Leuconostoc mesenteroides subsp. mesenteroides ftc m002, products and uses thereof to promote absorption of saponins and / or polysaccharides

Abstract

The application belongs to the technical field of microorganisms, and provides a Leuconostoc mesenteroides subsp. mesenteroides strain FTCM002 for promoting absorption of saponins and / or polysaccharides, products and applications thereof. The Leuconostoc mesenteroides subsp. mesenteroides strain FTCM002 has a preservation number of CGMCC No. 35016. The Leuconostoc mesenteroides subsp. mesenteroides strain FTCM002 has acid and bile salt resistance. In vitro experiments show that the strain can improve the bioavailability of total ginsenosides and dendrobium polysaccharides. Animal experiments show that the Leuconostoc mesenteroides subsp. mesenteroides strain FTCM002 can promote absorption of ginsenoside Rb1 and ginsenoside Rb2. The application also provides a method for preparing freeze-dried bacterial powder of the Leuconostoc mesenteroides subsp. mesenteroides strain FTCM002. The application also provides applications of the Leuconostoc mesenteroides subsp. mesenteroides strain FTCM002, which can be applied to products for promoting absorption of saponins and polysaccharides.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to Leuconostoc mesenteroides subsp. enterica FTCM002, a product of Leuconostoc mesenteroides that promotes the absorption of saponins and / or polysaccharides, and its applications. Background Technology

[0002] Saponins and polysaccharides are two important classes of bioactive substances widely found in nature. In many medicinal and edible products, saponins and polysaccharides serve as key active ingredients, exhibiting diverse physiological effects. For example, saponins possess significant pharmacological activities such as anti-tumor, anti-inflammatory, immunomodulatory, antiviral, antifungal, hepatoprotective, and immune-enhancing effects; while polysaccharides play positive roles in immunomodulation, anti-tumor, hypoglycemic, and antioxidant effects.

[0003] However, the absorption of saponins and polysaccharides in the human body is not optimistic, becoming a significant factor limiting their full efficacy. Saponins, due to their high polarity and low membrane permeability, are not readily absorbed and utilized by the body, thus limiting their effectiveness. Polysaccharides, on the other hand, have large molecular weights and complex structures, making them difficult for the body to digest and absorb. They require fermentation and decomposition by intestinal microorganisms to break down large molecules into smaller ones, thereby promoting absorption.

[0004] The microorganisms in the human gut possess various polysaccharide enzymes that can break down large molecules into usable, functional small molecules. Lactic acid bacteria, an important member of the gut microbiota, are active microorganisms that can produce beneficial effects on the host's health, playing a crucial role in human physiological metabolism. *Leuconostoc mesenteroides* subsp. *ménica*, belonging to the genus *Leuconostoc*, is a type of lactic acid bacteria that regulates gut microbiota balance, improves product flavor, and also possesses antioxidant and antagonistic abilities against pathogenic bacteria. Based on its physiological functions, *Leuconostoc mesenteroides* subsp. *ménica* may offer a new direction for solving the problem of low absorption and utilization rates of saponins and polysaccharides. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides Leuconostoc mesenteroides subsp. FTCM002, which promotes the absorption of saponins and / or polysaccharides.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] On the one hand, the present invention provides a Leuconostoc mesenchymal subsp. enterica ( Leuconostoc mesenteroides subsp. mesenteroides The accession number of Leuconostoc mesenteroides subsp. FTCM002 is CGMCC No. 35016.

[0008] Specifically, the Leuconostoc mesenteroides subsp. FTCM002 is a Gram-positive, facultatively anaerobic bacterium with an optimal growth temperature of 30–40°C and an optimal growth pH of approximately 5.5–6.2.

[0009] In another aspect, the present invention provides the application of Leuconostoc mesenteroides subsp. enterica FTCM002 or its culture in the preparation of products that promote the absorption of components.

[0010] Preferably, the enteromembranous subsp. enterica ( Leuconostoc mesenteroides subsp. mesenteroides The accession number of FTCM002 is CGMCC No.35016.

[0011] Preferably, it includes any one or more of the following applications:

[0012] (1) Promotes the absorption of ginsenosides;

[0013] (2) Promotes the absorption of Dendrobium officinale polysaccharides;

[0014] More preferably, the ginsenosides include one or more of saponins Rb1 and Rb2.

[0015] Preferably, the Leuconostoc mesenteroides FTCM002 culture is obtained by inoculating Leuconostoc mesenteroides FTCM002 into a culture medium.

[0016] In some embodiments, the culture medium for culturing Leuconostoc mesenteroides subsp. FTCM002 is MRS medium, which can be solid or liquid, and can be a commercially available medium or a self-prepared medium, the components of which can be routinely adjusted as needed.

[0017] More preferably, the composition of the above-mentioned MRS culture medium (per liter) is: 10g peptone, 5g beef extract powder, 4g yeast extract powder, 20g glucose, 1g Tween-80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate (MgSO4·7H2O), and 0.5g manganese sulfate (MnSO4·4H2O).

[0018] Preferably, the Leuconostoc mesenteroides FTCM002 culture includes one or more of the following: Leuconostoc mesenteroides FTCM002 fermentation broth, fermentation broth supernatant, fermentation broth precipitate, and live bacteria.

[0019] Specifically, the fermentation broth of Leuconostoc mesenteroides FTCM002 is a mixed liquid system obtained by artificially controlling the culture of Leuconostoc mesenteroides FTCM002 in a culture medium, which includes the bacterial cells themselves, intracellular and extracellular metabolites, unused culture medium, and fermentation by-products.

[0020] Specifically, the precipitate of the fermentation broth of Leuconostoc mesenteroides FTCM002 is the solid phase component separated from the fermentation broth of the strain after treatment such as standing, centrifugation or filtration. It mainly includes live / dead cells of the strain, cell fragments and insoluble substances produced in the fermentation system.

[0021] Specifically, the supernatant of the fermentation broth of Leuconostoc mesenteroides FTCM002 is a clear liquid phase component containing extracellular metabolites of the strain, soluble culture medium residues, and soluble fermentation by-products obtained after the fermentation broth of the strain has been allowed to stand, centrifuged, or filtered to remove solid phase precipitates such as bacterial cells.

[0022] Specifically, the live Leuconostoc mesenteroides subsp. FTCM002 is a bacterial cell with normal physiological activity, capable of carrying out life activities such as metabolism and reproduction.

[0023] Preferably, the viable count of Leuconostoc mesenteroides subsp. FTCM002 in the product is not less than 1×10⁻⁶. 11 CFU / g or 1×10 11 CFU / mL.

[0024] Preferably, the product is selected from one or more of the following: pharmaceuticals, functional foods, health products, microbial agents, and pharmaceutical raw materials.

[0025] Preferably, the dosage form of the product is selected from one or more of lyophilized powder, tablets, sprays, and granules.

[0026] More preferably, the freeze-dried powder is prepared by freeze-drying fermentation broth, fermentation broth supernatant, live bacteria, or fermentation broth precipitate of Leuconostoc mesenteroides FTCM002.

[0027] Specifically, the lyophilized powder also includes a lyophilization protectant.

[0028] More specifically, the freeze-drying protectant may be selected from one or more of pH buffers, stabilizers, fillers, or antioxidants.

[0029] More specifically, the pH buffer includes, but is not limited to, Tris, histidine, and citrate; the stabilizers include, but are not limited to, sugars, polyols, polymers, surfactants, certain proteins, and amino acids; the fillers include, but are not limited to, mannitol, glycine, bovine serum albumin, and milk powder; and the antioxidants include, but are not limited to, vitamin E, vitamin C, sodium thiosulfate, and protein hydrolysates.

[0030] In some embodiments, the freeze-drying protectant includes skim milk powder, trehalose, glycine, and sorbitol; wherein the freeze-drying protectant contains 10% skim milk powder, 8.00% trehalose, 3.13% glycine, and 3.57% sorbitol.

[0031] In another aspect, the present invention provides a product that promotes the absorption of ginsenosides and / or Dendrobium officinale polysaccharides, characterized in that it contains a live bacteria count of not less than 1×10⁻⁶. 11 CFU / g or 1×10 11 CFU / mL Leuconostoc mesenteroides subsp. FTCM002.

[0032] Preferably, it also includes excipients to maintain the activity of the strain.

[0033] The technical solution of the present invention has the following advantages:

[0034] 1. The Leuconostoc mesenteroides subsp. FTCM002 provided by this invention has strong acid and bile salt resistance and gastrointestinal tolerance, and can reach the intestine in a viable state.

[0035] 2. The Leuconostoc mesenteroides subsp. FTCM002 provided by this invention can improve the bioavailability of total ginsenosides and Dendrobium officinale polysaccharides. Furthermore, pharmacokinetic studies have shown that this bacterium can promote the bioavailability of ginsenosides Rb1 and Rb2 in vivo.

[0036] 3. The Leuconostoc mesenteroides subsp. FTCM002 provided by this invention can be used as a probiotic to develop functional foods, fermentation preparations, etc., and has good application value. Attached Figure Description

[0037] Figure 1 Graph showing the changes in the concentrations of ginsenoside Rb1 and ginsenoside Rb2 in rat plasma at different time points. Detailed Implementation

[0038] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0039] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0040] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of the present invention are obtained through conventional commercial channels. Products from different manufacturers do not have a significant impact on the effectiveness.

[0041] Preparation Example 1: Activation and Cultivation of Microbial Strains

[0042] This invention provides a strain of Leuconostoc mesenteroides subsp. FTCM002, which was deposited on June 26, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35016 and disclosed in Chinese patent CN121427751A.

[0043] Activation of Leuconostoc mesenteroides subsp. FTCM002: Leuconostoc mesenteroides subsp. FTCM002 was inoculated into MRS medium and cultured at 37℃ for 24 h. After three consecutive subcultures, the activated bacterial solution was obtained.

[0044] Preparation of *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 bacterial suspension: The activated bacterial suspension was inoculated into MRS liquid medium at a rate of 3% (v / v) and incubated at 37℃ for 24 h. The cells were then collected by centrifugation (4℃, 6000 r / min, 5 min). The concentration was adjusted to 1.0 × 10⁻⁶ using sterile physiological saline. 11 CFU / mL bacterial suspension.

[0045] The composition and preparation method of MRS culture medium are as follows: 10g peptone, 5g beef extract powder, 4g yeast extract powder, 20g glucose, 1g Tween-80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate (MgSO4·7H2O), 0.5g manganese sulfate (MnSO4·4H2O), 1L distilled water, pH 6.2±0.2, sterilized at 121℃ for 20min.

[0046] Preparation Example 2: Preparation of Leuconostoc mesenteroides subsp. enterica FTCM002 bacterial powder

[0047] The activated *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 bacterial suspension was centrifuged at 5000 rpm for 10 min at 4 °C, the supernatant was discarded, and the bacterial sludge was collected. The bacterial sludge was mixed with a cryoprotectant (10% skim milk powder, 8.00% trehalose, 3.13% glycine, and 3.57% sorbitol) at a ratio of 1:3 (V / V), allowed to stand at room temperature for 30 min, and then pre-frozen at -20 °C for 24 h. After pre-freezing, the mixture was freeze-dried in a vacuum freeze dryer at -50 °C for 24 h, resulting in a viable count of 1.0 × 10⁻⁶ cells / mL. 11 CFU / g.

[0048] Preparation Example 3: Preparation of Ginseng Aqueous Extract

[0049] Add 10g of untreated ginseng to a beaker, add 100mL of pure water, and cook in an induction cooker for 2 hours. Filter the residue, and the supernatant is the ginseng water extract.

[0050] Preparation Example 4: Preparation of water extract from Dendrobium officinale

[0051] Take an appropriate amount of Dendrobium officinale, add 12 times the amount of water and soak for 30 minutes, decoct for 1 hour, and filter; decoct the dregs again in the same way, combine the filtrates, and the filtrate is the water extract of Dendrobium officinale.

[0052] Experiment Example 1: Determination of Acid and Bile Salt Tolerance of Leuconostoc mesenteroides subsp. mesenteroides FTCM002

[0053] 1.1 Acid Resistance Test

[0054] The activated Leuconostoc mesenteroides FTCM002 bacterial suspension (Preparation Example 1) was centrifuged, and the supernatant was discarded. Sterile physiological saline with pH values ​​of 2.0, 4.0, and 6.0 was added to the bacterial cells, respectively. After mixing, the cells were incubated at 37°C for 3 hours. After adding sterile physiological saline again, the cultures were collected at 0 hours and 3 hours, diluted, and plated on MRS plates. After incubation at 37°C for 48 hours, the cells were counted, and the survival rate was calculated. The calculation formula is shown in Equation (1):

[0055]

[0056] Note: N2 represents the number of viable bacteria after the strain was incubated in sterile saline at different pH values ​​for 0 hours; N1 represents the number of viable bacteria after the strain was incubated in sterile saline at different pH values ​​for 3 hours.

[0057] 1.2 Bile salt tolerance test

[0058] The activated Leuconostoc mesenteroides subsp. FTCM002 bacterial suspension (Preparation Example 1) was inoculated at a 5% (V / V) inoculation rate into MRS liquid medium containing 0.01, 0.02, 0.03, and 0.04 g / L porcine bile salts, respectively. After incubation at 37°C for 18 h, the suspension was diluted and plated onto MRS plates. After incubation at 37°C for 48 h, the plates were counted, and the survival rate was calculated. The calculation formula is shown in Equation (2):

[0059]

[0060] Note: N0 represents the number of viable bacteria after culturing without bile salts; N1 represents the number of viable bacteria after the strain is inoculated with different concentrations of bile salts.

[0061] 1.3 Results and Analysis

[0062] Table 1. Survival rate of Leuconostoc mesenteroides subsp. FTCM002 at different pH values.

[0063]

[0064] Table 2. Survival rate of Leuconostoc mesenteroides subsp. mesenteroides FTCM002 at different bile salt concentrations.

[0065]

[0066] Table 1 shows that the survival rate of *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 decreased with increasing pH. At pH 2.0, the survival rate remained at 93.44%, indicating good acid tolerance. Table 2 shows that *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 exhibited varying degrees of tolerance to different concentrations of bile salt solutions, but its survival rate decreased with increasing bile salt content. At a bile salt concentration of 0.04 g / L, the survival rate remained at 82.24%, indicating good bile salt tolerance. In summary, *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 demonstrates good tolerance to both acid and bile salts.

[0067] Experimental Example 2: Test of gastrointestinal fluid tolerance of Leuconostoc mesenteroides subsp. mesenteroides FTCM002

[0068] 2.1 Solution Preparation

[0069] 2.2.1 Preparation of artificial gastric juice:

[0070] Accurately measure 16.4 mL of hydrochloric acid with a mass concentration of 100 g / L, dilute it to 800 mL with distilled water, adjust the pH value to 2.5, add 3.2 g of pepsin and 1.8 g of NaCl in sequence, and make up the volume to 1 L. Filter and sterilize for later use.

[0071] 2.1.2 Preparation of artificial intestinal fluid:

[0072] Weigh 6.8g of KH2PO4 and dissolve it in 800mL of distilled water. Then weigh 10g of trypsin and dissolve it in the KH2PO4 solution. Adjust the pH to 7.5 with a 40g / L NaOH solution and filter to sterilize before use.

[0073] 2.2 Experimental Methods

[0074] One mL of *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 suspension (Preparation Example 1) was added to a reaction tube containing 9 mL of artificial gastric fluid and incubated at 37°C with shaking at 180 rpm for 2 h to simulate human gastric peristalsis in vitro. One mL of the bacterial suspension treated with gastric fluid was added to a reaction tube containing 9 mL of artificial intestinal fluid and incubated at 37°C with shaking at 180 rpm for 2 h, 4 h, and 8 h to simulate changes in intestinal peristalsis. 100 μL of the isolated strain was extracted from the reaction solution at 0 h and 2 h in artificial gastric fluid, and at 0 h, 2 h, 4 h, and 8 h in artificial intestinal fluid, serially diluted, and inoculated onto MRS solid medium, with three replicates per group. After incubation at 37°C for 24 h, viable cell counts were performed, with conventional MRS liquid medium culture as the control group. The survival rate of *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 was calculated using the following formula:

[0075]

[0076] 2.3 Experimental Results

[0077] The simulated gastrointestinal survival rates of Leuconostoc mesenteroides subsp. FTCM002 are shown in Table 3. As the shaking culture time was extended, the survival rate of the Leuconostoc mesenteroides subsp. FTCM002 group showed slight changes. The survival rate reached 86.61% after 2 hours of culture in simulated gastric fluid and 73.36% after 8 hours of culture in simulated intestinal fluid, indicating that Leuconostoc mesenteroides subsp. FTCM002 has relatively good gastrointestinal tolerance.

[0078] Table 3. Gastrointestinal simulated survival rate (%) of Leuconostoc mesenteroides subsp. enterica FTCM002

[0079]

[0080] Experiment Example 3: Biological Accessibility Experiment

[0081] 3.1 Determination of total saponin bioavailability

[0082] 3.1.1 Sample to be tested: Ginseng water extract from Preparation Example 3

[0083] 3.1.2 Grouping and processing of test samples

[0084] Leuconostoc mesenteroides LM-G27 was purchased from Junyao Runying Biotechnology (Shanghai) Co., Ltd., with a viable count of 100 billion CFU / g; Leuconostoc mesenteroides FTCM002 bacterial powder (Preparation Example 2) had a viable count of 1×10⁻⁶. 11 CFU / g.

[0085] Normal group: 50 mL of sample from Preparation Example 3;

[0086] LM-G27 group: 0.2g of Leuconostoc mesenteroides LM-G27 bacterial powder was added to 50mL of the sample from Preparation Example 3 and stirred evenly to obtain the sample.

[0087] LM-G27 Fermentation Group: 0.2g of Leuconostoc mesenteroides LM-G27 bacterial powder was added to 50mL of sample from Preparation Example 3, and fermented at 37℃ for 48h to obtain fermentation product;

[0088] FTCM002 group: 0.2g of Leuconostoc mesenteroides FTCM002 bacterial powder was added to 50mL of sample from Preparation Example 3 and stirred evenly.

[0089] 3.1.3 Experimental Methods

[0090] Control group: Weigh 2 g of sample, add 3 mL of 0.5 mol / L NaHCO3; add pure water in the same volume as the digestion solution and the reagent used to adjust pH, simulate digestion for 3 h in a constant temperature shaker at 37℃ and 120 r / min in the dark, sonicate at 50℃ for 30 min, centrifuge at 8000 r / min for 10 min, and take the supernatant to obtain the sample to be tested.

[0091] In vitro simulated digestion group: Weigh 2 g of sample, add 2 mL of 1 mmol / L CaCl2 (containing 1 mg / mL α-amylase), and incubate at 37℃ for 5 min. Adjust the pH to 2 with 6 mol / L HCl, add 10 mL of 0.1 mol / L HCl (containing 50 mg / mL pepsin), and simulate digestion for 2 h in a constant temperature shaker at 37℃ and 120 r / min in the dark. Adjust the pH to 7 with 1 mol / L NaHCO3, add 3 mL of 0.5 mol / L NaHCO3 (containing 70 mg / mL porcine bile salts and 25 mg / mL trypsin), and simulate digestion for 3 h in a constant temperature shaker at 37℃ and 120 r / min in the dark. Sonicate at 50℃ for 30 min, centrifuge at 8000 r / min for 10 min, and collect the supernatant to obtain the sample to be tested.

[0092] 3.1.4 Determination method for total saponins

[0093] Take 10 mL of the sample to be tested and determine the total saponin content using the vanillin-glacial acetic acid method. Detect the total saponin content in the sample after simulated digestion.

[0094] The specific method for determining the total saponin content is as follows:

[0095] Take a 2 mL EP tube, pipette 300 µL of sample through a 0.45 µm filter membrane, dilute the sample by an appropriate factor, then pipette 100 µL of the diluted sample into a 10 mL EP tube, and place it in an oven at 60 °C until the solvent evaporates. Add 0.2 mL of 5% vanillin-glacial acetic acid (v:v) and 0.6 mL of perchloric acid (analytical grade) to the EP tube after solvent evaporation, stopper and seal, and incubate at 70 °C for 20 min. After the water bath, remove the EP tube and immediately cool it to room temperature in an ice-water bath, add 5 mL of glacial acetic acid (analytical grade), mix well and develop color. After color development, pipette 200 µL of the reaction solution into an ELISA plate, place it in an ELISA reader, and measure the OD560 nm value at an absorbance wavelength of 560 nm. Substitute the measured value into the standard curve to determine the total saponin content in the sample. Specific results are shown in Table 5.

[0096] Using ginsenoside Re (purchased from Sichuan Jingcui Tiancheng Pharmaceutical Technology Co., Ltd., CAS No.: 52286-59-6, purity 95-99%) as the standard, 12 mg was dissolved in 3 mL of methanol to obtain a ginsenoside Re solution with a concentration of 4 mg / mL. Then, standard sample solutions with concentrations of 0.8-4 mg / mL were prepared. The experimental steps were as described above. The standard concentration was plotted on the x-axis, and the OD value was plotted on the y-axis. The specific linear regression equation is shown in Table 4.

[0097] Table 4 Linear Regression Equations for Standards

[0098]

[0099] Formula for total saponin bioavailability:

[0100] Total saponin bioavailability (%) = (total saponin content in the sample after simulated digestion) / (total saponin content in the sample before simulated digestion) × 100%.

[0101] 3.2 Determination of polysaccharide bioavailability

[0102] 3.2.1 Sample to be tested: Aqueous extract of Dendrobium officinale from Preparation Example 4

[0103] 3.2.2 Grouping and processing of test samples

[0104] Leuconostoc mesenteroides LM-G27 was purchased from Junyao Runying Biotechnology (Shanghai) Co., Ltd., with a viable count of 100 billion CFU / g; Leuconostoc mesenteroides FTCM002 bacterial powder (Preparation Example 2) had a viable count of 1×10⁻⁶. 11 CFU / g.

[0105] Normal group: 50 mL of sample from preparation example 4;

[0106] LM-G27 group: 0.2g of Leuconostoc mesenteroides LM-G27 bacterial powder was added to 50mL of sample from Preparation Example 4 and stirred evenly to obtain the sample.

[0107] FTCM002 group: 0.2g of Leuconostoc mesenteroides FTCM002 bacterial powder was added to 50mL of sample from Preparation Example 4 and stirred evenly to obtain the sample.

[0108] 3.2.3 Experimental Methods

[0109] The test sample was obtained by performing the experimental operation according to method 3.1.3.

[0110] 3.2.4 Determination of polysaccharide content

[0111] The polysaccharide content was determined using the phenol-sulfuric acid method (with glucose as the standard curve): 1 mL of sample was added, followed by 1 mL of 5% phenol in each test tube. 5 mL of concentrated sulfuric acid was then added to each test tube, and the mixture was rapidly shaken to ensure homogeneity. The test tubes were placed in boiling water for 20 min, then cooled to room temperature under running water for 5 min. The OD value of the reaction system was measured at 490 nm. The total sugar content in the solution was calculated based on the standard curve. The specific polysaccharide content results are shown in Table 5. The blank control group used distilled water instead of the standard sugar solution.

[0112] Formula for the bioavailability of polysaccharides:

[0113] Polysaccharide bioavailability (%) = (polysaccharide content in the sample after simulated digestion) / (polysaccharide content in the sample before simulated digestion) × 100%.

[0114] 3.3 Experimental Results

[0115] The results of the bioavailability experiments for total saponins and polysaccharides are shown in Table 5. In in vitro simulated digestion, high bioavailability means that the target substances (saponins and polysaccharides) can be released from the traditional Chinese medicine in a simulated gastrointestinal environment and can be converted into a high proportion of substances that can be absorbed by intestinal epithelial cells.

[0116] Table 5 shows the bioavailability of Dendrobium officinale polysaccharides and total ginsenosides in the normal group, LM-G27 group, LM-G27 fermented group, and FTCM002 group. The results in Table 5 show that the bioavailability of total saponins in the FTCM002 group was 20.44%, which was 33% higher than the normal group and 16% higher than the unfermented LM-G27 group. The bioavailability of polysaccharides in the unfermented FTCM002 group was 36.21%, which was 120% higher than the normal group and 89% higher than the unfermented LM-G27 group. This indicates that the Leuconostoc mesenteroides subsp. mesenteroides FTCM002 powder can improve the bioavailability of total ginsenosides and Dendrobium officinale polysaccharides.

[0117] In this experiment, the simultaneous administration of the mycelial powder and the water extract of Dendrobium officinale polysaccharides (ginsenoside water extract) was merely a simple physical mixing; the active ingredients in the water extract remained naturally present in large molecular form. However, traditional Chinese medicine fermentation technology, through the metabolic fermentation of microorganisms, transforms large molecules in the water extract that are difficult for the body to absorb into smaller molecules. The fermentation products are enriched with a large number of small-molecule active components such as oligosaccharides, secondary saponins, and free phenols. In other words, before ingestion, the fermentation system has already completed component degradation and structural optimization, making it easier for small molecules to penetrate the intestinal mucosa and be absorbed and utilized by the body. Therefore, the absorption and utilization rate of the fermentation products is significantly better than that of the group where the mycelial powder and water extract were mixed.

[0118] While fermentation of the bacterial strain's water extract can effectively promote the absorption of active ingredients, this does not mean that direct administration of the bacterial powder and water extract together will have the same absorption-promoting effect. This is because the enzymatic metabolism and biotransformation processes of probiotics require specific conditions such as suitable temperature, pH, reaction time, and liquid matrix. When both are taken orally, the bacterial strain is rapidly subjected to the dual destruction and stress of gastric acid and digestive enzymes. This inhibits bacterial metabolism, and key enzyme systems such as extracellular hydrolases and glycosidases cannot be effectively synthesized and function, making it difficult to degrade and transform large molecular components in vivo. Therefore, the active ingredients cannot be modified into smaller molecules, and the absorption-promoting effect of in vitro fermentation cannot be achieved. This point was also confirmed in the unfermented and fermented groups of LM-G27 in this invention.

[0119] Compared to the normal group, the total saponin and polysaccharide bioavailability of the *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 group provided by this invention was significantly higher, indicating that simultaneous administration of *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 and *Dendrobium officinale* polysaccharide water extract (ginsenoside water extract) can also improve the bioavailability of *Dendrobium officinale* polysaccharide or ginsenosides, and the bioavailability of saponins and polysaccharides is not significantly different from that of the LM-G27 fermentation group. This shows that *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 can not only secrete functional enzymes such as glycoside hydrolases in the intestine to achieve in vivo degradation and structural transformation of various macromolecular active substances such as ginsenosides and *Dendrobium officinale* polysaccharides, but also optimize the intestinal mucosal barrier function and enhance the intestinal permeability and absorption capacity of plant-derived functional components; at the same time, the strain has excellent resistance to gastrointestinal stress and can stably exert its metabolic effects in the intestine.

[0120] Table 5 Results of the biological accessibility experiment

[0121]

[0122] Note: Compared with the normal group, * represents P<0.05, ** represents P<0.01, and *** represents P<0.001.

[0123] Experiment Example 4: Animal Experiment to Promote Ginsenoside Absorption

[0124] 4.1 Sample to be tested:

[0125] Normal group: 50 mL of solution from Example 3;

[0126] FTCM002 group: 0.2g of Leuconostoc mesenteroides FTCM002 bacterial powder and 50mL of the solution prepared in Example 3.

[0127] 4.2 Experimental Procedure

[0128] SPF-grade SD rats, half male and half female, 8-10 weeks old, weighing 200-250 g.

[0129] After 7 days of acclimatization, rats were randomly divided into two groups: a normal group and an FTCM002 group, with 8 rats in each group. Blank serum was collected from all animals after anesthesia with 3% sodium pentobarbital. Once the animals were awake, each group of rats was administered 4.5 mL / kg of the corresponding test sample via gavage. At 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h after administration, 0.5 mL of blood was collected from the fundus venous plexus of the rats, placed in EP tubes, incubated at 4℃ for 1 h, centrifuged at 2000 r / min for 10 min, and the supernatant serum was collected.

[0130] The method for calculating the amount of sample to be administered by gavage was as follows: 50 mL of the prepared solution (Example 3) was administered to humans daily, along with 0.2 g of Leuconostoc mesenteroides FTCM002 bacterial powder daily. Before gavage, 0.2 g of Leuconostoc mesenteroides FTCM002 bacterial powder was added to 50 mL of the prepared solution (Example 3) and stirred thoroughly. The gavage dose for rats was calculated to be 4.5 mL / kg according to the equivalent dose conversion method based on body surface area ratio, and this dose was administered by gavage.

[0131] 4.3 Detection of saponin content in serum

[0132] 4.3.1 The serum pretreatment method is as follows: Accurately pipette 200 μL of serum sample into a 1.5 mL EP tube, add 500 μL of acetonitrile, vortex mix for 3 min, centrifuge the suspension at 4 °C and 12000 r / min for 5 min, collect 500 μL of supernatant, blow dry with nitrogen, reconstitute the residue with 200 μL of 50% methanol, vortex mix again for 5 min, centrifuge at 4 °C and 12000 r / min for 5 min, and collect the supernatant, which is used to determine the saponin content.

[0133] 4.3.2 Determination of Ginsenoside Content

[0134] The saponin content was determined by chromatographic methods. The pharmaceutical concentration of each component was calculated using the standard curve method. The corresponding pharmacokinetic parameters of ginsenosides Rb1 and Rb2 in rats were then calculated, including peak concentration (Cmax) and area under the curve (AUC). 0→∞ )

[0135] The chromatographic column conditions were: Waters XBridge C 18 Column (250 mm × 4.6 mm, 5 μm), mobile phase: acetonitrile (A) - water (B), flow rate 1.0 mL / min; elution method: gradient elution, elution program shown in Table 6; column temperature: 30 °C; injection volume: 10 μL. Nebulizer gas: nitrogen (99.99% purity); nebulization temperature: 35 °C; power ratio: 1.0; sampling frequency: 5 Hz; filtration constant: 3.6 s.

[0136] Table 6 Mobile Phase Elution Procedure

[0137]

[0138] Standard curve method:

[0139] Standards: Ginsenoside Rb1, Ginsenoside Rb2

[0140] Ginsenoside Rb1 (CAS No.: 41753-43-9, purity 95-99%) and ginsenoside Rb2 (CAS No.: 11021-13-9, purity 95-99%) were both purchased from Sichuan Jingcui Tiancheng Pharmaceutical Technology Co., Ltd. Acetonitrile was mass spectrometry grade, methanol was chromatographic grade, and water was ultrapure water.

[0141] Mixed reference solution: Accurately weigh appropriate amounts of each reference standard and place them in the same 100 mL volumetric flask. Dissolve in methanol to prepare a mixed reference stock solution containing ginsenosides Rb1 and Rb2 at concentrations of 137.7 μg / mL and 6.1 μg / mL, respectively. Accurately measure 5 mL, 2.5 mL, 1.25 mL, 0.5 mL, 0.35 mL, and 0.25 mL, and place them in separate 5 mL volumetric flasks. Dilute to volume with methanol and shake well to obtain a series of mixed reference solutions with varying concentrations.

[0142] Standard curve preparation: Accurately weigh 200 μL of the mixed reference solution and add 200 μL of blank serum. Under the above chromatographic conditions, detect the saponin content. Plot the standard curve with the mass concentration of saponin on the x-axis and the peak area on the y-axis. The regression equation for the reference standard is shown in Table 7.

[0143] Table 7 Standard curves of individual ginsenosides

[0144]

[0145] Note: Y is the peak area, and X is the concentration of the reference standard (μg / mL).

[0146] 4.4 Experimental Results

[0147] Table 8 Pharmacokinetic parameters of ginsenosides (mean ± standard deviation)

[0148]

[0149] The plasma concentrations of each group of rats are shown in the figure. Figure 1 See Table 8. Figure 1 The table shows the blood concentration-time curves for ginsenosides Rb1 and Rb2, and Table 8 shows the pharmacokinetic parameters of ginsenosides. max This reflects the highest concentration that a substance can reach in the body. The area under the curve representing the change in blood drug concentration over time from the start of drug administration until the substance is completely eliminated from the body reflects the total amount of substance absorbed into the bloodstream. Both are closely related to the substance absorption process.

[0150] Depend on Figure 1 As shown in Table 8, the concentrations of ginsenosides Rb1 and Rb2 in the plasma of rats in the FTCM002 group were higher than those in the normal group, indicating that, as verified by in vivo rat experiments, *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 can better promote the absorption of saponins in rats. The concentration of ginsenoside Rb1 in the plasma of rats administered *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 powder by gavage reached its maximum at 2 hours, with a value of 34.56 μg / mL, which is 1.97 times that of the normal group; the concentration of ginsenoside Rb2 in the plasma reached its maximum at 4 hours, with a value of 4.52 μg / mL, which is 2.13 times that of the normal group; and the AUC of ginsenosides Rb1 and Rb2 in the plasma of rats in the FTCM002 group was also higher. 0→∞ The values ​​were all higher than those in the normal group, indicating that the combined administration of Leuconostoc mesenteroides FTCM002 bacterial powder and ginseng water extract resulted in a higher peak concentration in vivo compared to administration of ginseng water extract alone, and a greater overall exposure of ginsenosides in the blood. This suggests that the absorption rate of Leuconostoc mesenteroides FTCM002 bacterial powder and ginseng water extract was faster and the absorption degree was higher, meaning that Leuconostoc mesenteroides FTCM002 bacterial powder can promote the absorption of saponins Rb1 and Rb2 in rats.

[0151] Experiment Example 5: Calcium Ion Absorption Experiment

[0152] 5.1 Preparation of Leuconostoc mesenteroides subsp. enterica FTCM002 bacterial powder

[0153] Refer to Preparation Example 2.

[0154] 5.2 Culture of human colon cells Caco-2

[0155] Caco-2 cells were activated in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin, and then cultured at 37°C and 5% CO2. After the cells reached 80%–90% confluence, they were passaged or plated.

[0156] 5.3 Experiment on promoting calcium ion transport in Caco-2 cells

[0157] When the Caco-2 cells reached 80% confluence, the cells were collected and the concentration was adjusted to 1×10⁻⁶ cells using DMEM medium. 5 Cells / mL. Add 0.5 mL of cell suspension to the upper chamber of a Transwell plate and 1.5 mL of DMEM medium to the lower chamber. Incubate at 37°C and 5% CO2, changing the medium every other day. Measure the transmembrane resistance (TEER) of Caco-2 cells daily. When the TEER value is greater than 300 Ω / cm... 2 It can be used immediately for calcium ion transport experiments. A 2 mmol / L CaCl2 solution was prepared using D-Hanks solution (Beijing Solarbio), and 200 μL of CaCl2 solution was added to each well of the upper chamber of a Transwell plate. For the experimental group, 200 μg of FTCM002 bacterial powder was added to the upper chamber of the Transwell plate, while the control group received an equal volume of DMEM medium. The plates were incubated at 37℃ and 5% CO2 for 3 h, and the concentration of calcium ions in the lower chamber liquid was detected using a calcium ion assay kit. The calculation formula is as follows:

[0158] Relative growth rate = (Calcium ion concentration in experimental group / Calcium ion concentration in control group) × 100%

[0159] 5.4 Experimental Analysis

[0160] Compared with the control group, there was no significant difference in the relative growth rate of calcium ions in the FTCM002 group, indicating that Leuconostoc mesenteroides subsp. mesenteroides FTCM002 cannot promote the absorption of calcium ions.

[0161] In summary, in vitro experiments have shown that *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 can improve the bioavailability of total ginsenosides and *Dendrobium officinale* polysaccharides. Animal experiments have shown that *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 can promote the absorption of ginsenosides Rb1 and Rb2.

[0162] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. Application of Leuconostoc mesenteroides subsp. mesenteroides or its culture in the preparation of products that promote the absorption of ingredients.

2. The application according to claim 1, characterized in that, The Leuconostoc mesenteroides subsp. mesenteroides is Leuconostoc mesenteroides subsp. mesenteroides ( Leuconostoc mesenteroides subsp. mesenteroides FTCM002, with accession number CGMCC No.35016.

3. The application according to claim 1, characterized in that, Including any one or more of the following applications: (1) Promotes the absorption of ginsenosides; (2) Promotes the absorption of Dendrobium officinale polysaccharides.

4. The application according to claim 3, characterized in that, The ginsenosides include one or more of saponins Rb1 and Rb2.

5. The application according to claim 2, characterized in that, The culture of Leuconostoc mesenteroides subsp. FTCM002 was obtained by inoculating Leuconostoc mesenteroides subsp. FTCM002 into a culture medium.

6. The application according to claim 2, characterized in that, The viable count of Leuconostoc mesenteroides subsp. enterica FTCM002 in the product is not less than 1×10⁻⁶. 11 CFU / g or 1×10 11 CFU / mL.

7. The application according to claim 2, characterized in that, The product is selected from one or more of the following: pharmaceuticals, functional foods, health products, microbial agents, and pharmaceutical raw materials.

8. The application according to claim 2, characterized in that, The dosage form of the product is selected from one or more of the following: lyophilized powder, tablets, sprays, and granules.

9. A product that promotes the absorption of ginsenosides and / or Dendrobium officinale polysaccharides, characterized in that, Including a viable count of not less than 1×10 11 CFU / g or 1×10 11 CFU / mL Leuconostoc mesenteroides subsp. FTCM002.

10. The product according to claim 9, characterized in that, It also includes excipients to maintain the activity of the strain.

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