A probiotic with the functions of enriching calcium ions and synthesizing vitamin k, a probiotic composition and application thereof

Abstract

This invention provides a probiotic, a probiotic composition, and their applications, all possessing the functions of calcium ion enrichment and vitamin K synthesis. This invention belongs to the field of microbial technology. The probiotic provided by this invention is *Lactobacillus bulgaricus* subsp. Lactobacillus delbrueckii subsp. bulgaricus NWP-101 or Pyrococcus lactis ( Pediococcus acidilactici NWP-299 has the advantages of efficient calcium enrichment, high vitamin K production and excellent probiotic properties. It can be used to prepare probiotic preparations, functional foods, health foods or dietary supplements to prevent or improve osteoporosis and increase bone density.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a probiotic with the function of enriching calcium ions and synthesizing vitamin K, a probiotic composition, and its application. Background Technology

[0002] Calcium is a major mineral element that makes up human bones and teeth, and it is also essential for maintaining physiological functions such as nerve conduction, muscle contraction, and blood clotting. Insufficient calcium intake or malabsorption is a significant risk factor for bone loss, bone microstructure damage, increased bone fragility, and ultimately osteoporosis. Osteoporosis has become a global public health problem, posing a serious threat, especially to postmenopausal women and the elderly. Currently, common calcium supplements on the market are mainly divided into inorganic calcium (such as calcium carbonate and calcium phosphate) and organic calcium (such as calcium citrate, calcium gluconate, and calcium lactate). However, these traditional calcium sources all have their limitations: 1. Limited absorption rate: Although inorganic calcium has a high elemental calcium content, it has poor water solubility and requires the participation of gastric acid to dissociate calcium ions. Its absorption process is easily affected by factors such as gastric acid secretion levels, phytic acid, and oxalic acid, resulting in generally low bioavailability. Although organic calcium has better water solubility, its elemental calcium content is relatively low, and the improvement in absorption rate is limited. 2. Gastrointestinal side effects: Some people may experience gastrointestinal discomfort such as constipation, bloating, and nausea after taking inorganic calcium supplements like calcium carbonate, affecting long-term adherence. 3. Lack of synergistic effect: Traditional calcium supplements only provide elemental calcium and fail to simultaneously provide synergistic factors that promote calcium absorption and bone formation (such as vitamin K2 and vitamin D), resulting in a limited effect on improving bone health.

[0003] In recent years, the technology of enriching trace elements and preparing "biocalcium" using microorganisms has gradually emerged. Some microorganisms can absorb, transform, and integrate inorganic calcium ions from the environment into their cells or on their surface, forming organic "biocalcium." Theoretically, this biocalcium has better solubility, biocompatibility, and intestinal absorption efficiency. Furthermore, some probiotics themselves have the ability to produce bone-forming active substances such as vitamin K2 and short-chain fatty acids, demonstrating superior comprehensive application potential compared to traditional calcium supplements. For example, Chinese invention CN114391577A-High-Calcium Flavored Fermented Milk and its Preparation Method provides a *Lactobacillus delbrueckii* subspecies LB-81, which has the function of converting calcium carbonate into soluble calcium lactate, making the calcium in fermented milk more easily absorbed by the human body; however, it does not have the ability to produce vitamin K. Another Chinese invention, CN119999768A, describes a low-temperature fermented milk rich in vitamin K2, its preparation method, and its application. This invention records that adding *Lactobacillus delbrueckii* subsp. bulgaricus significantly affects the vitamin K2 content produced by *Lactococcus lactis* subsp. MO-3 fermentation, leading to a significant decrease in vitamin K2 content in the fermented milk. This demonstrates that different microorganisms exhibit differences in the conversion of calcium and vitamin K. Furthermore, the existing technology faces the following pressing problems: not all microorganisms capable of accumulating calcium are safe for human use; and there is a lack of high-performance, high-safety calcium-enriching microbial strains designed specifically for improving bone health and screened using precise quantitative methods. Therefore, providing a strain with the ability to accumulate calcium ions or synergistically promote calcium absorption (such as through vitamin K production) is a problem urgently needing to be solved in this field. Summary of the Invention

[0004] The purpose of this invention is to provide a probiotic and its composition that have the functions of enriching calcium ions and synthesizing vitamin K, thereby solving the problems of low absorption rate, obvious side effects and lack of highly efficient calcium-enriched probiotic formulas in the prior art.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a probiotic with the function of enriching calcium ions and synthesizing vitamin K, wherein the probiotic is *Lactobacillus bulgaricus* subsp. (Lactobacillus bulgaricus) Lactobacillus delbrueckii subsp. bulgaricus NWP-101 or Pyrococcus lactis ( Pediococcus acidilactici It consists of NWP-299.

[0006] Preferably, the Lactobacillus bulgaricus subsp. ( Lactobacillus delbrueckii subsp. bulgaricus NWP-101 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on November 10, 2025, with accession number GDMCC No:67256.

[0007] Preferably, the lactic acid cocci ( Pediococcus acidilactici NWP-299 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on November 10, 2025, with accession number GDMCC No:67257.

[0008] The present invention also provides a composition of the probiotics, wherein the probiotic composition is one or both of Lactobacillus bulgaricus subsp. NWP-101 and Pediococcus lactis NWP-299.

[0009] Preferably, when the probiotic composition consists of Lactobacillus bulgaricus subsp. NWP-101 and Pediococcus lactis NWP-299, they are compounded at a live bacteria ratio of 0.5~1.5:0.5~1.5.

[0010] The present invention also provides the use of the probiotic composition described herein in the preparation of products for preventing or improving osteoporosis and increasing bone density.

[0011] Preferably, the product is a probiotic preparation, a functional food, or a dietary supplement.

[0012] Beneficial effects: The *Lactobacillus bulgaricus* subsp. *bulgaricus* NWP-101 and *Pediococcus lactis* NWP-299 strains screened in this application exhibit outstanding calcium accumulation and vitamin K synthesis capabilities. Furthermore, the combination of these two strains significantly enhances both calcium accumulation and vitamin K synthesis capabilities, demonstrating a clear synergistic effect. These strains also exhibit high acid and bile salt tolerance and survival rates, strong antioxidant and antibacterial abilities, good gastrointestinal colonization and tolerance, high biosafety, and no gastrointestinal irritation. They can be directly used in the development of probiotic preparations, functional foods, health foods, dietary supplements, and pharmaceutical products. The process is mature, highly applicable, and has broad market and clinical application prospects. Attached Figure Description

[0013] Figure 1 This is a graph showing the calcium ion enrichment results of the strain in Example 1; Figure 2 This is a graph showing the vitamin K production results of the strain in Example 1; Figure 3 The graph shows the growth curve and pH change of the strain in Example 1.

[0014] Preservation Instructions

[0015] Lactobacillus bulgaricus subsp. ( Lactobacillus delbrueckii subsp. bulgaricusNWP-101, this strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on November 10, 2025, with accession number GDMCC No:67256.

[0016] Pediococcus acidilactici ( Pediococcus acidilactici NWP-299, this strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on November 10, 2025, with accession number GDMCC No:67257. Detailed Implementation

[0017] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0018] Example 1: Screening and Identification of Strains

[0019] 1. Experimental materials and solution preparation

[0020] (1) Culture medium formulation: MRS medium: 10.0 g peptone, 5.0 g beef extract, 5.0 g yeast extract, 20.0 g glucose, 5.0 g sodium acetate, 2.0 g diammonium citrate, 1.0 ml Tween-80, 2.0 g dipotassium hydrogen phosphate, 0.58 g magnesium sulfate, 0.25 g manganese sulfate monohydrate, 15.0 g agar (for solid media only) and 1.0 L distilled water.

[0021] LB solid medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, 15g agar powder, and 1L distilled water.

[0022] (2) Calcium chloride solution: Weigh 1.2g of anhydrous calcium chloride and dissolve it in 1L of distilled water to prepare a 1.2mg / ml calcium chloride solution, and sterilize it for later use.

[0023] (3) 0.01 mol / L EDTA solution: Accurately weigh an appropriate amount of disodium ethylenediaminetetraacetate, add about 800 ml of distilled water, heat to 70 °C to accelerate dissolution, stir until completely dissolved, cool to room temperature, add deionized water to 1 L, and shake well. A 0.01 mol / L EDTA solution is obtained.

[0024] 2. Strain activation and culture

[0025] (1) NWP-101 and NWP-299 glycerol bacteria were streaked on plates and incubated at 37°C for 24 h. Single colonies were picked and placed in 1 ml of MRS liquid medium and activated at 37°C for 24 h to obtain the first generation bacterial culture.

[0026] (2) Inoculate the first generation bacterial culture into 5 ml of MRS liquid medium at an inoculation rate of 2% (volume ratio) and incubate at 37°C for 24 h to prepare the second generation bacterial culture.

[0027] 3. Determination of calcium ion enrichment capacity

[0028] (1) Take the activated second-generation bacterial solution and divide it into NWP-101 group, NWP-299 group and NWP-101+NWP-299 group (mix NWP-101 and NWP-299 at a mass ratio of 1:1), centrifuge at 10000r / min for 5min to collect the bacterial cells, and wash twice with sterile PBS.

[0029] (2) The bacterial body was suspended in a sterile calcium chloride solution of 1.2 mg / ml and incubated at 37°C for 3 hours.

[0030] (3) After incubation, centrifuge at 10000 r / min for 5 min and collect the supernatant and bacterial cells respectively. Transfer the bacterial cells to sterile filter paper to absorb the moisture, place them in an oven at 40℃ for 2 h, and then dry them in an oven at 105℃ until constant weight (the difference between two consecutive weighings is <0.001 g), and weigh the dry weight of the bacterial cells.

[0031] (4) Determining the concentration of residual calcium ions in the supernatant using EDTA titration: Take the supernatant and adjust its pH to greater than 12.5 using NaOH (to eliminate interference from magnesium ions). Add calcium red indicator, and the solution turns wine red. Titrate with 0.01 mol / L EDTA standard solution until the solution turns pure blue and does not fade for 30 seconds, and record the volume of EDTA consumed, V. Use distilled water as a blank control and LGG as a positive control, and record the volume of EDTA consumed, V0. The results are as follows: Figure 1 As shown.

[0032] (5) Calculation: The calcium ion content (mg) in the supernatant is (V - V0) × C(EDTA) × M(Ca), where M(Ca) = 40 g / mol.

[0033] Calcium enrichment (mg / g) = (Total amount of calcium ions added - Calcium ion content in supernatant) / Dry weight of bacterial cells.

[0034] 4. Vitamin K synthesis capacity test

[0035] (1) Take the activated second-generation bacterial solution and divide it into NWP-101 group, NWP-299 group and NWP-101+NWP-299 group (mix NWP-101 and NWP-299 at a mass ratio of 1:1). Repeat the freeze-thaw cycle 10 times at -80℃ and 4℃ to break the cells.

[0036] (2) Centrifuge at 10000 r / min for 5 min and collect the supernatant. Use PBS as a blank control and LGG as a positive control. Use a vitamin K ELISA kit and strictly follow the instructions: add sample and enzyme-labeled antibody, incubate, wash plate, develop color, terminate reaction, and finally measure OD value on a microplate reader. Calculate vitamin K content based on the standard curve. The results are as follows: Figure 2 As shown.

[0037] 5. Detection of probiotic characteristics of bacterial strains

[0038] (1) Acid and bile salt tolerance characteristics: MRS liquid medium with pH 3.0 and MRS liquid medium containing 0.3% porcine bile salt were prepared respectively. The activated second-generation strain was inoculated into them and cultured for 3h and 6h respectively. The survival rate was calculated by plating and counting.

[0039] (2) Growth curve: The activated second-generation strain was inoculated into MRS liquid medium, and the OD of the bacterial solution was measured every 2 hours. 600 Based on the pH value, plot the growth curve and pH change curve.

[0040] (3) Antioxidant activity: The supernatant of the activated second-generation strain was tested using the ABTS free radical scavenging ability kit and the DPPH free radical scavenging ability kit.

[0041] (4) Antibacterial activity: Oxford cups were placed on LB plates. 150 μL of the supernatant of the activated second-generation strain was added to the wells formed in the Oxford cups. After incubation, the diameter of the inhibition zone was measured. The results are as follows: Figure 3 As shown in Table 1.

[0042] Table 1 Results of the detection of probiotic characteristics of the strains

[0043] Depend on Figure 1 It was found that the selected single strain, *Lactobacillus delbrueckii* subsp. bulgaricus NWP-101, had a calcium ion enrichment capacity of 160 mg / g, while *Pediococcus lactis* NWP-299 had a calcium ion enrichment capacity of 70 mg / g. When the two strains were combined in a 1:1 ratio, the calcium ion enrichment capacity of the composite strain was synergistically increased to 180 mg / g, significantly higher than that of the single strain, demonstrating an excellent synergistic calcium enrichment effect.

[0044] Depend on Figure 2 It is known that the vitamin K synthesis capacity of Lactobacillus delbrueckii subsp. bulgaricus NWP-101 reaches 790 pg / 10. 8 CFU; the vitamin K synthesis capacity of *Pediococcus lactis* NWP-299 reaches 430 pg / 10. 8 CFU. When combined, the vitamin K synthesis capacity of the composition is increased to 870 pg / 10.8 CFU.

[0045] Depend on Figure 3 As shown in Table 1, both strains exhibited excellent acid resistance (survival rate >95% at pH 3.0), bile salt tolerance (survival rate >85% at 0.3% bile salt), strong antioxidant capacity (ABTS and DPPH free radical scavenging rate), and inhibitory ability against pathogenic bacteria (Escherichia coli and Staphylococcus aureus), ensuring that they could effectively reach the intestines and perform their functions.

[0046] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A probiotic with the function of accumulating calcium ions and synthesizing vitamin K, characterized in that, The probiotic is Lactobacillus bulgaricus subsp. ( Lactobacillus delbrueckii subsp. bulgaricus NWP-101 or Pyrococcus lactis ( Pediococcus acidilactici NWP-299.

2. The probiotic as described in claim 1, characterized in that, The Lactobacillus bulgaricus subsp. ( Lactobacillus delbrueckii subsp. bulgaricus NWP-101 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on November 10, 2025, with accession number GDMCCNo:67256.

3. The probiotic as described in claim 1, characterized in that, The lactic acid cocci ( Pediococcus acidilactici NWP-299 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on November 10, 2025, with accession number GDMCC No:67257.

4. A composition containing the probiotics according to any one of claims 1 to 3, characterized in that, The probiotic composition is one or both of Lactobacillus bulgaricus subsp. NWP-101 and Pediococcus lactis NWP-299.

5. The probiotic composition as described in claim 4, characterized in that, When the probiotic composition consists of Lactobacillus bulgaricus subsp. NWP-101 and Pediococcus lactis NWP-299, it is formulated at a live bacteria ratio of 0.5~1.5:0.5~1.

5.

6. The use of a probiotic according to any one of claims 1 to 3 or a composition according to claim 4 or 5 in the preparation of a product for preventing or improving osteoporosis and increasing bone density.

7. The application as described in claim 6, characterized in that, The product is a probiotic preparation, functional food, or dietary supplement.

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