Probiotic agent containing akk11 strain for promoting calcium absorption and use thereof

Abstract

The present invention relates to a probiotic agent containing an Akk11 strain for promoting calcium absorption and the use thereof. The strain in the probiotic agent for promoting calcium absorption comprises an Akkermansia muciniphila Akk11 strain and a Bifidobacterium breve BBr16 strain. A new probiotic compounding mode is developed, and it is found that the Akk11 strain and the BBr16 strain have a potential interaction, and can cooperate with each other to synergize in promoting calcium absorption. Specifically, the probiotic agent can promote the overall metabolic capacity of calcium by means of improving intestinal health and enhancing the absorption capacity of the intestinal wall; the probiotic agent can increase a serum calcium level and a phosphorus level, and restore a normal electrolyte level; and the probiotic agent effectively reduces calcium loss in bones, thereby stabilizing the structure and function of the bones.

Description

Calcium absorption promoting probiotic agent containing Akk11 strain and application thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of probiotic agents, and relates to a calcium absorption promoting probiotic agent containing Akk11 strain and application thereof. BACKGROUND

[0002] Calcium is one of the most abundant minerals in the human body, and is essential for maintaining bone health, nerve conduction, muscle function, and blood clotting. Insufficient calcium absorption is associated with a variety of health problems, including osteoporosis, increased risk of fractures, and growth retardation. Calcium absorption mainly occurs in the small intestine and is influenced by a variety of factors, including the form of calcium in the diet, intestinal pH, the presence of other nutrients in the intestine, and the state of the intestinal microbial community.

[0003] Intestinal microbes, especially probiotics, have a potential promoting effect on calcium absorption. Probiotics can affect calcium absorption through a variety of mechanisms, including: changing the intestinal pH, promoting the expression of calcium-binding proteins, enhancing the integrity of the intestinal barrier, reducing inflammation, helping to improve the absorption of nutrients, regulating the composition of the intestinal microbial community, and possibly indirectly affecting calcium absorption and metabolism.

[0004] The application of probiotics in promoting calcium absorption is still in its early stages, and strategies are still relatively few. Therefore, developing more probiotic agents that can effectively promote calcium absorption not only provides new strategies for addressing the problem of insufficient calcium absorption, but also helps to better understand the interaction between intestinal microbes and host nutrient metabolism, providing scientific basis and technical support for preventing and improving insufficient calcium absorption.

[0005] SUMMARY

[0006] The present application provides a calcium absorption promoting probiotic agent containing Akk11 strain and application thereof, specifically relates to a calcium absorption promoting probiotic agent containing Akk11 strain and application thereof in preparing products for preventing or improving insufficient calcium absorption or in preparing products for preventing, improving or treating conditions caused by insufficient calcium absorption.

[0007] In a first aspect, the present application provides a probiotic agent for promoting calcium absorption containing an Akk11 strain, the probiotic agent for promoting calcium absorption comprising an Akkermansia muciniphila Akk11 strain and a Bifidobacterium breve BBr16 strain; wherein the Akkermansia muciniphila Akk11 strain has a preservation number of CCTCC NO: M2024119, a preservation date of January 15, 2024, and a preservation unit of China Center for Type Culture Collection, Wuhan University, Wuhan, China; and the Bifidobacterium breve BBr16 has a preservation number of CGMCC No. 24471, a preservation date of March 7, 2022, and a preservation unit of China General Microbiological Culture Collection Center, Beijing, China.

[0008] The present application develops a brand-new probiotic compound method, which is to compound the Akkermansia muciniphila Akk11 strain and the Bifidobacterium breve BBr16 strain. It is found that the two strains have potential interaction and can cooperate with each other to synergistically promote calcium absorption. Specifically, (1) it can improve intestinal health and enhance the absorption capacity of the intestinal wall to promote the overall metabolic capacity of calcium; (2) it can promote serum calcium levels and phosphorus content to restore normal electrolyte levels; (3) it can effectively reduce calcium loss in the skeleton to stabilize the structure and function of the skeleton.

[0009] Under the condition of using the same amount of bacteria, compared with a single Akk11 strain or a single BBr16 strain, the compound of the two strains significantly improves the above-mentioned effects. Therefore, the probiotic agent provides a new strategy for preventing, alleviating or treating diseases caused by insufficient calcium absorption. Since both Akkermansia muciniphila and Bifidobacterium breve are probiotics, they are safe and less likely to cause dependence when used to prepare related products.

[0010] Preferably, the ratio of viable bacteria of the Akk11 strain to the BBr16 strain is 1:10-10:1, such as 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1, and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0011] Preferably, the total number of viable bacteria in the probiotic agent is not less than 1 x 10 8 CFU / mL or 1 x 10 8 CFU / g, for example, 1 x 10 8 CFU / mL (CFU / g), 1 x 10 9 CFU / mL (CFU / g), 5 x 10 9 CFU / mL (CFU / g), 1 x 10 10 CFU / mL (CFU / g), 5 x 10 10 CFU / mL (CFU / g), 1 x 10 11 CFU / mL (CFU / g), 1 x 10 12 CFU / mL (CFU / g), or 1 x 10 13 CFU / mL (CFU / g), etc. Other specific point values within the numerical range can also be selected, which will not be repeated here.

[0012] Preferably, the dosage form of the probiotic agent includes a solution, a lyophilized powder, a capsule, a tablet, or a granule. The dosage form of the probiotic agent involved in the present application is not limited, including the most commonly used solution, lyophilized powder, or further prepared capsule, tablet, or granule. The lyophilized powder can be prepared by the following method, for example:

[0013] The Akk11 strain and the BBr16 strain are inoculated into the culture medium respectively for culture to obtain a culture solution; the culture solution is centrifuged to obtain bacterial cells; the bacterial cells are resuspended with a lyophilization protective agent to obtain a resuspension; the resuspension is lyophilized to obtain a lyophilized powder, and then the two are compounded in proportion.

[0014] Alternatively, the Akk11 strain and the BBr16 strain are inoculated into the culture medium respectively for culture to obtain a culture solution; the culture solution is centrifuged to obtain bacterial cells; the two kinds of bacterial cells are mixed in proportion and resuspended with a lyophilization protective agent to obtain a resuspension; the resuspension is lyophilized to obtain a lyophilized powder.

[0015] Preferably, the lyophilization is performed by vacuum freezing method.

[0016] Preferably, the probiotic agent further comprises a lyophilization protective agent and / or an auxiliary additive.

[0017] Preferably, the lyophilization protective agent comprises any one or a combination of at least two of skimmed milk, sucrose, lactose, trehalose, dextran, gelatin, dextrin, gum arabic, sodium alginate, polyvinylpyrrolidone, sorbitol, or xylitol.

[0018] Preferably, the auxiliary additive comprises any one or a combination of at least two of inulin, fructo-oligosaccharide, xylo-oligosaccharide, galacto-oligosaccharide, isomalto-oligosaccharide, soybean oligosaccharide, spirulina, arthrospira, grifola, stachyose, polydextrose, alpha-lactalbumin or lactoferrin.

[0019] In a second aspect, the present application provides use of the probiotic agent for promoting calcium absorption according to the first aspect in the preparation of a product for preventing or improving insufficient calcium absorption.

[0020] Preferably, the product further comprises an excipient.

[0021] The excipient comprises any one or a combination of at least two of an excipient, a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a colorant, a pH regulator, an antioxidant, a bacteriostatic agent or a buffer.

[0022] In a third aspect, the present application provides use of the probiotic agent for promoting calcium absorption according to the first aspect in the preparation of a product for preventing, improving or treating a disease caused by insufficient calcium absorption.

[0023] Preferably, the product further comprises an excipient.

[0024] The excipient comprises any one or a combination of at least two of an excipient, a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a colorant, a pH regulator, an antioxidant, a bacteriostatic agent or a buffer.

[0025] Compared with the prior art, the present application has the following beneficial effects:

[0026] The present application develops a brand-new probiotic compound mode, which is to compound Akkermansia muciniphila Akk11 strain and Bifidobacterium breve BBr16 strain. It is found that the two strains have potential interaction and can cooperate with each other to synergistically promote calcium absorption, which is specifically shown as follows: (1) the overall metabolic capacity of calcium can be promoted by improving intestinal health and enhancing the absorption capacity of intestinal wall; (2) the serum calcium level and phosphorus content can be promoted to restore normal electrolyte level; (3) the loss of calcium in bone can be effectively reduced, thereby stabilizing the structure and function of bone. BRIEF DESCRIPTION OF DRAWINGS

[0027] Fig. 1 is a graph of the body weight statistics of rats in each group.

[0028] Fig. 2 is a graph of the calcium apparent absorption rate statistics of rats in each group.

[0029] Figure 3 is a statistical result graph of the calcium storage rate of each group of rats.

[0030] Figure 4 is a statistical result graph of the serum calcium level of each group of rats.

[0031] Figure 5 is a statistical result graph of the serum phosphorus level of each group of rats.

[0032] Figure 6 is a statistical result graph of the serum alkaline phosphatase content of each group of rats.

[0033] Figure 7 is a statistical result graph of the bone calcium level of each group of rats. DETAILED DESCRIPTION

[0034] The technical solutions of the present application will be further illustrated by specific embodiments. Those skilled in the art should understand that the embodiments are only to help understand the present application and should not be regarded as specific limitations of the present application.

[0035] The medium formula involved in the following examples is as follows:

[0036] MRS medium: 10 g / L of proteose peptone, 10 g / L of beef extract, 20 g / L of glucose, 2 g / L of sodium acetate, 5 g / L of yeast powder, 2 g / L of diammonium hydrogen citrate, 2.6 g / L of K2PO4·3H2O, 0.1 g / L of MgSO4·7H2O, 0.05 g / L of MnSO4, 1 mL / L of polysorbate 80 (Tween 80), 0.5 g / L of cysteine hydrochloride.

[0037] The classification name of the Akk11 strain involved in the following examples is Akkermansia muciniphila Akk11, which is preserved in China Center for Type Culture Collection on January 15, 2024, with the preservation number of CCTCC NO: M2024119, and the address is: China. Wuhan. Wuhan University.

[0038] The classification name of the BBr16 strain involved in the following examples is Bifidobacterium breve, which is preserved in China General Microbiological Culture Collection Center on March 7, 2022, with the preservation number of CGMCC No. 24471, and the address is: Beijing City, Chaoyang District, Beichen West Road No. 1, No. 3.

[0039] The preparation method of the bacterial suspension is as follows: inoculate the required strain into a liquid culture medium, activate at 37°C for 24h, continuously activate twice, and obtain an activation liquid; inoculate the activation liquid into a liquid culture medium at an inoculation amount of 5% (v / v), culture at 37°C for 24h, and obtain a bacterial liquid; centrifuge the bacterial liquid at 5000rpm for 10min at 4°C, filter, obtain bacterial bodies, resuspend the bacterial bodies with a PBS solution, and obtain the bacterial suspension.

[0040] The test result data is statistically analyzed using R language ggplot2. Compared with the control group, ### represents p<0.001, ## represents p<0.01, and # represents p<0.05; compared with the model group, *** represents p<0.001, ** represents p<0.01, * represents p<0.05, and NS. represents no significant difference.

[0041] Example

[0042] This example investigates the symptom improvement ability of the strain on the calcium deficiency model of rats:

[0043] (1) Test animals: 4-week-old SD rats (56, body weight 75-85g, purchased from Shanghai Experimental Animal Center), half male and half female, these mice were raised in a controlled environment, the room temperature was maintained at 20-24°C, the humidity was 50-60%, and they followed a 12h light / dark cycle. They can eat and drink water at will. All experimental procedures involving mice comply with the animal care and use ethical guidelines set by the Shanghai Experimental Animal Care and Animal Experiment Center.

[0044] (2) Feed formula: corn starch 39.8%, casein 19.2%, malt dextrin 13.5%, sucrose 10%, cellulose 5%, soybean oil 7.5%, mineral mixture 3.5% (calcium carbonate as calcium source, the calcium content of the general feed is set to 5000mg / kg, and the calcium content of the calcium deficiency feed is reduced to 1000mg / kg), mixed vitamins 1%, DL-methionine 0.25%, and choline tartrate 0.25%.

[0045] (3) Animal grouping: after adaptive feeding for 1 week, the rats were randomly assigned into 7 groups: control group (CTL), calcium deficiency model group (MC group), Akk11 strain group (Akk11 group, referred to as S1 group), BBr16 strain group (BBr16 group, referred to as S2 group), commercially available mucinophilic Akkermansia group (BNCC341917 group, referred to as S3 group), composite bacteria group 1 (Akk11+BBr16 group, the live bacteria number ratio is 2:1, referred to as S4 group), and composite bacteria group 2 (BNCC341917+BBr16 group, the live bacteria number ratio is 2:1, referred to as S5 group).

[0046] (4) Animal modeling and intervention method:

[0047] CTL group was fed with normal feed + normal purified water, MC group was fed with calcium-deficient feed + normal purified water, S1-S5 group was fed with calcium-deficient feed + probiotic bacterial liquid (dose 10 8 CFU / day / each), and continuously fed for 8 weeks.

[0048] (5) Index analysis:

[0049] (5.1) Effect on rat weight:

[0050] The body weight of rats in each group was measured at the end of the experiment, and the results are shown in Figure 1. As can be seen from the figure, compared with the CTL group, the body weight of rats in the MC group decreased significantly; however, compared with the MC group, the body weight of calcium-deficient mice after probiotic intervention reversed and tended to be normal, indicating that the probiotic agent involved in the present application helps to alleviate the weight loss caused by calcium deficiency, especially the S4 group.

[0051] (5.2) Calcium metabolism balance study:

[0052] Three days before the end of the intervention, rats in each group were placed in a metabolic cage, and their food intake was recorded daily, while fecal and urine samples were collected. After digestion, the calcium content in the samples was determined by flame atomic absorption spectrometry. By calculating the amount of calcium ingested (Vi), the amount of calcium in the feces (Vf) and the amount of calcium in the urine (Vu), the apparent absorption rate of calcium and the calcium storage rate can be obtained. According to the amount of ingested calcium, fecal calcium and urinary calcium, the apparent absorption value of calcium (Vab) and the calcium storage value (Vac) can be obtained.

[0053] Calcium apparent absorption rate (%) = (Vab / Vi) x 100 = [(Vi-Vf) / Vi] x 100;

[0054] Calcium storage rate (%) = (Vac / Vi) x 100 = [(Vi-Vf-Vu) / Vi] x 100;

[0055] The results are shown in Figures 2 and 3. As can be seen from the figures, rats in the MC group showed significant changes in calcium metabolism compared with the CTL group, with significant increases in calcium apparent absorption rate and calcium storage rate, indicating that under the pressure of calcium deficiency, the regulatory mechanism in the body of rats was activated to optimize and improve the absorption of calcium and its retention in the body. This enhanced absorption efficiency and improved calcium storage strategy may be a biological adaptive response to maintain physiological calcium demand and support key functions; after probiotic intervention, the apparent absorption rate and storage rate of calcium were observed to gradually return to near normal levels, indicating that the probiotic agent involved in the present application can promote the overall metabolism of calcium by improving intestinal health and enhancing the absorption capacity of the intestinal wall, especially the S4 group.

[0056] (5.3) Blood biochemical index determination:

[0057] 200 μL of blood was collected from each rat after the intervention, and the collection time was 10-11 am. After centrifugation at 1200 x g for 10 min, the serum was extracted and stored at -80°C. The serum calcium level, serum phosphorus level and serum alkaline phosphatase content were quantitatively determined using an enzyme-linked immunosorbent assay kit (Wuhan Pure Biotechnology Co., Ltd.) according to the manufacturer's instructions.

[0058] The results are shown in Figures 4-6. As can be seen from the figures, compared with the CTL group, the serum calcium level and serum phosphorus level of the rats in the MC group were significantly reduced, which indicated that the calcium deficiency state had a negative impact on mineral metabolism, resulting in a decrease in the concentration of these key electrolytes in the blood. In addition, the serum alkaline phosphatase content of the rats in the MC group was significantly increased, which was usually related to the enhancement of bone metabolic activity, reflecting the attempt of the skeleton to release more minerals to compensate for the loss in the blood. After the intervention of probiotics, the serum calcium level and serum phosphorus level of the calcium-deficient rats were significantly increased, indicating that the intervention of the probiotic agent of the present application helped to restore normal electrolyte levels, and better utilization of minerals by improving intestinal absorption function. In addition, the serum alkaline phosphatase content of the calcium-deficient rats was significantly reduced, indicating that the bone metabolism process tended to be stable, and the skeleton no longer needed to excessively mobilize bone minerals to maintain the mineral balance in the blood, especially the S4 group, which had the most obvious effect.

[0059] (5.4) Rat femur index determination:

[0060] After the intervention, the muscles and connective tissues of the right femur of the rats were thoroughly stripped, and the treated femur was dried at 95°C for dry ashing treatment. The specific operation was as follows: ashing at 600°C for 8 hours, and then dissolving the ashed sample with 6 mol / L hydrochloric acid, and then determining the calcium content in the bone by atomic absorption spectrometry.

[0061] The results are shown in Figure 7. As can be seen from the figure, compared with the CTL group, the bone calcium level of the rats in the MC group was significantly decreased, which was a typical manifestation of the direct impact of calcium deficiency on bone mineral density. This decrease was probably due to the loss of calcium in the bone caused by insufficient calcium intake, which in turn affected the structure and function of the bone. However, after the intervention of the probiotics of the present application, the bone calcium content of the calcium-deficient rats was significantly increased, which indicated that the addition of probiotics had a positive effect on improving bone health, especially the S4 group, which could improve the health status of the intestinal tract and enhance the absorption function of the intestinal wall, thereby improving the bioavailability of calcium. This enabled more calcium to be absorbed into the blood for use in the body, including for the mineralization process of the bone, thereby improving the overall balance of calcium, helping to slow down the rate of bone loss, and enhancing the structural strength and density of the bone.

[0062] The applicant declares that the technical solutions of the present application are illustrated by the above-mentioned embodiments, but the present application is not limited to the above-mentioned embodiments, that is, the present application does not mean that it must rely on the above-mentioned embodiments to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc. fall within the protection scope and disclosure scope of the present application.

[0063] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above-mentioned embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application.

[0064] In addition, it should be noted that each specific technical feature described in the above-mentioned specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination manners.

Claims

1. A probiotic agent for promoting calcium absorption comprising an Akk11 strain, wherein the Akk11 strain comprises Akkermansia muciniphila Akk11 strain with a preservation number of CCTCC NO: M2024119 and Bifidobacterium breve BBr16 strain with a preservation number of CGMCC No. 24471.

2. The calcium absorption promoting probiotic agent according to claim 1, wherein, The ratio of viable cell count of the Akk11 strain to the BBr16 strain is 1:10-10:

1.

3. The calcium absorption promoting probiotic agent according to claim 1, wherein, In the probiotic agent, the total number of viable bacteria is not less than 1 x 10 8 CFU / mL or 1 x 10 8 CFU / g.

4. The calcium-absorption-promoting probiotic agent according to claim 1, wherein The dosage form of the probiotic agent comprises a solution, a lyophilized powder, a capsule, a tablet or a granule.

5. The calcium absorption promoting probiotic agent of claim 1, wherein, The probiotic agent further comprises a lyophilization protective agent and / or an auxiliary additive.

6. The calcium absorption promoting probiotic agent of claim 5, wherein, The lyophilization protective agent comprises any one or a combination of at least two of skim milk, sucrose, lactose, trehalose, dextran, gelatin, dextrin, acacia gum, sodium alginate, polyvinylpyrrolidone, sorbitol or xylitol.

7. The calcium absorption promoting probiotic agent of claim 5, wherein, The auxiliary additive comprises any one or a combination of at least two of inulin, fructooligosaccharide, xylooligosaccharide, galactooligosaccharide, isomaltooligosaccharide, soybean oligosaccharide, spirulina, arthrospira, grifola frondosa polysaccharide, stachyose, polydextrose, α-lactalbumin or lactoferrin. 8.Use of the probiotic agent for promoting calcium absorption according to any one of claims 1-7 in the preparation of a product for preventing or improving insufficient calcium absorption. 9.Use of the probiotic agent for promoting calcium absorption according to any one of claims 1-7 in the preparation of a product for preventing, improving or treating a disease caused by insufficient calcium absorption.

10. Use according to claim 9, wherein, The product further comprises an excipient; The excipient comprises any one or a combination of at least two of an excipient, a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a colorant, a pH regulator, an antioxidant, a bacteriostatic agent or a buffer.

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