CN115851506A

CN115851506A - Lactobacillus rhamnosus JYLR-219 for preventing osteoporosis as well as microbial inoculum and application thereof

Info

Publication number: CN115851506A
Application number: CN202211238115.5A
Authority: CN
Inventors: 潘仕城; 庞颂; 刘红亮; 王丽媛; 郄璐; 王雪娇; 刘鑫
Original assignee: Zhongke Jiayi Nutrition Medicine Shandong Micro Ecology Research Institute Co ltd
Current assignee: Zhongke Jiayi Nutrition Medicine Shandong Micro Ecology Research Institute Co ltd
Priority date: 2022-10-11
Filing date: 2022-10-11
Publication date: 2023-03-28

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus rhamnosus JYLR-219 for preventing osteoporosis, a microbial inoculum and application thereof, wherein the lactobacillus rhamnosus JYLR-219 comprises lactobacillus rhamnosus (Lactobacillus rhamnosus)Lactobacillus rhamnosus) JYLR-219 has been preserved in the China general microbiological culture Collection center of China general microbiological culture Collection center at 7.8.7.2019, the preservation address is No. 3 of West Lu No.1 of Beijing, korean-yang district, the preservation number is CGMCC NO.18097, and the Lactobacillus rhamnosus JYLR-219 can be used for preparing products for preventing osteoporosis. The Lactobacillus rhamnosus JYLR-219 has strong gastrointestinal survival rate and intestinal tract colonization ability, and can increase osteoclast density by proliferating osteoblast density and reducing osteoclast densityBone density, thereby preventing osteoporosis. The invention has the advantages of safety, effectiveness and no side effect.

Description

Lactobacillus rhamnosus JYLR-219 for preventing osteoporosis as well as microbial inoculum and application thereof

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to lactobacillus rhamnosus JYLR-219 for preventing osteoporosis, and a microbial inoculum and application thereof.

Background

Osteoporosis is a systemic bone disease characterized by low bone mass, damaged bone microstructure, increased bone fragility and easy fracture, and is most common in primary osteoporosis of menopausal women and old people, and the primary osteoporosis is mainly caused by that osteoclast-dominated bone resorption is greater than osteoblast-dominated bone formation, bone density is reduced, and fracture and disability are easily caused.

At present, common treatment methods aiming at osteoporosis comprise dietary supplement, health product taking and medicament treatment. 1. The calcium supplement is increased by eating a large amount of meat, eggs and the like, so that the effect of preventing osteoporosis is achieved; however, with the occurrence of 'rich diseases' of the current generation people, the supplement of the high meat and egg foods often causes 'three highs'. 2. Supplementing by taking health product such as calcium tablet and glucosamine chondroitin; however, the health care products on the market are uneven in quality, often have exaggerated efficacy propaganda, greatly weaken the digestive absorption system of the old, cannot effectively guarantee the absorption problem, even easily cause the phenomena of getting inflamed and constipation and the like, are expensive, and are difficult to be taken by common consumers for a long time. 3. Medication is carried out by taking vitamin D, calcium preparation, bone absorption resisting medicine, etc.; however, the common osteoporosis treatment drugs have side effects to a certain extent, damage the liver and kidney functions and the digestive tract functions of patients, or influence the endocrine metabolism level in the bodies of patients, and are not suitable for long-term administration.

Disclosure of Invention

Aiming at the defects of the osteoporosis treatment mode, the invention provides lactobacillus rhamnosus JYLR-219 for preventing osteoporosis, a microbial inoculum and application thereof, provides a new way for preventing and treating osteoporosis, and has the advantages of no side effect, safety and effectiveness.

In a first aspect, the invention provides lactobacillus rhamnosus JYLR-219 for preventing osteoporosis, wherein the lactobacillus rhamnosus JYLR-219 is prepared from lactobacillus rhamnosus (A), (B), (C) and (C)Lactobacillus rhamnosus) JYLR-219 has been deposited in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms at 7.8.7.2019, with the deposition address of No. 3 Hospital No.1 of Xilu, north Cheng, the rising area of Beijing, and the deposition number of CGMCC NO.18097.

In a second aspect, the present invention provides a microbial inoculum comprising lactobacillus rhamnosus JYLR-219 according to claim 1.

Further, the microbial inoculum comprises freeze-dried bacterial powder of lactobacillus rhamnosus JYLR-219.

Further, the preparation method of the freeze-dried bacterial powder comprises the following steps:

(1) Activating the preserved lactobacillus rhamnosus JYLR-219 on an MRS plate culture medium, inoculating the activated lactobacillus rhamnosus in an MRS liquid culture medium according to the inoculation amount of 1 percent for culturing for 24 hours to obtain a bacterial liquid;

(2) Centrifuging the bacterial liquid, collecting thalli, washing with sterile normal saline, and suspending in 15% by mass of recovered skim milk to obtain a suspension; adjusting the concentration of the Lactobacillus rhamnosus JYLR-219 in the suspension to be 1.0 to 2.0 multiplied by 10 ¹⁰ cfu/mL, obtaining a bacterial suspension; and (4) freeze-drying the bacterial suspension to obtain freeze-dried bacterial powder.

Further, the preparation method of the MRS plate medium in the step (1) comprises the following steps: mixing 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of dipotassium hydrogen phosphate heptahydrate, 1mL of tween-80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate, 15g of agar and 1000mL of distilled water, stirring the mixed solution at natural pH, sterilizing at 121 ℃ and 0.1MPa for 20min, pouring the sterilized mixed solution into a flat dish, and cooling for later use.

In a third aspect, the invention provides an application of lactobacillus rhamnosus JYLR-219 as set forth in claim 1 in preparing a product for enhancing bone density and preventing osteoporosis.

In a fourth aspect, the invention provides an application of the microbial inoculum according to claim 2 in preparing products for enhancing bone density and preventing osteoporosis.

The invention has the beneficial effects that:

1. the lactobacillus rhamnosus JYLR-219 for preventing osteoporosis is mainly applied to osteoporosis people and solves the problem of low bone density. The strain has stronger gastrointestinal survival rate and intestinal colonization ability, and can obviously increase the density of osteoblasts and obviously reduce the density of osteoclasts, thereby playing the roles of enhancing the bone density and preventing osteoporosis.

2. The invention utilizes the preserved strain lactobacillus rhamnosus JYLR-219 to prepare an oral product, is convenient for clinically treating osteoporosis, is safe and effective, and has no side effect.

Drawings

In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.

FIG. 1 is a microscopic observation result of osteoblasts of mice in the control group and the experimental group in example 5 of the present invention.

FIG. 2 is a microscopic observation result of osteoclasts in mouse of control group and experimental group in example 6 of the present invention.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 screening, purification and characterization of Lactobacillus rhamnosus JYLR-219

1. Bacterial source

Collected in 23 months 4 in 2018 from herdsmen in Ili, xinjiang by self-made fermented yogurt.

2. Screening of strains

(1) The yoghurt is sampled and diluted with a dilution gradient of 10 ^-1 、10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 、10 ^-7 The labels are 1#, 2#, 3#, 4#, 5#, 6#, and 7# respectively for standby;

(2) Preparing an MRS plate culture medium: mixing 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of dipotassium hydrogen phosphate heptahydrate, 1mL of tween-80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate, 15g of agar and 1000mL of distilled water, naturally adjusting the pH, stirring a bacterial solution, sterilizing at 121 ℃ and 0.1MPa for 20min, pouring the sterilized culture medium into a flat dish, and cooling for later use;

(3) Culturing: coating 0.1mL of the solutions 1#, 2#, 3#, 4#, 5#, 6# and 7# in MRS plate culture medium with a coater, and culturing at 37 deg.C under anaerobic condition for 48 hr;

(4) Selecting colonies: selecting bacterial colonies according to the characteristics of bacterial colonies with diameters of 1 to 2mm, round and smooth bacterial colonies, regular edges and slight white bulges in the middle;

(5) Separation and purification: and (4) selecting 5 single colonies according to the colony characteristics in the step (4), inoculating the single colonies to an MRS plate culture medium by adopting a scribing method, culturing for 48 hours at 37 ℃ under an anaerobic condition, selecting the single colonies, and placing the single colonies in a glycerol tube for preservation at-70 ℃.

2. Identification

And (3) sending the separated and purified single colony to identification, wherein the identification unit is as follows: biotechnology engineering (Shanghai) Ltd.

(1) In the identification process, the following primers were used:

27F：5'-AGAGTTTGATCCTGGCTCAG-3'；

1492R：5'- GGTTACCTTGTTACGACTT-3'。

(2) The gene sequences identified were as follows:

ATGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAGTTCTGATTATTGAAAGGTGCTTGCATCTTGATTTAATTTTGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAAATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTTTGATCACCTGAGAGATCAGGTTTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAGACCGCGAGGTCAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCGAAGCCGGTGGCGTAACCCTTTTAGGGAGCGAGCCGTCTAA。

(3) The bacterium is identified to be lactobacillus rhamnosus (lactobacillus rhamnosus:Lactobacillus rhamnosus) The strain is named as lactobacillus rhamnosus JYLR-219 and sent to the China Committee for culture Collection of microorganisms for culture preservation, and the preservation information is as follows:

the rhamnose is prepared from fructus HippophaeLactobacillus (A), (B), (C)Lactobacillus rhamnosus) JYLR-219 was deposited in the China general microbiological culture Collection center at 7/8.2019 under the following address: west road No.1, north chen, chaoyang district, beijing, zip code: 100101, accession number: CGMCC NO.18097.

Example 2 preparation of Lactobacillus rhamnosus JYLR-219 product

(1) Preparing an MRS liquid culture medium: mixing 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of dipotassium hydrogen phosphate heptahydrate, 80 mL of tween, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate and 1000mL of distilled water, adjusting the pH to 6.8, stirring a bacterial solution, and sterilizing at 121 ℃ and 0.1MPa for 20min to obtain the beef extract;

(2) Activating the preserved lactobacillus rhamnosus JYLR-219 on the MRS plate culture medium prepared in the embodiment 1, inoculating the activated lactobacillus rhamnosus into an MRS liquid culture medium according to the inoculation amount of 1%, and then culturing for 24h at 37 ℃ to obtain a bacterial liquid;

(3) Centrifuging the bacterial liquid, collecting thalli, washing with sterile normal saline, and suspending in 15% of recovered skim milk by mass ratio to obtain suspension; adjusting the concentration of the suspension to 1.0 to 2.0 × 10 ¹⁰ cfu/mL to obtain a bacterial suspension, and freeze-drying the bacterial suspension to obtain freeze-dried bacterial powder;

(4) Mixing the freeze-dried bacterial powder with isomaltooligosaccharide to prepare lactobacillus rhamnosus JYLR-219 microbial inoculum, wherein the isomaltooligosaccharide is purchased from Yongbao bio-resources Co., ltd;

in this example, the microbial inoculum was prepared in a cell count of 1X 10 ¹⁰ cfu/g，1.5×10 ¹⁰ cfu/g。

Example 3 gastrointestinal digestive juice resistance of Lactobacillus rhamnosus JYLR-219

1. Gastric juice preparation and manipulation

(1) Preparing artificial simulated gastric juice; weighing 16.4mL of 9.5% hydrochloric acid solution, adding distilled water to dilute until the pH value is 1.5, adding 1.0g of pepsin into each 100mL of hydrochloric acid solution, mixing uniformly, and filtering with a sterile filter membrane of 0.22 mu m to prepare the composition for use.

(2) Weighing 12 parts of the rhamnus frangula prepared in example 2The number of the lactobacillus saccharosus JYLR-219 cells is 1.5 multiplied by 10 ¹⁰ cfu/g of product was used as a sample, each sample weighing 1g, transferred to a pre-heated tube containing 9mL simulated gastric fluid and treated at 37 ℃ and 80r/min for 1h, 2h, 3h, 4h, 3 replicates each.

2. Preparation and operation of intestinal juice

(1) Preparing artificial simulated intestinal juice: mixing 6.8g KH ₂ PO ₄ Dissolving in 500mL of distilled water, adding 3g of cholate and 10g of trypsin, adjusting the pH value of the solution to 6.8 by using a NaOH solution with the concentration of 4g/L, diluting to 1L by using distilled water, mixing uniformly, and filtering by using a sterile filter membrane with the diameter of 0.22 mu m, wherein the cholate and the trypsin are used for preparation.

(2) 12 parts of Lactobacillus rhamnosus JYLR-219 strain prepared in example 2 are weighed to be 1 × 10 ¹⁰ cfu/g of the product was used as a sample, each sample weighing 1g, and transferred to a test tube containing 9mL of simulated intestinal fluid, and treated at 37 ℃ for 1h, 2h, 3h, and 4h at 80r/min, each for 3 replicates.

3. Statistics and analysis

And (4) carrying out gradient dilution on the treated sample solution, counting the viable bacteria by using a pouring method, and then calculating the survival rate.

Survival rate = (number of treated bacteria/number of original bacteria) × 100%

TABLE 1 JYLR-219 survival in simulated gastrointestinal environment in vitro

The survival rate data of the strain JYLR-219 in the in vitro simulated gastrointestinal environment is shown in Table 1, and the survival rate of the lactobacillus rhamnosus JYLR-219 in the in vitro simulated gastrointestinal environment is very high, and is still more than 90% in gastric juice and intestinal juice after 4h, so that the survival rate can lay a foundation for the subsequent strains to fix the intestinal tract and play a functional role.

Example 4 adhesion of Lactobacillus rhamnosus JYLR-219 to Caco-2 control experiment

(1) Test reagents:

DMEM medium, purchased from Gibco;

MRS plate medium prepared by the same method as example 1;

MRS liquid medium prepared by the same method as example 2;

weighing KH ₂ PO ₄ 0.27g、Na ₂ HPO ₄ 1.42g, naCl 8g and KCl 0.2g, adding about 800mL of deionized water, fully stirring and dissolving, adding concentrated hydrochloric acid to adjust the pH to 7.4, adding deionized water to fix the volume to 1L, and sterilizing at 121 ℃ for 20min to prepare the sterile PBS buffer solution with the pH = 7.4.

(2) Preparation of the test:

caco-2 cells were placed in DMEM medium containing 10% heat-inactivated newborn bovine serum and diabodies (penicillin concentration 100U/mL, streptomycin 1.0. Mu.g/mL) at 37 ℃ with 5% CO ₂ And incubating in carbon dioxide incubator with relative humidity of 95%, changing culture solution 1 time per day, subculturing 1 time for 3 days, inoculating cells in 24-well culture plate after 18 days, wherein the inoculation density is about 5 × 10 ⁵ cfu/mL, performing adhesion test after the cells grow to a monolayer;

activating the preserved lactobacillus rhamnosus JYLR-219 on an MRS plate culture medium, inoculating the activated lactobacillus rhamnosus JYLR-219 into an MRS liquid culture medium according to the inoculum size of 1% of the volume fraction, and culturing at 37 ℃ for 12h to obtain strain fermentation liquor;

centrifuging strain fermentation liquid, collecting thallus (4000 r/min, 4 deg.C, 10 min), washing with sterile PBS buffer solution for 3 times, and suspending thallus in sterile PBS buffer solution to obtain a concentration of 2 × 10 ⁸ cfu/mL JYLR-219 bacterial suspension.

(3) And (3) test operation:

the Caco-2 cells that had grown into monolayers were rinsed 2 times with sterile PBS buffer, 0.5mL of JYLR-219 bacterial suspension and 0.5mL of fresh DMEM medium were added to each well, and the CO was reduced 5% at 37 deg.C ₂ And incubated in a carbon dioxide incubator at 95% relative humidity for 1h, and then the cells were rinsed 5 times with sterile PBS buffer to remove non-adherent bacteria.

Carrying out gradient dilution on the rinsing liquid, counting live bacteria by using a pouring method, and calculating the adhesion rate: adhesion rate = (initial number of bacteria-number of bacteria in rinsing)/initial number of bacteria × 100%.

After rinsing, the number of bacteria adhered to 100 cells in 20 random fields was counted under an inverted microscope, fixed with formaldehyde, stained with gram, and examined under a microscope. The average number of bacteria per cell adhesion was calculated using no suspension cell culture as a control, and 3 replicates were run for each treatment.

TABLE 2 adhesion rate and number of adhesion of JYLR-219 on Caco-2 cells

Item	Rate of adhesion	Number of adhesion
			Results	85%	692 (all three)

The adhesion rate and the cell adhesion number of the strain JYLR-219 to Caco-2 cells are shown in Table 2, and the fact that the Lactobacillus rhamnosus JYLR-219 has strong capacity in the aspect of cell colonization can be seen.

Example 5 Effect of Lactobacillus rhamnosus JYLR-219 on mouse osteoblasts

Osteoblasts are the primary functional cells for bone formation and are responsible for the synthesis, secretion and mineralization of bone matrix. Bone is constantly being remodeled and osteoblasts are the key to maintaining normal bone mass.

A random grouping design is adopted, 40 inbred line male mice are randomly divided into 2 groups (20 mice in a control group and an experimental group respectively) in an adaptation period of 5 d. Keeping the environment temperature of the animal breeding at 21 +/-2 ℃, the humidity of 30-70%, alternately illuminating for 12h, freely drinking water and freely taking the feed. Changing padding every three days, and adding feed and water in time.

Taking the lactobacillus rhamnosus JYLR-219 prepared in the example 2The number of the cells was 1X 10 ¹⁰ cfu/g product, made by adding water to 1 × 10 ⁹ cfu/mL probiotic.

2mL of probiotic liquid is perfused into the experimental group every day, the same amount of sterile physiological saline is given to the control group, the perfusion is continuously carried out for 7 days, then 1 mouse is randomly drawn from each of the experimental group and the control group, the mouse is killed, and the right hind leg femur of the mouse is taken to detect the cell condition. The experiment was continued for another 7 days, after 7 days, 1 mouse each of the experimental group and the control group was randomly selected again, sacrificed, and the femoral bone of the right hind leg of the mouse was taken to examine the cell condition.

TABLE 3 MTT detection of osteoblast proliferation (OD 490 nm)

	Day 7	Day 14
			Control group	0.4637±0.0159	0.5010±0.0218
Experimental group	0.6029±0.0238**	0.7462±0.0199**

Note: * Expressed as having differential significance (p < 0.05).; as having very differential significance (p < 0.01)

In the research, the strain experimental group is lactobacillus rhamnosus JYLR-219, and as can be seen from Table 3, the experimental group obviously promotes the proliferation of osteoblasts (p is less than 0.01); the control group of mice has no difference, which shows that the mice in the experimental group really have the effect of enhancing the osteoblast proliferation after the mice are gavaged by the lactobacillus rhamnosus JYLR-219.

As can be seen from FIG. 1, the proliferation status of osteoblasts of mice after administration of Lactobacillus rhamnosus JYLR-219 is shown in the figure, wherein a is osteoblasts of mice in the control group after 7 days, b is osteoblasts of mice in the experimental group after 7 days, c is osteoblasts of mice in the control group after 14 days, and d is osteoblasts of mice in the experimental group after 14 days. As can be seen from FIG. 1, after 7 days, the osteoblast density of the control mice was less than that of the experimental mice after 7 days, i.e., the osteoblast density of the mice was increased by administering Lactobacillus rhamnosus JYLR-219; the osteoblast density of the control group mice after 7 days and the control group mice after 14 days is basically unchanged; compared with the control group mice after 14 days and the experimental group mice after 14 days, the osteoblast density of the experimental group mice after 14 days is obviously higher than that of the control group mice after 14 days, namely the density of the osteoblasts of the mice is increased by taking the lactobacillus rhamnosus JYLR-219; compared with the experimental group mice after 7 days and the experimental group mice after 14 days, the osteoblast density of the mice is further enhanced along with the continuous administration of the lactobacillus rhamnosus JYLR-219, which shows that the strain JYLR-219 can play the effects of enhancing the bone density and preventing osteoporosis when being administered.

Example 6 Effect of Lactobacillus rhamnosus JYLR-219 on mouse osteoclasts

The osteoclast consists of multinuclear giant cells with the diameter of 100 mu m, contains 2 to 50 closely packed nuclei, is mainly distributed on the surface of bone and around an intraosseous vascular channel, is formed by fusing a plurality of mononuclear cells, is basophilic, but becomes acidophilic along with the aging of the cells. Osteoclast releases lactic acid and citric acid to local part, and inorganic mineral in bone is swallowed from ruffled edge under acidic condition to form some swallow bubbles or phagocytic bubbles in ruffled edge matrix. Inside osteoclasts, inorganic substances are degraded and excreted into the bloodstream in the form of calcium ions, and therefore the level of osteoclast content can have a significant effect on the calcium content of bone, and can play an important role in the level of bone density and the formation of osteoporosis.

In example 5, the results of MTT assay for osteoclasts are shown in table 4 below.

TABLE 4 MTT assay osteoclast proliferation (OD 490 nm)

	Day 7	Day 14
			Control group	1.2396±0.0360	1.7869±0.0181
Experimental group	1.2166±0.0047*	1.2524±0.0306**

As is apparent from table 4, the osteoclast proliferation of the control group is faster at 7 days and 14 days, and does not show obvious inhibition, while the osteoclast proliferation of the mice after taking lactobacillus rhamnosus JYLR-219 shows a certain difference at 7 days, and the osteoclast proliferation of the mice in the experimental group is as low as 1.2524 ± 0.0306 after taking lactobacillus rhamnosus JYLR-219 continuously for 14 days, which shows a very significant difference, so that it can be determined that the osteoclast number of the mice in the experimental group is greatly reduced after taking lactobacillus rhamnosus JYLR-219, which is helpful for bone density maintenance, and plays an important role in preventing osteoporosis.

As can be seen from FIG. 2, the change of osteoclast was observed after mice were administered Lactobacillus rhamnosus JYLR-219. In the figure, e is osteoclast of a control group mouse after 7 days, f is osteoclast of an experimental group mouse after 7 days, g is osteoclast of a control group mouse after 14 days, and h is osteoclast of an experimental group mouse after 14 days. As can be seen from FIG. 2, the osteoclast number was increased in the control mice after 7 days compared with the control mice after 14 days; and the number of osteoclasts in the experimental group mice is reduced after 7 days compared with that in the experimental group mice after 14 days; comparing the osteoclast conditions of the control group of mice after 14 days and the experimental group of mice after 14 days, it is obvious that the osteoclast content of the mice after 14 days taking lactobacillus rhamnosus JYLR-219 is far lower than that of the control group of mice.

Although the present invention has been described in detail by referring to the drawings in connection with the preferred embodiments, the present invention is not limited thereto. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention.

Claims

1. Lactobacillus rhamnosus JYLR-219 for preventing osteoporosis, wherein the Lactobacillus rhamnosus (JYLR-219) is prepared from Lactobacillus rhamnosusLactobacillus rhamnosus) JYLR-219 has been deposited in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms at 7.8.7.2019, with the deposition address of No. 3 Hospital No.1 of Xilu, north Cheng, the rising area of Beijing, and the deposition number of CGMCC NO.18097.

2. A microbial inoculum comprising lactobacillus rhamnosus JYLR-219 of claim 1.

3. The microbial inoculum of claim 2, which comprises freeze-dried powder of lactobacillus rhamnosus JYLR-219.

4. The microbial inoculum of claim 3, wherein the preparation method of the freeze-dried bacterial powder comprises the following steps:

(2) Centrifuging the bacterial liquid, collecting thalli, washing with sterile normal saline, and suspending in 15% by mass of recovered skim milk to obtain a suspension; adjusting the concentration of the Lactobacillus rhamnosus JYLR-219 in the suspension to be 1.0 to 2.0 multiplied by 10 ¹⁰ cfu/mL to obtain a bacterial suspension; and (4) freeze-drying the bacterial suspension to obtain freeze-dried bacterial powder.

5. The microbial inoculum of claim 4, wherein the MRS plate medium in the step (1) is prepared by the following method: mixing 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of dipotassium hydrogen phosphate heptahydrate, 1mL of tween-80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate, 15g of agar and 1000mL of distilled water, stirring the mixed solution at natural pH, sterilizing at 121 ℃ and 0.1MPa for 20min, pouring the sterilized mixed solution into a flat dish, and cooling for later use.

6. The microbial inoculum according to claim 3, further comprising isomaltooligosaccharides.

7. The application of lactobacillus rhamnosus JYLR-219 of claim 1 in preparing products for increasing bone density and preventing osteoporosis.

8. Use of the bacterial agent of claim 2 in the preparation of a product for enhancing bone density and preventing osteoporosis.