Postbiotic strain with dietary cholesterol adsorption capacity in small intestine as well as preparation method and application thereof

Abstract

The invention discloses a metabiotic strain with dietary cholesterol adsorption capacity in small intestines as well as a preparation method and application of the metabiotic strain, the metabiotic strain comprises one of lactobacillus plantarum LSJ-TY-HYB-T7, lactobacillus fermentum LSJ-TY-HYB-L16 and lactobacillus pentosus TY-HYB-SYY-Y3, and the metabiotic strain comprises one of lactobacillus plantarum LSJ-TY-HYB-L16 and lactobacillus pentosus TY-HYB-SYY-Y3. And the lactobacillus plantarum LSJ-TY-HYB-T7, the lactobacillus fermentum LSJ-TY-HYB-L16 and the lactobacillus pentosus TY-HYB-SYY-Y3 are all in an inactivated state. Research finds for the first time that the metabiotic strain disclosed by the invention does not generate an adsorption effect in a gastric juice environment, but can specifically and efficiently adsorb dietary cholesterol in intestinal juice, efficiently block small intestines from absorbing the dietary cholesterol, reduce the levels of total cholesterol, low-density lipoprotein and triglyceride in mice, and inhibit the growth of the dietary cholesterol in the mice. And the adsorbed dietary cholesterol is discharged out of the body together with excrement after being fermented by the colon.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically concerning metagenic strains in the small intestine with the ability to adsorb dietary cholesterol, their preparation method, and their applications. Background Technology

[0002] In modern society, high cholesterol levels are closely linked to the occurrence of cardiovascular disease. Excessive accumulation of cholesterol in the body, especially in the blood and arteries, is a major cause of cardiovascular disease. Traditional management methods mainly rely on dietary control and drug treatment, but these methods often have limited effectiveness and numerous limitations or side effects. Therefore, exploring new, more effective, and safer ways to manage cholesterol is particularly important.

[0003] Against this backdrop, natural dietary cholesterol adsorbents have become a research focus. Epigenetics, as processed products of probiotics, can lower cholesterol levels, regulate lipid metabolism, and improve blood lipid profiles by adsorbing dietary cholesterol, thereby reducing the risk of cardiovascular disease. Simultaneously, epigenetics are in an inactive state, making them less susceptible to environmental conditions. This stability allows them to maintain their effective properties for a longer period, making them suitable for various applications. Therefore, developing epigenetic strains with stable and efficient dietary cholesterol adsorption capabilities in the gastrointestinal digestive environment has significant scientific and practical value.

[0004] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to provide a metabiotic strain with the ability to adsorb dietary cholesterol in the small intestine, its preparation method, and its application, which can solve the technical problems mentioned in the background art.

[0006] To achieve the above objectives, a specific embodiment of the present invention provides the following technical solution:

[0007] Metabiotic strains in the small intestine that have the ability to adsorb dietary cholesterol include

[0008] Lactobacillus plantarum LSJ-TY-HYB-T7, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 20596, classified as Lactobacillus plantarum, with a deposit date of September 4, 2020.

[0009] Lactobacillus fermentum LSJ-TY-HYB-L16, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 20595, classified as Lactobacillus fermentum, was deposited on September 4, 2020.

[0010] Lactobacillus pentosus TY-HYB-SYY-Y3 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 25127 and classified as Lactobacillus pentosus. The deposit date is June 20, 2022.

[0011] The *Lactobacillus plantarum* LSJ-TY-HYB-T7, *Lactobacillus fermentum* LSJ-TY-HYB-L16, and *Lactobacillus pentosaccharide* TY-HYB-SYY-Y3 were all in an inactivated state.

[0012] To achieve the above objectives, a specific embodiment of the present invention provides the following technical solution:

[0013] The method for preparing metagenic strains capable of adsorbing dietary cholesterol in the small intestine includes the following steps:

[0014] S1. Place Lactobacillus plantarum LSJ-TY-HYB-T7, Lactobacillus fermentum LSJ-TY-HYB-L16 and Lactobacillus pentosaccharide TY-HYB-SYY-Y3 in broth culture medium;

[0015] S2 was cultured at 30–45℃ for 12–48 h, and the live bacteria were collected, washed with sterile water, and sterilized at 121℃ for 20 minutes. Subsequently, it was freeze-dried under vacuum at 0–100℃ and 0.1–1 Pa for 24–72 h to obtain the postbiotic.

[0016] In one or more embodiments of the present invention, in step S2, the bacteria are cultured at 37°C for 24 hours, and after collection, they are washed three times with sterile water and sterilized at 121°C for 20 minutes. Subsequently, they are freeze-dried under vacuum at -50°C and 0.1 Pa for 72 hours to obtain the post-biotic.

[0017] To achieve the above objectives, a specific embodiment of the present invention provides the following technical solution:

[0018] The application of epigenetic strains capable of adsorbing dietary cholesterol in the small intestine in the preparation of products for adsorbing dietary cholesterol in the small intestine. The epigenetic strains have the ability to efficiently adsorb dietary cholesterol in the small intestine, thereby regulating cholesterol metabolism in the body, reducing cholesterol levels in the body, and the adsorbed dietary cholesterol is excreted with feces after fermentation in the colon.

[0019] To achieve the above objectives, a specific embodiment of the present invention provides the following technical solution:

[0020] The application of metabiotic strains with the ability to adsorb dietary cholesterol in the small intestine in the preparation of products for adsorbing dietary cholesterol in the small intestine, and for the preparation of drugs or foods that reduce cholesterol levels in animals or humans.

[0021] To achieve the above objectives, a specific embodiment of the present invention provides the following technical solution:

[0022] The application of metabiotic strains with the ability to adsorb dietary cholesterol in the small intestine in the preparation of products for adsorbing dietary cholesterol in the small intestine, and in the preparation of drugs or foods that reduce the level of low-density lipoprotein in animals or humans.

[0023] To achieve the above objectives, a specific embodiment of the present invention provides the following technical solution:

[0024] The application of metabiotic strains with the ability to adsorb dietary cholesterol in the small intestine in the preparation of products for adsorbing dietary cholesterol in the small intestine, and for the preparation of drugs or foods that improve triglyceride levels in animals or humans.

[0025] This study is the first to discover that the metabiotic strain of the present invention does not adsorb in the gastric juice environment, but can specifically and efficiently adsorb dietary cholesterol in the intestinal juice, effectively blocking the absorption of dietary cholesterol in the small intestine, reducing the levels of total cholesterol, low-density lipoprotein and triglycerides in mice, and the adsorbed dietary cholesterol is excreted with feces after colonic fermentation. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a comparison diagram of the adsorption capacity of different metagenic strains of gastric juice for cholesterol concentration in a solution in one embodiment of the present invention.

[0028] Figure 2 This is a comparison diagram of the adsorption capacity of different metagenic strains of bacteria in gastric juice for cholesterol in solution in one embodiment of the present invention;

[0029] Figure 3 This is a comparison diagram of the adsorption capacity of different metagenic strains in intestinal fluid for cholesterol concentration in a solution in one embodiment of the present invention;

[0030] Figure 4This is a comparison diagram of the adsorption capacity of different bacterial strains in intestinal fluid for postbiotics in solution in one embodiment of the present invention;

[0031] Figure 5 This is an adsorption equilibrium diagram of the simulated cholesterol adsorption isotherm in the gastric small intestine in an embodiment of the present invention;

[0032] Figure 6 This is an adsorption equilibrium diagram of the simulated cholesterol adsorption isotherm in the gastric and small intestine in one embodiment of the present invention;

[0033] Figure 7 This is a fluorescence microscope image acquired before and after cholesterol adsorption in one embodiment of the present invention;

[0034] Figure 8 This is an XPS data graph before and after cholesterol adsorption in one embodiment of the present invention;

[0035] Figure 9 This is a schematic diagram of the total cholesterol level in mouse serum in one embodiment of the present invention;

[0036] Figure 10 This is a schematic diagram of mouse serum low-density lipoprotein levels in one embodiment of the present invention;

[0037] Figure 11 This is a schematic diagram of mouse serum high-density lipoprotein levels in one embodiment of the present invention;

[0038] Figure 12 This is a schematic diagram of mouse serum triglyceride levels in one embodiment of the present invention;

[0039] Figure 13 This is a schematic diagram of the total cholesterol level in the liver of a mouse in one embodiment of the present invention;

[0040] Figure 14 This is a schematic diagram of the total cholesterol level in mouse feces in one embodiment of the present invention. Detailed Implementation

[0041] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0042] Example 1:

[0043] One embodiment of the present invention provides a metabiotic strain capable of adsorbing dietary cholesterol in the small intestine. Its main purpose is to provide a strain with highly efficient cholesterol adsorption capacity in the small intestine. For example, it can be used to prepare drugs or foods that lower cholesterol levels in animals or humans, drugs or foods that lower low-density lipoprotein levels in animals or humans, and drugs or foods that improve triglyceride levels in animals or humans. The aforementioned foods mainly involve health foods, weight-loss foods, and health supplements.

[0044] The metabiotic strains in the small intestine capable of adsorbing dietary cholesterol mainly consist of *Lactobacillus plantarum* LSJ-TY-HYB-T7, *Lactobacillus fermentum* LSJ-TY-HYB-L16, and *Lactobacillus pentosaccharide* TY-HYB-SYY-Y3. These three strains are deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession numbers CGMCC No. 20596, CGMCC No. 20595, and CGMCC No. 25127, respectively.

[0045] The preparation steps of metabiotic strains in the small intestine capable of adsorbing dietary cholesterol include:

[0046] S1. Place Lactobacillus plantarum LSJ-TY-HYB-T7, Lactobacillus fermentum LSJ-TY-HYB-L16 and Lactobacillus pentosaccharide TY-HYB-SYY-Y3 in broth culture medium;

[0047] S2 was cultured at 30–45℃ for 12–48 h, and the live bacteria were collected, washed with sterile water, and sterilized at 121℃ for 20 minutes. Subsequently, it was freeze-dried under vacuum at 0–100℃ and 0.1–1 Pa for 24–72 h to obtain the postbiotic.

[0048] In step S2, the bacteria were cultured at 37°C for 24 hours, and after collection, they were washed three times with sterile water and sterilized at 121°C for 20 minutes. Subsequently, they were freeze-dried under vacuum at -50°C and 0.1 Pa for 72 hours to obtain the postbiotic.

[0049] The mechanisms by which *Lactobacillus plantarum* LSJ-TY-HYB-T7, *Lactobacillus fermentum* LSJ-TY-HYB-L16, and *Lactobacillus pentosaccharide* TY-HYB-SYY-Y3 adsorb dietary cholesterol were systematically analyzed from the perspectives of kinetics, thermodynamics, and structural chemistry through in vitro digestive tract simulation experiments, confirming their highly efficient adsorption characteristics in the small intestine.

[0050] In vivo experiments further validated its regulatory effect on lipid metabolism, significantly reducing cholesterol levels in high-cholesterol mice. This epiphytic strain, through multi-dimensional experiments (covering adsorption kinetics, morphology, elemental chemistry, and in vivo lipid regulation), comprehensively elucidated its lipid-lowering mechanism, providing a novel epiphytic resource for cardiovascular disease prevention. Its technical solution can be directly applied to functional foods, health products, or pharmaceutical preparations. By efficiently adsorbing dietary cholesterol in the small intestine, it effectively reduces the risk of cardiovascular disease, demonstrating significant clinical application value and market potential.

[0051] Metabiotics are inactivated microorganisms that are beneficial to the health of the host. Because metabiotics are in an inactivated state and do not reproduce, they do not have the risk of acquiring antibiotic resistance genes and are not easily affected by environmental conditions. This stability allows metabiotics to maintain their effective characteristics for a long time, making them suitable for a variety of application scenarios.

[0052] Example 2:

[0053] A digestive tract simulation experiment was conducted to simulate the cholesterol adsorption kinetics of the stomach and small intestine. Simulating the gastric digestion stage, a 10% egg yolk solution (diluted with distilled water) was mixed with simulated gastric digestive fluid at a 1:1 ratio, with a pH of 3.0. The ratio of live bacteria to the simulated gastric digestive fluid was 1:80 (g / mL), and the ratio of postbiotics to the simulated gastric digestive fluid was 1:400 (g / mL). After incubating in a water bath at 37℃ and 100 rpm for 2 hours, samples were collected at 0 min, 5 min, 10 min, 20 min, 30 min, 60 min, 90 min, and 120 min. The cholesterol content was determined using the o-phthalaldehyde method.

[0054] In the simulated small intestinal digestion stage, the above-mentioned gastric simulated digestion mixture was mixed with the intestinal simulated digestion solution at a 1:1 ratio, with a pH of 7.0, and incubated in a water bath shaker at 37℃ and 100 rpm for 4 hours. Samples were taken at 0 min, 5 min, 10 min, 20 min, 30 min, 60 min, 90 min, 120 min, 150 min, 180 min, 210 min, and 240 min for subsequent analysis. The adsorption capacity of the T7, L16, and Y3 postbiotics is expressed as qA.

[0055]

[0056] In the formula, CA,L,0 represents the initial concentration of cholesterol in the digestion solution during adsorption (mg / L), CA,L represents the cholesterol concentration in the digestion solution during adsorption (mg / L), VA represents the solution volume during adsorption (L), and m represents the mass of the metagene during the experiment (g). Each experiment was repeated three times.

[0057] like Figures 1-4 We can obtain:

[0058]

[0059] Example 3:

[0060] Simulated cholesterol adsorption isotherms in the gastrointestinal tract: First, egg yolk solutions with concentrations of 4%, 7%, 10%, 13%, and 16% were prepared. The adsorption capacity of the metabiotic at different cholesterol concentrations was measured according to the adsorption method described in section 2.1 to ensure adsorption equilibrium was reached. After adsorption equilibrium, the relationship between the adsorption capacity of the metabiotic and the cholesterol concentration in the solution was fitted to the Langmuir isotherm model.

[0061]

[0062] In the formula, qm is the maximum adsorption capacity of the metabiotic predicted by the Langmuir model (mg / g), b is the Langmuir constant (L / mg), and CA,L,E are the cholesterol concentrations in the digestate at adsorption equilibrium (mg / L). The results are as follows: Figures 5-6 As shown.

[0063] Example 4:

[0064] Fluorescence microscopy before and after cholesterol adsorption: Before microscopic analysis, the metagenic elements before and after adsorption were freeze-dried. Subsequently, a fluorescence microscope (DM48, Leica Microsystems GmbH, Switzerland) was used to observe the freeze-dried egg yolk and the metagenic elements before and after adsorption under different conditions. The microscope was equipped with a fluorescence light source (EL6000, Leica Microsystems GmbH, Switzerland) for fluorescence illumination, and sample images were acquired and processed using Leica Application Suite V4.0.0 software. Results are as follows: Figure 7 As shown.

[0065] XPS before and after cholesterol adsorption: After pressing an appropriate amount of lyophilized sample into a tablet, the tablet was placed on a sample tray. The sample was then placed in an X-ray photoelectron spectrometer (K-Alpha type, manufactured by Thermo Fisher Scientific, USA). The pressure in the sample chamber was less than 2.0 × 10⁻⁶. - 7 At mbar, the sample was sent into the analysis chamber with a spot size of 400 μm, an operating voltage of 12 kV, and a filament current of 6 mA. The full-spectrum scan pass energy was 150 eV with a step size of 1 eV; the narrow-spectrum scan pass energy was 50 eV with a step size of 0.1 eV. The results are as follows: Figure 8 As shown.

[0066] Example 5:

[0067] Animal in vivo experiment: Fifty 4-week-old male C57BL / 6J mice were used. Husbandry conditions: 5 mice per cage, temperature 22±1℃, 12h light / 12h dark cycle, free access to food and water. After a 1-week environmental acclimatization period, the mice were randomly divided into five groups of 10 mice each and kept for 12 weeks. The control group had free access to a basal diet (mouse growth and reproduction feed, Beijing Keao Xieli Feed Co., Ltd.), the model group had free access to a high-cholesterol diet (ASHF4, high-cholesterol mouse food, Daizi Biotechnology Co., Ltd.), and the T7, L16, and Y3 metagenic groups had free access to a high-cholesterol diet mixed with 10% metagenic bacteria. After 12 weeks of animal experiment, mice were anesthetized with ether, euthanized by neck dissection, and serum and liver were collected. Serum total cholesterol, low-density lipoprotein, high-density lipoprotein, triglyceride levels, and liver total cholesterol levels were measured according to the kit instructions. Results are as follows: Figures 9-13 As shown in the image. The day before the end of the experiment, feces were collected from each group of mice, and the total cholesterol content in the feces was measured according to the kit instructions. The results are as follows. Figure 14 As shown.

[0068] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0069] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A probiotic bacterial strain having the specific ability to adsorb dietary cholesterol in the small intestine, characterized in that, include Lactobacillus plantarum LSJ-TY-HYB-T7, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNO.20596, or... Lactobacillus fermentum LSJ-TY-HYB-L16, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNO.20595, or... Lactobacillus pentosus TY-HYB-SYY-Y3 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 25127; The *Lactobacillus plantarum* LSJ-TY-HYB-T7, *Lactobacillus fermentum* LSJ-TY-HYB-L16, and *Lactobacillus pentosaccharide* TY-HYB-SYY-Y3 were all in an inactivated state.

2. A method for preparing a probiotic bacterial strain having the ability to adsorb dietary cholesterol in the small intestine, characterized in that, Includes the following steps: S1. Place Lactobacillus plantarum LSJ-TY-HYB-T7, Lactobacillus fermentum LSJ-TY-HYB-L16 and Lactobacillus pentosaccharide TY-HYB-SYY-Y3 in broth culture medium; S2 was cultured at 30–45℃ for 12–48 h, and the live bacteria were collected, washed with sterile water, and sterilized at 121℃ for 20 minutes. Subsequently, it was freeze-dried under vacuum at 0–100℃ and 0.1–1 Pa for 24–72 h to obtain the postbiotic.

3. The method for preparing the postbiotic bacterial strain having the ability to adsorb dietary cholesterol in the small intestine according to claim 2, characterized by, In step S2, the bacteria were cultured at 37°C for 24 hours, and after collection, they were washed three times with sterile water and sterilized at 121°C for 20 minutes. Subsequently, they were freeze-dried under vacuum at -50°C and 0.1 Pa for 72 hours to obtain the postbiotic.

4. Use of a probiotic bacterial strain having the ability to specifically adsorb dietary cholesterol in the small intestine for the preparation of a product for adsorbing dietary cholesterol in the small intestine, characterized in that, The metabiotic strains have the ability to efficiently adsorb dietary cholesterol in the small intestine.

5. The application of metabiotic strains capable of adsorbing dietary cholesterol in the small intestine in the preparation of products for adsorbing dietary cholesterol in the small intestine, characterized in that, Used to prepare medicines or foods that lower cholesterol levels in animals or humans.

6. The application of metabiotic strains capable of adsorbing dietary cholesterol in the small intestine in the preparation of products for adsorbing dietary cholesterol in the small intestine, characterized in that, Used to prepare drugs or food that reduce the level of low-density lipoprotein in animals or humans.

7. The application of metabiotic strains capable of adsorbing dietary cholesterol in the small intestine in the preparation of products for adsorbing dietary cholesterol in the small intestine, characterized in that, Used to prepare medicines or foods that improve triglyceride levels in animals or humans.

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