Lactobacillus plantarum PDG8 and application of lactobacillus plantarum PDG8 to treatment of cardiovascular diseases

Abstract

Provided are a strain of Lactobacillus plantarum, i.e., Lactobacillus plantarum PDG8, and compositions thereof. Also provided are methods of treating cardiovascular diseases, such as atherosclerosis, using Lactobacillus plantarum PDG8 or a composition thereof.

Description

[0001] Reference to an electronic sequence listing

[0002] The contents of the electronic sequence listing (F-CF240639_SEQUENCE_LISTING.xml; size: 3,263,438 bytes; creation date: November 22, 2024) are incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to a Lactobacillus plantarum strain, i.e., Lactobacillus plantarum PDG8, which can be used to treat cardiovascular diseases. Specifically, the present disclosure relates to a composition, including a pharmaceutical composition, which contains Lactobacillus plantarum PDG8 and a culture thereof. The present disclosure also relates to a method for treating cardiovascular diseases such as atherosclerosis using Lactobacillus plantarum PDG8 or a composition containing Lactobacillus plantarum PDG8. Background Art

[0004] Atherosclerosis is a global chronic cardiovascular disease and one of the main causes of human disability and even death. The current mainstream strategy for the clinical treatment of atherosclerosis is to take statins. Although statins can reduce the synthesis of cholesterol, long-term use may also cause great damage to the liver and may even increase the risk of heart disease. In more recent treatments, stem cells are used to promote tissue repair and angiogenesis, but the survival rate after stem cell transplantation is low, the tropism is relatively poor, and the treatment effect is not ideal. The formation of atherosclerotic plaques can also be inhibited by injecting various antibodies and vaccines. However, these treatment options are costly, the operation process is complicated, and the side effects after treatment are difficult to predict and control. Further research is needed to solve these problems. Therefore, other treatments for atherosclerosis are needed. Summary of the invention

[0005] The present disclosure provides a novel plant lactobacillus strain with neurological effects, namely plant lactobacillus PDG8. In addition, the present disclosure provides a composition comprising plant lactobacillus PDG8 and its culture. The present disclosure further discloses a method for treating cardiovascular diseases such as atherosclerosis using plant lactobacillus PDG8 or a composition containing plant lactobacillus PDG8.

[0006] In one aspect, the present disclosure provides an isolated Lactobacillus plantarum PDG8 strain, which is deposited in Guangdong Microbiological Culture Collection Center (GDMCC) with accession number 62261.

[0007] In another aspect, the present disclosure provides a composition comprising Lactobacillus plantarum PDG8 or a culture thereof and an excipient.

[0008] In another aspect, the present disclosure provides a method of treating cardiovascular disease (eg, atherosclerosis), the method comprising administering to a subject in need thereof a therapeutically effective amount of an isolated Lactobacillus plantarum PDG8 strain or a composition thereof.

[0009] In a related aspect, the present disclosure provides an isolated Lactobacillus plantarum PDG8 strain or a composition thereof for use in a method of treating the human or animal body by therapy.

[0010] In another related aspect, the present disclosure provides an isolated Lactobacillus plantarum PDG8 strain or a composition thereof for use in a method of treating cardiovascular disease (eg, atherosclerosis). BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly describe the technical solutions in the embodiments of the present invention, the drawings used in the embodiments are briefly described below.

[0012] Figure 1 ApoE in atherosclerosis as described in Example 1 is shown - / - Establishment of male mouse model.

[0013] Figure 2 Lactobacillus plantarum PDG8 showed the effect of ApoE - / - Results of the effects of abdominal fat weight in male mice.

[0014] Figure 3 Lactobacillus plantarum PDG8 showed the effect of ApoE - / - Effects of atherosclerotic lesion formation in male mice. Part A is the oil red staining of the aortic arch and thoracic aorta of mice. Part B is the statistical result of the proportion of lesion area in the aortic arch and thoracic aorta of mice.

[0015] Figure 4 Lactobacillus plantarum PDG8 showed the effect of ApoE - / - Effects on the aorta lipid levels of male mice. Part A is the statistical results of total cholesterol in mouse serum, Part B is the statistical results of triglycerides in mouse serum, and Part C is the statistical results of low-density lipoprotein in mouse serum.

[0016] Figure 5 Lactobacillus plantarum PDG8 showed the effect of ApoE - / - Results of the effects of inflammatory cytokine levels in male mice. Part A shows the statistical results of the pro-inflammatory factor IL-6 level, and Part B shows the statistical results of the tumor necrosis factor TNF-α level.

[0017] Figure 6 Lactobacillus plantarum showed that it can inhibit ApoE - / - Effects on intestinal barrier integrity in mice. DETAILED DESCRIPTION

[0018] Other aspects and advantages of the present disclosure will become apparent to those skilled in the art through the following detailed description, in which illustrative aspects of the present disclosure are shown and described. It should be appreciated that the present disclosure is capable of realizing other and different aspects, and that its several details can be modified in various aspects, all without departing from the present disclosure. Therefore, these descriptions should be regarded as illustrative in nature, rather than restrictive.

[0019] Where values ​​are described as a range, it should be understood that such disclosure includes disclosure of all possible sub-ranges within such range, as well as specific values ​​falling within such range, regardless of whether a specific value or a specific sub-range is explicitly stated.

[0020] The present disclosure provides a plant lactobacillus strain with neurological effects, namely plant lactobacillus PDG8. In addition, the present disclosure provides a composition, such as a pharmaceutical composition or a nutritional health care composition, comprising plant lactobacillus PDG8 and a culture thereof. The present disclosure further discloses a method for treating cardiovascular diseases (such as atherosclerosis) using plant lactobacillus PDG8 or a composition containing plant lactobacillus PDG8.

[0021] The Lactobacillus plantarum PDG8 disclosed herein has one or more of the following characteristics:

[0022] a) Acid resistance,

[0023] b) Bile resistance,

[0024] c) has no hemolytic activity on red blood cells,

[0025] d) weak or no antibiotic resistance,

[0026] e) is capable of reducing the number or size of atherosclerotic plaques when administered to a subject,

[0027] f) when administered to a subject, it can alleviate inflammation (for example, reduce the expression levels of pro-inflammatory factors including IL-6 and INF-α in serum),

[0028] g) when administered to a subject, is capable of reducing blood lipid levels (e.g., total cholesterol, triglycerides and / or low-density lipoprotein cholesterol),

[0029] h) capable of treating cardiovascular disease (e.g., atherosclerosis) in a subject in need thereof,

[0030] i) when administered to a subject, is capable of reducing body fat (e.g., abdominal fat),

[0031] j) when administered to a subject, is capable of maintaining or improving the integrity of the intestinal barrier,

[0032] k) can be used as an active ingredient in a pharmaceutical composition or a nutritional health composition.

[0033] Lactobacillus plantarum PDG8

[0034] In one aspect, the present disclosure provides a Lactobacillus plantarum strain, namely, Lactobacillus plantarum PDG8.

[0035] As used herein, the term "Lactobacillus" refers to a microorganism belonging to the genus of aerobic or facultative anaerobic Gram-positive rods that are widely distributed in nature. Microorganisms belonging to the genus Lactobacillus include Lactobacillus plantarum and the like.

[0036] The plant lactobacillus PDG8 disclosed in the present invention is obtained by isolation from fermented vegetables. After separation and purification, whole genome sequencing confirmed that the strain is plant lactobacillus, belonging to the Firmicutes, Lactobacillales, Lactobacillus, and plant lactobacillus. The newly isolated plant lactobacillus PDG8 was deposited in the Guangdong Microbial Culture Collection Center (GDMCC) of the Institute of Microbiology, Guangdong Academy of Sciences, Building 59, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou 510075, China on March 4, 2022, with a preservation number of 62261.

[0037] The term "separation" refers to the state that a material or microorganism is separated from its naturally occurring environment. In certain embodiments, at least 50% in the naturally occurring environment except the target substance or microorganism, for example at least 60%, 70%, 80%, 90%, 95%, 97%, 98% or 99% of the material has been removed to produce the separated material or microorganism. In certain embodiments, the separated material or microorganism are at least 50%, for example at least 60%, 70%, 80%, 90%, 95%, 97%, 98% or 99% pure. In other words, at least 50%, for example at least 60%, 70%, 80%, 90%, 95%, 97%, 98% or 99% (by weight) of the separated material or microorganism are target substances or microorganisms.

[0038] The present invention further provides mutants, variants and / or progeny of the deposited Lactobacillus plantarum PDG8 strain.

[0039] As used herein, the term "mutant" refers to any bacteria produced by modification of a parent (ie, deposited) strain. For example, a mutant can be a bacterium produced by genetically modifying a deposited strain.

[0040] As used herein, the term "variant" refers to a naturally occurring bacterium derived from a parent (ie, deposited) strain. For example, a variant may be a bacterium produced by adapting to specific cell culture conditions.

[0041] As used herein, the term "progeny" means any bacterium produced by the breeding or proliferation of a deposited strain. Therefore, "progeny" means any direct descendant of a deposited strain. Therefore, the progeny strain itself can be identified as the same strain as the parent (i.e., the deposited) strain. It will be clear to those skilled in the art that due to the process of asexual reproduction, the progeny strain will be almost identical to the parent strain genetically. Therefore, in one embodiment, the progeny can be identical to the parent strain genetically, and can be considered as a "clone" of the parent strain. Alternatively, the progeny can be substantially identical to the parent strain genetically.

[0042] Mutants, variants or progeny may have at least 90%, 95%, 98%, 99%, 99.5% or 99.9% sequence identity with their parental strains over the entire length of the bacterial genome. In addition, mutants, variants or progeny will retain the same phenotype as the deposited parental strain.

[0043] As used herein, "same" or "identity" refers to the similarity between a DNA, RNA, nucleotide, amino acid or protein sequence and another DNA, RNA, nucleotide, amino acid or protein sequence. Identity can be expressed as the percentage of sequence identity between a first sequence and a second sequence. The percentage (%) of sequence identity relative to a reference DNA sequence can be the percentage of DNA nucleotides in a candidate sequence that are identical to the DNA nucleotides in the reference DNA sequence after alignment of the sequences. The percentage (%) of sequence identity relative to a reference amino acid sequence can be the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference amino acid sequence after alignment of the sequences and introduction of gaps, if necessary, to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity. The algorithm used in this article to determine the percentage of sequence identity is the BLAST 2.0 algorithm, as described in Altschul et al. "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res. 2007, 25, 3389-3402, with parameters set to default values.

[0044] As shown in the following examples, Lactobacillus plantarum PDG8 can reduce the accumulation of fat (e.g., abdominal fat), the formation, number and / or size of atherosclerotic plaques, the amount of blood lipids (e.g., total cholesterol, triglycerides and low-density lipoprotein cholesterol) and the expression of pro-inflammatory factors (e.g., TNF-α, IL-6), and improve the intestinal barrier.

[0045] In some embodiments, Lactobacillus plantarum PDG8 may cause fewer side effects or less severe side effects in patients than conventional drugs (e.g., statins) for treating cardiovascular diseases (e.g., atherosclerosis). Side effects include headache, dizziness, feeling unwell, unusual tiredness or weakness, digestive problems, muscle pain, sleep problems, low platelet counts, etc.

[0046] In some embodiments, Lactobacillus plantarum PDG8 may be able to colonize the intestinal tract of a patient and may be resistant to acid and bile salts.

[0047] As used herein, the term "acid resistance" refers to the property of withstanding high acidity. If the probiotic is acid resistant, it can be consumed by oral administration to prevent it from being degraded or damaged even when exposed to the strong acidic conditions in the stomach.

[0048] As used herein, the term "bile resistance" refers to resistance to digestive enzymes in bile. Bile is produced in the liver and stored in the gallbladder, and is a weakly alkaline green-brown liquid that aids in the digestion of fats in the duodenum of the small intestine and contains various enzymes that aid in digestion and absorption by emulsifying fats. Bile is one of the main reasons for reducing the effectiveness of probiotic administration because it acts on probiotics ingested by oral administration.

[0049] The Lactobacillus plantarum PDG8 of the present disclosure may not exhibit hemolytic activity on red blood cells. Hemolysis refers to the destruction of red blood cells and the release of hemoglobin to the surrounding area, and is the act of hemolysis of red blood cells by enzymes produced by harmful bacteria in the body. Therefore, Lactobacillus plantarum PDG8 is considered to be a stable microorganism that does not cause hemolysis in blood vessels even if administered in vivo.

[0050] In addition, the disclosed Lactobacillus plantarum PDG8 may have weak or no resistance to antibiotics. No drug resistance genes were identified in the genome of Lactobacillus plantarum PDG8 using AMRFinderPlus (NCBI). Therefore, when Lactobacillus plantarum PDG8 is used in medicines, health functional foods, feed additives, etc., it has little or no resistance to antibiotics, so the possibility of causing related pharmacological effects or environmental problems is very low.

[0051] In some embodiments, the Lactobacillus plantarum PDG8 bacteria can be modified using recombinant technology to contain antibiotic sensitivity genes or other genes that aid in the destruction of the bacteria upon introduction into a patient.

[0052] In more specific embodiments, Lactobacillus plantarum PDG8 can be provided as a probiotic.

[0053] In order to stably maintain the Lactobacillus plantarum PDG8 of the present disclosure for a long term, the strain can be stored by dissolving the cells in a storage solution prepared by mixing a certain amount of glycerol in water at -70°C, or the strain can be freeze-dried by, for example, suspending the cells in sterilized 10% skim milk, but the method of maintaining the strain is not limited thereto.

[0054] Composition containing Lactobacillus plantarum PDG8

[0055] In another aspect of the present disclosure, provided is a composition (eg, a pharmaceutical composition or a nutraceutical composition) comprising Lactobacillus plantarum PDG8 bacteria, a culture thereof, a concentrate thereof, or a dry form thereof, and optionally an excipient.

[0056] In certain embodiments, the composition may comprise, consist essentially of, or consist of isolated Lactobacillus plantarum PDG8 bacteria. In other embodiments, the composition may be in a formulation for administration to a patient. In certain embodiments, the composition and / or excipient is not naturally occurring.

[0057] The term "excipient" refers to an inactive substance that is used as a vehicle, carrier or medium for an active substance (e.g., Lactobacillus plantarum PDG8 bacteria or a culture thereof). Excipients may be included in the composition for the purpose of long-term stability, filling a solid formulation containing a small amount of effective active ingredient, or imparting therapeutic enhancement to the active ingredient in the final dosage form, such as promoting drug absorption, reducing viscosity, or increasing solubility. Excipients can also be used in the manufacturing process to help deal with active substance issues, such as by promoting powder flowability or non-stickiness, and also help in vitro stability, such as preventing denaturation or aggregation during the expected shelf life.

[0058] Exemplary excipients include cryoprotectants (e.g., glycerol, trehalose, maltodextrin, skim milk powder, and starch), fillers, extenders, diluents, antiadherents (e.g., magnesium stearate), binders (e.g., sugars including sucrose, lactose, starch, cellulose, microcrystalline cellulose, hydroxypropyl cellulose, xylitol, sorbitol, or mannitol; proteins including gelatin; and synthetic polymers including polyvinyl pyrrolidone (PVP) and polyethylene glycol (PEG)), coatings (e.g., hydroxypropyl methylcellulose, fatty acids, waxes, shellac, and plant fibers), disintegrants (e.g., cross-linked polymers including cross-linked polyvinyl pyrrolidone, In some embodiments, the present invention provides the following: a) agarose gel (for example, propylene glycol, croscarmellose sodium, and sodium starch glycolate), b ...

[0059] In certain embodiments, the composition is in the form of a liquid, suspension, dry (e.g., freeze-dried) powder, tablet, capsule, suppository or enema, or a frozen liquid, suspension or enema. Dry compositions can be produced by air drying, natural drying, spray drying, freeze drying, freeze drying and any combination thereof. The composition in a freeze-dried state may include a protective agent, such as a cryoprotectant, which may include ethylene glycol, dimethyl sulfoxide (DMSO), glycerol and any combination thereof. Before oral, nasogastric or rectal administration, such compositions may be reconstructed with a solution (e.g., a buffer).

[0060] In the liquid or suspension composition, the concentration of Lactobacillus plantarum PDG8 bacteria may be 10 3 CFU / mL to 10 8 CFU / mL, 10 4 CFU / mL to 10 8 CFU / mL, 10 5 CFU / mL to 10 8 CFU / mL, 10 6 CFU / mL to 10 8 CFU / mL, 10 3 CFU / mL to 10 7 CFU / mL, 10 4 CFU / mL to 10 7 CFU / mL, 10 5 CFU / mL to 10 7 CFU / mL, 106 CFU / mL up to 10 7 CFU / mL, but unlimited. In the solid mixture, the weight of the mixture, the amount of Lactobacillus PDG8 is 10 5 CFU / g up to 10 13 CFU / g, 10 6 CFU / g up to 10 13 CFU / g, 10 7 CFU / g up to 10 13 CFU / g, 10 8 CFU / g up to 10 13 CFU / g, 10 5 CFU / g up to 10 12 CFU / g, 10 6 CFU / g up to 10 12 CFU / g, 10 7 CFU / g up to 10 12 CFU / g, 10 8 CFU / g up to 10 12 CFU / g, 10 5 CFU / g up to 10 11 CFU / g, 10 6 CFU / g up to 10 11 CFU / g, 10 7 CFU / g up to 10 11 CFU / g, 10 8 CFU / g up to 10 11 CFU / g, 10 5 CFU / g up to 10 10 CFU / g, 10 6 CFU / g up to 10 10 CFU / g, 10 7 CFU / g up to 10 10 CFU / g, 10 8 CFU / g up to 10 10 CFU / g, 10 5 CFU / g up to 10 9 CFU / g, 10 6 CFU / g up to 10 9 CFU / g, 10 7 CFU / g up to 10 9 CFU / g, 10 5 CFU / g up to 10 8 CFU / g or 10 6 CFU / g up to 10 8 CFU / g.

[0061] In certain embodiments, the composition is provided in a unit dosage form. The composition may include Lactobacillus plantarum PDG8 bacteria in an amount ranging from 10 5 CFU to 10 13 CFU, 10 6 CFU to 10 13 CFU, 10 7 CFU to 10 13 CFU, 10 8 CFU to 10 13 CFU, 10 5 CFU to 10 12 CFU, 10 6 CFU to 10 12 CFU, 10 7 CFU to 10 12 CFU, 10 8 CFU to 10 12 CFU, 10 5 CFU to 10 11 CFU, 10 6 CFU to 10 11 CFU, 10 7 CFU to 10 11 CFU, 10 8 CFU to 10 11 CFU, 10 5 CFU to 10 10 CFU, 10 6 CFU to 10 10 CFU, 10 7 CFU to 10 10 CFU, 10 8 CFU to 10 10 CFU, 10 5 CFU to 10 9 CFU, 10 6 CFU to 10 9 CFU, 10 7 CFU to 10 9 CFU, 10 5 CFU to 10 8 CFU, or 10 6 CFU to 10 8 CFU.

[0062] The compositions provided herein preferably contain Lactobacillus plantarum PDG8 bacteria or a culture thereof in an amount sufficient to colonize the intestinal tract of a subject when administered to the subject.

[0063] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an isolated Lactobacillus plantarum PDG8 bacterium and a pharmaceutically acceptable excipient.

[0064] Pharmaceutically acceptable excipients include pharmaceutically acceptable cryoprotectants, fillers, fillers, diluents, antiadherents, binders, coatings, disintegrants, flavoring agents, glidants, lubricants, preservatives, adsorbents, sweeteners, and vehicles or carriers.

[0065] In certain embodiments, the pharmaceutical composition comprises at least one cryoprotectant selected from the group consisting of glycerol, trehalose, maltodextrin, skim milk powder and starch. Based on the gross weight of the composition, the content of the cryoprotectant of the present disclosure can be 0.01 wt % to 20 wt % or 0.01 wt % to 10 wt %. Specifically, based on the gross weight of the composition, the content of glycerol in the composition can be 5 wt % to 20 wt %, the content of trehalose can be 2 wt % to 10 wt %, the content of maltodextrin can be 2 wt % to 10 wt %, the content of skim milk powder can be 0.5 wt % to 2 wt %, and the content of starch can be 0.1 wt % to 1 wt %.

[0066] In certain embodiments, the excipient may include at least one or any combination of the following: diluents with various pH values ​​and ionic strengths (Tris, citrate, acetate or phosphate buffers), solubilizers such as or polysorbate, a carrier such as human serum albumin or gelatin, a preservative such as thimerosal, parabens, benzalkonium chloride or benzyl alcohol, an antioxidant such as ascorbic acid or sodium pyrosulfite, and other ingredients such as lysine, glycine, glucose, dextrin, skim milk, mannitol or succinylated β-lactoglobulin. In addition, the content of the excipient may be 50 wt % to 95 wt %, 75 wt % to 95 wt % or 85 wt % to 95 wt % based on the total weight of the composition.

[0067] The choice of a particular pharmaceutical composition will depend on many factors, including the condition being treated, the route of administration, and the desired pharmacokinetic parameters. A more extensive survey of ingredients suitable for use in pharmaceutical compositions can be found in Remington's Pharmaceutical Sciences, 18th ed., A.R. Gennaro, ed., Mack, Easton, Pennsylvania (1980).

[0068] The route of administration of the pharmaceutical composition of final selection will depend on many factors and can be determined by those skilled in the art. In certain embodiments, the composition can be further formulated into a dosage suitable for oral, nasogastric or rectal administration using pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated into tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, enema formulations, etc., for oral, rectal or nasogastric administration to the patient to be treated. The formulation can include, for example, polyethylene glycol, cocoa butter, glycerol, etc.

[0069] In some embodiments, the pharmaceutical composition may further comprise one or more additional therapeutic agents, such as anti-atherosclerotic agents.

[0070] The pharmaceutical compositions can be administered in any sterile, biocompatible pharmaceutical carrier including saline, buffered saline, dextrose, and water.

[0071] In certain embodiments, the present disclosure provides a nutraceutical composition comprising isolated Lactobacillus plantarum PDG8 bacteria or a culture thereof as an active ingredient. "Nutraceutical composition" refers to a food product or food supplement that can not only provide nutritional effects and / or taste satisfaction, but also provide therapeutic (or other beneficial) effects for subjects who consume the composition. The nutraceutical composition can be in the form of a probiotic composition, a synbiotic composition, a functional food, a food supplement, or a food product (including a beverage).

[0072] The content of Lactobacillus plantarum PDG8 or a culture thereof in the nutritional health composition can be based on the age or weight of the subject, the desired effect, etc., and can be used, for example, in an amount of 0.0001% to 1% of the subject's weight, such as 0.0001% to 0.001%, 0.0001% to 0.01%, 0.0001% to 0.1%, 0.001% to 0.01%, 0.001% to 0.1%, 0.001% to 0.1%, 0.01% to 0.1%, 0.01% to 1%, or 0.1% to 1% of the subject's weight.

[0073] The nutritional health-care composition of the present invention may further comprise a mixture of one or more of the following: organic acids, such as citric acid, fumaric acid, adipic acid, lactic acid, etc.; phosphates, such as potassium phosphate, sodium phosphate, polyphosphates, etc.; or natural antioxidants, such as polyphenols, catechins, tocopherols, vitamin C, green tea extract, chitosan, tannic acid, etc.

[0074] In certain embodiments, the isolated Lactobacillus plantarum PDG8, its culture or its composition can be used as a probiotic or probiotic composition. As used herein, the term "probiotic" refers to live bacteria that enter the body and provide health benefits, such as improving the body's microbial balance by, for example, interacting with the resident flora in the digestive tract and / or reducing or eliminating the symptoms or severity of disorders (e.g., atherosclerosis) by, for example, affecting the host physiology and immune system in a positive way.

[0075] The probiotic composition provided herein may further comprise one or more other probiotics, such as other lactobacilli, bifidobacteria and enterococci in the form of fermented milk, granules, powders, and the like.

[0076] In certain embodiments, the present disclosure provides a symbiotic composition comprising isolated Lactobacillus plantarum PDG8 or a culture thereof and one or more prebiotics. "Prebiotics" refers to substances that have a beneficial effect on probiotics, such as food for Lactobacillus plantarum PDG8. Exemplary prebiotics include inulin, pectin, and resistant starch.

[0077] In certain embodiments, the present disclosure provides a food supplement comprising isolated Lactobacillus plantarum PDG8 or a culture thereof and one or more prebiotics. As used herein, "food supplement", "supplement composition" or "dietary supplement" refers to a composition containing dietary ingredients (e.g., isolated Lactobacillus plantarum PDG8 or a culture thereof) intended to increase nutritional value or health benefits to supplement a diet. A food supplement can be added to food, but it is not food or intended to be food itself, or can be taken independently of food. The food supplement disclosed herein may also include one or more dietary ingredients, such as vitamins, minerals, herbs or other botanicals, and amino acids, in addition to isolated Lactobacillus plantarum PDG8 or a culture thereof.

[0078] The food supplement of the present disclosure can be a quick-release or sustained-release formulation, and can further include a carrier, such as an edible carrier. The edible carrier can include corn starch, lactose, sucrose or propylene glycol. Solid carriers can be used for tablets, powders, lozenges, etc., and liquid carriers can include syrups, liquid suspensions, emulsions, solutions, etc. In addition, the food supplement can include a preservative, a lubricant, a solution promoter or a stabilizer.

[0079] The present disclosure also provides a functional food containing Lactobacillus plantarum PDG8 or a culture thereof. As used herein, the term "functional food" refers to a human food that has a beneficial effect (eg, a medical or physiological benefit other than a pure nutritional effect) in addition to providing nutrition.

[0080] In certain embodiments, the present disclosure provides a food product comprising isolated Lactobacillus plantarum PDG8 or a culture thereof and one or more prebiotics. "Food product" means a product or composition for human or non-human animal consumption. Such food products include any food, feed, snack, food supplement, liquid, beverage, sweet, toy (chewable and / or edible toy), meal substitute or meal replacement.

[0081] In the functional food or food product of the present invention, Lactobacillus plantarum PDG8 or its culture may be present in an amount of 0.0001 wt % to 1 wt %, particularly 0.001 wt % to 0.1 wt %, based on the raw material composition comprising Lactobacillus plantarum PDG8. However, in the case of long-term administration, the amount may be less than the above range, for example, 0.001 wt % to 0.01 wt % based on the raw material composition comprising Lactobacillus plantarum PDG8.

[0082] There is no particular limitation on the type of food that can contain Lactobacillus plantarum PDG8 or its culture. Examples of foods to which Lactobacillus plantarum PDG8 or its culture can be added include meat, sausages, bread, chocolate, candy, snacks, desserts, pizza, instant noodles, other noodles, chewing gum, dairy products (including ice cream, yogurt, curd and whole milk), various soups, beverages, teas, drinks, alcoholic beverages, vitamin complexes, etc., and all functional foods.

[0083] Beverages or drinks containing plant lactobacillus PDG8 or its culture may further contain various flavorings or natural carbohydrates such as in traditional beverages. The above-mentioned natural carbohydrates may include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose; polysaccharides such as dextrin, cyclodextrin, etc.; and sugar alcohols such as xylitol, sorbitol, erythritol, etc. Natural sweeteners such as thaumatin, stevia extract, etc.; and synthetic sweeteners such as saccharin, aspartame, etc. can be used as sweeteners. Based on 100 parts by weight of plant lactobacillus PDG8 or its culture, the ratio of the additional ingredients may be in the range of 0.01 to 0.04 parts by weight, specifically 0.02 to 0.03 parts by weight.

[0084] The present disclosure further includes a method for preparing a composition as described herein, which may include the step of mixing an additive with Lactobacillus plantarum PDG8, a culture thereof, a concentrate thereof, or a dry form thereof. The additive may be an excipient as described above, such as a cryoprotectant, or a carrier.

[0085] Lactobacillus plantarum PDG8 can be cultured by traditional methods of culturing lactobacillus strains. For the culture medium, a natural culture medium or a synthetic culture medium can be used. For the carbon source of the culture medium, for example, glucose, sucrose, dextrin, glycerol, starch, etc. can be used. For the nitrogen source, peptone, meat extract, yeast extract, dry yeast, soybean, ammonium salts, nitrates and other organic or inorganic nitrogen-containing compounds can be used, but the nitrogen source is not limited thereto. For the inorganic salts contained in the culture medium, magnesium, manganese, calcium, iron, potassium, etc. can be used, but the inorganic salts are not limited thereto. In addition to the components of the carbon source, nitrogen source and inorganic salt, amino acids, vitamins, nucleic acids and related compounds can also be added to the culture medium. Lactobacillus plantarum PDG8 can be cultured in a temperature range of 20°C to 40°C for 12 hours to 4 days.

[0086] Specifically, the culture solution of Lactobacillus plantarum PDG8 can be a crude culture solution containing cells, or cells or concentrated cells with culture supernatant removed. The composition of the culture solution can not only contain additional components required for traditional culture of lactobacillus, but also contain components that play a synergistic role in the growth of lactobacillus, and its composition can be easily selected by those skilled in the art.

[0087] Treatment using Lactobacillus plantarum PDG8

[0088] In another aspect, the present disclosure provides a method for treating cardiovascular disease (e.g., atherosclerosis), the method comprising administering to a subject in need thereof a therapeutically effective amount of an isolated Lactobacillus plantarum PDG8 strain provided herein or a composition (e.g., a pharmaceutical composition) comprising an isolated Lactobacillus plantarum PDG8 strain or a culture thereof.

[0089] In a related aspect, the present disclosure provides an isolated Lactobacillus plantarum PDG8 strain as described above, or a composition (e.g., a pharmaceutical composition) comprising the isolated Lactobacillus plantarum PDG8 strain or a culture thereof, for use in a method of treating the human or animal body by therapy, for example, for use in a method of treating cardiovascular disease (e.g., atherosclerosis).

[0090] Cardiovascular diseases (heart and blood vessel diseases) that may be treated by administering Lactobacillus plantarum PDG8 or compositions thereof include, but are not limited to, atherosclerosis, aortic disease, coronary artery disease, peripheral arterial disease, and rheumatic heart disease.

[0091] As used herein, "atherosclerosis" refers to arterial disease characterized by the formation of fatty deposits, called "atherosclerotic plaques," on the walls of arteries. Atherosclerosis, including the number and size of plaques, can be detected by imaging, but there are usually no symptoms until the plaques are large enough to obstruct blood flow. At that time, depending on the location and severity of the plaques, blood clots, carotid artery disease, chronic kidney disease, coronary artery disease, heart attack, peripheral artery disease, stroke, or other cardiovascular-related events may occur.

[0092] Atherosclerosis is characterized by endothelial dysfunction, massive accumulation of low-density lipoprotein (LDL), necrotic debris and the presence of immune cells in the endothelial space. The pathogenesis is complex and may be related to endothelial cell damage caused by harmful external stimuli. If the concentration of LDL in the blood is too high, it may be deposited in the endothelial space. After oxidative modification, LDL becomes oxLDL and activates endothelial cells. Activated endothelial cells begin to secrete leukocyte adhesion molecules to capture monocytes in the blood and trigger a series of immune responses, including the maturation of monocytes into macrophages, and then the scavenger receptors on the surface of macrophages recognize oxLDL and activate the NF-κB signaling pathway, thereby enhancing the release of proinflammatory factors such as IL-1β and TNF-α. At the same time, this process promotes the further differentiation of macrophages into foam cells, ultimately forming atherosclerotic plaques (Herrero-Fernandez, B. et al. (2019). "Immunobiology of Atherosclerosis: A Complex Net of Interactions." Int J Mol Sci 20(21)).

[0093] This pathological process also develops with the migration of smooth muscle cells from the arterial media to the intima, the increase in the synthesis of extracellular matrix macromolecules (such as collagen, elastin and proteoglycans), and the increase in the fibrous cap covering the necrotic core. (Libby, P. et al. (2011). "Progress and challenges in translating the biology of atherosclerosis." Nature [Nature] 473 (7347): 317-325). In addition, the complications of atherosclerosis also seriously endanger human health. Proinflammatory cytokines stimulate the production of connective tissue enzymes and accumulate in plaques, leading to plaque rupture, thereby triggering acute cardiovascular disease.

[0094] An "individual," "subject," or "patient" herein is a vertebrate, such as a human or a non-human animal, such as a mammal. Mammals include, but are not limited to, humans, non-primates, farm animals, sport animals, rodents, and pets. Examples of non-human animal subjects include rodents, such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cows; horses; and non-human primates, such as apes and monkeys.

[0095] As used herein, a "therapeutically effective amount" of a substance refers to an amount sufficient to produce a beneficial or desired result (including a clinical result), and therefore, an "effective amount" depends on the context of its application. In some embodiments, in the context of administering a composition to treat cardiovascular disease, when a therapeutically effective amount is administered, the severity, frequency, or duration of at least one symptom of the cardiovascular disease may be reduced.

[0096] As used herein, and as understood in the art, "treatment" or "treating" is a method of obtaining a beneficial or desired result (including a clinical result). For the purposes of this subject matter, a beneficial or desired clinical result includes, but is not limited to, preventing, alleviating or ameliorating one or more symptoms, reducing the scope of the disease, stabilizing (i.e., not worsening) the disease state, preventing the disease, delaying or slowing the progression of the disease, reducing the risk of disease recurrence, and / or improving or alleviating the disease state.

[0097] Plant lactobacillus PDG8, compositions, or foods or supplement compositions as described herein can be administered to a subject. In particular, a therapeutically effective amount of plant lactobacillus PDG8, compositions, or foods or supplement compositions as described herein can be administered to a subject suffering from cardiovascular disease (e.g., atherosclerosis). The therapeutically effective amount of a composition, or a food or supplement composition can depend on many factors, such as the age, weight, and severity of cardiovascular disease of the subject, duration of treatment, other therapeutic drugs administered to the subject at the same time, and the gastrointestinal condition of the subject.

[0098] In some embodiments, Lactobacillus plantarum PDG8, compositions, or food or supplement compositions can be administered in an amount sufficient to detect colonization of the intestinal tract of a subject by Lactobacillus plantarum PDG8. Colonization can be tested within a period of time after administration of Lactobacillus plantarum PDG8 or a composition comprising Lactobacillus plantarum PDG8 bacteria or a culture thereof, for example, after one day, one week, two weeks, three weeks, or one month.

[0099] In a particular embodiment, Lactobacillus plantarum PDG8 or a composition thereof can be administered together with another therapeutic drug for the treatment of cardiovascular disease (e.g., atherosclerosis). The administration of other therapeutic drugs can be performed simultaneously with, before, or after the administration of Lactobacillus plantarum PDG8 or a composition thereof. When Lactobacillus plantarum PDG8 is colonized in the intestinal tract of a subject, the dosage of other therapeutic drugs can be reduced over time.

[0100] Lactobacillus plantarum PDG8 or a composition thereof can be administered to the subject once or more times, for example, daily, weekly, every two weeks, every three weeks, monthly, every two months, every six months, yearly, or when Lactobacillus plantarum PDG8 is not detected in the subject's intestine.

[0101] The subject can be a human, but in some embodiments can be another animal susceptible to cardiovascular disease (eg, atherosclerosis), such as a dog, cat, cow, goat, or horse.

[0102] Lactobacillus plantarum PDG8 or a composition thereof can be administered to a subject orally, nasally or rectally. Preferably, the administration is oral administration.

[0103] In some embodiments, Lactobacillus plantarum PDG8 or a composition thereof may be administered such that the dose of Lactobacillus plantarum PDG8 is 10 5 CFU / day or higher, 10 6 CFU / day or higher, 10 7 CFU / day or higher, 10 8 CFU / day or higher, 10 9 CFU / day or higher, 10 10 CFU / day or higher, 10 11 CFU / day or higher, 10 12 CFU / day or higher, or 10 13 CFU / day or more, or within a range between any of these amounts (e.g., 10 5 CFU to 10 13 CFU / day).

[0104] Lactobacillus plantarum PDG8 or a composition thereof can be administered repeatedly until at least one symptom of cardiovascular disease (eg, atherosclerosis) in the subject is improved.

[0105] In certain embodiments, Lactobacillus plantarum PDG8 or its compositions can be used to reduce the formation of atherosclerotic plaques, body fat inflammation and / or blood lipids to a subject. Specifically, using Lactobacillus plantarum PDG8 or its compositions can result in one or more of the following: reduce the accumulation of fat (e.g., abdominal fat), reduce the formation, quantity and / or size of atherosclerotic plaques, reduce the amount of blood lipids (e.g., total cholesterol, triglycerides and low-density lipoprotein cholesterol), reduce the expression of proinflammatory factors (e.g., TNF-α, IL-6), and improve the integrity of intestinal barrier. The method for measuring the accumulation of fat formation, the quantity and / or size of atherosclerotic plaques, the amount of blood lipids (e.g., total cholesterol, triglycerides and low-density lipoprotein cholesterol), the expression level of proinflammatory factors (e.g., TNF-α, IL-6) and the integrity of intestinal barrier is known, and its example is described in Example 3-7.

[0106] Example

[0107] The presently disclosed subject matter will be better understood by reference to the following examples, which are provided by way of illustration of the invention and not by way of limitation.

[0108] Example 1: Identification of Lactobacillus plantarum PDG8

[0109] The bacterial strain from the homemade fermented vegetables was isolated and purified to form the isolated Lactobacillus plantarum PDG8. Specifically, the slurry was diluted to 10 -3 or 10 -4 , and inoculated in a modified MRS medium, and then cultured anaerobically at 30°C for 48 hours. The colonies were counted and then picked. Each picked colony was inoculated in MRS liquid medium and cultured under anaerobic conditions for 48 hours. The strain was purified by repeated streaking. The isolated and purified strain was cultured anaerobically at 30°C for 48 hours, and the cells were collected by centrifugation.

[0110] DNA was extracted from isolated bacteria using the Biospin Bacterial Genomic DNA Extraction Kit. The purity and concentration of the DNA were determined. Covaris M220 was used to fragment genomic DNA, and Invitrogen Qubit 4.0 Fluorometer was used for concentration quantification. KAPA Hyper Prep Kit was used for library construction, and Illumina NovaSeq was used for sequencing. 42 contigs were obtained (SEQ ID NO: 1-42).

[0111] The obtained raw sequences were classified, the genome was assembled, and compared with 63,400 assembled gene sequences from NCBI by MASH (version: 2.2). Identification was based on mash distance information. The similarity with Lactobacillus plantarum was 99.68%. Therefore, the newly isolated strain was named Lactobacillus plantarum PDG8.

[0112] Example 2: ApoE - / - Establishment of mouse atherosclerosis model

[0113] like Figure 1 As shown, 6-week-old ApoE - / - Male mice were prepared for one week (12 / 12 light / dark alternation, free access to water and food), and then randomly divided into 3 groups: control group, model group, and probiotic group. - / - The mice received a normal diet and were gavaged with PBS. - / - The mice received a high-fat diet and were gavaged with PBS. - / - Mice were fed a high-fat diet and fed 1×10 8 CFU / day / mouse was intragastrically administered with Lactobacillus plantarum PDG8. The test was conducted for 10 weeks, and the results are as described in Examples 3-7.

[0114] Example 3: Lactobacillus plantarum PDG8 reduces ApoE - / - Abdominal fat weight in male mice

[0115] At the end of 10 weeks and after completion of other experiments, the animals were sacrificed and fixed on a dissecting board. The abdominal cavity was opened to isolate the white adipose tissue, which was then weighed. Figure 2 As shown, the abdominal fat accumulation weight in the model group was significantly higher than that in the control group (p<0.05). Compared with the model group, abdominal fat weight was reduced after 10 weeks of treatment with Lactobacillus plantarum PDG8 (p=0.0538). These results indicate that a high-fat diet causes fat weight gain in mice, but Lactobacillus plantarum PDG8 reduces fat weight gain to a level similar to that of mice not fed a high-fat diet.

[0116] Example 4: Lactobacillus plantarum PDG8 reduces ApoE - / - Atherosclerosis in male mice

[0117] In the sacrificed animals, the anterior chest skin was incised, the sternum and the entire thorax were removed, and the entire abdominal and thoracic cavities were exposed. 10 ml of 4°C pre-cooled PBS solution (autoclaved) was placed in a 50 mL infusion needle / syringe. A small opening was made in the right atrial appendage and the left ventricle (apex) was punctured with an infusion needle. PBS was injected slowly and evenly until the outflowing fluid was clear. After perfusion, the lungs were removed from the pedicles lobe by lobe, leaving only the heart, thymus and aorta in the thoracic cavity. The entire aortic tree was removed from the heart, the blood vessels were cut and fixed with 4% paraformaldehyde. After fixation for more than 24 hours, Oil Red O staining was performed.

[0118] After staining, pictures were taken with a common microscope, and the aortic lesion area was measured using image J software. Figure 3 , as shown in part A. Compared with the model group, Lactobacillus plantarum PDG8 significantly reduced the formation of atherosclerotic plaques.

[0119] like Figure 3 As shown in Part B, aortic lesions were significantly increased in the model group compared with the control group (p < 0.01). Although the level of aortic lesions in the Lactobacillus plantarum PDG8-treated group was not as low as that in the control group, it still showed a significant decrease compared with the model group (p < 0.05). This suggests that Lactobacillus plantarum PDG8 can treat aortic diseases, such as high-fat diet-induced atherosclerosis.

[0120] Example 5: Effect of Lactobacillus plantarum PDG8 on ApoE - / - Effects of lipid profiles in male mice

[0121] Collect whole orbital blood from mice and measure total cholesterol ( Figure 4 , Part A), triglycerides ( Figure 4 , part b) and low-density lipoprotein cholesterol ( Figure 4 , Part C). Figure 4 As shown, a high-fat diet increased the blood lipid levels of mice in the model group, but after 10 weeks of treatment with Lactobacillus plantarum PDG8, the blood lipid levels of all tested types decreased. This suggests that Lactobacillus plantarum PDG8 improves the imbalance of blood lipid levels induced by a high-fat diet.

[0122] Example 6: Effect of Lactobacillus plantarum PDG8 on ApoE - / - Effects of inflammatory cytokine levels in male mice

[0123] Orbital whole blood was collected, and the expression levels of TNF-α and IL-6 in serum were measured. Figure 5 As shown, high-fat diet increased the inflammatory factor IL-6 ( Figure 5 , Part A) and TNF-α( Figure 5 , Part B). After 10 weeks of treatment with Lactobacillus plantarum PDG8, the levels of both inflammatory factors decreased. This suggests that Lactobacillus plantarum PDG8 alleviates atherosclerosis-induced inflammation in mice.

[0124] Example 7: Effect of Lactobacillus plantarum PDG8 on ApoE - / - Effects on intestinal barrier integrity in male mice

[0125] Four hours before the mice were killed, they were gavaged with 0.6 mg / g of FITC-dextran. After the mice were killed, serum samples were obtained and the FITC content in the serum was measured using a fluorescence spectrophotometer. The results are shown in Figure 2. Figure 6As shown. Compared with the control group, the concentration of glucan in the serum of the model group was significantly increased (p < 0.001), while the previous treatment with Lactobacillus plantarum PDG8 significantly reduced the concentration of glucan in the serum of mice. (P < 0.01). This indicates that Lactobacillus plantarum PDG8 reduces the intestinal barrier damage induced by high-fat diet.

[0126] The various embodiments described above can be combined to provide additional embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification and / or listed in the application data sheet (including Chinese patent application No. 202210585567.4 filed on May 26, 2022, and U.S. Provisional Patent Application No. 63 / 347,281 filed on May 31, 2022) are incorporated herein by reference in their entirety. If necessary, the various aspects of the embodiment can be modified to provide further embodiments using the concepts of each patent, application, and publication.

[0127] In light of the above detailed description, these and other changes can be made to the embodiments. Generally, in the following claims, the terms used should not be interpreted to limit the claims to the specific embodiments disclosed in the specification and claims, but should be interpreted to include all possible embodiments and the entire range of equivalents to which such claims are entitled. Therefore, the claims are not limited by this disclosure.

Claims

1. An isolated Lactobacillus plantarum PDG8 strain, wherein the isolated Lactobacillus plantarum PDG8 strain is deposited in Guangdong Microbiological Culture Collection Center (GDMCC) with deposit number 62261.

2. A composition comprising the isolated Lactobacillus plantarum PDG8 strain or a culture thereof as claimed in claim 1 and an excipient.

3. The composition of claim 2, wherein the composition is not naturally occurring.

4. The composition of claim 2 or 3, wherein the composition is a pharmaceutical composition.

5. The composition of claim 4, wherein the composition is formulated for oral, nasogastric or rectal administration.

6. The composition of claim 2 or 3, wherein the composition is a nutritional health composition.

7. The composition of claim 6, wherein the composition is in the form of a probiotic composition, a synbiotic composition, a food supplement, a functional food, a food product or a beverage.

8. The composition of any one of claims 2-7, wherein the composition is a liquid.

9. The composition of any one of claims 2-7, wherein the composition is dry.

10. The composition of claim 9, wherein the composition is lyophilized or freeze-dried.

11. The composition of any one of claims 2-10, comprising Lactobacillus plantarum PDG8 in an amount sufficient to colonize the intestinal tract of the subject when administered to the subject.

12. The composition according to any one of claims 2 to 11, comprising Lactobacillus plantarum PDG8, wherein the amount of Lactobacillus plantarum PDG8 is in the range of 10 3 CFU / mL to 10 8 CFU / mL or 10 5 CFU / g to 10 13 CFU / g.

13. The composition of any one of claims 2-11, wherein the composition is in unit dosage form.

14. The composition of claim 13, wherein Lactobacillus plantarum PDG8 is present in an amount of 10 5 CFU to 10 13 The amount of CFU is present in the composition.

15. A method for treating cardiovascular disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of the isolated Lactobacillus plantarum PDG8 strain of claim 1 or the composition of any one of claims 2-14.

16. The method of claim 15, wherein the cardiovascular disease is atherosclerosis.

17. The method of claim 15 or 16, wherein administration comprises oral, nasogastric or rectal administration.

18. The method of any one of claims 15-17, comprising administering a therapeutically effective amount of the isolated Lactobacillus plantarum PDG8 strain or the composition in multiple doses.

19. The method of claim 18, comprising administering the therapeutically effective amount daily for at least one week.

20. The method of any one of claims 15-19, comprising administering the therapeutically effective amount until the intestinal tract of the subject is detectably colonized with Lactobacillus plantarum PDG8.

21. The method of any one of claims 15-20, wherein the therapeutically effective amount ranges from 10 5 CFU to 10 13 CFU.

22. The method of any one of claims 15-21, wherein the number or area of ​​atherosclerotic plaques in the subject is reduced.

23. The method of any one of claims 15-22, wherein the expression level of pro-inflammatory factors in the serum of the subject is reduced.

24. The method of claim 23, wherein the proinflammatory factor is IL-6, IL-8, IL-1β or TNF-α.

25. The method of any one of claims 15-24, wherein the subject has reduced lipid levels in the serum.

26. The method of claim 25, wherein the lipid is total cholesterol, triglycerides or low density cholesterol.

27. The method of any one of claims 15-26, wherein the subject's body fat mass is reduced.

28. The method of any one of claims 15-27, wherein the amount of abdominal fat is reduced.

29. The isolated Lactobacillus plantarum PDG8 strain of claim 1 or the composition of any one of claims 2-14 for use in a method of treating the human or animal body by therapy.

30. The isolated Lactobacillus plantarum PDG8 strain of claim 1 or the composition of any one of claims 2-14, for use in a method for treating cardiovascular disease.

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