A composition having a function of promoting bone health and use thereof

Abstract

This invention discloses a composition with bone-health-promoting functions and its applications, belonging to the field of microbial engineering. The composition comprises milk calcium, colostrum basic protein, and Bifidobacterium breve i1088 powder. The composition provided by this invention, which has a synergistic effect between milk calcium, colostrum basic protein, and Bifidobacterium breve i1088 powder, enhances the conversion rate of calcium from the blood to bone through the action of probiotics, thereby improving the actual utilization rate of calcium and effectively promoting bone health. Experimental data show that this composition significantly increases bone mineral density, bone volume fraction, and trabecular thickness in a female rat osteoporosis model, and significantly reduces trabecular spacing in the same model. The composition provided by this invention is suitable for promoting bone health and preventing and / or improving osteoporosis.

Description

Technical Field

[0001] This invention belongs to the field of microbial engineering technology and relates to a composition containing Bifidobacterium breve i 1088, specifically a composition with bone health promoting function and its application. Background Technology

[0002] With the increasing aging of the global population, bone health has gradually become a focus of social concern. Bone diseases, such as osteoporosis, not only affect the quality of life of the elderly but also place a heavy burden on the healthcare system. Therefore, developing new products and technologies that can promote bone health has significant social implications and market potential. Currently, there are various products and methods on the market for improving bone health, including calcium supplements, vitamin D, and certain drug therapies. However, these methods often have limitations, such as potential side effects from long-term use, low absorption rates, and limited applicability to specific populations.

[0003] Studies have shown that probiotics can regulate the balance of gut microbiota and may indirectly promote bone health by affecting the absorption of minerals such as calcium through the gut-bone axis. Epigenetics refers to preparations of non-living microorganisms and / or their components that are beneficial to the host's health, continuing to provide health benefits even after the probiotics die. In recent years, epigenetics prepared from probiotics have gradually become a new trend in products promoting bone health. However, probiotics or epigenetics have certain limitations in promoting bone health, such as relatively weak effects and a long time required to achieve significant efficacy.

[0004] Milk calcium, also known as whey calcium, is a type of mineral salt obtained from milk through dehydration, defatting, and spray drying. Although milk calcium has high bioavailability and is easily absorbed, its bioavailability in the body is affected by factors such as dietary fiber, fat digestion, vitamin D deficiency, and an unsuitable calcium-to-phosphorus ratio.

[0005] Colostrum basic protein (CBP) is a protein found in bovine colostrum that promotes bone growth and repair. However, the content of CBP in bovine colostrum is very low, only 0.04%, which limits its large-scale production and application. Therefore, how to efficiently utilize the functions of raw materials has become the primary bottleneck in product manufacturing and nutritional health. Summary of the Invention

[0006] The purpose of this invention is to provide a composition and its application that promotes bone health, in order to solve the problems of various drawbacks and poor calcium supplementation effects of current bone health promotion products.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A composition for promoting bone health, the composition comprising Bifidobacterium breve i 1088 bacterial powder, milk calcium, and colostrum basic protein.

[0009] Among them, *Bifidobacterium breve* i 1088 was deposited on May 8, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNO.27281. This strain was first disclosed in Chinese invention patent CN117143778A, entitled "*Bifidobacterium breve* i 1088 and its applications".

[0010] As a limitation, by weight, the amount of Bifidobacterium breve i 1088 bacterial powder is 0.2-1000 parts, the amount of milk calcium is 600-2500 parts, and the amount of colostrum basic protein is 40-200 parts.

[0011] As another limitation, the Bifidobacterium breve i 1088 bacterial powder includes live bacterial powder and / or inactivated bacterial powder.

[0012] As a further limitation, the viable count of the *Bifidobacterium breve* i 1088, or its viable count before inactivation, is 3 × 10⁻⁶. 8 -5×10 11 CFU / g or CFU / mL.

[0013] The present invention also provides the application of the above-described composition having a bone health promoting function, the composition being used to prepare a product having a bone health promoting function.

[0014] As a limitation, the bone health-promoting function includes the prevention and / or improvement of osteoporosis.

[0015] The present invention also provides a formula milk powder for middle-aged and elderly people, comprising the above-mentioned composition with bone health promotion function.

[0016] The present invention also provides a low-temperature yogurt comprising the above-described composition having bone health-promoting functions.

[0017] The present invention also provides a room-temperature liquid milk comprising the above-described composition having bone health-promoting functions.

[0018] By adopting the above technical solution, the technical progress achieved by this invention compared with the prior art is as follows:

[0019] ① This invention provides a composition that promotes bone health. Bifidobacterium breve i 1088, milk calcium, and CBP have a synergistic effect. While supplementing calcium, the composition increases the conversion rate of calcium in the blood to calcium in the bones through the action of probiotics, thereby improving the actual utilization rate of calcium and effectively promoting bone health. According to experimental data, compared with i 1088 bacterial powder or a combination of milk calcium and CBP, this composition can significantly increase bone mineral density, bone volume fraction, and trabecular thickness in a female rat osteoporosis model, and significantly reduce trabecular spacing in a female rat osteoporosis model.

[0020] ②The present invention provides a composition that promotes bone health. Even if the Bifidobacterium breve i 1088 bacterial powder is an inactivated bacterial powder, it can effectively improve osteoporosis. Moreover, the inactivated bacterial powder has good stability and will not produce problems such as post-fermentation and acid production. There is no need to consider the maintenance of the number of live bacteria. Detailed Implementation

[0021] The present invention will be further described in detail below through specific embodiments. It should be understood that the described embodiments are only for explaining the present invention and do not limit the present invention.

[0022] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art.

[0023] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0024] Example 1

[0025] This embodiment provides a composition that promotes bone health. The composition comprises 10g of Bifidobacterium breve i 1088 live bacteria powder, 792g of milk calcium, and 60g of CBP, wherein the live bacteria count of the Bifidobacterium breve i 1088 live bacteria powder is 1×10⁻⁶. 11 CFU / g.

[0026] Example 2

[0027] This embodiment provides a formula milk powder for middle-aged and elderly people, which contains a composition (added at 5.7 wt%) that promotes bone health. This composition comprises 0.2 g of Bifidobacterium breve i 1088 live bacteria powder, 600 g of milk calcium, and 40 g of CBP. The live bacteria count of the Bifidobacterium breve i 1088 live bacteria powder is 3 × 10⁻⁶. 11 CFU / g.

[0028] Example 3

[0029] This embodiment provides a low-temperature yogurt containing a composition (1 wt%) that promotes bone health. This composition comprises 1 g of Bifidobacterium breve i 1088 live bacteria powder, 250 g of milk calcium, and 40 g of CBP. The live bacteria count of the Bifidobacterium breve i 1088 live bacteria powder is 2 x 10^6 bacteria. 11 CFU / g.

[0030] Example 4

[0031] This embodiment provides a composition that promotes bone health, comprising 10g of inactivated Bifidobacterium breve i 1088 bacterial powder, 792g of milk calcium, and 60g of CBP.

[0032] The inactivated Bifidobacterium breve i1088 bacterial powder is obtained by placing the live i1088 bacterial powder in a glass or metal container with good thermal conductivity, sealing it, and placing it in an electric heating drying oven at 80°C for 30 minutes.

[0033] The viable count of the *Bifidobacterium breve* i 1088 before inactivation was 1 × 10⁻⁶. 11 CFU / g, the number of viable bacteria after inactivation is less than 3000 CFU / g.

[0034] Example 5

[0035] This embodiment provides a room-temperature liquid milk containing a composition (0.2 wt%) that promotes bone health. This composition comprises 1000 g of inactivated Bifidobacterium breve i 1088 bacterial powder, 2500 g of milk calcium, and 200 g of CBP. The viable count of the Bifidobacterium breve i 1088 before inactivation is 3 x 10⁻⁶. 8 CFU / g, the number of viable bacteria after inactivation is less than 3000 CFU / g.

[0036] Example 6

[0037] This embodiment provides a formula milk powder for middle-aged and elderly people, which contains a composition (added at 3 wt%) that promotes bone health. This composition comprises 10g of inactivated Bifidobacterium breve i 1088 bacterial powder, 2000g of milk calcium, and 50g of CBP. The viable count of the Bifidobacterium breve i 1088 before inactivation is 5 x 10⁻⁶. 11 CFU / g, the number of viable bacteria after inactivation is less than 3000 CFU / g.

[0038] Example 7

[0039] This embodiment provides an application of a composition that promotes bone health, uses the composition to improve osteoporosis, and provides a control experiment, specifically including the following steps:

[0040] S1. Preparation of Experimental Materials

[0041] 1) Sample preparation

[0042] Sample 1: Probiotic powder containing live Bifidobacterium breve i 1088 was prepared into a solution with a certain volume of physiological saline and recorded as active i 1088.

[0043] Sample 2: Take the probiotic powder of inactivated Bifidobacterium breve i 1088 and prepare a solution with the same volume of physiological saline, which is denoted as inactivated i 1088;

[0044] Sample 3: Take the composition from Example 1 and prepare a solution with the same volume of physiological saline, denoted as Complex 1;

[0045] Sample 4: Take the composition from Example 4 and prepare a solution with the same volume of physiological saline, denoted as Complex 2;

[0046] Sample 5: Take 1584g of milk calcium and 120g of CBP, and prepare a solution with the same volume of physiological saline, which is denoted as complex 3;

[0047] Anesthetic (5% chloral hydrate): Dissolve 5g of chloral hydrate in about 60ml of physiological saline, add physiological saline to make up to 100ml, and store at room temperature away from light.

[0048] 2) Laboratory animals

[0049] Sprague Dawley rats (SD rats), female, 10-12 weeks old.

[0050] The laboratory animals were provided by Spifort (Suzhou) Biotechnology Co., Ltd. (China), certificate of conformity: SCXK(Su)2022-0006.

[0051] 3) Instruments, consumables and reagents

[0052] Disposable 5ml syringes (Shanghai Kangdelai, China), electronic analytical balance Precisa12A (Precisa, Switzerland), pyrogen-free ultrapure water system Millipo3150 (Millipore, USA), three-section rat gavage needle (Zhongke Life, China), electric thermostatic water bath HW-SY11-KP2 (Beijing Changfeng Instrument Co., Ltd., China), 1.5ml centrifuge tubes (Wuxi Nice Life Science & Technology Co., Ltd., China), ultra-low temperature freezer Sanyo-382AT (Sanyo Corporation, Japan), 6-0 absorbable sutures (Shanghai Jinhuan Medical Instrument Co., Ltd., China), Micro-CT scanner Skyscan1276 (Bruker, USA).

[0053] 0.9% sodium chloride injection (L222042401, Sichuan Kelun Pharmaceutical Co., Ltd., China), chloral hydrate (C104202-250g, Shanghai Aladdin Biochemical Technology Co., Ltd., China), paraformaldehyde fixative (G1101, Wuhan Saiweier Biotechnology Co., Ltd., China), alendronate sodium (A115345-25g, Shanghai Aladdin Biochemical Technology Co., Ltd., China).

[0054] S2. Experimental Methods

[0055] 1) Animal grouping and osteoporosis modeling

[0056] After 7 days of acclimatization, 80 SD rats were divided into 8 groups: blank group, model group, positive drug group, active i 1088 group, inactivated i 1088 group, and complex group (including complex group 1, complex group 2, and complex group 3). Each group consisted of 10 rats.

[0057] Except for the control group, all SD rats underwent ovariectomy: After anesthesia, the ventral skin was prepared, hair removed, and disinfected. A 2-cm incision was made along the midline of the abdomen using sterile surgical scissors to expose the ovaries. Both ovaries were then removed after ligation with sutures. After the surgery, the uterus was returned to the abdominal cavity, and the muscles and skin were sutured layer by layer. The wound was disinfected with iodine and the rats were placed on a heating pad until they recovered and were then fed normally.

[0058] The control group underwent sham surgery: only anesthesia was used to make an incision, the ovary was exposed and removed without ligation, placed back into the abdominal cavity and the wound was sutured, and the animals were fed normally after waking up.

[0059] 2) Samples were administered via gavage.

[0060] The day after surgery, SD rats in each group were administered daily by gavage. The blank control group and the model group were given a certain volume of physiological saline daily. The positive drug group was given the same volume of 40 mg / kg alendronate sodium (solvent is physiological saline) once a day for 28 consecutive days.

[0061] SD rats in the active i 1088 group were administered an equal volume of active i 1088 via gavage daily (the live bacteria intake of the rats was 1.135 x 10^6 bacteria). 8 CFU, when converted to human dosage, is 5 x 10⁻⁶ CFU per day. 9 CFU, once daily for 28 consecutive days.

[0062] SD rats in the inactivated i 1088 group were administered an equal volume of inactivated i 1088 via gavage daily (the amount of inactivated bacteria ingested by the rats was 1.135 x 10^6). 8 CFU, when converted to human dosage, is 5 x 10⁻⁶ CFU per day. 9CFU, once daily for 28 consecutive days.

[0063] SD rats in the complex group were administered equal volumes of Complex 1 and Complex 2 via gavage daily (the rats ingested 18 mg of milk calcium, 1.36 mg of colostrum basic protein, and 2.27 x 10^6 bacteria daily). 7 CFU, when converted to human dosage, corresponds to daily intakes of 792 mg, 60 mg, and 1 x 10 mg, respectively. 9 CFU) and complex 3 (rats ingested 36 mg of milk calcium and 2.72 mg of colostrum basic protein daily, which translates to 1584 mg and 120 mg of human daily intake, respectively) once daily for 28 consecutive days.

[0064] 3) Femoral bone sampling and scanning detection

[0065] 24 hours after the last gavage, the lower femur of each group of SD rats was taken, the muscles and connective tissue around the lower femur were removed, and the femur was fixed and preserved in paraformaldehyde fixative.

[0066] The lower femur was removed from the fixation solution, rinsed three times with PBS buffer, dried, and wrapped with sealing film to obtain the lower femur sample.

[0067] Start the Skyscan1276 Micro-CT scanner program. After initialization and warm-up, open the sample chamber and place the lower femur sample along the length of the semi-cylindrical sample chamber. Use foam and paper to elevate the position of the lower femur sample to ensure that it is in the center of the semi-circle and fix it with 3M tape. Close the sample chamber door and start scanning. The scanning resolution is 18μm.

[0068] After scanning, 3D reconstruction was performed using CTVOX software to export the lower femur morphology image. CTAn software was used to analyze the bone mineral density, bone volume / total tissue volume (bone volume fraction), trabecular thickness, and trabecular separation / spacing in the femoral condyle region. The results are shown in Table 1, and the meanings of each data point are as follows:

[0069] BMD: Bone mineral density, this indicator reflects the condition of bones, and a decrease in the index indicates osteoporosis;

[0070] BV / TV: Bone volume fraction. This indicator reflects bone metabolism. A decrease in the index means that bone breakdown is greater than bone synthesis, resulting in decreased bone mass.

[0071] Tb.Th: Trabecular thickness, an indicator that reflects the morphology and structure of the bone. A decrease in this indicator indicates that the porosity within the bone has increased.

[0072] Tb.Sp: Trabecular spacing, an indicator that reflects the morphology and structure of the bone. An increase in this indicator indicates that there are more pores within the bone.

[0073] S3. Experimental Results

[0074] Table 1 Experimental Results

[0075]

[0076] Note: *** indicates p < 0.001 compared to the control group; ## indicates p < 0.05 compared to the model group; ### indicates p < 0.001 compared to the model group; different lowercase letters indicate significant differences between experimental groups, p < 0.05.

[0077] Table 1 shows that both live and inactivated Bifidobacterium breve i 1088 probiotics have a certain degree of effect in improving osteoporosis. Specifically, compared with the model group, live i 1088 significantly increased bone mineral density, bone volume fraction, and trabecular thickness in female rats with osteoporosis, and significantly reduced trabecular spacing. Among them, the live bacterial preparation was significantly better than the inactivated bacterial preparation in improving trabecular thickness and trabecular spacing, while both were comparable in improving bone volume fraction and bone mineral density.

[0078] It can also be seen that compositions containing live or inactivated Bifidobacterium breve i 1088 bacteria have the effect of improving osteoporosis, and the effect is better than that of simple bacterial agents or simple compositions of milk calcium and CBP. This indicates that Bifidobacterium breve i 1088, milk calcium and CBP have a synergistic effect in improving osteoporosis, especially the composition with live bacteria, which is more effective than the composition with dead bacteria (specifically in reducing the intertrabecular spacing). Specifically, compared with complex 3 and active i 1088, complex 1 has significant differences in increasing bone mineral density, bone volume fraction and trabecular thickness, and reducing intertrabecular spacing.

Claims

1. A composition having a function of promoting bone health, characterized in that, The composition comprises Bifidobacterium breve i1088 bacterial powder, milk calcium, and colostrum basic protein; The Bifidobacterium breve i1088 bacterial powder includes live bacterial powder and / or inactivated bacterial powder; The accession number for Bifidobacterium breve i1088 is CGMCC NO.27281; By weight, the amount of Bifidobacterium breve i1088 bacterial powder is 0.2-1000 parts, the amount of milk calcium is 600-2500 parts, and the amount of colostrum basic protein is 40-200 parts.

2. The composition for promoting bone health according to claim 1, characterized in that, The viable cell count of the Bifidobacterium breve i1088 or the viable cell count before inactivation thereof is 3 x 10 8 - 5 x 10 11 CFU / g.

3. The application of a composition with bone health-promoting function according to claim 1 or 2, characterized in that, The composition is used to prepare products that promote bone health.

4. A formula milk powder for middle-aged and elderly people, characterized in that, The composition comprising the bone health promoting function as described in claim 1 or 2.

5. A type of low-temperature yogurt, characterized in that, The composition comprising the bone health promoting function as described in claim 1 or 2.

6. A room-temperature liquid milk, characterized in that, The composition comprising the bone health promoting function as described in claim 1 or 2.

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