A strain of Lactobacillus plantarum capable of promoting mineral absorption and transport and its application

Abstract

The present invention provides a strain of Lactobacillus plantarum that can promote mineral absorption and transport, and its uses. The present invention provides a strain of Lactobacillus plantarum with a deposit number of CGMCC No. 32404. The present invention also provides a culture containing the Lactobacillus plantarum, a sterilized solution, a supernatant, a filtrate, a diluent, a precipitate or a freeze-dried powder, a composition, a probiotic preparation, and related uses and methods.

Description

Technical Field

[0001] The present invention belongs to the field of food microorganisms, and more particularly relates to a Lactobacillus plantarum strain capable of promoting mineral absorption and transport and application thereof. Background Art

[0002] Minerals (such as calcium, iron, and zinc) are essential nutrients for maintaining normal human physiological functions and are involved in numerous life processes, including bone development, nerve conduction, blood production, and immune regulation. However, inefficient mineral absorption is a widespread nutritional problem worldwide, easily leading to mineral deficiencies, which in turn can cause a variety of diseases, including osteoporosis and iron-deficiency anemia, posing serious health risks.

[0003] Osteoporosis is a bone metabolic disorder characterized by damage to the microstructure of bone tissue, a continuous decrease in the ratio of bone mineral content to bone matrix, bone thinning, a decrease in the number of trabeculae, increased bone brittleness, and an increased risk of fracture. A deficiency in minerals, particularly calcium, can easily lead to osteoporosis. However, current treatment options for osteoporosis are limited. While medication-based interventions have some effectiveness, they cannot completely cure the condition, and long-term medication use can also cause numerous side effects, hindering effective treatment.

[0004] Iron deficiency anemia (IDA) is a common type of anemia characterized by microcytic and low hemoglobin levels due to iron deficiency. Early symptoms of iron deficiency anemia are usually subtle, but as the disease progresses, symptoms such as dizziness, fatigue, and pale complexion gradually appear, affecting the blood's oxygen-carrying capacity and, in turn, physical fitness, learning ability, and immunity. However, current treatments for iron deficiency anemia still primarily rely on iron supplements such as ferrous sulfate, ferrous gluconate, ferrous succinate, compound polysaccharide iron, and sucrose iron, lacking effective means of early intervention and treatment.

[0005] Since minerals cannot be synthesized in the human body, they must be supplemented through the diet. Dietary deficiencies of minerals such as calcium, iron, and zinc can lead to various diseases such as osteoporosis and iron-deficiency anemia, threatening human health. Therefore, it is necessary to explore methods to promote the absorption of minerals in the diet.

[0006] Studies have shown that certain probiotic strains, such as Lactiplantibacillus plantarum Lp299v, can promote mineral absorption and transport, improving osteoporosis and iron-deficiency anemia. However, this strain's ability to secrete organic acids, promote the absorption and transport of certain minerals, improve trabecular number and trabecular spacing in osteoporosis, and increase hemoglobin levels all need further improvement.

[0007] Therefore, there is a need in the art to develop a new strain of Lactobacillus plantarum to solve the above problems. Summary of the Invention

[0008] The purpose of the present invention is to provide a Lactobacillus plantarum strain capable of promoting mineral absorption and transport and application thereof.

[0009] The first aspect of the present invention provides a Lactobacillus plantarum strain with a deposit number of CGMCC No. 32404.

[0010] The second aspect of the present invention provides a culture containing Lactobacillus plantarum with a deposit number of CGMCC No. 32404.

[0011] In one or more embodiments, the culture contains Lactobacillus plantarum with a deposit number of CGMCC No. 32404 and a culture medium.

[0012] In one or more embodiments, the culture medium is MRS medium.

[0013] The third aspect of the present invention provides a sterilized liquid, supernatant, filtrate, diluent, precipitate or lyophilized powder, which is obtained by sterilizing, centrifuging, filtration, ultrafiltration, dilution, precipitation and / or lyophilizing the culture described in any embodiment of the present invention.

[0014] A fourth aspect of the present invention provides a composition comprising the plant lactobacillus described in any embodiment of the present invention, or the culture described in any embodiment of the present invention, or the sterilized liquid, supernatant, filtrate, diluent, precipitate or lyophilized powder described in any embodiment of the present invention.

[0015] In one or more embodiments, the composition is a food composition.

[0016] In one or more embodiments, the food composition includes finished food products, semi-finished food products, food additives, food supplements and health food compositions.

[0017] In one or more embodiments, the composition is a pharmaceutical composition.

[0018] In a fifth aspect, the present invention provides a probiotic preparation, which contains the plant lactobacillus described in any embodiment of the present invention, or the culture described in any embodiment of the present invention, or the sterilized liquid, supernatant, filtrate, diluent, precipitate or freeze-dried powder described in any embodiment of the present invention, or the composition described in any embodiment of the present invention.

[0019] In one or more embodiments, the probiotic preparation further comprises one or more of dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0020] A sixth aspect of the present invention provides a use of the plant lactobacillus described in any embodiment of the present invention, or the culture described in any embodiment of the present invention, or the sterilized liquid, supernatant, filtrate, diluent, precipitate or lyophilized powder described in any embodiment of the present invention in an aspect selected from the following aspects:

[0021] (1) Application in promoting mineral absorption and transport, or in preparing a composition or probiotic preparation for promoting mineral absorption and transport;

[0022] (2) Application in promoting the production of short-chain fatty acids and increasing phytase, or in preparing compositions or probiotic preparations for promoting the production of short-chain fatty acids and increasing phytase;

[0023] (3) Application in improving osteoporosis, or in preparing compositions or probiotic preparations for treating or preventing osteoporosis;

[0024] (4) Application in improving iron deficiency anemia, or in preparing a composition or probiotic preparation for treating or preventing iron deficiency anemia.

[0025] In a seventh aspect, the present invention provides a use of the pharmaceutical composition described in any embodiment of the present invention, or the probiotic preparation described in any embodiment of the present invention in an aspect selected from the following aspects:

[0026] (1) Application in promoting mineral absorption and transport;

[0027] (2) Promote the production of short-chain fatty acids and increase the application of phytase;

[0028] (3) Application in improving osteoporosis;

[0029] (4) Application in improving iron deficiency anemia.

[0030] In an eighth aspect, the present invention provides the use of the food composition described in any embodiment of the present invention in an aspect selected from the following aspects:

[0031] (1) Application in promoting mineral absorption and transport;

[0032] (2) Promote the production of short-chain fatty acids and increase the application of phytase;

[0033] (3) Application in improving osteoporosis for non-therapeutic purposes;

[0034] (4) Application for non-therapeutic purposes to improve iron deficiency anemia.

[0035] In a ninth aspect, the present invention provides a method for promoting mineral absorption and transport, promoting the production of short-chain fatty acids, increasing phytase, improving osteoporosis and / or improving iron deficiency anemia, the method comprising: culturing the plantarum Lactobacillus described in any embodiment of the present invention in a culture medium, or

[0036] Using the culture described in any embodiment of the present invention, or the sterilized liquid, supernatant, filtrate, dilution, precipitate or lyophilized powder described in any embodiment of the present invention to prepare a composition or probiotic preparation, or

[0037] The pharmaceutical composition according to any embodiment of the present invention, or the probiotic formulation according to any embodiment of the present invention is administered to an individual.

[0038] In one or more embodiments, the minerals include calcium, iron, and zinc, preferably calcium and iron.

[0039] In one or more embodiments, the promoting of mineral absorption and transport includes promoting the absorption of calcium ions, promoting the transport of calcium ions, and promoting the absorption of iron ions.

[0040] In one or more embodiments, the short-chain fatty acids include acetic acid, propionic acid, and butyric acid.

[0041] In one or more embodiments, the improvement of osteoporosis includes an increase in the number of trabeculae and / or a decrease in the spacing between trabeculae.

[0042] In one or more embodiments, the improving iron deficiency anemia comprises increasing the hemoglobin content.

[0043] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 , Gram staining of Lactobacillus plantarum Fe-01 strain, the stained area is Gram-positive.

[0045] Figure 2 , plate streak results of Lactobacillus plantarum Fe-01 strain.

[0046] Figure 3A , the whole genome map of Lactobacillus plantarum Fe-01.

[0047] Figure 3B, Lactobacillus plantarum Fe-01 hemolytic plate test results. Figure 1 shows the negative control bacteria: Listeria innocua CICC 10417; 2 shows the positive control bacteria: Staphylococcus aureus CICC 10473; 3 shows Lactobacillus plantarum Fe-01. These results and reports were generated and issued by the China Center for Industrial Culture Collection (CICC). CICC 10417 and CICC 10473 are CICC's own strains. The test report number is: 25-0282-00498.03-00906.

[0048] Figure 4 , tolerance of Lactobacillus plantarum Fe-01 to artificial gastric juice (A), artificial intestinal juice (B) and artificial bile salts (C).

[0049] Figure 5 An in vitro simulated intestinal fermentation model was used, and Lactobacillus plantarum Fe-01 was fermented in the simulated intestine for 24 hours. The effects of acid production (pH value) (A), L-lactic acid production (B), intracellular phytase activity (C), extracellular phytase activity (D), total short-chain fatty acids (total SCFAs) (E), acetic acid (F), propionic acid (G) and butyric acid (H) were studied. Lp299v was used as the positive control strain.

[0050] Figure 6 , Lactobacillus plantarum Fe-01 can promote the absorption of Ca, Fe and Zn by cells. Caco-2 cells were used to study the effects of Lactobacillus plantarum Lp299v and Fe-01 on cell activity (A, D, H) and their effects on cell mineral Ca 2+ (BC), Fe 2 (EF) + and Zn 2+ (IJ) Influence of absorption and transport.

[0051] Figure 7 Effects of 347-384 HgCl2 on the body weight (A) and uterus (B) of ovariectomized osteoporosis model mice.

[0052] Figure 8 Effects of Lactobacillus plantarum Fe-01 on bone metabolism blood indicators TRACP-5b (A), PTH (B), 1,25-(OH)2D3 (C), serum phosphorus content (D), BALP (E) and serum calcium content (F).

[0053] Figure 9MicroCT bone microstructure and analysis. (A) Micro-CT images of longitudinal and transverse femoral sections of mice in each group. Scale bar: 1 mm. (B-D) Statistical results of bone mineral density (BMD) (B), trabecular number (Tb.N) (C), and trabecular spacing (Tb.Sp) (D) in each group.

[0054] Figure 10 Analysis of total short-chain fatty acids (total SCFAs) (A), acetate (B), butyrate (C) and propionate (D) in feces of mice in the normal control group (Control), sham operation group (Sham), OVX group, Lp299v group and Fe-01 group.

[0055] Figure 11 Analysis results of routine blood indicators including hemoglobin (HGB) (A), red blood cell count (RBC) (B), hematocrit (HCT) (C) and mean corpuscular volume (MCV) (D) in the normal control group (Control), iron deficiency anemia mouse model group (IDA), Lp299v group and Fe-01 group. DETAILED DESCRIPTION

[0056] After in-depth research, the inventors discovered a strain of Lactobacillus plantarum Fe-01 that can promote the absorption and transport of minerals. Its deposit number is CGMCC No. 32404. This strain has good tolerance to artificial gastric juice, artificial intestinal juice, and artificial bile salts. It can significantly reduce the pH value under conditions of in vitro simulated human intestinal fermentation and produce short-chain fatty acids, especially beneficial fatty acids such as acetic acid, propionic acid, and butyric acid. The strain itself produces a high amount of phytase, which promotes the cell's absorption of minerals, especially Ca. 2+ 、Fe 2+ Using the ovariectomized osteoporosis mouse model and the iron-deficiency anemia mouse model, it was found that the plant lactobacillus also has excellent functions in improving osteoporosis and iron-deficiency anemia.

[0057] Lactobacillus plantarum Fe-01

[0058] The present invention provides a Lactobacillus plantarum strain that can promote mineral absorption or transport and improve osteoporosis and iron-deficiency anemia, and is named Fe-01 or LP-M56. It should be understood that in the present invention, Fe-01 and LP-M56 are used interchangeably, both referring to Lactobacillus plantarum, which was deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 30, 2024, with a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 32404 and a classification name of Lactiplantibacillus plantarum.

[0059] The colonies of Lactobacillus plantarum Fe-01 are usually round, with a diameter of 1-2 mm and neat edges; the front is yellow and opaque, the surface of the colony is smooth, moist, sticky, and easy to pick up, and the center of the colony is convex, semicircular or convex.

[0060] Lactobacillus plantarum Fe-01 was isolated from the feces of healthy and long-lived elderly people in Xinxiang, Henan Province. The isolation, purification, detection, and identification of this strain are described in detail in the Examples below. Because this strain has good tolerance to artificial gastric juice, artificial intestinal juice, and artificial bile salts, it can significantly reduce the pH value under in vitro simulation of human intestinal fermentation and produce short-chain fatty acids, especially beneficial fatty acids such as acetate, propionate, and butyrate. The strain can also produce high levels of phytase, which can promote the cell's absorption of minerals, especially Ca. 2+ Absorption and transport of minerals. Using ovariectomized osteoporosis and iron-deficiency anemia mouse models, this Lactobacillus plantarum strain was found to have excellent benefits in improving osteoporosis and iron-deficiency anemia. Therefore, this strain and its application in promoting in vitro mineral absorption or transport, producing short-chain fatty acids, and improving osteoporosis and / or iron-deficiency anemia are unexpected.

[0061] Culture of Lactobacillus plantarum Fe-01

[0062] The present invention also provides a culture of Lactobacillus plantarum with a deposit number of CGMCC No. 32404. The culture may contain the Lactobacillus plantarum described herein and a culture medium. The culture medium may be a culture medium commonly used in the art for culturing Lactobacillus plantarum.

[0063] Typically, the culture medium that is suitable for cultivating the plant lactobacillus contains nitrogen source, carbon source, vitamins and mineral substances. A non-limiting example of the culture medium that is suitable for cultivating the plant lactobacillus is MRS culture medium. Preferably, the culture medium contains peptone, beef extract, yeast extract, glucose, triammonium citrate, sodium acetate, magnesium sulfate, manganese sulfate, Tween 80 etc.

[0064] The fermentation process of the present invention can be carried out using Lactobacillus plantarum fermentation processes well known in the art, such as in vitro simulated human intestinal fermentation. An exemplary fermentation involves inoculating a culture medium with a Lactobacillus plantarum bacterial solution at an inoculum rate of 1-20% (e.g., 1-5%, 2-4%, or 2-3%) by weight, and fermenting under anaerobic conditions at, for example, 37°C for 24 hours. The fermentation can be carried out in a culture device, such as a vial.

[0065] The fermented liquid produced by the plant lactobacillus fermentation of the present invention can be directly used, or thalline can be removed by steps such as sterilization, centrifugation, filtration, ultrafiltration and / or dilution, and its sterilizing solution, supernatant, filtrate or diluent can be used. The sterilizing solution, supernatant, filtrate or diluent of the plant lactobacillus of the present invention can also be further obtained by steps such as precipitation and / or freeze-drying to obtain precipitate or freeze-dried powder of this plant lactobacillus.

[0066] Composition

[0067] The present invention provides a composition, comprising the Lactobacillus plantarum Fe-01 described in any embodiment of the present invention, or a culture of the Lactobacillus plantarum Fe-01 described in any embodiment of the present invention (such as its fermentation broth, sterilized liquid, supernatant, filtrate, dilution, precipitate or freeze-dried powder).

[0068] The compositions of the present invention can be formulated into a food composition using standard techniques well known to those of ordinary skill in the art. For example, the composition can be added directly to a food-acceptable material, or it can be used to prepare an intermediate composition (e.g., a food additive or premix) suitable for subsequent addition to a food-acceptable material.

[0069] As used herein, the term "food composition" refers to an article or substance that can be ingested by an individual, including finished food products, semi-finished food products, food additives, food supplements, and health food compositions. Exemplary food compositions include, but are not limited to, fluid dairy products, such as milk and condensed milk, fermented dairy products, such as yogurt, sour milk, and frozen yogurt, powdered milk, ice cream, cheese, cottage cheese, soy milk, fermented soy milk, vegetable juice, fruit juice, sports drinks, jelly, biscuits, energy bars, health foods, animal feed, and dietary supplements.

[0070] The composition of the present invention can also be formulated into a pharmaceutical composition, which can further include a pharmaceutically acceptable carrier.

[0071] As used herein, "pharmaceutically acceptable carriers" refer to carriers that have no significant irritating effect on an organism and do not impair the biological activity and properties of the agent in the administered pharmaceutical composition, such as, but not limited to, solvents, buffers, emulsifiers, suspending agents, decomposers, disintegrating agents, dispersing agents, binding agents, excipients, stabilizers, chelating agents, diluents, gelling agents, preservatives, wetting agents, lubricants, absorption delaying agents, or liposomes. In some embodiments, a pharmaceutically acceptable carrier can be an inert substance added to the pharmaceutical composition to further facilitate the administration of the agent, such as calcium carbonate, calcium phosphate, various sugars and starches, cellulose derivatives, gelatin, vegetable oils, polyethylene glycol, and the like.

[0072] The pharmaceutical composition of the present invention can be formulated into any suitable dosage form for oral administration or the like. Suitable dosage forms for oral administration include, but are not limited to, sterile powders, tablets, troches, lozenges, pellets, capsules, dispersible powders or granules, solutions, suspensions, drops, emulsions, syrups, elixirs, or slurries. The composition or pharmaceutical composition of the present invention can also be stored in a sterile apparatus suitable for injection or instillation.

[0073] The Lactobacillus plantarum Fe-01 or its culture in the pharmaceutical composition is present in a therapeutically or prophylactically effective amount. An effective amount is an amount sufficient to ameliorate or in some way alleviate symptoms associated with a disease, for example, to effectively ameliorate or eliminate one or more symptoms. This amount can be determined based on the subject's age, gender, and physical condition. While an administered amount may cure a disease, administration is typically intended to ameliorate disease symptoms. Repeated administration is generally required to achieve the desired symptom improvement.

[0074] In a specific embodiment of the present invention, mice are used as experimental animals, and a dosage regimen of Lactobacillus plantarum Fe-01 or its culture for osteoporosis or iron deficiency anemia is proposed. When mice are used as experimental animals, the concentration of Lactobacillus plantarum Fe-01 can be 10 6 CFU / mL or more, for example 10 6 ~10 11 CFU / mL. It should be understood that the conversion of the mouse dosage to a dosage suitable for human use is easily made by those skilled in the art, for example, by using the Meeh-Rubner formula: A = k × (W 2 / 3 ) / 10000. Where A is body surface area, measured in m2; W is body weight, measured in g; and K is a constant that varies with the species: 9.1 for mice and rats, 9.8 for guinea pigs, 10.1 for rabbits, 9.9 for cats, 11.2 for dogs, 11.8 for monkeys, and 10.6 for humans.

[0075] If necessary, the Lactobacillus plantarum Fe-01 or its culture can be administered in combination with other active ingredients or drugs. Exemplary active ingredients or drugs for improving osteoporosis include, but are not limited to, Lactobacillus plantarum Lp299v, calcium supplements (such as calcium carbonate, vitamin D and its active metabolites), vitamin K and its derivatives, bone resorption inhibitors, and other traditional Chinese medicines and their active ingredients. Exemplary active ingredients or drugs for treating iron deficiency anemia include, but are not limited to, Lactobacillus plantarum Lp299v and iron supplements (such as ferrous sulfate, ferrous gluconate, ferrous succinate, compound polysaccharide iron, iron sucrose, etc.).

[0076] Probiotic preparations

[0077] The present invention also provides a probiotic preparation, comprising the Lactobacillus plantarum Fe-01 described in any embodiment of the present invention, or comprising a culture of the Lactobacillus plantarum Fe-01 described in any embodiment of the present invention (such as its fermentation liquid, sterilized liquid, supernatant, filtrate, dilution, precipitate or freeze-dried powder), or comprising the composition described in any embodiment of the present invention.

[0078] The probiotic preparation may further include nutritionally acceptable nutritional additives, such as one or more of dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0079] The probiotic preparation may also include a medicinal and edible adjuvant or food additive, thereby developing and preparing a food that promotes mineral absorption and transport, promotes the production of short-chain fatty acids, increases phytase, a special-purpose functional food, a special medical food or a health product, all of which are within the scope of protection of the present invention.

[0080] In the probiotic preparation, the viable bacteria count of Lactobacillus plantarum Fe-01 can be 10 6 CFU / mL(g) or more, for example 10 8 CFU / mL (g) or more, 10 9 CFU / mL(g) or above.

[0081] The probiotic preparation may be in the form of tablets, capsules, soft capsules, granules, pills, gel candies, powders (including freeze-dried powders), oral liquids, drops, and the like.

[0082] The present invention also provides a food or health product box, which contains the Lactobacillus plantarum Fe-01 described in any embodiment of the present invention, or the culture of Lactobacillus plantarum Fe-01 described in any embodiment of the present invention (such as its fermentation liquid, sterilized liquid, supernatant, filtrate, dilution, precipitate or freeze-dried powder), or the composition described in any embodiment of the present invention (such as a food composition, a pharmaceutical composition), or the probiotic preparation described in any embodiment of the present invention.

[0083] The food or health product box may contain: a container 1 , and the Lactobacillus plantarum Fe-01 or a culture thereof of the present invention placed in the container 1 ; and a container 2 , and a nutritionally acceptable carrier placed in the container 2 .

[0084] The food or health product box may also contain some auxiliary materials, such as measuring instruments and containers such as syringes required for using or administering the composition in various dosage forms. The food or health product box may also contain instructions for use, thereby explaining and illustrating the method for promoting in vitro mineral absorption or transport, producing short-chain fatty acids, increasing phytase, etc.

[0085] Application / Method

[0086] The present invention found that Lactobacillus plantarum Fe-01 can significantly promote the cell's absorption of minerals, especially Ca in minerals. 2+ absorption and transport, and its role in increasing cellular Ca 2+In terms of transport rate, it is significantly better than Lactobacillus plantarum Lp299v.

[0087] The present invention also found that in an in vitro fermentation model simulating human intestinal flora, fermentation with Lactobacillus plantarum Fe-01 significantly lowered the pH value and produced large amounts of L-lactic acid and short-chain fatty acids (such as acetic acid, propionic acid, and butyric acid). The total amount of short-chain fatty acids produced, as well as the amounts of propionic acid and butyric acid, were significantly higher than those of Lactobacillus plantarum Lp299v. The present invention also found that Lactobacillus plantarum Fe-01 is rich in phytase. During simulated in vitro fermentation, large amounts of intracellular phytase (18.62 U / mg) and extracellular phytase (0.14 U / mg) were detected, both of which were higher than those of Lactobacillus plantarum Lp299v.

[0088] The present invention further found that Lactobacillus plantarum Fe-01 has a superior ability to significantly improve the number and trabecular spacing of osteoporotic mice compared to Lactobacillus plantarum Lp299v, and can improve osteoporosis caused by ovariectomy.

[0089] The present invention also found that Lactobacillus plantarum Fe-01 can significantly improve iron deficiency anemia. Compared with Lactobacillus plantarum Lp299v, Lactobacillus plantarum Fe-01 can significantly increase the hemoglobin content in mice with iron deficiency anemia.

[0090] Based on the above findings of the present invention, the present invention provides the use of Lactobacillus plantarum Fe-01, its culture, or its fermentation liquid, sterilized liquid, supernatant, filtrate, dilution, precipitate or lyophilized powder in the following aspects:

[0091] (1) Application in promoting mineral absorption and transport, or in preparing a composition or probiotic preparation for promoting mineral absorption and transport;

[0092] (2) Application in promoting the production of short-chain fatty acids and increasing phytase, or in preparing compositions or probiotic preparations for promoting the production of short-chain fatty acids and increasing phytase;

[0093] (3) Application in improving osteoporosis, or in preparing compositions or probiotic preparations for treating or preventing osteoporosis;

[0094] (4) Application in improving iron deficiency anemia, or in preparing a composition or probiotic preparation for treating or preventing iron deficiency anemia.

[0095] In the present invention, the minerals include calcium, iron, and zinc. In some preferred embodiments, the mineral is calcium or iron.

[0096] In the present invention, the terms "short-chain fatty acids" and "SCFA" are used interchangeably. The short-chain fatty acids include acetic acid, propionic acid, and butyric acid.

[0097] In the present invention, the "improvement of osteoporosis" includes, for example, an increase in the number of trabeculae, a decrease in the distance between trabeculae, and the like.

[0098] In the present invention, the "improvement of iron deficiency anemia" includes, for example, increasing the content of hemoglobin.

[0099] The present invention also provides a method for promoting mineral absorption and transport, promoting short-chain fatty acid production, increasing phytase, improving osteoporosis and / or improving iron deficiency anemia, the method comprising: culturing the plantarum lactobacillus Fe-01 described in any embodiment of the present invention in a culture medium, or

[0100] Using the Lactobacillus plantarum Fe-01 culture described in any embodiment of the present invention to prepare a composition or probiotic preparation for promoting mineral absorption and transport, promoting short-chain fatty acid production, increasing phytase, improving osteoporosis and / or improving iron deficiency anemia, or

[0101] The pharmaceutical composition according to any embodiment of the present invention, or the probiotic formulation according to any embodiment of the present invention is administered to an individual.

[0102] As used herein, the term "subject" refers to any animal of interest. In some embodiments, the subject is a mammal, such as humans, monkeys, cows, sheep, horses, pigs, goats, dogs, cats, mice, or rats.

[0103] In the present invention, the term "administering" refers to introducing, providing or delivering a substance to a subject through any appropriate route to achieve its intended function.

[0104] Advantages of the present invention include:

[0105] The present invention provides a plant lactobacillus Fe-01 that can promote the absorption and transport of minerals, and its deposit number is CGMCC 32404. The plant lactobacillus Fe-01 has good tolerance to artificial gastric juice, artificial intestinal juice and artificial bile salts, can significantly reduce the pH value under the condition of in vitro fermentation of the intestine, produce short-chain fatty acids, especially beneficial fatty acids such as acetic acid, propionic acid and butyric acid, increase the activity of phytase, and promote the absorption and transport of minerals, especially Ca by cells. 2+ 、Fe 2+In addition, the plant lactobacillus also has excellent functions of improving osteoporosis and iron deficiency anemia.

[0106] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Examples of the embodiments are shown in the accompanying drawings. Unless otherwise indicated, the same numbers in different drawings represent the same or similar elements.

[0107] The methods described in the examples are not intended to be exhaustive and are merely illustrative of systems and methods consistent with certain aspects of the present invention as detailed in the claims. Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions or those recommended by the manufacturer.

[0108] In the embodiments of the present invention, experimental data are expressed as mean ± SEM. The data were statistically analyzed using PRISM version 10.0 (GraphPad, San Diego, CA, USA). The differences between the groups were analyzed using one-way ANOVA followed by Tukery's multiple comparison test. Statistical significance was considered when p < 0.05.

[0109] Example 1: Cultivation, identification and preservation of Lactobacillus plantarum Fe-01

[0110] 1.1 Isolation and cultivation of bacterial strains

[0111] Feces from a healthy, elderly volunteer from Xinxiang, Henan Province, was collected and prepared into a 10% (wt / vol) fecal suspension using anaerobic phosphate-buffered saline (PBS). After mixing, the suspension was filtered using a fecal processor to remove large food debris. A serial dilution method was used to obtain dilutions. 100 μL of the dilution was spread onto a plate containing MRS medium supplemented with 0.5% cysteine. After anaerobically incubating at 37°C for 48–72 hours, colonies of varying shapes were picked onto new MRS medium plates based on their shape, size, and color. These colonies were then streaked and purified to obtain isolated strains. MRS medium was used.

[0112] The isolated strains were subjected to Gram staining and hydrogen peroxide enzymatic test, and then potential strains were selected based on the characteristics of Gram staining positivity and absence of catalase. Figure 1 ).

[0113] The DNA of the strain was extracted and amplified by PCR using universal primers 27F / 1492R for 16s rDNA fragment of lactic acid bacteria, followed by sequencing. The homology comparison was then performed with the gene sequence in the NCBI gene library to obtain Lactobacillus plantarum Fe-01.

[0114] Sequencing results of the Lactobacillus plantarum Fe-01 gene (SEQ ID NO: 1)

[0115]

[0116] 1.2 Strain preservation and activation

[0117] Prepare a bacterial solution of Lactobacillus plantarum Fe-01, add glycerol to a final concentration of 20%, mix well and place in a -80°C refrigerator for storage, thaw in an ice bath, use a sterile inoculation loop to inoculate the bacterial solution into a pre-prepared MRS culture dish, place in an anaerobic incubator and grow for about 48 hours until complete colonies grow in the culture dish, then you can proceed to subculture.

[0118] Colony status: The colonies are round, usually 1-2mm in diameter, with neat edges; the front is yellow and opaque, the surface of the colonies is smooth, moist, sticky, and easy to pick up, and the center of the colony is convex, semicircular or convex ( Figure 2 ).

[0119] 1.3 Strain preservation

[0120] Lactobacillus plantarum Fe-01 (isolated from the feces of healthy and long-lived elderly people) was deposited in the General Microbiology Center of the China Microorganism Collection Administration on October 30, 2024. It was classified as Lactobacillus plantarum, the preserved strain was named LP-M56, and the preservation number was CGMCC No. 32404.

[0121] Example 2: Tolerance of Lactobacillus plantarum Fe-01 to artificial gastric juice, artificial intestinal juice and artificial bile salts

[0122] This example tests the acid resistance of the Lactobacillus plantarum Fe-01 strain of the present invention to artificial gastric juice, artificial intestinal juice, and artificial bile salts. Meanwhile, Lactobacillus plantarum Lp299v (purchased from the commercial bacterial powder Probi®) with good tolerance and the ability to promote mineral absorption is used as a positive control strain.

[0123] The test method is as follows: the rejuvenated lactic acid bacteria strain was cultured in MRS liquid medium at 37℃ for 18 hours, and then centrifuged at 4℃ and 2500 rpm for 10 minutes to collect the bacteria.

[0124] The strains were incubated in artificial gastric fluid (SGJ, Shanghai Yuanye, Catalog No. R28616) at pH 1.5, pH 2.5, and pH 3.5 for 3 h, artificial intestinal fluid (Shanghai Yuanye, Catalog No. R22156) at pH 6.8 for 4 h, or in 0.1% (m / v), 0.3% (m / v), and 1% (m / v) artificial bile salts (ox bile salts, Qingdao Haibo, HB8290) for 0 and 24 h. Viable bacterial plate counts were performed, and the survival rate was used to evaluate the strains' resistance to acid, intestinal fluid, and bile salts. Survival rate = (number of viable bacteria after treatment / number of viable bacteria at time 0) × 100%.

[0125] The survival rate of the strain in artificial gastric fluid is as follows Figure 4 As shown in A, the survival rate of Lactobacillus plantarum Fe-01 after treatment in artificial gastric juice at pH 2.5 for 3 hours was 67.8%, and the survival rate after treatment in artificial gastric juice at pH 3.5 for 3 hours was 89.3%, which was not significantly different from the positive strain (Lp299v) (p>0.05), indicating that the Lactobacillus plantarum Fe-01 of the present invention has excellent gastric acid resistance and can smoothly pass through the stomach and reach the gastrointestinal tract.

[0126] The survival rate of strains in artificial intestinal fluid is as follows Figure 4 As shown in B, the survival rate of Lactobacillus plantarum Fe-01 after being treated in artificial intestinal fluid at pH 6.8 for 4 hours was 59.2%, which was not significantly different from the positive strain (Lp299v) (p>0.05) ( Figure 4 B).

[0127] The survival rate of strains in artificial bile salts is as follows Figure 4 As shown in Figure C, the survival rate of Lactobacillus plantarum Fe-01 after 24 hours of co-culture with 0.3% artificial bile salts was 61.9%, and the survival rate after 24 hours of co-culture with 1% artificial bile salts was 20.3%, showing no significant difference from the positive strain (Lp299v) (p>0.05). This indicates that the Lactobacillus plantarum Fe-01 of the present invention is resistant to intestinal fluid and bile salts, and can survive and colonize in the intestine.

[0128] Example 3: Whole genome sequencing and safety analysis of Lactobacillus plantarum Fe-01

[0129] Lactobacillus plantarum is not only widely distributed in nature, but has also been found in the oral cavity and digestive organs of humans and other mammals. It is a recognized probiotic with a long history of safe use in various foods. It is included in the European Food Safety Authority (EFSA) Qualified Presumption of Safety (QPS) list and the U.S. Food and Drug Administration (FDA) Generally Recognized as Safe (GRAS) list. It is also included in my country's "List of Bacteria Used in Food" and "List of Bacteria Used in Infant Food."

[0130] In this example, whole genome sequencing and safety analysis were performed on Lactobacillus plantarum Fe-01.

[0131] Genome assembly

[0132] Bacterial genome scans are generated by splicing optimized sequences from second-generation sequencing (NGS) using the short sequence assembly software SOAPdenovo2 (http: / / soap.genomics.org.cn / ) using multiple Kmer parameters to generate optimal contig assembly results. Reads are then aligned to the contigs, and the assembly results are locally assembled and optimized based on paired-end and overlap relationships, forming scaffolds. Complete bacterial genomes are generated by assembling NGS sequences using the assembly software Unicycler. Sequence correction is performed using Pilonjin software during the assembly process. If overlap of a certain length or greater exists between the ends of the final assembled sequence, the sequence is looped and the overlapping sequence at one end is truncated. This ultimately yields complete chromosome and plasmid sequences.

[0133] Assembly result:

[0134]

[0135]

[0136] The FMIC-QO01-022-2020 bacterial average nucleotide identity (ANI) identification test method was used to identify the bacterial average nucleotide identity of the Lactobacillus plantarum Fe-01 genome, and the strain was identified as Lactobacillus plantarum. The genome circle map is shown in Figure 3A .

[0137] The strain's safety was analyzed using the FMIC-QO01-024-2020 bacterial antibiotic resistance and virulence gene detection method. The results showed that no antibiotic resistance-related genes or virulence-related genes were detected in the Lactobacillus plantarum Fe-01 genome. The SH-QO01-042-2022 whole-genome sequence-based bacterial pathogenicity analysis method confirmed that Lactobacillus plantarum Fe-01 is not a potential human pathogen.

[0138] The antimicrobial sensitivity test of Lactobacillus plantarum Fe-01 was carried out using the EFSA 5206-2018 "Guidelines on the microbial characterization of feed additives or fermented products" 2.2.1 antimicrobial sensitivity method. The results showed that Lactobacillus plantarum Fe-01 was sensitive to ampicillin, gentamicin, kanamycin, erythromycin, clindamycin, tetracycline, and chloramphenicol.

[0139] The strains were tested on hemolysis plates using the FMIC-QO01-043-2024 microbial strain hemolysis detection method. The results showed that the hemolysis reaction of Lactobacillus plantarum Fe-01 was negative ( Figure 3B ).

[0140] After culturing the strain, biogenic amine production was tested according to the National Food Safety Standard (GB 5009.208-2016) for the determination of biogenic amines in food (Method 1). Lactobacillus plantarum Fe-01 was activated for one generation in MRS medium and then inoculated into MRS liquid medium. The cells were anaerobically cultured at 37°C for 48 hours, followed by centrifugation. The wet cells were weighed and extracted in a boiling water bath with 10% trichloroacetic acid for 1 hour. The supernatant was then derivatized and chromatographically determined for putrescine, cadaverine, histamine, and tyramine. Results were calculated based on the wet cells. The detection limits for the samples were 1.25 mg / kg for putrescine, histamine, and tyramine; and 0.75 mg / kg for cadaverine. The results showed that no putrescine, cadaverine, histamine, or tyramine were detected in Lactobacillus plantarum Fe-01.

[0141] Based on the results of this example, it can be seen that Lactobacillus plantarum Fe-01 is a safe probiotic.

[0142] Example 4: Performance of Lactobacillus plantarum Fe-01 in in vitro fermentation

[0143] In this example, an in vitro fermentation method was used to evaluate the fermentation performance of Lactobacillus plantarum Fe-01, with Lactobacillus plantarum Lp299v as a positive control strain and a control group without lactic acid bacteria (control group).

[0144] Select a simulated intestinal culture medium (Table 1) based on experimental needs and prepare it according to the medium formula / instructions. Heat and stir until dissolved, then dispense the medium into vials. Aerate the vials and close the cap. Sterilize in an autoclave. For the rejuvenated bacterial solution, inoculate the vial with a sterile 1mL syringe at 2-3% (v / v) of the solution. Gently shake to mix, then incubate in a 37°C incubator for 24 hours. After incubation, sample and test for pH and short-chain fatty acids (SCFAs).

[0145] Table 1. Formula of simulated intestinal culture medium (1L)

[0146] Element content Tryptone 10g yeast extract 2.5g L-Cysteine 1g Hemoglobin 2ml Sodium chloride 0.9g <![CDATA[CaCl2·6H2O]]> 0.009g <![CDATA[KH2PO4]]> 0.45g <![CDATA[K2HPO4]]> 0.45g <![CDATA[MgSO4·7H2O]]> 0.09g Vitamin I 200 μl Resazurin (1 mg / ml) 1ml starch 8g

[0147] Note: In Table 1, before heating, resazurin was prepared by adding 5 mg / mL hemin in 1 M NaOH and 100 μL of vitamin II solution in 1 L of PBS. The formulas for the vitamin I solution in Table 1 and the vitamin II solution added to resazurin are shown in Tables 2 and 3, respectively.

[0148] Table 2. Vitamin I solution formula (40 ml, stored at -30°C)

[0149] Element content Biotin (VH) 2mg <![CDATA[Cobalamin (VB 12 )]]> 2mg p-Aminobenzoic acid 6mg folic acid 10mg <![CDATA[Pyridoxamine (VB6)]]> 30mg

[0150] Table 3. Vitamin II solution formula (1 ml, stored at -30°C)

[0151] Element content <![CDATA[Thiamine (VB1)]]> 5mg <![CDATA[Riboflavin (VB2)]]> 5mg

[0152] Use a handheld pH meter to test the pH value of the sample. Before the measurement, clean the electrode three times with ultrapure water. Use lab paper to rinse off the residual water and then place the electrode in the sample solution. Wait for the reading to stabilize and then record the reading.

[0153] Short-chain fatty acids in fermentation broth samples were quantitatively assessed using a gas chromatography external standard calibration method: 500 μL of the fermentation broth to be measured was added with 100 μL of 15.0168 μmol / mL crotonic acid. The mixture was then centrifuged at 16,000 rpm for 5 minutes. The supernatant was collected and filtered into a sampling bottle equipped with a water system filter. 0.5 μL of the supernatant was analyzed using a gas chromatography system equipped with a DB-FFAP column. Chromatographic conditions were as follows: the column temperature was increased at a rate of 20°C / min to 180°C for 1 minute, then at a rate of 50°C / min to 220°C for 1 minute. The split ratio was 10:1, and the flow rate was 2.8 mL / min.

[0154] Figure 6Results showed that using an in vitro model simulating human intestinal fermentation, Lactobacillus plantarum Fe-01 significantly reduced the pH of the fermentation medium and produced significant amounts of L-lactic acid and beneficial short-chain fatty acids, such as acetate, propionate, and butyrate. The amounts of total SCFAs (p<0.001), propionate (p<0.05), and butyrate (p<0.05) produced by Lactobacillus plantarum Fe-01 were significantly higher than those produced by the positive strain Lactobacillus plantarum Lp299v.

[0155] Based on the results of this embodiment, the simulated in vitro fermentation of the human intestine shows that the fermentation of Lactobacillus plantarum Fe-01 in vivo can significantly reduce the pH value and produce short-chain fatty acids, especially beneficial fatty acids such as acetate, propionate and butyrate, which can promote the absorption and transport of minerals.

[0156] Example 5: High phytase production by Lactobacillus plantarum Fe-01

[0157] To test whether Lactobacillus plantarum Fe-01 contains phytase activity, 100 μL of the culture medium was added to 10 mL of MRS medium after passage and fermented at 37°C and 200 rpm for 24 hours. After fermentation, the cells were centrifuged to obtain the supernatant containing phytase. The extracellular phytase content was determined using a phytase assay kit (Beijing Box Biotechnology Co., Ltd.).

[0158] The isolated cells were weighed and rapidly frozen and thawed three times to disrupt the cells. The cells were then centrifuged at 4°C and 8,000 rpm for 10 minutes. The supernatant was collected to obtain a crude extract containing phytase. The intracellular phytase content was determined using a phytase assay kit (Beijing Box Biotechnology Co., Ltd.).

[0159] The results showed that the ability of Lactobacillus plantarum Fe-01 to produce intracellular phytase (18.62 U / mg) and extracellular phytase (0.14 U / mg) was higher than that of the positive strain Lactobacillus plantarum Lp299v (p<0.01).

[0160] Example 6: Lactobacillus plantarum Fe-01 promotes cellular absorption and transport of minerals

[0161] Caco-2 human colon cancer cell lines were purchased from Wuhan Punosai Life Science Co., Ltd. The cell line was cultured in Dulbecco Eagle Medium (DMEM) (Servicebio, G4511), a modified version of Dulbecco Eagle Medium enriched with 10% fetal bovine serum (FBS, Gibco, 10,270,106) and supplemented with 1% (v / v) penicillin-streptomycin (Beyotime, C0222). Cell lines were used between passages 10 and 25. The medium was changed every two days after the cells reached confluence.

[0162] 6.1 Cytotoxicity assay

[0163] Bacteria were cultured and collected as follows:

[0164] The cytotoxicity of the strains was assessed on Caco-2 cells: Caco-2 cells were incubated in culture medium for 24 h, washed in HBSS buffer, and then the strain density was adjusted to 10 in DMEM medium. 5 Cells were re-plated in 96-well tissue culture plates and grown for approximately 7 days until confluence. The old medium was discarded, the cells were washed twice with PBS buffer, and then 300 μL of the different bacterial suspensions were added to the cell monolayer. PBS buffer served as a negative control group (Control group). After incubation for 24 hours at 37°C and 5% CO2, 50 μL of cell supernatant was collected and lactate dehydrogenase (LDH) activity was measured using an LDH-based in vitro toxicology assay kit (Sigma-Aldrich, St. Louis, USA). Results are expressed as a percentage of LDH activity relative to the negative control cells.

[0165] 6.2 Ca 2+ 、Fe 2+ 、Zn 2+ Absorption and transport cell experiments

[0166] The cells were seeded at 50,000 cells / well on transwell permeable supports (0.4 µm, polyester membrane) in 12-well tissue culture plates and incubated in an incubator at 37°C with 95% O2 and 5% CO2 for 15–20 days. The transepithelial electrical resistance (TEER) of the Caco-2 monolayer was measured using a voltmeter and ohmmeter MilliCell resistance system (Merck Millipore, Burlington, USA). The TEER value was calculated according to the following equation: A TEER value greater than 600 was considered to indicate monolayer formation.

[0167] A bacterial suspension was prepared, and the Caco-2 cell monolayer was washed twice with HBSS. Then, 500 μL of the bacterial suspension (DMEM medium was used as a control) and CaCl2 (250 mM), ZnSO4 (50 μM), and FeSO4 (50 μM) were added to the upper chamber (apical side) of the cells. The cells were then incubated at 37°C, 5% CO2 for 24 hours. The culture medium in the lower chamber was collected, and calcium content was measured by inductively coupled plasma optical emission spectrometry (iCAP7400, Thermo Fisher Scientific, USA). The Caco-2 monolayer was washed with HBSS, washed with ice-cold buffer, and collected. The intracellular fluid was then collected by rapid freeze-thaw, and the collected intracellular fluid was also used to measure cellular calcium uptake by inductively coupled plasma optical emission spectrometry.

[0168] 6.3 Experimental Results

[0169] Caco2 cells were used to study the absorption and transport of minerals by the strain. The results are as follows Figure 6 As shown in the figure, the strains (Lp299v and Lactobacillus plantarum Fe-01) and the added minerals (CaCl2, FeSO4, ZnSO4) did not cause toxicity to the cells. Both Lp299v and Fe-01 strains significantly promoted the cell's absorption of CaCl2. 2+ 、Fe 2+ and Zn 2+ absorption or transport of Ca(P<0.05), and Fe-01 promoted the 2+ The transport rate of Fe-01 was significantly higher than that of Lp299v. 2+ The absorption rate of Lactobacillus plantarum Fe-01 is significantly higher than that of Lp299v. This shows that Lactobacillus plantarum Fe-01 has the function of significantly promoting the absorption and transport of minerals, especially promoting the absorption of Ca 2+ 、Fe 2+ The absorption and transport are most efficient.

[0170] Example 7: Intervention effect of Lactobacillus plantarum Fe-01 on ovariectomized osteoporosis mouse model

[0171] In this example, the intervention effect of Lactobacillus plantarum Fe-01 on the ovariectomized osteoporosis mouse model was investigated.

[0172] 7.1 Experimental Methods

[0173] Ovariectomy: After hair removal and skin disinfection with 10% povidone-iodine, the mice were anesthetized and sterile surgery was performed. Ovarian tissue was identified and removed bilaterally at the intersection of the femur and spine.

[0174] Fifty four-week-old female C57BL / 6J mice were randomly divided into groups according to body weight after acclimation and feeding for one week. Thirty mice underwent ovariectomy (OVX), 10 mice were assigned to the sham group (skin incision was immediately sutured), and the remaining 10 mice were assigned to the normal control group (Control). After one week of recovery, the ovariectomized mice were randomly divided into the following three groups: (1) model control group (OVX group, n=10), (2) OVX + Lactobacillus plantarum Lp299v (Lp299v group, n=10), and (3) OVX + Lactobacillus plantarum Fe-01 (Fe-01 group, n=10). After grouping, the test substance was administered orally once a day. The Lp299v group and the Fe-01 group were given 10 9 The mice were gavaged with Lactobacillus plantarum Lp299v and Lactobacillus plantarum Fe-01 at a dose of 100 cfu / d / mouse, respectively. The sham group (Sham), model group (OVX) and normal control group (Control) were gavaged with PBS.

[0175] After 10 weeks of intervention, all mice were killed, and samples of uterus, femur, tibia, etc. were collected, and osteoporosis-related indicators such as uterine coefficient, bone microstructure, bone structure model parameters, blood, colon and feces were measured.

[0176] 7.2 Results of Weight and Uterine Changes

[0177] The changes in animal body weight before and after intervention are shown in Figure 7 (A) After successful model establishment, the body weight of the sham group was significantly lower than that of the ovariectomized group in all other groups, consistent with the normal control group. The body weight of the ovariectomized group was significantly higher than that of both the control and sham groups, consistent with the significant weight gain seen in postmenopausal women. Intervention with Lactobacillus plantarum Lp299v and Lactobacillus plantarum Fe-01 had no significant effect on body weight.

[0178] The mouse uterine coefficient is an important indicator for evaluating estrogen-like effects or drug effects. It is calculated by multiplying the ratio of uterine wet weight to body weight by 100% (i.e., uterine coefficient = uterine weight / body weight × 100%). Figure 7 Results from test B showed that the uterine coefficient of the ovariectomized (OVX) group was significantly lower than that of the sham and control groups (p<0.0001), indicating that after ovariectomy, the uterus shrinks significantly as estrogen levels decrease. After intervention with Lactobacillus plantarum Lp299v and Lactobacillus plantarum Fe-01, the uterine coefficients of the Lp299v and Fe-01 groups were no different from those of the OVX group, indicating that Lactobacillus plantarum Fe-01 does not improve ovarian atrophy caused by ovariectomy.

[0179] 7.3 Hematological Indicators Related to Osteoporosis

[0180] Hematological analysis results such as Figure 8 As shown:

[0181] After ovariectomy, serum TRACP-5b and PTH (parathyroid hormone) levels were significantly higher in the OVX group than in the control group (p<0.001) and the sham group (p<0.0001). BALP, serum vitamin D3 (1,25-(OH)2D3), and phosphorus levels were significantly lower than in the control and sham groups, while serum calcium levels remained unchanged. This suggests that the sudden drop in estrogen levels after ovariectomy triggers a high-turnover state of bone metabolism, manifested by enhanced bone resorption (TRACP-5b ↑) and compensatory increases in PTH secretion. PTH maintains serum calcium homeostasis and reduces serum phosphorus by regulating bone calcium release and renal phosphate excretion. While the reduction in vitamin D3 exacerbates the imbalance in calcium and phosphorus metabolism, the compensatory effect of PTH prevents significant fluctuations in serum calcium.

[0182] After intervention with Lactobacillus plantarum Lp299v and Lactobacillus plantarum Fe-01, TRACP-5b and PTH levels were significantly decreased in both the Lp299v and Fe-01 groups, while VD3 (1,25-(OH)2D3) levels were significantly increased. The Fe-01 group was significantly more effective than the Lp299v group in reducing TRACP-5b levels. Furthermore, BALP levels in the Fe-01 group were significantly higher than those in the OVX group (p<0.01), and VD3 (1,25-(OH)2D3) levels were significantly higher than those in the Sham group (p<0.05). This suggests that Lactobacillus plantarum Fe-01 can significantly ameliorate bone destruction caused by ovariectomy (estrogen deficiency) and promote calcium-phosphorus balance.

[0183] 7.4 Osteoporosis Micro-CT Bone Tissue Morphology Analysis

[0184] Micro-CT results showed that ( Figure 9 In the OVX group, trabeculae were sparsely arranged, with prominent cavities at their borders, and bone microarchitecture was impaired, as evidenced by significant decreases in bone mineral density (BMD) and trabecular number (Tb.N), and a significant increase in trabecular spacing (Tb.Sp). These characteristics are typical manifestations of bone loss caused by estrogen deficiency.

[0185] The therapeutic efficacy of osteoporosis can be measured by increases in bone density and improvements in trabecular bone parameters. Trabecular alignment was enhanced in the Fe-01 group compared with the OVX group, and border cavities were significantly reduced. Trabecular nulling (Tb.N) and trabecular spacing (Tb.Sp) in the Lp299v group were not significantly different from those in the OVX group (p>0.05). However, trabecular nulling (Tb.N) (p<0.01) and trabecular spacing (Tb.Sp) (p<0.001) were significantly increased in the Fe-01 group, and trabecular spacing (Tb.Sp) was significantly smaller in the Fe-01 group than in the Lp299V group (p<0.05). This suggests that Lactobacillus plantarum Fe-01 can significantly improve trabecular number and spacing in osteoporotic mice, thereby alleviating ovariectomy-induced osteoporosis.

[0186] 7.5 Detection of Fecal Metabolites in Ovariectomized Osteoporosis Mouse Model

[0187] Ovariectomy may cause intestinal microbial metabolic dysfunction, thereby affecting bone metabolism. Figure 10 As shown:

[0188] The total short-chain fatty acids (SCFAs) content in the feces of mice in the OVX group was significantly lower than that in the Sham group (p<0.0001) and the Control group (p<0.001). Compared with the OVX group, the total SCFAs content in the feces of mice in the Lactobacillus plantarum Lp299v and Lactobacillus plantarum Fe-01 groups was significantly higher (p<0.0001).

[0189] The fecal acetic acid content of mice in the OVX group was significantly lower than that in the Sham and Control groups (p<0.01). Compared with the OVX group, the fecal acetic acid content of mice in the Lactobacillus plantarum Lp299v and Lactobacillus plantarum Fe-01 groups was significantly higher than that in the OVX group (p<0.001 and p<0.0001, respectively), and the acetic acid content in the Fe-01 group was significantly higher than that in the normal control group (p<0.05).

[0190] The propionate content in the feces of mice in the OVX group was significantly lower than that in the Sham group and the Control group (p<0.0001). Compared with the OVX group, the acetic acid and propionate content in the feces of mice in the Lactobacillus plantarum Lp299v group and the Lactobacillus plantarum Fe-01 group were significantly higher than those in the OVX group (p<0.01 and p<0.001, respectively).

[0191] The fecal butyrate content of mice in the OVX group was lower than that in the Sham group (p<0.05) and the Control group (p<0.01). Compared with the OVX group, the fecal butyrate content of mice in the probiotic Lp299v and Fe-01 groups was significantly higher (p<0.01 and p<0.001, respectively).

[0192] It can be seen that the mechanism by which Lactobacillus plantarum Fe-01 promotes bone health may be to promote the fermentation of the strain itself to produce short-chain fatty acids, especially beneficial short-chain fatty acids such as acetic acid, butyric acid and propionic acid.

[0193] Example 8: Effect of Lactobacillus plantarum Fe-01 on blood count in iron deficiency anemia mouse model

[0194] 8.1 Model Construction Method

[0195] After one week of acclimatization, the mice were randomly divided into the following groups using SPSS:

[0196] Normal control group (Control): fed with low-iron diet and gavaged with 3 mg / kg bw FeSO4;

[0197] Model group (IDA group): iron-deficient diet, oral administration of 200 μL normal saline;

[0198] Positive strain control group (Lp299v group): iron-deficient diet, oral administration of 200 μL at a dose of 1×10 9 cfu / mL of Lactobacillus plantarum Lp299v and 3 mg / kg bw of FeSO4;

[0199] Lactobacillus plantarum Fe-01 group (Fe-01 group): iron-deficient diet, oral administration of 200 μL Lactobacillus plantarum Fe-01 (1×10 9 cfu / mL) and FeSO4 (3 mg / kg bw).

[0200] During the experiment, blood was collected from the tail vein of mice at a fixed time every week for biochemical testing. After 4 weeks, if the hemoglobin (HGB) of the model group was <90g / L, the model was considered to be complete and the experiment could be stopped to collect blood for testing.

[0201] Figure 11Routine blood count results showed that the hemoglobin (HGB), red blood cell count (RBC), hematocrit (HCT), and mean corpuscular volume (MCV) of the model group (IDA group) were significantly lower than those of the control group (control), indicating that the IDA model successfully induced typical anemia. HGB, HCT, and MCV levels were significantly higher in the Lp299v and Fe-01 groups compared to the IDA group, and the hemoglobin content in the Fe-01 group was significantly higher than that in the Lp299v group (p < 0.05). Furthermore, Fe-01 treatment significantly increased the RBC count of anemic mice (p < 0.01), with no significant difference compared to the control group (p > 0.05). This suggests that Lactobacillus plantarum Fe-01 can significantly improve iron deficiency anemia.

[0202] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the scope of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be based on the appended claims. At the same time, all documents mentioned in this application are cited as references in this application, just as if each document was cited as a reference individually.

Claims

1. A plant lactobacillus with a deposit number of CGMCC No. 32404.

2. The culture of Lactobacillus plantarum according to claim 1, characterized in that The culture contains the Lactobacillus plantarum according to claim 1 and a culture medium.

3. A diluent, precipitate or freeze-dried powder, characterized in that: The culture medium is obtained by centrifuging, filtering, ultrafiltration, diluting, precipitating and / or freeze-drying the culture medium according to claim 2.

4. A composition, characterized in that The composition contains the Lactobacillus plantarum according to claim 1, or the culture according to claim 2, or the dilution, precipitate or freeze-dried powder according to claim 3.

5. The composition according to claim 4, wherein The composition is a food composition.

6. The composition according to claim 5, wherein The food composition is a finished food product, a semi-finished food product, a food additive, a food supplement or a health food composition.

7. The composition according to claim 4, wherein The composition is a pharmaceutical composition.

8. A probiotic preparation, characterized in that The probiotic preparation contains the Lactobacillus plantarum according to claim 1, or the culture according to claim 2, or the dilution, precipitate or freeze-dried powder according to claim 3, or the composition according to claim 4.

9. The probiotic preparation according to claim 8, wherein The probiotic preparation further comprises one or more of dietary fiber, prebiotics, protein, lipids, minerals and vitamins.

10. Use of the Lactobacillus plantarum according to claim 1, or the culture according to claim 2, or the dilution, precipitate or lyophilized powder according to claim 3 in an aspect selected from the following: (1) Application of promoting intestinal absorption and transport of minerals, or application of preparing a composition or probiotic preparation for promoting intestinal absorption and transport of minerals, wherein the mineral is Fe 2+ or Ca 2+ ; (2) Application in the production of short-chain fatty acids, L-lactic acid or phytase, or in the preparation of a composition or probiotic preparation for the production of short-chain fatty acids, L-lactic acid or phytase, wherein the short-chain fatty acid is acetic acid, propionic acid or butyric acid; (3) Application in the preparation of pharmaceutical compositions for the treatment of osteoporosis; (4) Application of the preparation of a pharmaceutical composition for treating iron deficiency anemia.

11. Use of the probiotic preparation according to claim 8 or 9 in an aspect selected from the following: (1) Application of promoting intestinal absorption and transport of minerals, wherein the mineral is Fe 2+ or Ca 2+ ; (2) Application in the production of short-chain fatty acids, L-lactic acid or phytase, wherein the short-chain fatty acid is acetic acid, propionic acid or butyric acid.

12. Use of the composition according to claim 4 in an aspect selected from the group consisting of: (1) Application of promoting intestinal absorption and transport of minerals, wherein the mineral is Fe 2+ or Ca 2+ ; (2) Application in the production of short-chain fatty acids, L-lactic acid or phytase, wherein the short-chain fatty acid is acetic acid, propionic acid or butyric acid.

13. A method for producing short-chain fatty acids, L-lactic acid or phytase, the method comprising: The Lactobacillus plantarum of claim 1 is cultured in a culture medium, wherein the short-chain fatty acid is acetic acid, propionic acid or butyric acid.

14. The use according to claim 10, characterized in that It also has one or more of the following characteristics: (A) The improvement of osteoporosis includes an increase in the number of trabeculae and / or a decrease in the spacing between trabeculae; (B) The improvement of iron deficiency anemia includes increasing the hemoglobin content.

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