A compound microbial agent that promotes calcium absorption and transport

Abstract

This invention discloses a compound microbial agent that promotes calcium absorption and transport. By constructing a cell model of calcium absorption and transport, it is demonstrated that this compound microbial agent can promote calcium absorption and transport, and can be used to improve calcium utilization and alleviate osteoporosis. Probiotics primarily enhance the body's active absorption of natural calcium from food by improving the intestinal microenvironment. This is a slow, stable, and physiologically regulated quantitative absorption process that does not cause excessive fluctuations in blood calcium ion concentration, thus reducing the pathological risks of vascular calcification and gallstones.

Description

Technical Field

[0001] This invention relates to a compound microbial agent that promotes calcium absorption and transport. It belongs to the field of microbial fermentation technology. Background Technology

[0002] Calcium is a massive essential element that the human body cannot function normally. It is widely involved in core physiological areas including blood clotting, muscle contraction, bone development, and nerve signal transmission. When the body is deficient in calcium, it not only directly leads to osteoporosis, but also over-activates osteoclasts, thereby accelerating bone resorption and worsening bone loss.

[0003] Clinically, existing methods for improving calcium deficiency primarily rely on administering calcium supplements to increase calcium uptake. Common supplements include calcium carbonate, calcium oxide, calcium phosphate, and calcium lactate gluconate. However, these conventional calcium supplements generally suffer from low bioavailability, meaning that free calcium cannot be effectively absorbed and utilized in the intestines. Furthermore, long-term or high-dose intake can easily lead to systemic side effects such as hypercalcemia or hypercalciuria, significantly increasing the pathological risk of calcium-containing kidney stones. Therefore, researchers in this field are dedicated to developing methods that can effectively improve calcium absorption without producing undesirable side effects. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a compound microbial agent that promotes calcium absorption and calcium transport.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A compound microbial agent that promotes calcium absorption and transport is made from *Lactobacillus fermentum* LF553 and *Leuconostoc membranaceus* subsp. *enteromorpha* LEM989. The Latin name of *Lactobacillus fermentum* LF553 is... Limosilactobacillus fermentum It was deposited on April 20, 2026 at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNO.38315; The Latin name of *Leuconostoc membranaceus* subsp. *enteromorpha* LEM989 is... Leuconostoc mesenteroides subsp. mesenteroidesIt was deposited on April 17, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.38314.

[0006] Preferably, the compound bacterial agent is obtained by mixing Lactobacillus fermentum LF553 and Leuconostoc membranaceus subsp. LEM989 in a CFU ratio of 3:2.

[0007] More preferably, in the compound microbial agent, the viable bacterial concentrations of *Lactobacillus fermentum* LF553 and *Leuconostoc membranaceus* subsp. *enteromorpha* LEM989 are respectively: (A) Promotes calcium absorption: 2.25 × 10 5 CFU / mL and 1.50×10 5 CFU / mL; (B) Promotes calcium transport: 6.00 × 10 5 CFU / mL and 4.00×10 5 CFU / mL.

[0008] The aforementioned compound microbial agent is used in the preparation of drugs that promote calcium absorption and calcium transport.

[0009] The aforementioned compound microbial agent is used in the preparation of osteoporosis treatment drugs.

[0010] The beneficial effects of this invention are: This invention discloses a compound microbial agent that promotes calcium absorption and transport. By constructing a cell model of calcium absorption and transport, it was demonstrated that this compound microbial agent, at a 3:2 ratio, effectively promotes calcium absorption and transport, and can be used to improve calcium utilization and alleviate osteoporosis. Probiotics primarily enhance the body's active absorption of natural calcium from food by improving the intestinal microenvironment. This is a slow, stable, and physiologically regulated quantitative absorption process that does not cause excessive fluctuations in blood calcium ion concentration, thus reducing the pathological risks of vascular calcification and gallstones. Attached Figure Description

[0011] Figure 1 Cellular model experiments on calcium absorption and calcium transport; (A) Results of the calcium uptake cell model experiment. Caco-2 cells were cultured into a monolayer, and calcium chloride solution and potential strains were added. After 24 hours, Caco-2 cell lysates were collected, and the calcium ion concentration was measured using a commercially available calcium ion kit. The NC group did not contain calcium chloride solution or potential strains, the Ca group only contained calcium chloride solution and no potential strains, and the other groups contained potential strains and calcium chloride solution. The calcium content in the cells was standardized to the total protein concentration, and the values ​​of each group were standardized to the values ​​of the Ca group. The values ​​were presented as ratios, with the values ​​representing Mean ± SD. (B) Results of the calcium transport cell model experiment. Caco-2 cells were cultured into a monolayer, and calcium chloride solution and potential strains were added. After 24 hours, the cell culture medium penetrating below the Caco-2 cell layer was collected, and the calcium ion concentration was measured using a commercially available calcium ion kit. The NC group received no calcium chloride solution or potential strains; the Ca group received only calcium chloride solution, without any potential strains; the other groups received both potential strains and calcium chloride solution. Values ​​for each group were standardized to those of the Ca group and presented as ratios, with values ​​expressed as Mean ± SD.

[0012] Figure 2 Results of the calcium uptake cell model experiment. Caco-2 cells were cultured into a monolayer, and calcium chloride solution and potential bacterial strains were added. After 24 hours, Caco-2 cell lysates were collected, and calcium ion concentrations were measured using a commercially available calcium ion kit. The NC group received no calcium chloride solution or potential bacterial strains, the Ca group received only calcium chloride solution without potential bacterial strains, and the other groups received both potential bacterial strains and calcium chloride solution. Cellular calcium content values ​​were standardized to total protein concentration, and the values ​​for each group were standardized to those for the Ca group. Values ​​were presented as ratios, with mean ± SD. Detailed Implementation

[0013] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its content.

[0014] 1. Materials and Methods

[0015] (1) Culture of strains

[0016] LF553, LEM984 (Leuconostoc membranaceus subsp. enterica, purchased from Biostime Biotechnology (Yangzhou) Co., Ltd.), and LEM989 were inoculated into MRS (Becton, Dickinson and Company, catalog number 288130) and cultured at 37°C for 16 hours. This inoculation-culture step was then repeated twice to activate LF553, LEM984, and LEM989. The bacterial culture was then centrifuged at 4,695 g for 5 minutes, the supernatant was removed, and the cells were washed three times with 1X PBS (UniRegion Bio-Tech, catalog number UR-PBS001-5L). The cells were then dispersed with an appropriate amount of PBS, and the OD600nm absorbance was adjusted to 1.00-1.04, at which point the bacterial concentration was approximately 1×10⁻⁶. 9 CFU / mL.

[0017] (2) Calcium absorption experiment

[0018] Referencing Raveschota et al. and making minor adjustments [1] 4 × 10 4 Caco-2 cells (BCRC60182, purchased from the Bioresource Conservation and Research Center of the Food Industry Research Institute, Taiwan) were seeded in 24-well plates and cultured in DMEM medium (Gibco™, #10569010) [containing 10% FBS (fetal bovine serum, Gibco™, A52567-01) and 1% penicillin-streptomycin-amphoteric acid B mixture (Biological Industries, 03-033-1B)] for 21 days before calcium absorption experiments were performed.

[0019] The experimental group was prepared by adding 800 μL of DMEM medium (Gibco™, #10569010) to Caco-2 cells, containing 2% FBS and a total bacterial count of 3 × 10⁻⁶ cells / mL. 5 CFU bacterial suspension and 4 mM calcium chloride solution (Sigma Aldrich, C7902). The ratio of the two bacterial strains added was 1:1, therefore the concentration of each strain was 1.5 × 10⁻⁶. 5 CFU / well, the ratio of the two strains added was 3:2, therefore the concentrations of the two strains were 1.8 × 10⁻⁶. 5 CFU / well (LF553) and 1.2×10 5 CFU / well (LEM984 or LEM989), after conversion, the concentrations of the two strains are 2.25 × 10⁻⁶. 5 CFU / mL and 1.50×10 5CFU / mL.

[0020] In addition, the control group consisted of Caco-2 cells supplemented with 800 μL of DMEM medium (Gibco™, #10569010) containing 2% FBS and 4 mM calcium chloride solution (Sigma Aldrich, C7902).

[0021] After 24 hours of cell treatment, cells were washed twice with PBS, and then lysed with 200 μL of CelLytic™ M cell lysis reagent (Sigma Aldrich, C2978). The cells were lysed by shaking at room temperature for 10–15 minutes, followed by centrifugation at 13,000 g at 4°C for 15 minutes. The supernatant was collected. Calcium content in cells was measured using a calcium ion detection kit (Abcam, ab102505), following the kit's standard operating procedure. Protein concentration was simultaneously detected using a protein analysis dye concentration reagent (Bio-Rad, #5000006). Cellular calcium content was normalized to total protein concentration, and the relative fold increase in calcium ion uptake was calculated using the following formula: A = B / C; Where: A = relative multiple of calcium ion absorption; B = calcium ion concentration measured in each group; C = calcium ion concentration measured in the control group; (3) Calcium transport test Referencing Raveschota et al. and making minor adjustments [1] Caco-2 cells were cultured in DMEM medium (Gibco™, #10569010) [containing 10% FBS (Gibco™, A52567-01) and 1% penicillin-streptomycin-amphotericidal B mixture (Biological Industries, 03-033-1B)]. 4 × 10⁶ cells were cultured at a concentration of [missing information]. 4 400 μL of Caco-2 cells were seeded in a cell culture chamber, placed in a 24-well plate, and 700 μL of DMEM medium was placed outside the chamber for 21 days of growth. The Caco-2 cells were washed twice with PBS, and the bacterial concentration was adjusted to 1 × 10⁻⁶ cells / well. 6 CFU / mL was dissolved in DMEM (containing 2% FBS by mass). The experimental group consisted of 300 μL of DMEM medium containing 2% FBS by mass and a total bacterial count of 3 × 10⁻⁶. 5 The bacterial culture at CFU / well and 4 mM calcium chloride solution (Sigma Aldrich, C7902) were added to the chamber, with the ratio of the two bacterial strains being 3:2. Therefore, the concentrations of the two strains were 1.8 × 10⁻⁶.5 CFU / well and 1.2×10 5 CFU / well, after conversion, the concentrations of the two strains are 6.00 × 10⁻⁶. 5 CFU / mL and 4.00×10 5 CFU / mL.

[0022] In addition, the control group consisted of 300 μL of DMEM medium containing 2% FBS and 4 mM calcium chloride solution. The small room was placed with 600 μL of DMEM medium (containing 2% FBS).

[0023] After culturing at 37°C and 5% CO2 for 24 hours, the culture medium outside the cell was removed. The calcium concentration of the culture medium sample was measured using a calcium ion detection kit (Abcam, ab102505), following the kit's standard operating procedure. The relative fold of calcium ion transport was calculated by substituting the measured calcium ion concentration into the following formula: A = B / C Where: A = relative fold of calcium ion transport; B = calcium ion concentration measured in the experimental group; C = calcium ion concentration measured in the control group;

[0024] 2 Results

[0025] A calcium absorption cell model was established by culturing Caco-2 cells into a monolayer, adding calcium chloride solution and potential bacterial strains, and then measuring the intracellular calcium ion concentration in Caco-2 cells. Cells with higher calcium ion concentrations were considered to have the ability to promote calcium absorption in Caco-2 cells.

[0026] In the calcium transport cell model, Caco-2 cells were cultured to form a monolayer, and calcium chloride solution and potential strains were added. Then, the cell culture medium that penetrated below the Caco-2 cell layer was collected, and the calcium ion concentration was measured. Cells with higher calcium ion concentrations were considered to have the ability to promote calcium transport in Caco-2 cells.

[0027] The experimental results are shown in Figure 1 Calcium absorption and transport experiments were conducted using the LF553+LEM984 and LF553+LEM989 combinations at a ratio of 3:2. The calcium ion concentration of the LF553+LEM989 combination was higher than that of the single strain and the LF553+LEM984 combination.

[0028] The experimental results are shown in Figure 2 Calcium absorption experiments were conducted on the LF553+LEM989 combination at ratios of 1:1 and 3:2, respectively. The calcium ion concentration of the LF553+LEM989 combination at the 3:2 ratio was higher than that of the single strain and the LF553+LEM989 combination at the 1:1 ratio.

[0029] Therefore, it can be concluded that LF553+LEM989 in a 3:2 ratio has a good effect on calcium absorption and transport, and has a good calcium utilization rate, which can be used to improve osteoporosis.

[0030] While the specific embodiments of the present invention have been described above in conjunction with the accompanying drawings, this is not intended to limit the scope of protection of the present invention. Based on the technical solutions of the present invention, various modifications or variations that can be made by those skilled in the art without creative effort are still within the scope of protection of the present invention.

[0031] References

[0032] 【1】Raveschot, C., Coutte, F., Frémont, M., Vaeremans, M., Dugersuren,J., Demberel, S., ... & Cudennec, B. (2020). Probiotic Lactobacillus strains from Mongolia improve calcium transport and uptake by intestinal cells invitro. Food Research International, 133, 109201.

Claims

1. A compound microbial agent that promotes calcium absorption and transport, characterized in that, It is made from *Lactobacillus fermentum* LF553 and *Leuconostoc membranaceus* subsp. *enteromorpha* LEM989. The Latin name of *Lactobacillus fermentum* LF553 is... Limosilactobacillus fermentum It was deposited on April 20, 2026 at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNO.38315; The Latin name of *Leuconostoc membranaceus* subsp. *enteromorpha* LEM989 is... Leuconostoc mesenteroides subsp. mesenteroides It was deposited on April 17, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.38314.

2. The compound microbial agent according to claim 1, characterized in that, The compound bacterial agent is prepared by mixing Lactobacillus fermentum LF553 and Leuconostoc membranaceus subsp. LEM989 in a CFU ratio of 3:

2.

3. The compound microbial agent according to claim 2, characterized in that, In the compound microbial agent, the viable bacterial concentrations of *Lactobacillus fermentum* LF553 and *Leuconostoc membranaceus* subsp. *enteromorpha* LEM989 are respectively: (A) Promotes calcium absorption: 2.25 × 10 5 CFU / mL and 1.50×10 5 CFU / mL; (B) Promotes calcium transport: 6.00 × 10 5 CFU / mL and 4.00×10 5 CFU / mL.

4. The use of the compound microbial agent according to claim 1 in the preparation of a drug that promotes calcium absorption and calcium transport.

5. The use of the compound microbial agent according to claim 1 in the preparation of osteoporosis treatment drugs.

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