WO2019210762A1

WO2019210762A1 - Lactobacillus plantarum for improving osteoporosis

Info

Publication number: WO2019210762A1
Application number: PCT/CN2019/081367
Authority: WO
Inventors: 艾连中; 于婧; 夏永军; 王光强; 熊智强; 张汇
Original assignee: 上海理工大学
Priority date: 2018-05-03
Filing date: 2019-04-04
Publication date: 2019-11-07
Also published as: CN108467843A; CN108467843B

Abstract

Disclosed is a Lactobacillus plantarum strain AR495 for improving osteoporosis. The Lactobacillus plantarum strain AR495 is deposited in China General Microbiological Culture Collection Center, the deposit number being CGMCC No.14004.

Description

Lactobacillus plantarum for improving osteoporosis

Technical field

The invention belongs to the field of microorganisms, and particularly relates to a Lactobacillus plantarum which improves osteoporosis.

Background technique

Osteoporosis is a bone metabolism in which the microstructure of bone tissue is damaged, the ratio of bone mineral components and bone matrix is decreasing, bone is thinned, the number of trabecular bone is reduced, bone fragility is increased, and fracture risk is increased. Obstructive disease. About 200 million people in the world suffer from osteoporosis. China's osteoporosis patients rank first in the world, with about 90 million, accounting for 7.1% of the total. In the United States, one in three women over the age of 50 is diagnosed with osteoporosis, with an average of 150 million people suffering from osteoporosis each year.

Osteoporotic fracture is a serious consequence of the disease. Due to the reduced bone strength, hip fractures are easily caused by accidental falls or collisions in daily life, including femoral neck and intertrochanteric fractures, and lumbar vertebral compression fractures. During the treatment of fractures, due to bed rest, limited activity, etc., it is prone to a series of complications such as hypostatic pneumonia, pressure sores, deep vein thrombosis, urinary tract infection and constipation. According to statistics, 50% of women and 20% of men have at least one osteoporotic fracture during their lifetime. The onset of this condition is determined by the peak bone mass reached at 20-30 years of age and the bone loss that occurs with age. The peak bone mass of human body is 50%-85%, which is determined by genetics, and environmental factors play a more important role in the reduction of bone mass with age. Osteoporosis has become one of the major diseases that threaten the normal life of middle-aged and postmenopausal women. Therefore, it is of great significance to develop a Lactobacillus plantarum that can effectively improve osteoporosis.

Summary of the invention

The present invention has been made to solve the above problems, and an object thereof is to provide a Lactobacillus plantarum which improves osteoporosis.

The present invention provides a Lactobacillus plantarum AR495 strain for improving osteoporosis, which has the characteristic that the Lactobacillus plantarum strain AR495 has been deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee, and the preservation number is CGMCC No. .14004.

The present invention also provides the use of the Lactobacillus plantarum AR495 strain in the fermentation preparation of a lactic acid bacteria product for improving osteoporosis.

The role and effect of the invention

According to the present invention, the Lactobacillus plantarum which improves osteoporosis can effectively improve osteoporosis in mice caused by ovariectomy, and statistically analyzes the trabecular bone density, the number of trabecular bones and the small bone. The thickness of the beam, which significantly increases the modulus of elasticity to reduce the risk of fracture.

DRAWINGS

1 is a μCT image of a femoral trabecular bone of a SHAM group in an embodiment of the present invention;

2 is a femoral trabecular μCT image of the OVX group in the embodiment of the present invention;

Figure 3 is a femoral trabecular μCT image of the AR495 group in an embodiment of the present invention;

4 is a femoral trabecular μCT image of the E2 group in the embodiment of the present invention.

detailed description

In order to make the technical means and effects of the present invention easy to understand, the present invention will be specifically described below in conjunction with the embodiments and the accompanying drawings.

In the following examples, the reagents used were obtained from general commercial routes unless otherwise specified, and the unspecified experimental conditions were based on conventional experimental conditions or in accordance with the conditions recommended by the supplier.

Example 1: Isolation, culture and preservation of Lactobacillus plantarum AR495

1,1 isolation and cultivation of strains

In step 1, the rice wine trough is collected and diluted by a concentration gradient dilution method to obtain a diluent.

Step 2: 100 uL of the diluted solution was applied to the MRS medium plate for 40-56 h of anaerobic culture at 37 ° C, and the different traits were selected to the new MRS culture according to the shape, size, color, and the like of the colony. The base plate was subjected to scribing separation and purification to obtain an isolated strain.

Among them, each liter of MRS medium is prepared by taking 10 g of beef leaching powder, 10 g of casein mash, 5 g of yeast dipping powder, 20 g of glucose, 8.3 g of sodium acetate trihydrate, 2 g of dipotassium hydrogen phosphate, 0.58 g of magnesium sulfate heptahydrate, Manganese hydrate monohydrate 0.25g, Tween-801mL, dilute to 1L with deionized water, and then sterilized at 115 ° C for 20 min.

In step 3, the isolated strain is subjected to Gram staining and catalase touch test, and then Gram staining positive and catalase negative reaction are selected according to the characteristics of lactic acid bacteria Gram staining and no catalase. Strain.

In step 4, the DNA of the strain is extracted, and the universal primer 27F/1492R of the lactic acid bacteria 16sr DNA fragment is used, and amplified by PCR, and then homologously compared with the gene sequence in the NCBI gene bank to obtain the Lactobacillus plantarum AR237 strain.

Among them, 27F is 5'-AGAGTTTGATCCTGGCTCAG-3', and 1492R is 5'-GGTTACCTTGTTACGACTT-3'.

2. Culture preservation and activation

Lactobacillus plantarum AR495 strain is configured as Lactobacillus plantarum AR495 liquid solution, 20% of glycerin is added to the bacterial liquid, mixed and placed in a -80 ° C refrigerator, which can be stored for 2-3 years, and the glycerol is frozen during activation. The tube is taken out and thawed under ice bath conditions. The inoculum is inoculated into the prepared MRS culture dish with a sterile inoculating loop, and placed in an anaerobic incubator for about 40 hours until the whole colony grows in the culture dish. Passage.

In addition, the Lactobacillus plantarum AR495 strain has been deposited at the China General Microorganisms Collection and Management Center on April 7, 2017, and its deposit number is CGMCC No. 14004.

Example 2: Evaluation of the efficacy of Lactobacillus plantarum AR495 in improving osteoporosis

1. Establish an animal model

One week after SPF female C57BL/6 mice were acclimated to the environment, 32 mice weighing 18 g±1 g were taken, fasted for 12 hours (not allowed for water), randomized into groups, 8 rats in each group, intraperitoneal injection of each group of mice. 70mg/kg anesthetic sodium pentobarbital, and 8 of them were only incision in the corresponding site, no ovary was removed as sham operation group, and the other 24 were bilateral ovarian ablation. Drinking water, normal wound healing, no redness, exudation, suppuration and so on.

1.1 method of bilateral ovarian ablation

After anesthesia was performed on the mice and the anesthesia was successful, the dorsal fixation was performed, and the hair was cut at a position about 1 cm below the last rib, and the skin of about 1.5-2 cm was cut, and the back muscles within 2 cm from the left and right sides of the incision were The skin is separated, and the white fat group under the back muscle can be seen in the visual field, and the ovary is embedded therein; the elliptical ophthalmology is used to gently pull the cellulite to the outside of the incision, and the pink honeycomb ovary can be seen; Tightly connect the oviduct to the fallopian tube to prevent bleeding, and then completely remove the ovaries. The same method is used to remove the other side of the ovary and finally return the uterus to the original position, and suture the back muscle and skin in turn.

2. Animal grouping and administration

The sham-operated mice were recorded as SHAM group, and the mice that returned to normal after surgery were randomly divided into 3 groups, which were recorded as OVX group, AR495 group and E2 group, SHAM group and OVX group were given 0.9% normal saline, and AR495 group was given. Lactobacillus plantarum AR495 suspension, E2 group was given 0.02% 17β-estradiol solution, and then administered by intragastric administration, the dosage volume was 100 μL/10 g body weight, administered daily for 8 weeks, and during the administration, The body weight was weighed once a day, and the amount of stomach was adjusted according to the body weight.

Among them, the preparation method of the Lactobacillus plantarum AR495 suspension is: centrifugation to the third generation of the bacterial liquid at a temperature of 4 ° C and a speed of 6000 rpm, the inoculation amount of each generation is 2%, the growth time is 16 h, discarded The supernatant was resuspended in 0.9% saline, and the above steps were repeated 3 times until the supernatant after centrifugation was colorless and transparent. According to the relationship between the absorbance of AR495 OD ₆₀₀ and CFU/mL, the concentration of bacteria was adjusted by UV-visible spectrophotometer. The final concentration of the bacterial suspension was 10^9 CFU/mL.

3, sample collection

All the mice in the 4 groups were fasted for 12 hours after the last administration (not allowed to drink water), then weighed, collected urine and feces, stored in a refrigerator at -80 ° C, and then intraperitoneally injected with 70 mg / kg anesthetic sodium pentobarbital. The mice were bled by heart and the mice were sacrificed. The bilateral femur and tibia were completely isolated and fixed in 10% formalin solution.

4, femur bone trabecular parameter determination

1 is a femur trabecular μCT image of the SHAM group in the embodiment of the present invention, and FIG. 2 is a femoral trabecular μCT image of the OVX group in the embodiment of the present invention, and FIG. 3 is an embodiment of the present invention. Femur trabecular μCT images of the AR495 group; Fig. 4 is a femur trabecular μCT image of the E2 group in the embodiment of the present invention.

The left femur was scanned with micro-CT, and the femur bone density, bone volume fraction, trabecular bone number, trabecular thickness, and trabecular bone separation were measured. The results are shown in Table 1 and Figures 1 to 4.

Table 1: Femoral trabecular parameters

5. Determination of mechanical strength of femur materials

A three-point bending test was performed on the right femur using a universal material testing machine to determine the maximum bending stress, maximum bending load, and elastic modulus of the femur. The results are shown in Table 2.

Table 2: Mechanical parameters of femoral materials

The role and effect of the embodiment

In the above embodiments of the present invention, the femoral trabecular parameters before and after intervention of Lactobacillus plantarum AR495 were determined by a mouse osteoporosis model: bone mineral density, bone volume fraction, number of trabecular bone, trabecular thickness, and trabecular bone separation. Degree; mechanical strength of femur material: maximum bending stress, maximum bending load and elastic modulus.

Compared with the SHAM group, the OVX group had significant differences in trabecular bone parameters and elastic modulus. Compared with the OVX group, AR495 significantly improved femur bone mineral density, bone volume fraction, trabecular bone number, and trabecular bone. Thickness and elastic modulus, and the thickness of trabecular bone returned to the level of sham operation group, the elastic modulus was significantly better than that of SHAM group. At the same time, the positive control group was significantly improved compared with the model group in the above parameters. Lactobacillus plantarum AR495 is effective in improving osteoporosis caused by estrogen deficiency.

Therefore, the Lactobacillus plantarum AR495 screened in the examples of the present invention can effectively improve osteoporosis in mice caused by ovariectomy, and statistically analyzes the trabecular bone density, the number of trabecular bones and the trabecular bone. The thickness, which significantly increases the modulus of elasticity to reduce the risk of fracture, so the Lactobacillus plantarum AR495 can be used to prepare lactic acid bacteria products for improving osteoporosis, thereby ensuring the health of the body.

The above embodiments are preferred examples of the invention and are not intended to limit the scope of the invention.

Claims

A strain of Lactobacillus plantarum AR495 for improving osteoporosis, characterized in that the Lactobacillus plantarum strain AR495 has been deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee, and the preservation number is CGMCC No. 14004.
The use of the Lactobacillus plantarum AR495 strain according to claim 1 for the fermentation preparation of a lactic acid bacteria product for improving osteoporosis.