Application of bifidobacterium longum B188 in product for promoting calcium absorption and skeletal development

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to application of bifidobacterium longum B188 in products for promoting calcium absorption and skeletal development. The preservation number of the bifidobacterium longum B188 is GDMCC No: 67123, and the bifidobacterium longum B188 can play roles through multiple mechanisms: the intestinal calcium absorption efficiency is improved so as to repair the calcium metabolism steady state and regulate serum related indexes; bone formation is promoted, bone resorption is inhibited, and bone health related markers are regulated and controlled; growth and development disorders caused by low-calcium diet are improved, organ indexes are adjusted, bone trabecula structures are repaired, bone mineral density is improved, and cortical bone thickness is increased. The strain can be used for preparing functional foods, health foods or dietary supplements for promoting calcium absorption and improving skeletal development, and a new scheme is provided for intervention of related problems of calcium metabolism.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to application of Bifidobacterium longum B188 in products for promoting calcium absorption and bone development. BACKGROUND

[0002] Calcium, as a macroelement essential for human body, is the core component of bones and teeth, and is also involved in key physiological processes such as nerve signal transmission, muscle contraction and blood clotting. The absorption efficiency and homeostasis of calcium directly determine the quality of bone development and the health of systemic metabolism. At present, the problem of bone health caused by insufficient dietary calcium intake, intestinal absorption disorder or calcium metabolism disorder is increasingly prominent, especially in children and adolescents during the growth and development period, pregnant women and the elderly. If children and adolescents have long-term insufficient calcium intake or absorption, it is easy to lead to growth and development retardation, insufficient bone mass accumulation, and increase the risk of osteoporosis in adulthood. The elderly have reduced intestinal function and vitamin D synthesis capacity, which reduces calcium absorption efficiency and accelerates bone loss rate, resulting in a high incidence of osteoporotic fractures. In addition, some people have lactose intolerance and intestinal flora imbalance, which further exacerbates intestinal calcium absorption disorder. Although traditional intervention methods such as calcium supplements (such as calcium carbonate and calcium citrate) can improve calcium intake in the short term, they have low bioavailability and can cause gastrointestinal discomfort such as constipation and abdominal distension. Moreover, they cannot fundamentally improve the active absorption mechanism of calcium in the intestine.

[0003] In the prior art, the intervention means for calcium absorption and bone development mainly focus on dietary calcium supplementation, vitamin D synergistic supplementation and calcium dosage form optimization. Although these methods can alleviate the problem of calcium deficiency to some extent, they have obvious limitations. On the one hand, simple calcium supplement relies on passive absorption in the intestine, which is greatly affected by intestinal pH value, dietary oxalate / phytate and other anti-nutritional factors. The actual absorption efficiency is usually very low, and long-term excessive supplementation can increase the metabolic burden on the kidneys and even induce the risk of kidney stones. On the other hand, although vitamin D can promote the synthesis of intestinal calcium-binding proteins and enhance the active transport of calcium, it needs to be obtained through sun exposure or exogenous supplementation. Some people (such as indoor workers and the elderly) have insufficient vitamin D synthesis or conversion disorders, and separate supplementation cannot regulate the synergistic effect of the intestinal microecological environment on calcium absorption. In recent years, research on probiotics in regulating intestinal flora and improving nutrient absorption has gradually deepened. Some studies have confirmed that some Bifidobacterium and Lactobacillus can improve the solubility and absorption rate of calcium in the intestine by reducing intestinal pH, promoting bile salt hydrolysis and synthesizing short-chain fatty acids. However, the functions of different strains have high specificity. Most of the reported strains have not been systematically verified in terms of calcium absorption promotion effect, bone development regulation mechanism and safety, especially the intervention research on growth and development disorders induced by low-calcium diet. Therefore, it is difficult to meet the needs of different populations for high-efficiency and safe bone health intervention products.

[0004] In this context, developing new intervention means with clear calcium absorption promoting function and bone protection effect has important theoretical significance and application value. Compared with traditional calcium agents and vitamin D supplements, functional intervention based on intestinal microecological regulation can optimize the intestinal absorption environment of calcium from the source by reshaping the balance of intestinal flora, and can achieve whole-process regulation of bone development through multiple mechanisms such as regulating calcium metabolism related hormones (such as parathyroid hormone, calcitonin), regulating osteoblast and osteoclast activity, promoting growth factor secretion, etc., with the advantages of high bioavailability and low side effects. The development of such intervention means not only fills the gap in the field of the correlation between intestinal microecology and bone health of existing technologies, provides technical support for the development of new functional foods and health products, but also provides a new solution for dietary intervention for calcium metabolism related diseases such as growth retardation in children and senile osteoporosis, which has important practical significance for promoting the application of biotechnology in the field of functional foods and improving the level of population bone health. SUMMARY

[0005] In view of the above deficiencies, the present application provides a long bifidobacterium B188, which can play a role through multiple mechanisms: improving intestinal calcium absorption efficiency to repair calcium metabolism homeostasis, regulating the level of calcium metabolism related hormones in serum; promoting bone formation and regulating the level of osteogenesis related markers and growth factors; inhibiting bone resorption and regulating the expression of bone resorption related markers; and also improving growth and development disorders caused by low calcium diet, regulating organ index, and repairing trabecular bone structure, increasing bone mineral density, and increasing cortical bone thickness. The strain can be used for preparing functional foods, health foods or dietary supplements for promoting calcium absorption and improving bone development, providing a new solution for calcium metabolism related problem intervention.

[0006] The technical solution of the present application is: In one aspect, the present application provides a long bifidobacterium B188, the preservation number of which is GDMCC No: 67123.

[0007] In another aspect, the present application provides a preparation method of the aforementioned long bifidobacterium B188, which comprises inoculating the long bifidobacterium B188 into a culture medium for culture.

[0008] Specifically, the culture medium includes but is not limited to MRS, BL, TPY or M17.

[0009] Preferably, the culture medium comprises MRS.

[0010] In another aspect, the present application provides the use of the aforementioned long bifidobacterium B188 in the preparation of products for promoting calcium absorption and bone development.

[0011] Specifically, the product comprises fermentation broth of Bifidobacterium longum B188, fermentation broth supernatant, fermentation broth precipitate, lyophilized powder and / or bacterial suspension.

[0012] More specifically, the fermentation broth refers to the liquid in which the microbial strain is inoculated into a culture medium and cultured for a period of time.

[0013] More specifically, the supernatant of the fermentation broth refers to the clear liquid at the top after centrifugation of the fermentation broth; it contains abundant metabolic products from the bacterial growth and reproduction process, as well as some bacterial cell fragments. The acidic substances and bacteriocins secreted by the bacteria have antagonistic and bactericidal effects on harmful bacteria. The amino acids and vitamins synthesized by the bacteria after decomposing food are also in the culture medium, as well as enzymes secreted by the bacteria that are useful to the human body. Some of the bacterial cell components also have an immune-boosting effect on the human body.

[0014] More specifically, the fermentation broth sediment refers to the liquid sediment obtained after centrifugation, including free proteins, residual bacterial cells, broken cells, and culture medium residues, mainly proteins and intracellular matrix.

[0015] More specifically, the lyophilized powder is obtained by freeze-drying culture medium; the lyophilized powder generally also includes a lyophilization protectant. The lyophilization protectant includes, but is not limited to: pH buffers, fillers, sugars, nonionic surfactants, ligands, etc. The pH buffers include, but are not limited to, any one or more of Tris, amino acids or their salts, citric acid or its salts, acetic acid or its salts. The fillers include, but are not limited to, any one or more of mannitol, glycine, and bovine serum albumin. The sugars can be disaccharides, such as sucrose or trehalose, any one or more. The nonionic surfactants include, but are not limited to, Tween, such as Tween-20, Tween-60, Tween-80, etc. The lyophilization protectant may also include antioxidants, etc. Specifically, the lyophilization protectant may also include albumin, polyethylene glycol, etc.

[0016] More specifically, the bacterial suspension is a homogeneous suspension formed by discarding the supernatant after centrifuging the culture medium, adding water, culture medium or buffer solution, and suspending the lower layer of bacteria by shaking or blowing.

[0017] Specifically, the products include, but are not limited to, health supplements or medicines.

[0018] In another aspect, the present invention provides a health product comprising the aforementioned Bifidobacterium longum B188.

[0019] Specifically, this also includes food or medically acceptable excipients.

[0020] More specifically, the food or medically acceptable excipient is selected from at least one of fillers, stabilizers, prebiotics, flavoring agents, and coating materials.

[0021] Preferably, the filler is selected from one or more of maltodextrin, fructooligosaccharides, erythritol, and microcrystalline cellulose; the stabilizer is selected from one or more of xanthan gum, pectin, trehalose, and vitamin E; the prebiotic is selected from one or more of galactooligosaccharides, inulin, and stachyose; the flavoring agent is selected from one or more of steviol glycosides, erythritol, and lemon powder flavoring; and the coating material is selected from one or more of hydroxypropyl methylcellulose, shellac, and gum arabic.

[0022] In another aspect, the present invention provides a medicine comprising the aforementioned Bifidobacterium longum B188.

[0023] Specifically, the viable count of Bifidobacterium longum B188 in the drug is not less than 1×10⁻⁶. 7 CFU / g.

[0024] Specifically, the drug also includes pharmaceutically acceptable excipients.

[0025] The beneficial effects of this invention are as follows: The Bifidobacterium longum B188 of this invention can effectively improve calcium metabolism homeostasis, reduce bone calcium mobilization dependence by improving intestinal calcium absorption efficiency, reverse the decrease in serum calcium caused by low-calcium diet, regulate the abnormal expression of calcium metabolism-related hormones, restore metabolic balance, and avoid the drawbacks of traditional calcium supplements. It can bidirectionally regulate bone health, both by promoting osteoblast differentiation, bone matrix deposition, and osteogenic-related factor expression to enhance osteogenic activity, and by regulating osteoprotegerin and type I collagen C-terminal peptide expression to improve the ratio imbalance and inhibit osteoclast activation to reduce bone loss. It can improve growth and development disorders in rats caused by low-calcium diet, regulate abnormal indices of organs such as kidneys and testes, and reduce the negative effects of low calcium on the body. Micro-CT verification shows that it can also repair bone microstructure, increase bone volume fraction, trabecular thickness and number, reduce trabecular separation, increase bone density and increase cortical bone thickness, and enhance bone mechanical stability.

[0026] Preservation instructions: ; Category Naming: Bifidobacterium longum ; Accession number: GDMCC No: 67123; Preservation period: October 17, 2025; Preservation institution: Guangdong Provincial Center for Microbial Culture Collection; Abbreviation of depositary institution: GDMCC; Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0027] Figure 1 Changes in rat body weight and body length.

[0028] Figure 2 The effect of different lowercase letters on the calcium metabolism regulation indicators in rat serum indicates significant differences between groups. P <0.05, Duncan's multiple comparison method was used); the same lowercase letter indicates no significant difference between groups.

[0029] Figure 3 The effect of different lowercase letters on bone formation markers in rat serum indicates significant differences between groups. P <0.05, Duncan's multiple comparison method was used); the same lowercase letter indicates no significant difference between groups.

[0030] Figure 4 The effect of different lowercase letters on bone resorption markers in rat serum indicates significant differences between groups. P <0.05, Duncan's multiple comparison method was used); the same lowercase letter indicates no significant difference between groups.

[0031] Figure 5 Micro-CT of rat femur. Detailed Implementation

[0032] The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0033] Example 1 1.1 Materials and Reagents Table 1 Experimental Materials and Reagents

[0034] 1.2 Instruments and Equipment Table 2 Instruments and Equipment

[0035] 1.3 Experimental Methods 1.3.1 Preparation of experimental strains The experimental strain used in this invention is Bifidobacterium longum B188, which was isolated from healthy breast milk. The provided preserved strain was inoculated into MRS liquid medium at a 2% inoculum size and cultured at 37°C for 16-24 hours, followed by a second activation for subsequent experiments.

[0036] 1.3.2 Animal Model Fifty three-week-old male SD rats (weighing 60 ± 5 g) were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. The rats were kept in an acclimatization environment at room temperature (25 ± 2℃) and relative humidity (50 ± 5%), with a 12-hour light / dark cycle. They were fed standard rat feed and water without restriction, and their bedding was changed every two days for one week. The rats were randomly divided into five groups: normal group, model group, positive group, high B188 group, and low B188 group. The experiment lasted for 8 weeks. The samples administered via gavage and the groupings were as follows: Normal group: AIN93-standard G diet (Nantong Trofi Feed Technology Co., Ltd., LAD 0022) + 0.5 mL / 100g 0.9% saline via gavage; Model group: AIN93-low calcium G diet (Nantong Trofi Feed Technology Co., Ltd., TP 790-02) + 0.5 mL / 100g (rat body weight) 0.9% saline via gavage; Positive control group (positive group): AIN93-low calcium G diet + calcium carbonate (1.6667 g / 100 mL) via gavage; High concentration of Bifidobacterium longum B188 group (B188 high group): AIN93-low calcium G diet + high concentration of (10 g / 100g) calcium carbonate via gavage. 9 CFU / kg) bacterial suspension; low concentration group of Bifidobacterium longum B188 (B188 low group), AIN93-low calcium G feed + low concentration (10 CFU / kg) by gavage 7 CFU / kg bacterial suspension. The weight and length of the rats were recorded weekly during the experiment, and blood was collected from the rats' eyeballs and femurs at the end of the experiment. All animal experiments were reviewed by the Animal Ethics Committee of the Chongqing Collaborative Innovation Center for Functional Foods (Ethics No.: 2025011701A).

[0037] 1.3.3 Detection of serum markers in rats Rat blood was centrifuged at 4000 rpm for 10 minutes at 4°C to separate and collect rat serum, which was then stored at -80°C for later use. The levels of Ca, 1,25(OH)2D3, PTH, CT, BGP, PINP, β-ALP, IGF-1, CTX-I, and OPG in the serum were determined using appropriate biochemical kits according to the manufacturer's recommended procedures.

[0038] 1.3.4 Micro-CT Analysis of Rat Femur The obtained rat femurs were fixed in centrifuge tubes containing universal tissue fixative until Micro-CT analysis. After removing the samples from the fixative, excess liquid was wiped off with gauze, and the samples were placed on the instrument's scanning bed for scanning (scanning parameters are shown in Tables 3 and 4). Raw images were obtained after scanning. The raw images were reconstructed using the 3D reconstruction software Recon. Finally, the target region was analyzed using the data analysis software Avatar. The same region was selected for analysis of all samples. The required parameter values ​​were obtained using the data acquisition software Cruiser, and the data was exported.

[0039] Table 3 Scanning Parameters

[0040] Table 4 Scanning Parameters

[0041] 1.3.5 Data Analysis Serum and tissue parameters of each rat were performed in triplicate or at least in parallel, and the average values ​​were taken. Data were statistically analyzed using statistical software. Experimental results are expressed as mean ± standard deviation (SD). Differences between the means of each group were assessed using one-way ANOVA. p Differences <0.05 are considered statistically significant.

[0042] 1.4 Results and Analysis 1.4.1 Changes in rat body weight and length, and organ indices Low-calcium diet intervention significantly inhibited weight gain and body length development in rats. Figure 1 The model group rats showed a significantly lower rate of weight gain during the experimental period compared to the normal group. p <0.05, and the rate of increase in height also decreased significantly ( p <0.05, indicating a negative impact of a low-calcium diet on overall growth and development. The intervention results in the positive group showed a significant improvement in weight and height growth compared to the model group. p <0.05), with some indicators recovering to near-normal levels, validating the effectiveness of the model and the basic effect of drug intervention. Organ index analysis showed (Table 5) that, compared with the normal group, the kidney index in the model group was significantly increased ( p <0.05); Testicular index significantly decreased ( p <0.05 indicates that low calcium may affect reproductive function by inhibiting testosterone synthesis or germ cell differentiation. After intervention with Bifidobacterium longum B188, the weight gain rate and height gain in the high-concentration group were close to normal levels, and the kidney index and testicular index were significantly improved. p<0.05), suggesting that B188 alleviates systemic developmental inhibition caused by low calcium by regulating calcium metabolism and endocrine function.

[0043] Table 5. Organ Index of Rats

[0044] 1.4.2 Effects of B188 on serum calcium metabolism regulation indicators in rats Effects of calcium metabolism regulation-related indicators in rat serum, such as Figure 2 As shown, compared with the normal group, the serum Ca and CT levels in the model group were significantly reduced ( p <0.05%, accompanied by a compensatory increase in PTH and 1,25(OH)2D3 ( p <0.05 indicates that low calcium triggers the body to maintain serum calcium homeostasis through bone calcium mobilization and vitamin D activation. The positive group effectively increased serum calcium levels (…). p <0.05), and significantly reduced compensatory increases in PTH and 1,25(OH)2D3 ( p <0.05 indicates its mechanism of action: directly supplementing calcium or promoting absorption. After B188 intervention, compared with the model group, the serum calcium in the high-concentration group returned to a level not significantly different from that in the normal group ( p >0.05), CT levels significantly increased ( p <0.05), PTH level decreased to 60.15 ± 4.86 pg / mL ( p <0.05), and 1,25(OH)2D3 decreased to 106.48 ± 3.19 ng / mL, close to the normal group. Its regulatory trend was consistent with that of the positive group, and some indicators (such as CT enhancement) showed potential to be superior to the positive group, indicating that B188 restores calcium metabolism homeostasis by enhancing intestinal calcium absorption efficiency and reducing dependence on bone calcium mobilization.

[0045] 1.4.3 Effects of B188 on bone formation markers in rat serum The results are as follows Figure 3 As shown, compared with the normal group, the serum levels of BGP and PINP in the model group rats were significantly reduced ( p The level <0.05 indicates impaired collagen synthesis and bone matrix deposition. B-ALP activity in the model group rats was significantly lower than that in the normal group ( p <0.05, further indicating impaired mineralization function. After intervention, the levels of BGP, PINP, and B-ALP in the positive group were significantly higher than those in the model group ( p<0.05, confirming its bone-forming effect. After B188 intervention, the levels of BGP (25.69 ± 0.88 ng / mL), PINP (241.95 ± 9.85 ng / mL), and B-ALP (27.55 ± 2.41 ng / mL) in the high-concentration group were significantly higher than those in the model group. p <0.05, and its increase was not significantly different from that of the positive group ( p >0.05), suggesting that it restores bone formation capacity by promoting osteoblast differentiation and function. Insulin-like growth factor-1 (IGF-1) levels were decreased in the model group (3543.00 ± 202.50 ng / mL vs. normal group 5369.17 ± 100.52 ng / mL). p <0.05), after B188 intervention, it recovered to 4716.00 ± 167.13 ng / mL ( p The result was <0.05, indicating that IGF-1 may mediate the promoting effect of B188 on osteogenic activity.

[0046] 1.4.4 Effects of B188 on bone resorption markers in rat serum The results are as follows Figure 4 As shown, a low-calcium diet significantly enhanced osteoclast activity. Compared with normal rats, the serum CTX-I level in the model group rats was significantly increased ( p <0.05%, OPG levels decreased significantly ( p <0.05 indicates that an imbalance in the RANKL / OPG ratio leads to increased osteoclast differentiation. The positive group can effectively reduce the elevated CTX-I level in the model group ( p <0.05), and increase OPG level ( p <0.05, indicating an inhibitory effect on bone resorption. Compared with the model group rats, after B188 intervention, the high concentration group CTX-1 decreased to 13.85 ± 0.75 ng / mL ( p <0.05), OPG recovered to 154237.5 ± 2350.49 pg / mL ( p <0.05), indicating that it reduces bone resorption by regulating OPG expression.

[0047] 1.4.5 Micro-CT Analysis of Rat Femur rat femur micro-CT images ( Figure 5 The results of the quantitative analysis (Table 6) showed that the low-calcium diet successfully induced osteoporosis in rats, as evidenced by the fact that the model group had significantly higher bone volume fraction (BV / TV, 0.131 vs. 0.383) and trabecular bone number (Tb.N, 0.798 mm) compared to the normal group. -1 vs. 1.557 mm-1 Bone mineral density (BMD, 47.928 mg / cm³) 3 vs. 166.694 mg / cm 3 All parameters were significantly reduced, while trabecular separation (Tb.Sp, 1.104 ± 0.752 mm vs. 0.450 ± 0.351 mm) and structural pattern index (SMI, 1.196 vs. 0.662) were significantly increased. 3D images clearly showed sparse and fractured trabeculae and thinning of cortical bone. Positive drug intervention significantly improved all parameters (e.g., BV / TV recovered to 0.320). Bifidobacterium longum B188 intervention showed a positive effect on bone microstructure repair, especially in the high-dose group, where BV / TV (0.387) and SMI (0.396) recovered to levels superior to the normal group. Trabecular continuity was also significantly improved in the images. The low-dose group showed improved BMD (83.601 mg / cm²). 3 The results showed outstanding performance in areas such as calcium absorption, indicating that Bifidobacterium longum B188 can effectively reverse bone loss and bone structure degeneration caused by a low-calcium diet.

[0048] Table 6. Micro-CT analysis of bone mechanical strength in rats

[0049] Bifidobacterium longum B188 demonstrated a significant ameliorative effect on systemic growth and development disorders in a low-calcium diet model, with mechanisms encompassing multiple dimensions including calcium metabolism regulation, growth factor secretion, and organ function protection. A low-calcium diet induces metabolic disorders by restricting calcium absorption, leading to persistently elevated parathyroid hormone (PTH), suppressed insulin-like growth factor-1 (IGF-1) secretion, and impaired bone mineralization, resulting in stagnant weight gain, limited height development, and abnormal organ indices. B188 intervention effectively alleviated these pathological changes by coordinating calcium absorption and endocrine balance: it improved intestinal calcium absorption efficiency, inhibited excessive PTH secretion, and reduced compensatory renal burden; simultaneously, B188 promoted IGF-1 production, stimulating growth plate chondrocyte proliferation, thereby supporting longitudinal bone growth. Furthermore, its upregulation of osteoprotegerin (OPG) inhibited osteoclast activity, while enhanced bone-specific alkaline phosphatase (B-ALP) activity promoted bone mineralization, ultimately restoring bone mineral density and cortical bone thickness.

[0050] In conclusion, Bifidobacterium longum B188 comprehensively improves growth and development disorders induced by low-calcium diet by coordinating calcium metabolism, promoting growth factor secretion, and protecting organ function, and has the potential to be used in the intervention of growth retardation in children or metabolic syndrome in the elderly.

[0051] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A strain of Bifidobacterium longum B188, characterized in that, The preservation number of the Bifidobacterium longum B188 is GDMCC No:67123.

2. The method for preparing Bifidobacterium longum B188 according to claim 1, characterized in that, This includes inoculating Bifidobacterium longum B188 onto a culture medium for cultivation.

3. The preparation method according to claim 2, characterized in that, The culture medium includes MRS, BL, TPY, or M17.

4. The use of Bifidobacterium longum B188 as described in claim 1 in the preparation of products that promote calcium absorption and bone development.

5. The application according to claim 4, characterized in that, The product comprises fermentation broth of Bifidobacterium longum B188, fermentation broth supernatant, fermentation broth precipitate, lyophilized powder and / or bacterial suspension.

6. The application according to claim 4, characterized in that, The product in question is a health supplement or a medicine.

7. A health product, characterized in that, Includes Bifidobacterium longum B188 as described in claim 1.

8. The health product according to claim 7, characterized in that, It also includes food or medically acceptable excipients.

9. A drug, characterized in that, Includes Bifidobacterium longum B188 as described in claim 1.

10. The medicament according to claim 9, characterized in that, The viable count of Bifidobacterium longum B188 in the drug is not less than 1×10⁻⁶. 7 CFU / g.

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