CN116904378B - Lactobacillus johnsonii JYLO-010 for promoting bone growth, microbial inoculum and application - Google Patents
Lactobacillus johnsonii JYLO-010 for promoting bone growth, microbial inoculum and application Download PDFInfo
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- CN116904378B CN116904378B CN202311182507.9A CN202311182507A CN116904378B CN 116904378 B CN116904378 B CN 116904378B CN 202311182507 A CN202311182507 A CN 202311182507A CN 116904378 B CN116904378 B CN 116904378B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention relates to the technical field of microorganism application, in particular to Lactobacillus johnsonii JYLO-010 for promoting bone growth, a microbial inoculum and application. Lactobacillus johnsoniiLactobacillus johnsonii) JYLO-010 is preserved in 2022 at 3-month 17 to China general microbiological culture Collection center with the preservation number of CGMCC No.24538 and the preservation address of North Chen Xiyu No. 1 and 3 in the Chaoyang area of Beijing city. The lactobacillus johnsonii JYLO-010 provided by the invention can obviously improve the content of 25-hydroxy vitamin D in a mouse body, thereby promoting the apparent absorption rate of the mouse to calcium. Lactobacillus johnsonii JYLO-010 is effective in promoting osteoblast proliferation, differentiation and maturation and inhibiting osteoclast formation. The administration of the Lactobacillus johnsonii JYLO-010 bacterial agent can obviously improve the femur bone density of the mice.
Description
Technical Field
The invention relates to the technical field of microorganism application, in particular to Lactobacillus johnsonii JYLO-010 for promoting bone growth, a microbial inoculum and application.
Background
The bone development of infants is faster during growth, if calcium deficiency exists, the bone development can be affected, calcium is generally supplemented by eating foods with high calcium content or drinking calcium-containing oral liquid, however, only a small part of the calcium is absorbed by the body, and the majority of the calcium is discharged from the body, so that the absorption rate is lower. Current methods for promoting bone growth in children include the following: 1. performing proper motion: bone development can be stimulated by bouncing, jumping and other movements, so that bone growth of children is promoted. 2. Keep sufficient sleep: bone mainly grows at night, and growth hormone is more secreted at night, so that the bone growth of children can be promoted by keeping sufficient sleep and keeping good habit of early sleep and early onset. 3. The purpose of promoting the growth of the bone of children is achieved by adding substances such as protein, vitamins, minerals and the like into the children milk powder. Most of the health care products for promoting bone growth in the market at present are compounded by a plurality of nutrient substances, and the effect of promoting bone growth and development is not obvious due to the lack of low absorption rate and utilization rate of the nutrient substances by children.
With the increasing research of bone growth and development, intestinal probiotics are found to have a certain promotion effect on bone growth and development. Short chain fatty acid generated by probiotics in the intestinal tract can reduce the environmental pH value of the intestinal tract, and reduce calcium and phosphorus in the intestinal tract to form a compound, so that the absorption of calcium is promoted; the probiotics stimulate intestinal cells to produce incretins, which comprise a series of hormones secreted from intestinal tracts and have glucose concentration-dependent insulin secretion promoting effect, including glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide 1 (GLP-1), and can also promote calcium absorption and bone formation. Therefore, development of intestinal probiotics and development of probiotics to promote long-term production are becoming research hotspots.
Disclosure of Invention
Aiming at the problem that the effect of the product for promoting the bone growth of children is not obvious in the market, the invention provides lactobacillus johnsonii JYLO-010 for promoting the bone growth, a microbial inoculum and application. The lactobacillus johnsonii JYLO-010 can remarkably improve the vitamin D content in the body, promote the absorption of calcium by intestinal tracts and increase the bone density of femur, and can realize the effect of promoting the bone development of children by improving the vitamin D content, the calcium content and the bone density level in the body.
In a first aspect, the invention provides a Lactobacillus johnsonii JYLO-010 for promoting bone growth, lactobacillus johnsoniiLactobacillus johnsonii) JYLO-010 is preserved in China general microbiological culture Collection center (CGMCC) in 2022, 3 and 17 days, with a preservation number of CGMCC No.24538 and a preservation address of North Chen Xiyu No. 1 and 3 in the Chaoyang area of Beijing city.
In a second aspect, the invention provides a Lactobacillus johnsonii JYLO-010 microbial inoculum, comprising the bacterial cells of Lactobacillus johnsonii JYLO-010.
Further, the preparation method specifically comprises the following steps:
(1) Preparing MRS liquid culture medium and MRS plate culture medium;
(2) Activating preserved Lactobacillus johnsonii JYLO-010 on MRS flat-plate culture medium, inoculating activated bacteria into MRS liquid culture medium, and culturing to obtain bacterial liquid;
(3) Centrifuging the bacterial liquid, collecting bacterial cells, washing the bacterial cells by using sterile physiological saline, and then, re-suspending the bacterial cells in reconstituted skim milk with the mass concentration of 15% to obtain suspension; adjusting the concentration of the suspension to obtain a bacterial suspension, and freeze-drying the bacterial suspension to obtain bacterial powder;
(4) Mixing the bacterial powder with isomaltooligosaccharide to prepare the bacterial agent.
Further, in the step (1), the method for preparing the MRS liquid culture medium comprises the following steps: mixing peptone 10g, beef powder 5g, sodium acetate trihydrate 5g, dipotassium phosphate heptahydrate 2g, tween 80 1mL, manganese sulfate tetrahydrate 0.05g, triammonium citrate 2g, glucose 20g, magnesium sulfate heptahydrate 0.2g and distilled water 1000mL, regulating pH to 6.8 with HCl, stirring, and sterilizing at 121deg.C under 0.1MPa for 20 min.
In the step (2), the inoculation amount of the activated bacteria inoculated to the MRS liquid culture medium is 1% -2%.
Further, in the step (2), the culture temperature is 35-38 ℃ and the culture time is 24-36 h.
Further, in the step (3), the concentration of the suspension was adjusted to 1.0X10 10 ~2.0×10 10 cfu/mL。
In a third aspect, the invention provides an application of the lactobacillus johnsonii JYLO-010 in preparing a product for promoting bone growth of children.
The invention has the beneficial effects that:
the lactobacillus johnsonii JYLO-010 provided by the invention can obviously improve the content of 25-hydroxy vitamin D in a mouse body, thereby promoting the apparent absorption rate of the mouse to calcium. The detection result shows that the femoral bone density of the mice can be obviously improved by taking the Lactobacillus johnsonii JYLO-010 bacterial agent. Further, experiments prove that the lactobacillus johnsonii JYLO-010 can effectively promote proliferation, differentiation and maturation of osteoblasts and inhibit formation of osteoclasts. Thus, the Lactobacillus johnsonii JYLO-010 has important significance in preparing products for promoting the bone growth of children.
Detailed Description
In order to better understand the technical solutions of the present invention, the following description will clearly and completely describe the technical solutions of the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
Example 1 isolation and identification of species
1. Strain screening and purification
(1) Bacterial strain source: and collecting samples from the self-made pickled vegetables by the Yunnan Jing farmers 10 months in 2017, and putting the samples into a sample transfer box to be brought back to a laboratory for standby.
(2) Preparing a sample:
(1) placing sterilized normal saline (0.85%) into a sterile triangular flask, adding 1g of the sample collected in the step (1) into the sterilized normal saline, and oscillating for later use;
(2) diluting the solution obtained in the step (1) to obtain samples with different concentration gradients, respectively 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 The reference numerals are 1#, 2#, 3#, 4#, 5#, 6#, 7#, respectively, for standby.
(3) Preparation of MRS plate medium:
10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of dipotassium 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; mixing the above materials, naturally adjusting pH, stirring, sterilizing at 121deg.C under 0.1MPa for 20min, pouring sterilized culture medium into a plate, and cooling.
(4) Culturing the strain: coating the No. 1, no.2, no. 3, no. 4, no. 5, no. 6 and No. 7 solutions in MRS plate culture medium by using a coater, and culturing for 48h under anaerobic condition at 37 ℃;
(5) Colonies were selected according to the following colony characteristics:
the diameter is 1-2 mm, the colony is round, the edge is neat, the middle of the micro white is provided with a bulge, the calcium dissolving ring is large, and the bacterial strain with negative catalase and positive gram is selected.
(6) Separation and purification
Selecting 5 single colonies according to the colony and biochemical characteristics of the step (5), inoculating the single colonies to an MRS flat plate culture medium by a streaking method, purifying for 2-3 times, culturing for 48 hours at 37 ℃ under anaerobic conditions, selecting the single colonies, and preserving the single colonies in a glycerol tube at-80 ℃.
2. Authentication
Single colony obtained after separation and purification is sent to identification unit: the primers used in the identification process of the biological engineering (Shanghai) Co., ltd were as follows:
primer sequence:
27F：5'-AGAGTTTGATCCTGGCTCAG-3'
1492R：5'-GGTTACCTTGTTACGACTT-3'
the gene sequences of the strains obtained in the identification process are as follows:
CACCGGCTTTGGGTGTTACAGACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAACGGCTTTAAGAGATCCGCTTGCCTTCGCAGGTTCGCTTCTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCAACTTAGTGATGGCAACTAATGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCTCAGCGTCCCCGAAGGGAACACCTAATCTCTTAGGTTTGCACTGGATGTCAAGACCTGGTAAGGCTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAACCTCCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGAGAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGTTCAACAGTTTCTGATGCAATTCTCCGGTTGAGCCGAAGGCTTTCACATCAGACTTATTGAACCGCCTGCACTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTAAGTAATTACCGTCAAATAAAGGCCAGTTACTACCTCTATCTTTCTTCACTACCAACAGAGCTTTACGAGCCGAAACCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATTGCCTTGGTAAGCCGTTACCTTACCAACTAGCTAATGCACCGCAGGTCCATCCAAGAGTGATAGCAGAACCATCTTTCAAACTCTAGACATGCGTCTAGTGTTGTTATCCGGTATTAGCATCTGTTTCCAGGTGTTATCCCAGTCTCTTGGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTCGCTTGTATCTAGTTTCATTTAGTGCAAGCACCAAAATCATCTAGGCAAGCTCGCTCGACTGCAGTA
the identification result is as follows: the strain is Lactobacillus johnsoniiLactobacillus johnsonii) The classifying units are as follows:Bacteria;Firmicutes;Bacilli;Lactobacillales;Lactobacillaceae;Lactobacillus。
the identified strain is named as Lactobacillus johnsonii JYLO-010, and is sent to China general microbiological culture Collection center for preservation, and the preservation information is as follows;
classification naming: lactobacillus johnsoniiLactobacillus johnsoniiPreservation date: 2022, 3, 17, deposit address: beijing, chaoyang area, north Chenxi way No. 1, no. 3, post code: 100101, accession number: CGMCC No.24538.
EXAMPLE 2 preparation of Lactobacillus johnsonii JYLO-010 microbial inoculum
(1) Preparation of MRS liquid medium and MRS plate medium: mixing 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of dipotassium 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 and 1000mL of distilled water, regulating the pH to 6.8 by using HCl, stirring uniformly, and sterilizing for 20min at 121 ℃ and 0.1MPa to prepare an MRS liquid culture medium; adding 15g/L agar into the prepared MRS liquid culture medium, and cooling to obtain the MRS flat culture medium.
(2) And (3) activating the preserved lactobacillus johnsonii JYLO-010 on an MRS flat-plate culture medium for 1 time, picking single bacterial colony, inoculating the single bacterial colony into the MRS liquid culture medium, and then culturing for 24 hours at 37 ℃ to obtain bacterial liquid.
(3) After the bacterial liquid is centrifuged, bacterial cells are collected, the bacterial cells are obtained by washing with sterile normal saline and centrifuging again, the bacterial cells are resuspended in 15% (w/w) reconstituted skim milk to obtain bacterial suspension, and the bacterial suspension is put into a vacuum freeze dryer to be freeze-dried to obtain bacterial powder.
(4) The microbial powder is mixed with fructo-oligosaccharide to prepare the microbial agent, and the fructo-oligosaccharide is purchased from Boehringer Biotechnology Co., ltd.
In this example, the prepared Lactobacillus johnsonii JYLO-010 bacterial agent had a bacterial count of 200 hundred million/g.
EXAMPLE 3 preparation of Lactobacillus johnsonii JYLO-010 microbial inoculum
(1) Preparation of MRS liquid medium and MRS plate medium: mixing 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of dipotassium 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 and 1000mL of distilled water, regulating the pH to 6.8 by using HCl, stirring uniformly, and sterilizing for 20min at 121 ℃ and 0.1MPa to prepare an MRS liquid culture medium; adding 15g/L agar into the prepared MRS liquid culture medium, and cooling to obtain the MRS flat culture medium.
(2) And (3) activating the preserved lactobacillus johnsonii JYLO-010 on an MRS flat-plate culture medium for 1 time, picking single bacterial colony after activation, inoculating the single bacterial colony into the MRS liquid culture medium, and culturing for 24 hours at 37 ℃ to obtain bacterial liquid.
(3) After the bacterial liquid is centrifuged, bacterial cells are collected, the bacterial cells are obtained by washing with sterile normal saline and centrifuging again, the bacterial cells are resuspended in 15% (w/w) reconstituted skim milk to obtain bacterial suspension, and the bacterial suspension is put into a vacuum freeze dryer to be freeze-dried to obtain bacterial powder.
(4) The microbial powder is mixed with fructo-oligosaccharide to prepare the microbial agent, and the fructo-oligosaccharide is purchased from Boehringer Biotechnology Co., ltd.
In this example, the amount of cells in the prepared Lactobacillus johnsonii JYLO-010 bacterial agent was 100 hundred million/g.
EXAMPLE 4 Effect of Lactobacillus johnsonii JYLO-010 on 25-hydroxyvitamin D content in mice
Kunming mice (KM mice) purchased 4 weeks old were randomly divided into 2 groups of 20 mice each, one group being a control group and the other group being an experimental group. The test group added the Lactobacillus johnsonii JYLO-010 bacterial powder (the dosage is 1 g/d) prepared in example 2 into the daily feed, and the control group did not added the Lactobacillus johnsonii JYLO-010 bacterial powder into the daily feed, and the daily quantitative feeding was carried out. After 14 days of feeding, 25-hydroxyvitamin D content in KM mouse serum was determined using a mouse 25-hydroxyvitamin D (25-OH-VD) enzyme-linked immunosorbent assay kit (purchased from cernuikan biotechnology (beijing) inc.) with the following results:
TABLE 1 serum 25-hydroxyvitamin D content of KM mice of different groups
According to the data in the table, the variance analysis shows that the experimental group and the control group have obvious difference (P=0.021), which indicates that the Lactobacillus johnsonii JYLO-010 can obviously improve the content of 25-hydroxy vitamin D in the mice.
Example 5 influence of Lactobacillus johnsonii JYLO-010 on apparent absorption Rate of mouse calcium
Kunming mice (KM mice) purchased 4 weeks old were randomly divided into 2 groups of 20 mice each, one group being a control group and the other group being an experimental group. The control group was fed with a low-calcium feed (calcium content: 0.4 mg/g) supplemented with calcium lactate particles (calcium content: 20 mg/g), and the experimental group was fed with a low-calcium feed (calcium content: 0.4 mg/g) supplemented with calcium lactate particles (calcium content: 20 mg/g) and with Lactobacillus johnsonii JYLO-010 powder (1 g/d) prepared in example 2. After 14 days of feeding, all mice were transferred into metabolic cages, and 3 days of calcium metabolism experiments were performed, and the daily food intake of each mouse was recorded; and collecting the feces for 72 hours, drying at 80 ℃, weighing, and recording the feces quantity. Grinding the collected mouse dung every day into fine powder, sieving with a 40-mesh sieve, uniformly mixing, preparing a sample solution after digestion according to the method described in the calcium absorption experiment standard in the technical Specification for inspection and evaluation of health food, measuring the calcium content in the mouse dung by using a flame atomic absorption method, and calculating the apparent absorption rate of calcium.
TABLE 2 apparent absorption of calcium by KM mice of different groups
Note that: in the same column, superscript contains the same letter indicating that the difference is statistically significant (P > 0.05), and superscript contains the different letter indicating that the difference is statistically significant (P < 0.05).
From the results in Table 2, there was a significant difference (P < 0.05) between the apparent absorption rates of calcium in the experimental and control groups KM mice by analysis of variance, indicating that Lactobacillus johnsonii JYLO-010 was able to significantly increase the apparent absorption rate of calcium in the mice.
EXAMPLE 6 influence of Lactobacillus johnsonii JYLO-010 on femoral bone Density in mice
The femur of the blank mice and the experimental mice treated in example 4 were subjected to experiments, and the bone density of the femur of the mice was measured using a dual-energy X-ray bone densitometer.
TABLE 3 femur bone Density for KM mice of different groups
According to the data in the table, the experimental group and the control group have significant differences (P < 0.05) through analysis of variance, which indicates that the Lactobacillus johnsonii JYLO-010 can significantly improve the femoral bone density of the mice.
EXAMPLE 7 influence of Lactobacillus johnsonii JYLO-010 on osteoblasts
First aboutPreparation of bacterial suspension (1×10) from JYLO-010 bacterial powder of Lactobacillus gasseri 9 CFU/mL). Digesting osteoblast MC3T3-E1 Subclone 14 to prepare single cell suspension, and regulating single cell suspension concentration to 5×10 after counting 4 cell/mL, inoculated in 96-well plate, 100 [ mu ] L per well. After culturing for 24 hours, the culture solution is discarded, the culture solution is replaced by a newly prepared lactobacillus johnsonii-containing suspension whole culture medium or a blank whole culture medium (containing 10% FBS and 1% of green-streptomycin), the culture is continued in an incubator for 48 hours, 10 mu L of MTT solution of 5.0mg/mL is added into each hole, liquid in each hole is sucked and removed after 4 hours, 150 mu L of DMSO is added into each hole, the crystals are fully dissolved by shaking for 10 minutes, the absorbance value is measured at 490nm, and the proliferation rate is calculated.
TABLE 4 influence of Lactobacillus johnsonii JYLO-010 on osteoblasts
From the results in Table 4, it is clear that Lactobacillus johnsonii JYLO-010 significantly promoted proliferation, differentiation and maturation of osteoblasts compared with the blank group.
EXAMPLE 7 influence of Lactobacillus johnsonii JYLO-010 on osteoclasts
Obtaining marrow cell suspension from femur and tibia of hindlimb of rabbit with birth time less than 48 hr, sieving with 200 mesh cell sieve, counting, and regulating cell suspension concentration to 2×10 6 Each well was inoculated with 500. Mu.L/mL of the culture medium in a 24-well plate. After 24h, the culture solution was aspirated, washed with PBS 1 time, and then added with a suspension of JYLO-010 bacteria of Lactobacillus johnsonii or a blank osteoclast induction medium (alpha-MEM whole medium added with 10) -8 mol/L25-hydroxy vitamin D) and culturing, and changing the culture medium every 2 days. After 8 days of incubation, the medium was discarded, and after light washing with PBS, TRAP staining was performed, and specific staining methods were referred to the instructions attached to the anti-tartaric acid phosphatase staining kit (purchased from Beijing Soy Biotech Co., ltd.). After staining, the cells were observed under a microscope, and the numbers of TRAP positive cells in each culture well were counted.
TABLE 5 influence of Lactobacillus johnsonii JYLO-010 on osteoclasts
According to the data in the table above, the TRAP positive cell count was significantly reduced (P < 0.01) in the experimental group compared to the blank group, indicating that Lactobacillus johnsonii JYLO-010 was able to significantly inhibit osteoclast formation.
Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims.
Claims (7)
1. A Lactobacillus johnsonii JYLO-010 for promoting bone growth is characterized in thatLactobacillus johnsonii) JYLO-010 is preserved in 2022 at 3-month 17 to China general microbiological culture Collection center with the preservation number of CGMCC No.24538 and the preservation address of North Chen Xiyu No. 1 and 3 in the Chaoyang area of Beijing city.
2. A lactobacillus johnsonii JYLO-010 bacterial agent, comprising the bacterial body of the lactobacillus johnsonii JYLO-010 of claim 1.
3. The lactobacillus johnsonii JYLO-010 microbial agent as claimed in claim 2, wherein the preparation method comprises the following steps:
(1) Preparing MRS liquid culture medium and MRS plate culture medium;
(2) Activating preserved Lactobacillus johnsonii JYLO-010 on MRS flat-plate culture medium, inoculating activated bacteria into MRS liquid culture medium, and culturing to obtain bacterial liquid;
(3) Centrifuging the bacterial liquid, collecting bacterial cells, washing the bacterial cells by using sterile physiological saline, and then, re-suspending the bacterial cells in reconstituted skim milk with the mass concentration of 15% to obtain suspension; adjusting the concentration of the suspension to obtain a bacterial suspension, and freeze-drying the bacterial suspension to obtain bacterial powder;
(4) Mixing the bacterial powder with isomaltooligosaccharide to prepare the bacterial agent.
4. The lactobacillus johnsonii JYLO-010 bacteria agent of claim 3, wherein in the step (1), the method for preparing MRS liquid culture medium comprises the following steps: mixing peptone 10g, beef powder 5g, sodium acetate trihydrate 5g, dipotassium phosphate heptahydrate 2g, tween 80 1mL, manganese sulfate tetrahydrate 0.05g, triammonium citrate 2g, glucose 20g, magnesium sulfate heptahydrate 0.2g and distilled water 1000mL, regulating pH to 6.8 with HCl, stirring, and sterilizing at 121deg.C under 0.1MPa for 20 min.
5. The lactobacillus johnsonii JYLO-010 microbial inoculum of claim 3, wherein in the step (2), the inoculation amount of the activated bacteria inoculated in MRS liquid culture medium is 1% -2%.
6. The lactobacillus johnsonii JYLO-010 microbial inoculum of claim 3, wherein in the step (2), the culture temperature is 35-38 ℃ and the culture time is 24-36 h.
7. The Lactobacillus johnsonii JYLO-010 bacteria of claim 3, wherein in step (3), the concentration of the suspension is adjusted to 1.0X10 10 ~2.0×10 10 cfu/mL。
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