Bifidobacterium breve and preparation for promoting bone development and application thereof

Abstract

The present invention belongs to the field of microbial technology, and specifically relates to a Bifidobacterium breve and a preparation and application for promoting bone development. The preservation unit of the Bifidobacterium breve is the China Center for Type Culture Collection; the preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; the preservation date is July 10, 2024, and the preservation number is CCTCC NO: M20241534; the taxonomic name of the Bifidobacterium breve is Bifidobacterium breve YBT36 ( Bifidobacterium breve Compared with the original strain, the Bifidobacterium breve and the microcapsule preparation have a good effect on promoting bone development and are tolerant to aerobic culture conditions.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a Bifidobacterium breve and a preparation for promoting bone development and application thereof. Background Art

[0002] Bifidobacterium is a kind of beneficial microorganism in the intestines of mammals, which helps digestion and absorption, and has health-promoting functional activities such as regulating immune function and inhibiting the growth of intestinal pathogens. Properly increasing the intake of bifidobacteria in the diet has been proven to improve intestinal health, enhance immune function, and promote the absorption of nutrients.

[0003] Bone development is a complex biological process that involves the formation, growth, and remodeling of bones. Many drugs used to treat bone-related diseases, such as osteoporosis, can cause serious side effects. Bisphosphonates (such as alendronic acid and etidronate) increase bone density by inhibiting bone resorption, but they can cause esophageal inflammation, gastrointestinal upset, and even rare cases of osteonecrosis of the jaw and increased risk of fractures. Selective estrogen receptor modulators (such as raloxifene) can mimic the protective effects of estrogen on bones, but may increase the risk of blood clots and hot flashes. In contrast, as a natural biological product, probiotics are more mild in their activities in the body, and some probiotics can promote bone development. For example, the literature "Human breastmilk-derived Bifidobacterium longum subsp. infantisCCFM1269 regulates bone formation by the GH / IGF axis through PI3K / AKTpathway" (GUT ICROBES, 2024, 16 (1): 2290344) reported that a longum subsp. infantis CCFM1269 can promote bone development. Therefore, the demand for probiotic products that promote bone development is relatively strong.

[0004] Bifidobacterium has a variety of health-promoting effects, but it belongs to anaerobic bacteria, is sensitive to oxygen and dissolved oxygen, and often needs to use expensive anaerobic fermentation equipment in actual fermentation and culture process, and the operation process is complicated and inefficient. In addition, under anaerobic conditions, harmful bacteria represented by sunshine bacteria may also breed, posing a threat to food safety. In addition, the survival rate of bifidobacteria in microecological preparations is also affected by oxygen, because it is sensitive to oxygen, so that the shelf life and effectiveness of such preparations are limited. Therefore, how to effectively improve the tolerance of bifidobacteria to oxygen, so as to improve its fermentation efficiency, product quality and effectiveness, persistence, become a problem that needs to be solved urgently in this field. Document CN112980719A discloses a method for oxygen-resistant domestication of animal bifidobacteria, requires repeated hydrogen peroxide treatment and repeated stirring, anaerobic circulation, required operation is complicated, and the strain is not Bifidobacterium breve. Summary of the invention

[0005] In view of the above technical problems, the object of the present invention is to provide a Bifidobacterium breve, and to provide a product and application containing the Bifidobacterium breve for promoting bone development.

[0006] First, the present invention provides a breve bifidobacterium, wherein the breve bifidobacterium is named as Bifidobacterium breve YBT36, and its Latin name is Bifidobacterium breve YBT36. The preservation unit of the Bifidobacterium breve is: China Center for Type Culture Collection; the preservation address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; the preservation date is: July 10, 2024, and the preservation number is: CCTCC NO: M20241534. The taxonomic name of the Bifidobacterium breve is Bifidobacterium breve YBT36 ( Bifidobacterium breve YBT36).

[0007] Secondly, the invention provides a preparation for promoting bone development, which contains the aforementioned Bifidobacterium breve (i.e. Bifidobacterium breve YBT36).

[0008] Preferably, the preparation for promoting bone development is a microcapsule preparation.

[0009] Preferably, the microcapsule wall material of the microcapsule preparation is any one of alginate, sodium alginate and calcium alginate.

[0010] Preferably, the microcapsule wall material of the microcapsule preparation is sodium alginate.

[0011] Preferably, the preparation method of the microcapsule preparation comprises the following steps:

[0012] S1: Take a suspension of oxygen-tolerant Bifidobacterium breve with a deposit number of CCTCC NO: M20241534, mix it with a sodium alginate solution with a weight volume concentration of 3g / 100mL at a volume ratio of 1:2.5, add calcium carbonate; then add soybean oil containing 1% Tween 80 by volume at a water / oil volume ratio of 1:1, and homogenize at 400xg for 15min to form a water-in-oil emulsion;

[0013] S2: Add glacial acetic acid to the emulsion, stir, and add CaCl 2 The solution is stirred, the precipitate is collected, and the oil phase is removed by washing to obtain microcapsules.

[0014] Preferably, in step S2, CaCl 2 The concentration of the solution is 0.8 mol / L.

[0015] Preferably, in step S2, the volume ratio of the emulsion to the glacial acetic acid is 1:1.

[0016] Preferably, in step S2, 0.85 g / 100 mL NaCl solution is used to wash and remove the oil phase.

[0017] The Bifidobacterium breve YBT36 microcapsule preparation prepared by the present invention can resist the acidic environment of gastrointestinal mucus and help the colonization of Bifidobacterium breve.

[0018] Again, the present invention provides the use of the aforementioned Bifidobacterium breve (Bifidobacterium breve YBT36) and the preparation for promoting bone development in the preparation of a product for promoting bone development.

[0019] The present invention has the following beneficial effects:

[0020] (1) The Bifidobacterium breve obtained by the present invention has better activity under aerobic conditions and can adapt to the conditions that are not strictly anaerobic in large-scale industrial fermentation.

[0021] (2) The Bifidobacterium breve microcapsule preparation obtained by the present invention can help Bifidobacterium breve resist the harsh environment of the gastrointestinal tract and promote the colonization of Bifidobacterium breve.

[0022] (3) The oxygen-resistant Bifidobacterium breve and microcapsule preparation obtained by the present invention have the advantage of promoting bone development, such as increasing the length of the femur and tibia of growing rats and increasing bone density. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The growth curves of Bifidobacterium breve with different treatments under aerobic conditions;

[0024] Figure 2 Comparison of morphological characteristics between Bifidobacterium breve YBT36 and the original strain;

[0025] Figure 3 Comparison of the levels of two bone markers in the serum of rats in different treatment groups. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with specific embodiments. The concentration of the strain suspension involved in the microcapsule preparation in the following examples is only used for comparative studies and is not considered to be a limitation on the preparation method. For those skilled in the art, the strain suspension of the required concentration can be prepared according to the dosage requirements to prepare the microcapsule preparation. For example, 10 6 CFU / mL ~10 16 CFU / mL of strain suspension.

[0027] Example 1 Acclimation and treatment of Bifidobacterium breve KLDS 2.0007

[0028] In the present invention, the Latin name of Bifidobacterium breve YBT36 is Bifidobacterium breve YBT36, deposited in China Center for Type Culture Collection; the deposit address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; the deposit date is: July 10, 2024, and the deposit number is: CCTCC NO: M20241534. The taxonomic name of this Bifidobacterium breve is Bifidobacterium breve YBT36 ( Bifidobacterium breve YBT36).

[0029] 1 Materials and methods

[0030] 1.1 Experimental Materials

[0031] 1.1.1 Experimental strains

[0032] The original strain Bifidobacterium breve KLDS 2.0007 was isolated from infant feces and preserved by the Industrial Microbial Culture Collection Center of the Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University.

[0033] 1.1.2 Experimental strain culture medium

[0034] Modified MRS medium (MRS): 12 g peptone, 6 g yeast extract, 6 g beef extract, 18 g glucose, 5 g sodium acetate, 2.15 g ammonium citrate, 1 g Tween, 0.58 g magnesium sulfate, 0.05 g manganese sulfate, 2 g dipotassium hydrogen phosphate, 0.5 g L-cysteine ​​salt, make up to 1 L with pure water, adjust the pH to 6.5, sterilize in a high-pressure steam autoclave at 121°C for 15 min for use.

[0035] Improved MRS medium (X-MRS) with added oxygen-tolerant acclimation promoter: 12 g peptone, 6 g yeast extract, 6 g beef extract, 18 g glucose, 5 g sodium acetate, 2.15 g ammonium citrate, 1 g Tween, 0.58 g magnesium sulfate, 0.05 g manganese sulfate, 2 g dipotassium hydrogen phosphate, 0.5 g L-cysteine ​​salt, 0.1 g zinc sulfate, 0.01 g sodium selenite. Use pure water to make up to 1 L, adjust the pH to 6.5, and sterilize in a high-pressure steam autoclave at 121°C for 15 min for later use.

[0036] 1.1.3 Experimental instruments and equipment

[0037] LDZF-50KB-II vertical pressure steam sterilizer; VD-1320 clean bench; DHP-9272 electric constant temperature incubator; Defendor AMW1000 anaerobic workstation; HPG-9245 oven; vortex oscillator; Lambda Bio35 UV-visible spectrophotometer; Model 680 microplate reader; PHS-25 pH meter; GL-21M high-speed refrigerated centrifuge; 0.22μm filter membrane. 1 / 10,000 electronic balance, Beijing Sartorius Instrument System Co., Ltd.; electric constant temperature water bath, Tianjin Lester Instrument Co., Ltd.; refrigerator, Qingdao Haier Co., Ltd.; vortex oscillator, Weiwosuoke Technology (Beijing) Co., Ltd.; centrifuge, Shanghai Chemical Machinery Company; ultra-low temperature refrigerator, ThermoFisher, USA.

[0038] 1.2 Experimental methods

[0039] 1.2.1 Activation of Bifidobacterium breve

[0040] Bifidobacterium breve KLDS 2.0007 at -80°C, 20% glycerol (vv −1 ) before the experiment. Before the experiment, the glycerol tube was taken out and dissolved naturally. The strain was inoculated in sterile modified MRS medium in a sterile clean bench. Incubated in a constant temperature incubator at 37°C for 24 hours. After incubation, Bifidobacterium breve KLDS 2.0007 was centrifuged at 2000rpm for 10min, the precipitate was harvested after removing the medium, and washed twice with sterile phosphate buffered saline (PBS). The washed precipitate was diluted with PBS to a cell concentration of 10 7 CFU / mL of Bifidobacterium breve KLDS 2.0007 suspension.

[0041] 1.2.2 Effects of zinc sulfate and sodium selenite on strain growth activity

[0042] The suspension of Bifidobacterium breve KLDS 2.0007 was inoculated into the modified MRS liquid culture medium and the modified MRS culture medium with oxygen tolerance acclimation promoter at a 5% inoculation rate by aseptic operation and cultured anaerobically at 37°C for 24 hours. The initial pH of the culture medium was adjusted to 5.9, and the viable bacteria were counted to determine the activity in log CFU / mL.

[0043] 1.2.3 Pre-culture of strains

[0044] From the obtained Bifidobacterium breve suspension (concentration 10 7 CFU / mL) and inoculate it into a new sterile modified MRS medium. Usually, the inoculation volume is 5% of the total volume of the medium. The inoculated medium is placed in a 37°C constant temperature anaerobic incubator for 24 hours. Ensure that the incubation conditions are constant to promote the optimal growth of Bifidobacterium breve. After the first generation of culture is completed, the cultured Bifidobacterium breve suspension is transferred to a new sterile modified MRS medium according to a 5% inoculation volume to continue the next generation of culture. Repeat this step until 6 to 8 generations of subculture are completed. After each subculture, incubate at 37°C for 24 hours. Before subculture, the viability is determined by live bacteria technology in log CFU / mL. The Bifidobacterium breve KLDS2.0007 with the highest strain activity was taken as the subsequent treatment strain and named Bifidobacterium breve YBT33.

[0045] 1.2.4 UV treatment

[0046] Take 3 ml of the last generation of strain pre-culture solution (see 1.2.3 of this example) and dilute to 10 8 CFU / mL, placed in a sterile culture dish with a diameter of 6 cm on a sterile operating table, placed the culture dish 28 cm away from the ultraviolet lamp (20W), and gently shake the culture dish so that the bacterial liquid can be evenly irradiated by the ultraviolet lamp. The irradiation time is 0s, 5s, 10s, 20s, 40s, 80s, and 160s. After irradiation, dilute and plate, culture at 37°C for 24h, count and calculate the lethality. From the strains with a lethality of 90-99.9%, the strain with the fastest growth rate and the largest colony morphology under the culture conditions of 1.2.3 was selected as the subsequent treatment strain, named Bifidobacterium breve YBT34.

[0047] 1.2.5 Oxygen tolerance acclimation

[0048] The strains obtained after UV treatment (see 1.2.4 of this embodiment) or pre-culture (see 1.2.3 of this embodiment) were inoculated into modified MRS medium containing an oxygen-tolerant acclimation promoter. The inoculated culture medium was placed in an anaerobic workstation, and the initial oxygen concentration was set to 1%. During the aerobic acclimation process, the oxygen concentration was gradually increased, and the amplitude of each increase was controlled at 2%, and each oxygenation was incubated for at least 24 hours to allow Bifidobacterium breve to adapt. After the oxygen concentration reached 21%, aerobic acclimation was terminated. The strain that was pre-cultured and aerobic-tolerant was named Bifidobacterium breve YBT35; the strain that was pre-cultured, UV-treated and aerobic-tolerant was named Bifidobacterium breve YBT36. The temperature should be maintained at 37°C during the entire aerobic process.

[0049] 1.2.6 Oxygen resistance test

[0050] Bifidobacterium breve YBT33, YBT34, YBT35, and YBT36 were inoculated into the modified MRS medium and incubated at 37°C under conventional aerobic conditions. The optical density (OD) of the samples at 600 nm was measured every 2 hours. 600 value).

[0051] 1.2.7 Microscopic observation of morphological characteristics before and after domestication

[0052] Take a clean slide, mark the front and back of one side with a marker, and then drop a small drop of distilled water in the center of the other side of the slide. Sterilize the inoculation loop over the flame, wait for it to cool, dip a little bacterial solution from the bacterial solution next to the alcohol lamp and mix it in sterile distilled water (while covering the plate upside down and putting it aside), use the inoculation loop to apply a bacterial film with a diameter of about 1.5cm, and then pass it over the flame of the alcohol lamp at a pendulum speed to fix the bacteria, wait for cooling and stain. First, add oxalic acid crystal violet solution to stain for 1~2min, then wash with water; then add Gram iodine solution to stain for 1~2min, then wash with water; then add 95% alcohol to decolorize for 30~60s, then wash with water; finally, add diluted fuchsin to counterstain for 30~60s, then wash with water; wait for drying or absorbent paper to dry and then examine under a microscope. Place the dried slide under a 1000x oil lens, add a drop of pine cypress oil for microscopic examination, and observe its morphological characteristics.

[0053] 1.2.8 Physiological and biochemical identification

[0054] Gram staining and microscopic examination methods are shown in 1.2.7. The biochemical identification tube used in the sugar fermentation experiment is a ready-made bacterial biochemical micro-identification tube. The bacterial solution is appropriately diluted, and then a certain amount of dilution is injected into the biochemical identification tube under a sterile environment. The inoculated identification tube is placed in a 37°C constant temperature incubator for culture. After 24 hours, the color change of the liquid in each tube is observed. If the color changes, it is positive, otherwise it is negative. The characteristic table of identifying species within the genus Bifidobacterium is compared with the national standard GB 4789.34-2016 "National Food Safety Standard Food Microbiology Test Bifidobacterium Test", that is, its species is determined.

[0055] 1.2.9 Data Processing

[0056] The experimental data were expressed as mean ± standard deviation and repeated at least 3 times. SPSS18.0 software was used to perform one-way ANOVA on the data between different groups. The Spearman correlation coefficient was used to evaluate the correlation between two statistical variables, and GraphPad Prism 5.0 and Origin 9.0 software were used for plotting. p < 0.05 was considered statistically significant.

[0057] 2 Results and analysis

[0058] 2.1 Effects of zinc sulfate and sodium selenite on strain growth activity

[0059] In order to understand whether the addition of oxygen-tolerant acclimation promoters to the modified MRS medium has a negative impact on the growth activity of the strain, the effects of zinc sulfate and sodium selenite on the growth activity of the strain were studied. Zinc sulfate and sodium selenite were added or not added to the modified MRS medium, and anaerobic culture was performed after inoculation of Bifidobacterium breve. After 24 hours, the number of viable bacteria with and without the addition of zinc sulfate and sodium selenite was 7.05±0.12 Log of CFU / mL and 7.12±0.08 Log of CFU / mL (P>0.05), respectively, which was not statistically significant, indicating that the zinc sulfate and sodium selenite selected by the present invention as oxygen-tolerant acclimation promoters added to the modified MRS medium had no negative impact on the growth activity of the strain.

[0060] 2.2 Activity of strains after pre-culture

[0061] After 8 generations of Bifidobacterium breve KLDS 2.0007, the strain activity of each generation was measured. The results are shown in Table 1. Pre-culture before aerobic acclimation can promote the efficiency of aerobic acclimation.

[0062] Table 1 Effect of pre-culture generation of strains on strain activity

[0063]

[0064] Note: The unit of strain activity is Log of CFU / mL

[0065] 2.3 Ultraviolet treatment mortality rate

[0066] After UV treatment, the lethality of strains at different treatment times is shown in Table 2. In this study, the bacterial solution after 15 seconds of irradiation was selected as the optimal treatment time. The strains counted on the plate were screened, and the strains with the fastest growth rate and the largest colony morphology were selected and cultured in sterile modified MRS medium for 24 hours, and used as the strains to be acclimated to oxygen tolerance.

[0067] Table 2 Effect of different irradiation time on the mortality rate

[0068]

[0069] 2.4 Determination of oxygen tolerance activity

[0070] The results of oxygen resistance test are as follows Figure 1 As shown, compared with Bifidobacterium breve YBT33 and YBT34, Bifidobacterium breve YBT35 and YBT36 entered the logarithmic growth phase the fastest, and during the stable period, their live cell counts were higher, significantly higher than Bifidobacterium breve YBT33 and YBT34.

[0071] 2.5 Morphological characteristics and physiological and biochemical identification of strains before and after treatment

[0072] The morphological characteristics of the strains before and after treatment are as follows Figure 3 As shown, Bifidobacterium breve YBT36 and Bifidobacterium breve KLDS2.0007 have similar morphological structures, while the arrangement of Bifidobacterium breve YBT36 is denser than that of Bifidobacterium breve KLDS 2.0007.

[0073] The physiological and biochemical characteristics of Bifidobacterium breve YBT36 were identified, and the results are as follows:

[0074] Gram staining is positive, short rod, slender rod or spherical, can form various branches or bifurcations, not acid-resistant, no spores, no power. The optimal growth temperature is 37℃, and the optimal growth pH is 6.75.

[0075] The results of the sugar fermentation experiment are shown in the following table

[0076] Table 3 Results of sugar alcohol fermentation experiments of strains

[0077]

[0078] Note: + indicates positive strain; - indicates negative strain.

[0079] The above results indicate that the strain belongs to Bifidobacterium breve ( Bifidobacterium breve ).

[0080] Example 2 Preparation of microcapsule preparation of Bifidobacterium breve and in vitro digestion simulation

[0081] 1 Materials and methods

[0082] 1.1 Experimental materials and equipment.

[0083] Sodium alginate, calcium carbonate, glacial acetic acid, sodium chloride (NaCl), potassium chloride (KCl), mucin, α-amylase, bile salts, trypsin, lipase, etc.

[0084] LDZF-50KB-II vertical pressure steam sterilizer; VD-1320 clean bench; DHP-9272 electric constant temperature incubator; HPG-9245 oven; vortex oscillator; Lambda Bio35 UV-visible spectrophotometer; Model 680 microplate reader; PHS-25 pH meter; GL-21M high-speed refrigerated centrifuge; 0.22μm filter membrane. 1 / 10,000 electronic balance, Beijing Sartorius Instrument System Co., Ltd.; electric constant temperature water bath, Tianjin Lester Instrument Co., Ltd.; refrigerator, Qingdao Haier Co., Ltd.; vortex oscillator, Weiwosuoke Technology (Beijing) Co., Ltd.; centrifuge, Shanghai Chemical Machinery Company; ultra-low temperature refrigerator, ThermoFisher, USA; constant temperature shaker, etc.

[0085] 1.2 Experimental methods

[0086] 1.2.1 Preparation of microcapsule preparations of Bifidobacterium breve

[0087] Bifidobacterium breve YBT36 was used to prepare a Bifidobacterium breve suspension (concentration: 10 7 CFU / mL). Sodium alginate was used as the wall (carrier) material. The suspension of Bifidobacterium breve was mixed with a sodium alginate solution (3%, w / v; i.e., 3 g / 100 ml) at a volume ratio of 1:2.5 (v / v), and calcium carbonate (W (Ca): W (Alg) = 0.1) was added, where Alg is sodium alginate. The mixture was then added to soybean oil containing 1% (v / v) Tween 80 (emulsifier) ​​at a water / oil ratio (1:1, v / v) and homogenized at 400xg for 15 min to form a water-in-oil (W / O) emulsion. Glacial acetic acid was added at a volume ratio of 1:1 and stirred for 30 min to promote CaCO 3 Release of calcium ions. Adding CaCl 2 (0.8 mol / L) solution and gently stirred for 20 minutes. After the washed and solidified microcapsules were deposited at the bottom of the solution, the precipitate was collected and washed with NaCl solution (0.85%, w / v; i.e. 0.85 g / 100 ml) to remove the oil phase until no oil remained. Finally, the harvested microcapsules were stored at 4°C.

[0088] 1.2.2 In vitro digestion simulation

[0089] The control group used a non-microcapsule preparation, namely, the suspension of Bifidobacterium breve prepared according to 1.2.1 of this Example. The experimental group used a microcapsule preparation prepared according to 1.2.1 of this Example. Artificial saliva, gastric juice, and intestinal juice were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0090] 2g of microcapsule preparation and 2mL of Bifidobacterium breve suspension were inoculated into a shake flask containing 20mL of artificial saliva, incubated in a 37°C incubator for 2 minutes, and then centrifuged (1000rpm, 2min) to obtain the Bifidobacterium breve suspension and microcapsule preparation after saliva digestion. The Bifidobacterium breve suspension and microcapsule preparation after saliva digestion were added to a shake flask containing 20mL of artificial gastric juice and placed in a constant temperature shaker (37°C, 3rpm, 2h). Centrifuged (1000rpm, 2min) to obtain the Bifidobacterium breve suspension and microcapsule preparation digested by gastric juice, and added to a shake flask containing 20mL of artificial intestinal juice, placed in a constant temperature shaker (37°C, 2rpm, 3h), and then the Bifidobacterium breve suspension and microcapsule preparation were harvested. At the end of each step, the survival rate was determined by the viable bacteria count method, in units of log CFU / mL.

[0091] 1.2.3 Data processing

[0092] The experimental data were expressed as mean ± standard deviation and repeated at least 3 times. SPSS18.0 software was used to perform one-way ANOVA on the data between different groups. The Spearman correlation coefficient was used to evaluate the correlation between two statistical variables, and GraphPad Prism 5.0 and Origin 9.0 software were used for plotting. p < 0.05 was considered statistically significant.

[0093] 2 Results

[0094] Bifidobacterium breve is difficult to pass through the stomach in its natural state because the pH value of the stomach environment is extremely low and highly acidic, while bile salts are present in the small intestine, both of which have a killing or inhibitory effect on microorganisms. The shell of the microcapsule can temporarily protect the bifidobacterium, reducing or preventing direct contact between acid and bile salts and the bacteria. In addition, microcapsule packaging helps to maintain the activity and stability of bifidobacteria during storage and transportation, ensuring that the strain is active when it reaches the consumer and when it is actually consumed. The prepared microcapsule preparation was subjected to in vitro digestion simulation, and the results are shown in Table 4. After digestion in saliva, gastric juice, and intestinal juice, the activity of the microcapsule preparation group was significantly increased compared to the suspension group (p<0.05), indicating that the microcapsule preparation can resist the acidic environment of the gastrointestinal tract, help Bifidobacterium breve to better colonize in the large intestine, and exert its probiotic effect.

[0095] Table 4 Effects of microcapsule preparations on simulated gastrointestinal digestion

[0096]

[0097] Note: Data are expressed as mean ± standard deviation; different lowercase letters in the same column indicate significant differences (p<0.05).

[0098] Example 3 Bifidobacterium breve YBT36 microcapsule preparation for promoting bone development

[0099] 1 Materials and methods

[0100] 1.1 Experimental Materials

[0101] According to the method of section 1.2.1 of Example 2, Bifidobacterium breve YBT35 and Bifidobacterium breve YBT36 were taken to prepare suspensions of Bifidobacterium breve YBT35 and Bifidobacterium breve YBT36 (concentration: 2×10 7 CFU / mL).

[0102] The microcapsule preparation of Bifidobacterium breve YBT36 was prepared according to the method in Section 1.2.1 of Example 2, wherein the concentration of the Bifidobacterium breve YBT36 suspension was 2×10 7 CFU / mL.

[0103] 1.2 Experimental Reagents

[0104] Calcium carbonate D3 chewable tablets (National Medicine Standard: J20140153, Imported Drug Registration Number: H20140573), 10% paraformaldehyde (Shanghai Yuanye Biotechnology Co., Ltd., China), CTX-1 and PINP ELISA kits (Shanghai ELISA Biotechnology Co., Ltd., China)

[0105] 2 Animal experiments

[0106] 2.1 Animal Experiment Design

[0107] Three-week-old female SD rats were purchased from the Second Affiliated Hospital of Harbin Medical University and housed in a standard specific pathogen-free (SPF) laboratory: 25±2°C, 50±5% humidity, 12h / 12h light-dark cycle. After one week of adaptive feeding, they were randomly assigned to a blank group, a YBT35 suspension group, a YBT36 suspension group, a YBT36 microcapsule group, and a positive control group (calcium carbonate D3), with 6 rats in each group. After adaptive feeding, equal volumes of gavage were performed. The blank group was gavaged with normal saline; the YBT35 suspension group was gavaged with 10 7 CFUYBT35;YBT36 suspension group was gavaged daily for 10 7 CFU YBT36; the microcapsule preparation group was intragastrically gavaged with YBT36 microcapsules (10 7CFUYBT36, fully dissolve the preparation in physiological salt); positive control group was intragastrically administered with calcium carbonate D 3 Chewable tablets, the dosage was converted to a rat dosage of 72 mg / 100 g / d according to the body surface area conversion method; after 4 weeks, all rats were humanely euthanized after fasting for 24 h, and the serum of the rats was collected and stored at -80 °C for further use. Tibias and femurs were collected and fixed in 10% buffered formaldehyde for histological analysis and micro-computed tomography.

[0108] 2.2 Micro CT scanning of rat femur

[0109] The femur to be tested was placed on the sample stage of the micro-CT instrument for micro CT three-dimensional image acquisition. After the microscopic image system collected data, the image was automatically generated using 3Dmed-version2.0 image analysis software. The entire femur was fixed on the micro CT stage for scanning. The micro-CT tube parameters were set according to the following parameters: micro-focus X-ray source, focal spot 5 μm, working voltage 60 KVP, working power 40 W, beam cone angle 45°; spatial resolution: 15 μm, image matrix 4096′4096; field of view: single scan 10 mm′10 mm (min), continuous cross-sectional μCT images were obtained at a 360° angle (scanning the distal femur along the femoral shaft of the rat), and the computer software automatically reconstructed the three-dimensional image of the bone tissue. The main bone density parameters were detected.

[0110] 2.3 Bone marker content in rats

[0111] An appropriate amount of serum was taken and the bone resorption marker (CTX-1) and bone formation marker (PINP) in the rat serum samples were determined using an enzyme-linked immunosorbent assay kit according to the manufacturer's instructions.

[0112] 3 Data processing

[0113] The experimental data were expressed as mean ± standard deviation and repeated at least 3 times. SPSS18.0 software was used to perform one-way ANOVA on the data between different groups, and GraphPad Prism 5.0 and Origin 9.0 software were used for plotting. P < 0.05 was considered statistically significant.

[0114] 4 Results

[0115] 4.1 Femur and tibia length

[0116] Changes in femur length are one of the important indicators for assessing bone development and growth status. The femur is the longest bone in the human body, and the increase in its length reflects the overall growth of the individual, especially during childhood and adolescence. During this period, the growth plate (growth cartilage) is active and is a key area for bone growth. As the body secretes growth hormone and sex hormones to promote bone growth, the length of the femur increases until the growth plate closes and bone development is complete. Therefore, measuring the length of the femur can intuitively reflect the bone development status of the organism.

[0117] As shown in Table 5, the changes in femur and tibia length increased the femur and tibia length of rats to a certain extent after Bifidobacterium breve intervention. YBT35 suspension had a tendency to promote the development of femur and tibia of rats (compared with the blank group, p>0.05); YBT36 suspension and YBT36 microcapsule groups increased significantly (compared with the blank group, p<0.05). Among them, the effect brought by the YBT36 microcapsule group was more significant (compared with the YBT35 suspension group, p<0.05), and exceeded the positive control group.

[0118] Table 5 Effects of different treatments on the length of femur and tibia of rats

[0119]

[0120] Note: Data are expressed as mean ± standard deviation; different lowercase letters in the same column indicate significant differences (p<0.05).

[0121] 4.2 Bone density and bone microstructure

[0122] To evaluate the bone development of rats in each treatment group, Micro-CT scans were used to analyze bone density and bone microstructure. Bone density is a measure of the content of calcium salts and other minerals in bones. High bone density usually indicates stronger bones, while low bone density may lead to osteoporosis. Therefore, bone density is a key indicator for assessing the risk of osteoporosis and bone development.

[0123] The bone density results are shown in Table 6. Compared with the blank group, the YBT35 suspension group increased the bone density of rats at the mean level (p>0.05 compared with the blank group). The increase in the YBT36 suspension and YBT36 microcapsule groups was more obvious (p<0.05 compared with the blank group). It is worth noting that the bone density of the microcapsule preparation group was significantly higher than that of the YBT36 suspension (P<0.05), reaching the level of the positive control group (p>0.05).

[0124] Table 6 Effects of different treatments on bone density in rats

[0125]

[0126] Note: Data are expressed as mean ± standard deviation; different lowercase letters in the same column indicate significant differences (p<0.05).

[0127] 4.3 Determination of bone markers

[0128] CTX-1 (C-terminal peptide of type I collagen) and PINP (N-terminal propeptide of type I collagen) are two key biomarkers of bone metabolism. CTX-1 mainly reflects the degree of bone resorption, while PINP indicates the rate of bone formation. Changes in the levels of these two can provide important information for bone development. PINP is an early product of type I collagen synthesis during new bone formation, and its increased level generally means increased bone formation activity. During the growth and development stages of children and adolescents, as well as during fracture healing or certain bone disease treatments in adults, PINP levels may increase significantly, reflecting accelerated bone remodeling and growth. CTX-1 is a product of collagen degradation during bone resorption, and its increased level indicates an intensified bone resorption process. In osteoporosis, bone metastatic cancer, or other diseases that cause bone loss, CTX-1 levels often increase, indicating excessive bone resorption.

[0129] Bone marker results Figure 3 In the figure, ns means no significant difference; * means p < 0.05; ** means p < 0.001.

[0130] Compared with the blank group, the YBT35 suspension group increased the PINP content in rat serum at the mean level. The PINP content in the YBT36 suspension (p<0.05) and YBT36 microcapsule groups was significantly higher than that in the blank group (p<0.001). Among them, the microcapsule system reached a level equivalent to that of the positive control.

[0131] In addition, the YBT35 suspension group, YBT36 suspension group, and positive control group also reduced the mean level of CTX-1 in rat serum. The YBT36 microcapsule group brought about a significant reduction (p<0.05), even exceeding the positive control group.

[0132] These data indicate that the regulatory effect of YBT36, especially YBT36 microcapsules, on bone growth is related to two bone metabolism markers, CTX-1 and PINP.

[0133] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention. For example, in the preparation of probiotic microcapsule preparations, wall materials (carriers) are used to encapsulate probiotics to reduce the impact of the adverse gastrointestinal environment on probiotics. Without departing from the principle of the present invention, ordinary technicians in this technical field can change the wall materials, preparation processes, etc. of the microcapsule preparations, or change the amount of Bifidobacterium breve YBT36 in the microcapsule preparations, etc., and these changes should also be regarded as the protection scope of the present invention.

Claims

1. A Bifidobacterium breve, characterized in that The preservation unit of the Bifidobacterium breve is the China Center for Type Culture Collection; the preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; the preservation date is July 10, 2024, and the preservation number is CCTCC NO: M20241534; the taxonomic name of the Bifidobacterium breve is Bifidobacterium breve ( Bifidobacterium breve )YBT36.

2. A preparation for promoting bone development, characterized in that: The preparation contains Bifidobacterium breve with a deposit number of CCTCC NO: M20241534.

3. A preparation for promoting bone development according to claim 2, characterized in that: The preparation is a microcapsule preparation.

4. A preparation for promoting bone development according to claim 3, characterized in that: The microcapsule wall material of the microcapsule preparation is any one of alginic acid, sodium alginate and calcium alginate.

5. A preparation for promoting bone development according to claim 3, characterized in that: The microcapsule wall material of the microcapsule preparation is sodium alginate.

6. A preparation for promoting bone development according to claim 5, characterized in that: The preparation method of the microcapsule preparation comprises the following steps: S1: Take a suspension of Bifidobacterium breve with a preservation number of CCTCC NO: M20241534, mix it with a sodium alginate solution with a weight volume concentration of 3g / 100ml at a volume ratio of 1:2.5, add calcium carbonate; then add soybean oil containing 1% Tween 80 at a volume percentage concentration of 1% at a water / oil volume ratio of 1:1, and homogenize at 400×g for 15 minutes to form a water-in-oil emulsion; S2: Add glacial acetic acid to the emulsion, stir, add CaCl2 solution and stir, collect the precipitate, wash and remove the oil phase to obtain microcapsules.

7. A preparation for promoting bone development according to claim 6, characterized in that: The concentration of the CaCl2 solution in step S2 is 0.8 mol / L.

8. A preparation for promoting bone development according to claim 6, characterized in that: In step S2, the volume ratio of the emulsion to the glacial acetic acid is 1:

1.

9. A preparation for promoting bone development according to claim 6, characterized in that: In the step S2, 0.85 g / 100 mL of NaCl solution is used to wash and remove the oil phase.

10. Use of the Bifidobacterium breve according to claim 1 or the preparation for promoting bone development according to any one of claims 2 to 9 in the preparation of a product for promoting bone development.

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