Application of bifidobacterium longum subsp. Longum i772 in products for promoting bone health

Abstract

The invention discloses application of bifidobacterium longum subsp. Longum i772 in a product for promoting bone health, and belongs to the field of microbial engineering. The effective components of the product for promoting bone health comprise live bifidobacterium longum subsp. Longum i772 and / or inactivated bifidobacterium longum subsp. Longum i772. The product provided by the invention can effectively promote bone health no matter whether the product contains viable bacteria or inactivated bacteria, and experimental data shows that the probiotic agent containing the bifidobacterium longum subsp. Longum i772 can significantly improve the bone density, bone volume fraction and bone trabecula thickness in a female rat osteoporosis model, and has a good application prospect. The bone trabecula spacing in the female rat osteoporosis model is reduced; meanwhile, the strain has an intestinal colonization effect and can continuously play a role in promoting bone health in the intestinal tract, and the trouble of daily medication can be reduced. The food provided by the invention is suitable for promoting bone health and preventing and / or improving osteoporosis.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial engineering, and relates to a new application of Bifidobacterium longum subspecies longum i772, and specifically to the application of Bifidobacterium longum subspecies longum i772 in products for promoting bone health. Background Art

[0002] In recent years, bone health problems in my country have become increasingly prominent, and the corresponding medical treatment and care have consumed a lot of manpower, material resources, and financial resources, causing a heavy burden on families and society. According to the "Research Report on the Current Status of Height Management of Chinese Children" released by the China Children and Teenagers' Foundation, 30.8% of children are of medium to low height or have growth retardation, which is equivalent to 1 in every 3 children having height problems. 54.2% of children's current height level does not reach the genetic height. The incidence of knee deformity in children is about 5%. In 2019, the disease burden of low back pain in children and adolescents aged 5 to 14 in my country ranked tenth; the number of adolescent scoliosis patients exceeded 5 million, and increased by 300,000 people each year. At the same time, about 200,000 people in the middle-aged and elderly population in my country suffer from hip fractures each year, of which about 30% die as a result. The above data also reveal that the existing bone health preparations are insufficient in efficacy and function, and how to effectively promote bone health has become a major issue.

[0003] Conventional calcium supplementation methods have always focused on increasing calcium intake and optimizing the form of calcium, thereby facilitating the body's absorption of calcium. However, there is controversy over whether calcium can be truly used by the body for bone growth and bone strengthening after absorption. Some scholars believe that calcium absorption only increases the concentration of calcium in the blood to prevent calcium loss in the bones, but does not promote bone strength and growth. How to convert absorbed calcium into calcium in the bones is the key to the preparation of preparations that promote bone health.

[0004] Bifidobacterium longum subsp. longum is a type of Gram-positive, strictly anaerobic, non-spore-forming microorganism. Its same strains often have certain effects in improving motor ability, relieving constipation, and improving cognitive ability, and have good application prospects. However, there is currently no application of Bifidobacterium longum subsp. longum i772 and its inactivated bacteria in promoting bone health. Summary of the invention

[0005] The purpose of the present invention is to provide an application of Bifidobacterium longum subspecies longum i772 in a product that promotes bone health, so as to solve the problem that the current conventional calcium supplementation methods cannot convert the absorbed calcium into calcium in the bones, and thus cannot promote bone health well, while broadening the application scope of Bifidobacterium longum subspecies longum i772.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is: A use of Bifidobacterium longum subspecies longum i772 in a product for promoting bone health, wherein the active ingredient of the product for promoting bone health comprises live bacteria of Bifidobacterium longum subspecies longum i772 and / or inactivated bacteria thereof.

[0007] Among them, Bifidobacterium longum subspecies longum i772 (Latin name: Bifidobacterium longum subsp.longum ), whose strain was deposited in the General Microbiological Center of China Microbiological Culture Collection Committee on May 20, 2020, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.19856. The strain was first disclosed in the Chinese invention patent with publication number CN112458015B and the name "Bifidobacterium longum subsp. longum i772, its isolation and purification method and application".

[0008] As a limitation, the product includes a probiotic, a dairy product or a health food.

[0009] As a further limitation, in the product, the amount of live bacteria of Bifidobacterium longum subspecies longum i772 or the amount of live bacteria before inactivation thereof is 1×10 8 -2x10 10 CFU / g.

[0010] As another limitation, promoting bone health includes preventing and / or improving osteoporosis.

[0011] The present invention also provides milk, comprising live bacteria of Bifidobacterium longum subspecies longum i772 and / or inactivated bacteria thereof.

[0012] The present invention also provides a milk powder comprising live bacteria of Bifidobacterium longum subspecies longum i772 and / or inactivated bacteria thereof.

[0013] Due to the adoption of the above technical solution, the present invention has the following technical advances compared with the prior art: ① The product containing Bifidobacterium longum subspecies longum i772 provided by the present invention can effectively promote bone health. According to experimental data, the probiotic containing Bifidobacterium longum subspecies longum i772 can significantly increase the bone density, bone volume fraction and trabecular thickness in the osteoporosis model of female rats, and significantly reduce the trabecular spacing in the osteoporosis model of female rats; ② The product containing Bifidobacterium longum subspecies longum i772 provided by the present invention can effectively improve osteoporosis even if it is an inactivated bacterium of Bifidobacterium longum subspecies longum i772, and the inactivated bacterium agent has good stability, will not produce post-acid problems after fermentation, and does not need to consider the maintenance of the number of live bacteria; ③ The product containing Bifidobacterium longum subspecies longum i772 provided by the present invention has a strain that has an intestinal colonization effect and can continuously exert its effect of promoting bone health in the intestine, thereby reducing the trouble of daily medication. DETAILED DESCRIPTION

[0014] The present invention is further described in detail below by specific examples. It should be understood that the described examples are only used to explain the present invention, and are not intended to limit the present invention.

[0015] Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art.

[0016] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0017] In the following embodiments: Bifidobacterium longum subspecies longum i772, the strain was deposited in the China General Microbiological Culture Collection Center on May 20, 2020, with the deposit number CGMCC NO.19856, and the Latin name is Bifidobacterium longum subsp.longum ; Bifidobacterium longum subsp. longum JM-0847, which is deposited in the strain collection laboratory of the applicant and can be obtained through purchase or other channels; Bifidobacterium longum subsp. longum JM-1107, the bacterial strain is deposited in the strain collection laboratory of the applicant and can be obtained through purchase and other channels.

[0018] In the following embodiments: The formula of high-density culture medium is: soy peptone 10g / L, beef extract 10g / L, yeast extract powder 5g / L, dihydrogen citrate 2g / L, anhydrous sodium acetate 5g / L, dipotassium hydrogen phosphate 2g / L, Tween 80 1g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.054g / L, calcium carbonate 5g / L, anhydrous glucose 30g / L, L-cysteine ​​hydrochloride 5g / L, pH value is 7.2; The formula of the freeze-drying protective agent is: in weight percentage, 12% skimmed milk powder, 1% trehalose, 3% glycerol, 1% sodium glutamate, and the pH value is 5.6.

[0019] Example 1 This embodiment provides a probiotic containing live bacteria of Bifidobacterium longum subspecies longum i772, and the preparation method thereof is as follows: Bifidobacterium longum subspecies longum i772 was inoculated in a high-density culture medium at an inoculation rate of 2% by mass, and cultured for 20 hours at an initial pH of 7.2 and a temperature of 39°C, then the culture was terminated, and low-temperature high-speed centrifugation was performed to obtain bacterial sludge, and the bacterial sludge and a lyophilization protective agent were mixed at a weight ratio of 1:2, and freeze-dried bacterial cells were prepared by freeze-drying process; The parameters of the freeze-drying process were set as follows: the temperature was lowered to -40°C at a cooling rate of 4°C / min and maintained for 24 minutes to obtain freeze-dried bacteria; The freeze-dried bacteria were put into a grinder for pulverization. The pulverized product was sieved through a 40-mesh sieve to obtain a probiotic agent containing live bacteria of Bifidobacterium longum subspecies i772. After testing, the live bacteria amount of Bifidobacterium longum subspecies i772 in the bacterial powder was 1.6 x 10 10 CFU / g.

[0020] The following is a study on the intestinal colonization effect of Bifidobacterium longum subspecies longum i772 through cell adhesion experiments: Bifidobacterium longum subsp. longum i772 was inoculated into MRS liquid medium at a mass fraction of 2% and activated for 3 generations. The cells were cultured at 37°C for 20 h, centrifuged, and the cells were collected and washed twice with sterile PBS buffer, resuspended in DMEM medium, and the cell concentration was adjusted to 10 7 CFU / mL for later use; Caco-2 cells were directly plated in a 24-well plate and the concentration of the cell suspension was adjusted to 10 5 / mL, put it in a CO2 incubator, observe until the cells are fully grown, and wash it twice with sterile PBS buffer to remove excess non-adherent Caco-2 cells; The prepared bacterial suspension was added dropwise to a 24-well plate (320 μL of bacterial suspension was added to each well), placed in a CO2 incubator, incubated at 37°C for 3 h, washed 3 times with sterile PBS buffer to remove unbound bacteria, 1 mL of 1% (v / v) Triton×100 was added to each well, and the plates were thoroughly beaten to separate the adherent bacteria from the cells. After gradient dilution, the plates were placed on an MRS plate for culture and counting. The results are shown in Table 1: Table 1 Bacterial cell adhesion results Adhesion rate calculation: Adhesion rate = initial addition amount / amount after 3h adhesion and elution As shown in Table 1, the intestinal epithelial cell adhesion ability of Bifidobacterium longum subspecies longum i772 is 2.2%, and it can colonize well in the intestine.

[0021] Example 2 This embodiment provides a milk containing live bacteria of Bifidobacterium longum subspecies longum i772, wherein live bacteria powder of Bifidobacterium longum subspecies longum i772 is added to the milk. After testing, the live bacteria amount of Bifidobacterium longum subspecies longum i772 in the bacteria powder is 1×10 8 CFU / g.

[0022] Example 3 This embodiment provides a milk powder containing live bacteria of Bifidobacterium longum subspecies longum i772. Live bacteria powder of Bifidobacterium longum subspecies longum i772 is added to the milk powder. After testing, the live bacteria amount of Bifidobacterium longum subspecies longum i772 in the bacteria powder is 2×10 10 CFU / g.

[0023] Example 4 This embodiment provides a probiotic containing inactivated bacteria of Bifidobacterium longum subspecies longum i772, and the preparation method thereof is as follows: According to the inoculum amount of 2% by mass, Bifidobacterium longum subspecies longum i772 was inoculated in a high-density culture medium. Under the conditions of initial pH value of 7.2 and temperature of 39°C, the culture was cultured for 20 hours, and the culture was terminated. The culture was centrifuged at low temperature and high speed to obtain bacterial mud. The live bacterial count of Bifidobacterium longum subspecies longum i772 in the bacterial mud was 1.6×10 10 CFU / g, the bacterial sludge was heated at 95°C for 30 min to obtain inactivated bacterial sludge, the inactivated bacterial sludge and the lyophilization protective agent were mixed at a weight ratio of 1:2, and freeze-dried bacterial cells were prepared by freeze-drying process; The parameters of the freeze-drying process were set as follows: the temperature was lowered to -40°C at a cooling rate of 4°C / min and maintained for 24 minutes to obtain freeze-dried bacteria; The freeze-dried bacteria were put into a grinder for grinding, and the ground products were passed through a 40-mesh sieve to obtain a probiotic preparation of inactivated bacteria of Bifidobacterium longum subspecies longum i772, wherein the amount of live bacteria in the inactivated probiotic preparation was less than 3000 CFU / g.

[0024] Example 5 This embodiment provides a milk containing inactivated bacteria of Bifidobacterium longum subspecies longum i772, wherein inactivated bacteria powder of Bifidobacterium longum subspecies longum i772 is added to the milk. After testing, the amount of live bacteria of Bifidobacterium longum subspecies longum i772 before inactivation is 1×10 8 CFU / g.

[0025] Example 6 This embodiment provides a milk powder containing inactivated bacteria of Bifidobacterium longum subspecies longum i772. Inactivated bacteria powder of Bifidobacterium longum subspecies longum i772 is added to the milk powder. After testing, the amount of live bacteria of Bifidobacterium longum subspecies longum i772 before inactivation is 2×10 10 CFU / g.

[0026] Example 7 This embodiment provides the use of a probiotic containing Bifidobacterium longum subspecies longum i772 that promotes bone health, uses the probiotic to improve osteoporosis, and provides a control experiment, which specifically includes the following steps: S1. Preparation of experimental materials 1) Sample preparation Bifidobacterium longum subspecies longum i772 live bacteria sample: Take the probiotic agent of Bifidobacterium longum subspecies longum i772 live bacteria prepared in Example 1, and use physiological saline to prepare a bacterial powder solution, which is recorded as active i772; Bifidobacterium longum subspecies longum i772 inactivated bacteria sample: Take the probiotic agent of Bifidobacterium longum subspecies longum i772 inactivated bacteria prepared in Example 4, and use physiological saline to prepare a bacterial powder solution, which is recorded as inactivated i772; Bifidobacterium longum subspecies longum JM-0847 live bacteria sample: Bifidobacterium longum subspecies longum JM-0847 was used to prepare a probiotic agent according to the method of Example 1 (live bacteria amount was 1.6×10 10 CFU / g), and the bacterial powder solution was prepared with normal saline, which was recorded as activity 0847; Bifidobacterium longum subspecies longum JM-0847 inactivated bacteria sample: Bifidobacterium longum subspecies longum JM-0847 was used to prepare a probiotic according to the method of Example 4, and a bacterial powder solution was prepared with physiological saline, which was recorded as inactivated 0847; Bifidobacterium longum subspecies longum JM-1107 live bacteria sample: Bifidobacterium longum subspecies longum JM-1107 was used to prepare a probiotic agent according to the method of Example 1 (live bacteria amount was 1.6×10 10 CFU / g), using physiological saline to prepare a bacterial powder solution, recorded as activity 1107; Inactivated bacteria sample of Bifidobacterium longum subspecies longum JM-1107: Bifidobacterium longum subspecies longum JM-1107 was used to prepare a probiotic according to the method of Example 4, and a bacterial powder solution was prepared with physiological saline, which was recorded as inactivated 1107; Anesthetic (5% chloral hydrate): Dissolve 5 g of chloral hydrate in about 60 ml of normal saline, add normal saline to make up to 100 ml, and store at room temperature away from light.

[0027] 2) Experimental animals Sprague Dawley rats (SD rats), female, 10-12 weeks old.

[0028] The experimental animals were provided by Sbefor (Suzhou) Biological Co., Ltd. (China), certificate: SCXK(Su)2022-0006.

[0029] 3) Instruments, consumables and reagents Disposable 5 ml syringe (Shanghai Kangdeli, China), electronic analytical balance Precisa12A (Plissex, Switzerland), depyrogenation ultrapure water system Millipo3150 (Millipore, USA), three-section rat gavage needle (Zhongke Life Science, China), electric constant temperature water bath HW-SY11-K P2 (Beijing Changfeng Instrument Co., Ltd., China), 1.5 ml centrifuge tube (Wuxi Nice Life Science Co., Ltd., China), ultra-low temperature refrigerator Sanyo-382AT (Sanyo Company, Japan), 6-0 absorbable suture (Shanghai Jinhuan Medical Instrument Co., Ltd., China), Micro-CT scanner Skyscan1276 (Bruker, USA).

[0030] 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), and alendronate sodium (A115345-25g, Shanghai Aladdin Biochemical Technology Co., Ltd., China).

[0031] S2. Experimental methods 1) Animal grouping and osteoporosis modeling After 7 days of adaptive feeding, 90 SD rats were divided into 9 groups: blank control group, model control group, positive drug group, inactivated control group (including inactivated i772 group, inactivated 0847 group, inactivated 1107 group), experimental group (including active i772 group, active 0847 group, active 1107 group), with 10 rats in each group.

[0032] Except for the blank control group, all SD rats underwent ovariectomy: after anesthesia, the ventral skin was prepared, hair removed, and disinfected. A 2-cm incision was made in the midline of the abdomen using sterile surgical scissors to separate and expose the ovaries, and then the ovaries were removed after ligation with sutures. After the operation, the uterus was returned to the abdominal cavity, the muscles and skin were sutured layer by layer, and the wound was disinfected with iodine and placed on a heating pad for normal feeding after waking up.

[0033] The blank control group underwent sham surgery: only anesthesia was performed to make the incision, and the ovaries were exposed but not ligated and removed. The ovaries were placed back into the abdominal cavity and the wounds were sutured. The mice were fed normally after waking up.

[0034] 2) Sample administration by intragastric gavage The SD rats in each group were gavaged daily starting the day after surgery. The SD rats in the blank control group and the model control group were given an equal volume of normal saline daily; the SD rats in the positive drug group were given the same volume of 40 mg / kg alendronate sodium (solvent was normal saline) once a day for 28 consecutive days.

[0035] The SD rats in the experimental group were gavaged with equal volumes of active i772, active 0847 or active 1107 daily (the amount of live bacteria ingested by the rats was 1.135x10 8 CFU, the dosage is converted to human body, which is 5x10 per day 9 CFU), once a day for 28 consecutive days.

[0036] The SD rats in the inactivated control group were gavaged with an equal volume of inactivated i772, inactivated 0847, or inactivated 1107 daily (the amount of inactivated bacteria ingested by the rats was 1.135 x 10 8 CFU, the dosage is converted to human body, which is 5x10 per day 9 CFU), once a day for 28 consecutive days.

[0037] 3) Femoral sampling and scanning 24 hours after the last intragastric administration, the femurs of SD rats in each group were taken, the muscles and connective tissues around the femurs were removed, and the femurs were fixed and preserved in paraformaldehyde fixative. The lower femur was taken out from the fixative, rinsed with PBS buffer for 3 times, wiped dry, and wrapped with a sealing film to obtain the lower femur sample; Start the Micro-CT scanner Skyscan1276 program, initialize and preheat, open the sample chamber, put the lower femur sample along the length direction of the semi-cylindrical column of the sample chamber, raise the position of the lower femur sample with the help of foam and paper to ensure that it is at the center of the semicircle, and fix it with 3M tape, close the sample chamber door, and scan with a scanning resolution of 18 μm; After scanning, CTvox software was used for 3D reconstruction, and the femoral morphology was exported. CTAn software was used to analyze the bone density, bone tissue volume / total tissue volume (bone volume fraction), trabecular thickness, and trabecular separation / spacing in the femoral condyle area. The results are shown in Table 2. The meanings of each data are as follows: BMD: bone density, which reflects the condition of bones. A decrease in the index indicates osteoporosis. BV / TV: Bone volume fraction, which reflects bone metabolism. A decrease in the index means that bone decomposition is greater than synthesis, and bone mass decreases. Tb.Th: trabecular thickness, which reflects the morphological structure of bones. A decrease in the index indicates that the pores in the bone become larger. Tb.Sp: Trabecular spacing, this index reflects the morphological structure of bones. An increase in the index means more pores in the bone.

[0038] S3. Experimental Results Table 2 Experimental results Note: * indicates that there is a significant difference with the model control group, p < 0.05; *** indicates that there is a very significant difference with the model control group, p < 0.001; Different lowercase letters indicate significant differences among experimental groups, p < 0.05.

[0039] As shown in Table 2, the probiotics prepared from the live bacteria or inactivated bacteria of Bifidobacterium longum subspecies longum i772 all have the effect of improving osteoporosis, and compared with JM-0847 and JM-1107 of the same genus and species, the effect of i772 on improving osteoporosis is significantly different. Specifically, compared with the model control group, the live bacteria or inactivated bacteria of i772 can significantly inhibit the decrease of bone density, bone volume fraction and trabecular thickness in the rat osteoporosis model, and significantly increase the trabecular spacing in the rat osteoporosis model; while compared with the model control group, the live bacteria or inactivated bacteria of 0847 and the inactivated bacteria of 1107 can not significantly inhibit the decrease of bone density, bone volume fraction and trabecular thickness in the rat osteoporosis model, nor can they significantly increase the trabecular spacing. Compared with groups 0847 and 1107, group i772 showed significant differences in inhibiting the decrease of bone density, bone volume fraction and trabecular thickness in the rat osteoporosis model, and increasing the trabecular spacing in the rat osteoporosis model.

Claims

1. A use of Bifidobacterium longum subspecies longum i772 in a product for promoting bone health, characterized in that: The active ingredients of the product for promoting bone health include live bacteria of Bifidobacterium longum subspecies longum i772 and / or inactivated bacteria thereof.

2. The use according to claim 1, characterized in that: The products include probiotics, dairy products or health foods.

3. The use according to claim 2, characterized in that: In the product, the amount of live bacteria of Bifidobacterium longum subspecies longum i772 or the amount of live bacteria before inactivation thereof is 1×10 8 -2x10 10 CFU / g.

4. The use according to any one of claims 1 to 3, characterized in that: The promotion of bone health includes preventing and / or improving osteoporosis.

5. A milk, characterized in that: The invention comprises live bacteria of Bifidobacterium longum subspecies longum i772 and / or inactivated bacteria thereof.

6. A milk powder, characterized in that: The invention comprises live bacteria of Bifidobacterium longum subspecies longum i772 and / or inactivated bacteria thereof.

Images