DHG). [00178] Endocannabinoidome mediator profiling was not performed for HF-CHY hamsters, as plasma and tissue biopsies were not collected. All 2 -MAG congeners were increased in the plasma following HF feeding (FIG. 7A). These elevations were exacerbated by some probiotic formulations. Greater 2-AG, 2-DHG and 2-DPG levels were observed in the plasma following the supplementation with L. acidophilus CL1285 but not by its combinations with L. plantarum CHOL-200. In contrast, HF feeding decreased plasma levels of NAEs, especially AEA, OEA and LEA, and the supplementations with L. acidophilus CL1285, alone or in combination with L. plantarum CHOL-200, prevented such decreases in LEA levels. Positive associations were noted between most 2 -MAG congeners and levels of fasting insulin, triglycerides, total cholesterol and non-HDL cholesterol. These parameters were generally negatively associated with plasma NAEs (FIG. 7B).
Figure imgf000075_0001
[00179] HF-induced changes in plasma levels of 2 -MAGs were of greater amplitude than those observed in the ileum and both adipose tissue depots (FIG. 7 A). NAE and 2- MAG levels in the ileum were mostly not responsive to the HF feeding and probiotic interventions. Indeed, only 2-DPG levels in the ileum were increased by HF feeding supplemented with either or both L. acidophilus CL1285 and L. plantarum CHOL-200 (FIG. 7A).
[00180] Some NAE congeners, but not 2 -MAGs, were significantly influenced by the interventions in the inguinal adipose tissue (FIG. 7 A). Namely, AEA levels tended to be higher in HF-Milkthan in LF-Milk hamsters and were increased by the intervention with both L. acidophilus CL1285 and L. plantarum CHOL-200. The OEA decreases in the subcutaneous adipose tissue of HF-fed hamsters were also attenuated by the combination of L. acidophilus CL1285 and L. plantarum CHOL-200, and instead somewhat enhanced in the subcutaneous adipose tissue when L. plantarum was administered alone.
[00181] As with the subcutaneous adipose tissue, only some NAE congeners were modulated in the epididymal adipose tissue depot. DHEA was lower in HF-Milk hamsters than LF-Milk hamsters and its levels were partly restored by the intervention with either L. plantarum CHOL-200 alone or combined with L. acidophilus CL1285.
DHEA levels in this visceral adipose tissue depot were completely restored by the
Figure imgf000076_0001
supplementation with L. acidophilus CL1285 alone (FIG. 7A). Similarly, OEA levels in the epididymal adipose tissue were decreased by the HF diet and were partially restored closer to those of the LF-Milk hamsters by L. acidophilus CL1285 and L. plantarum CHOL- 200 interventions, alone or in combination.
[00182] As with metabolic parameters, two opposite patterns were obtained for association of gut bacterial families with NAEs and 2-MAGs (FIG. 8). Indeed, most circulating NAEs were positively associated to the relative abundance of key bacterial families such as Eubacteriaceae and Deferribacteraceae in association with beneficial metabolic effect. Conversely, most circulating 2-MAGs - linked to the dysmetabolic states in HF-fed hamsters - were inversely associated to the relative abundance of these bacterial families. Interestingly, some of these associations were also observed in the visceral and subcutaneous adipose tissue (z.e., Eubacteriaceae). Finally, the Erysipelotrichaceae family, which was reported in obesity and inflammation and for which we observed an increased in gut microbiota of HF-La hamsters, was positively associated with circulating 2-MAGs.
[00183] These results show that L. acidophilus CL1285, alone or in combination with L. plantarum CHOL-200, is effective at normalizing the HF diet-induced metabolic alterations, and at producing variations in the gut microbiota composition and the plasma/adipose tissue NAE levels. Therefore, if a sequential relationship exists among
Figure imgf000077_0001
probiotic-induced changes in gut microbiota composition, endocannabinoidome signaling, and beneficial metabolic effects, this is likely mediated by NAEs. Additionally, the magnitude of the cholesterol-lowering effect of both L. acidophilus CL1285-based interventions i.e., HF-La and HF-LaLp) was comparable to that observed in cholestyramine-treated hamsters.
Figure imgf000078_0001

Claims

1. A composition for reducing cholesterol and/or treating dyslipidemia, wherein said composition comprises one or more strains of Lactobacillaceae, and wherein at least one of said one or more strains exhibits bile salt hydrolase (BSH) activity, and wherein at least one of said one or more strains exhibits feruloyl esterase (EF) activity.
2. The composition according to claim 1, wherein at least one of said one or more strains exhibits BSH activity and EF activity.
3. The composition according to claim 1, wherein at least two of said one or more strains exhibits BSH activity.
4. The composition according to any one of claims 1-3, wherein at least one of said one or more strains exhibits cholesterol assimilation activity.
5. The composition according to any one of claims 1-4, wherein at least one of said one or more strains exhibits gut microbiome modulatory activity and/or endocannabinoidome modulatory activity.
-78-
6. The composition according to claim 1, wherein said composition comprises
Lactiplantibacillus plantarum CHOL-200.
7. The composition according to claim 1, wherein said composition comprises Lactobacillus acidophilus CL1285.
8. The composition according to claim 1, wherein said composition comprises Lactobacillus acidophilus CL1285 and Lactiplantibacillus plantarum CHOL-200.
9. The composition according to claim 8, wherein said composition comprises one or more additional probiotic strains.
10. The composition according to claim 9, wherein at least one of said one or more additional probiotic strains is selected from Lactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacillus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus,
-79- Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus,
Lentilactobacillus, Convivina, Frucctobacillus, Oenococcus, Weissella, Saccharomyces, Streptococcus, Bacteroides, Pediococcus, Enterococcus, Leuconostoc, and/or Bifidobacterium.
11. The composition according to claim 10, wherein at least one of said one or more additional probiotic strains is selected from Lacticaseibacillus casei LBC80R, Lacticaseibacillus casei ATCC 393, Lacticaseibacillus rhamnosus ATCC 53103, Lactiplantibacillus pentosus BK1034, Lactiplantibacillus plantarum BK300, Lactiplantibacillus plantarum BK303, Lactiplantibacillus plantarum BK324, Lactiplantibacillus plantarum BK1135, Lactiplantibacillus plantarum BK1039, Lactiplantibacillus plantarum BK1040, Lactobacillus acidophilus BK1197, Lactobacillus acidophilus ATCC 314, Lactobacillus acidophilus ATCC 4355, Lactobacillus acidophilus ATCC 4356, Lactobacillus acidophilus ATCC 4796, Lactobacillus acidophilus ATCC 53544, Lactobacillus acidophilus ATCC 53671, Lactobacillus acidophilus ATCC 9224, Lactobacillus acidophilus LMG 11466, Lactobacillus acidophilus FERM BP-4980, Lactobacillus acidophilus FERM BP-4981, Lactobacillus crispatus BK343, Lactobacillus crispatus BK482, Lactobacillus crispatus ATCC 55221, Lactobacillus gallinarum 9435, Lactobacillus asseri 33323, Levilactobacillus (Lactobacillus') namurensis BK214,
-80- Limosilactobacillus (Lactobacillus) vaginalis BK337, Limosilactobacillus (Lactobacillus) vaginalis BK372, Limosilactobacillus (Lactobacillus) vaginalis BK373, Limosilactobacillus (Lactobacillus) vaginalis BK349, Limosilactobacillus (Lactobacillus) vaginalis BK488, and Limosilactobacillus (Lactobacillus) vaginalis BK384.
12. The composition according to any one of claims 1-11, wherein said composition is a pharmaceutical, nutraceutical, feed, foodstuff, food supplement, or beverage, composition.
13. The composition according to claim 12, wherein said composition comprises at least one pharmaceutically acceptable carrier or additive.
14. The composition according to claim 12, wherein said composition is a feed, foodstuff, or food supplement.
15. The composition according to any one of claims 1-14, wherein at least one of said one or more strains is in a lyophilized or spray-dried form.
-81-
16. The composition according to any one of claims 1-15, wherein said composition further comprises at least one additional agent for reducing cholesterol or treating dyslipidemia.
17. The composition according to claim 16, wherein said additional agent is selected from the group consisting of Atorvastatin, Fluvastatin, Lovastatin, Pitavastatin, Pravastatin, Rosuvastatin, Simvastatin, Ezetimibe, Bempedoic acid, Cholestyramine, Colesevelam, Colestipol, Amlodipine, Fenofibrate, Gemfibrozil, niacin, omega-3 fatty acids, fibers, soluble fibers, beta-glucans, and oat beta glucan.
18. A method for reducing cholesterol and/or treating dyslipidemia, wherein said method comprises administering, to a subject in need thereof, one or more strains of Lactobacillaceae, wherein at least one of said one or more strains exhibits bile salt hydrolase (BSH) activity, and wherein at least one of said one or more strains exhibits feruloyl esterase (EF) activity.
19. The method according to claim 18, wherein at least one of said one or more administered strains exhibits BSH activity and EF activity.
-82-
20. The method according to claim 18, wherein at least two of said one or more administered strains exhibits BSH activity.
21. The method according to any one of claims 18-20, wherein at least one of said one or more administered strains exhibits cholesterol assimilation activity.
22. The method according to any one of claims 18-21, wherein at least one of said one or more administered strains exhibits gut microbiome modulatory activity and/or endocannabinoidome modulatory activity.
23. The method according to claim 18, wherein said subject is administered Lactiplantibacillus plantarum CHOL-200 deposited under Accession Number CNCM I- 5742.
24. The method according to claim 18, wherein said subject is administered Lactobacillus acidophilus CL1285 deposited under Accession Number LALC 1285.
25. The method according to claim 18, wherein said subject is administered Lactobacillus acidophilus CL1285 and Lactiplantibacillus plantarum CHOL-200.
-83-
26. The method according to claim 25, wherein Lactobacillus acidophilus CL1285 and Lactiplantibacillus plantarum CHOL-200 are administered to the subject at the same or different times.
27. The method according to claim 25, wherein said subject is administered one or more additional probiotic strains.
28. The method according to claim 27, wherein at least one of said one or more additional probiotic strains is selected from Lactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacillus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus, Lentilactobacillus, Convivina, Frucctobacillus, Oenococcus, Weissella, Saccharomyces, Streptococcus, Bacteroides, Pediococcus, Enterococcus, Leuconostoc, and/or
Bifidobacterium.
-84-
29. The method according to claim 28, wherein at least one of said one or more additional probiotic strains is selected from Lacticaseibacillus casei LBC80R, Lacticaseibacillus casei ATCC 393, Lacticaseibacillus rhamnosus ATCC 53103, Lactiplantibacillus pentosus BK1034, Lactiplantibacillus plantarum BK300, Lactiplantibacillus plantarum BK303, Lactiplantibacillus plantarum BK324, Lactiplantibacillus plantarum BK1135, Lactiplantibacillus plantarum BK1039, Lactiplantibacillus plantarum BK1040, Lactobacillus acidophilus BK1197, Lactobacillus acidophilus ATCC 314, Lactobacillus acidophilus ATCC 4355, Lactobacillus acidophilus ATCC 4356, Lactobacillus acidophilus AT CC 4796, Lactobacillus acidophilus AT CC 53544, Lactobacillus acidophilus ATCC 53671, Lactobacillus acidophilus ATCC 9224, Lactobacillus acidophilus LMG 11466, Lactobacillus acidophilus FERM BP-4980, Lactobacillus acidophilus FERM BP-4981, Lactobacillus crispatus BK343, Lactobacillus crispatus BK482, Lactobacillus crispatus A CC 55221, Lactobacillus gallinarum 9435, Lactobacillus asseri 33323, Levilactobacillus (Lactobacillus') namurensis BK214, Limosilactobacillus (Lactobacillus) vaginalis BK337, Limosilactobacillus (Lactobacillus) vaginalis BK372, Limosilactobacillus (Lactobacillus) vaginalis BK373, Limosilactobacillus (Lactobacillus) vaginalis BK349, Limosilactobacillus (Lactobacillus) vaginalis BK488, and Limosilactobacillus (Lactobacillus) vaginalis BK384.
Figure imgf000086_0001
30. The method according to any one of claims 18-29, wherein said one or more strains of Lactobacillaceae are administered to said subject in a pharmaceutical, nutraceutical, feed, foodstuff, food supplement, or beverage, composition.
31. The method according to any one of claims 18-30, wherein said administration is oral or enteral administration.
32. The method according to claim 30, wherein said administered composition comprises at least one pharmaceutically acceptable carrier or additive.
33. The method according to claim 30, wherein said administered composition is a feed, foodstuff, or food supplement.
34. The method according to any one of claims 18-33, wherein at least one of said one or more administered strains is in a lyophilized or spray-dried form.
35. The method according to any one of claims 18-34, wherein said subject has, or is, receiving at least one additional agent for reducing cholesterol or treating dyslipidemia.
Figure imgf000087_0001
36. The method according to any one of claims 18-34, wherein said method further comprises administering at least one additional agent for reducing cholesterol or treating dyslipidemia.
37. The method according to claim 35 or 36, wherein said additional agent is selected from the group consisting of Atorvastatin, Fluvastatin, Lovastatin, Pitavastatin, Pravastatin, Rosuvastatin, Simvastatin, Ezetimibe, Bempedoic acid, Cholestyramine, Colesevelam, Colestipol, Amlodipine, Fenofibrate, Gemfibrozil, niacin, omega-3 fatty acids, fibers, soluble fibers, beta-glucans, and oat beta glucan.
-87-
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