CN108467843A - A kind of lactobacillus plantarum improving osteoporosis - Google Patents
A kind of lactobacillus plantarum improving osteoporosis Download PDFInfo
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- CN108467843A CN108467843A CN201810414459.4A CN201810414459A CN108467843A CN 108467843 A CN108467843 A CN 108467843A CN 201810414459 A CN201810414459 A CN 201810414459A CN 108467843 A CN108467843 A CN 108467843A
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- lactobacillus plantarum
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- bone trabecula
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- 240000006024 Lactobacillus plantarum Species 0.000 title claims abstract description 30
- 235000013965 Lactobacillus plantarum Nutrition 0.000 title claims abstract description 30
- 229940072205 lactobacillus plantarum Drugs 0.000 title claims abstract description 29
- 208000001132 Osteoporosis Diseases 0.000 title claims abstract description 23
- 230000001580 bacterial effect Effects 0.000 claims abstract description 13
- 244000005700 microbiome Species 0.000 claims abstract description 4
- 241000894006 Bacteria Species 0.000 claims description 15
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 10
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
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- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 description 1
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Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
-
- 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
- A61K2035/11—Medicinal preparations comprising living procariotic cells
- A61K2035/115—Probiotics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12R2001/25—Lactobacillus plantarum
Abstract
The invention discloses a kind of lactobacillus plantarum (Lactobacillus plantarum) AR495 bacterial strains improving osteoporosis, lactobacillus plantarum AR495 bacterial strains have been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.14004.The lactobacillus plantarum AR495 of the present invention can be effectively improved the mouse osteoporosis because of caused by removal ovary, its trabecular bone density, bone trabecula number and bone trabecula thickness can be dramatically increased through statistical analysis, to dramatically increase elasticity modulus to reduce risk of bone fracture.
Description
Technical field
The invention belongs to microorganism fields, and in particular to a kind of lactobacillus plantarum improving osteoporosis.
Background technology
Osteoporosis is a kind of impaired with bone tissue microstructure, and bone mine ingredient and bone matrix ratio are constantly reduced, bone
Qualitative change is thin, and bone trabecula quantity is reduced, and bone brittleness increases and risk of fractures increases the dysostosis disease for symptom.The whole world
There are about 200,000,000 or more crowds to suffer from osteoporosis, and China's sufferers of osteoporosis face occupy first place in the world, and existing about 90,000,000, it accounts for total
The 7.1% of number.In the U.S., 50 years old or more women has one third to be diagnosed as patients with osteoporosis, there is 1.5 hundred million every year on average
People suffers from osteoporosis.
Osteoporotic fracture is the serious consequence of the illness, due to bone strength lower, in daily life accidentally fall or
Easily occur Hip Fracture, including neck of femur and intertrochanteric fracture, compression fracture of lumbar vertebra after colliding.In the fractures phase
Between, due to bed, limitation of activity etc., easily there is hypostatic pneumonia, pressure sore, deep vein thrombosis, urinary infection and constipation etc.
A series of complication.According to statistics, 50% women and 20% male they in life, an at least concurrent osteoporotic bone
Folding.The peak bone mass and two side of bone loss occurred with age growth that the morbidity of the illness is reached when being by 20-30 Sui
What face factor determined.Human body peak bone mass 50%-85% be by genetic determination, and environmental factor with the age increase and occur
Bone amount plays the role of more importantly in reducing.Osteoporosis, which has become, threatens the middle-aged and the old and menopausal women normal life
One of principal disease.Therefore, a kind of lactobacillus plantarum that can be effectively improved osteoporosis is developed, there is very important meaning
Justice.
Invention content
The present invention is to carry out to solve the above-mentioned problems, and it is an object of the present invention to provide a kind of plant breast improving osteoporosis
Bacillus.
The present invention provides a kind of lactobacillus plantarums (Lactobacillus plantarum) improving osteoporosis
AR495 bacterial strains, have the feature that：Lactobacillus plantarum AR495 bacterial strains have been preserved in Chinese microorganism strain preservation management committee
Member's meeting common micro-organisms center, deposit number are CGMCC No.14004.
The present invention also provides lactobacillus plantarum AR495 bacterial strains to prepare the lactic acid bacteria production for improving osteoporosis in fermentation
Application in product.
The effect of invention
According to the lactobacillus plantarum according to the present invention for improving osteoporosis, it can be effectively improved and cause because of removal ovary
Mouse osteoporosis, can dramatically increase its trabecular bone density, bone trabecula number and bone trabecula thickness through statistical analysis, to
Elasticity modulus is dramatically increased to reduce risk of bone fracture.
Description of the drawings
Fig. 1 is the femur bone trabecula μ CT images of the SHAM groups in the embodiment of the present invention；
Fig. 2 is the femur bone trabecula μ CT images of the OVX groups in the embodiment of the present invention；
Fig. 3 is the femur bone trabecula μ CT images of the AR495 groups in the embodiment of the present invention；
Fig. 4 is the femur bone trabecula μ CT images of the E2 groups in the embodiment of the present invention.
Specific implementation mode
In order to which the technological means for making the present invention realize is easy to understand with effect, with reference to embodiments and attached drawing is to this
Invention is specifically addressed.
In following embodiments, used reagent is obtained from general commercial sources unless otherwise specified, the reality being not specified
Condition is tested with reference to conventional laboratory conditions or the condition suggested in accordance with supplier.
Embodiment one：Separation, culture and the preservation of lactobacillus plantarum AR495
1,1, strain separating and culture
Step 1, yellow rice wine vinasse is acquired, is diluted with concentration gradient dilution method, obtains dilution.
Step 2, the dilution of 100uL is taken to be coated on 37 DEG C of Anaerobic culturel for carrying out 40-56h on MRS culture medium flat plates
Afterwards, being chosen respectively to new MRS culture medium flat plates for various trait is crossed according to the shape of bacterium colony, size, color etc.
It isolates and purifies, obtains isolated strains.
Wherein, the production method of every liter of MRS culture medium is：Take extracted beef powder 10g, casein peptone 10g, yeast extract
5g, glucose 20g, sodium acetate trihydrate 8.3g, dipotassium hydrogen phosphate 2g, bitter salt 0.58g, Manganous sulfate monohydrate 0.25g,
Tween-80 1mL is settled to 1L using deionized water, and the sterilizing of 20min is then carried out under conditions of 115 DEG C.
Step 3, isolated strains are subjected to Gram's staining and catalase touches experiment, then according to lactic acid bacteria gram
The characteristics of stained positive and non-catalase, chooses Gram-positive and catalase touches negative bacterial strain.
Step 4, the DNA for extracting bacterial strain, using lactic acid bacteria 16srDNA segment universal primer 27F/1492R, through PCR amplification
After be sequenced, then in NCBI gene pools gene order carry out Homology search, obtain lactobacillus plantarum AR237 bacterial strains.
Wherein, 27F 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R 5'-GGTTACCTTGTTACGACTT-
3'。
2, culture presevation and activation
Lactobacillus plantarum AR495 bacterial strains are configured to lactobacillus plantarum AR495 bacterium solutions, final concentration 20% is added in bacterium solution
Glycerine, mixing is placed on -80 DEG C of refrigerators, can save 2-3, takes out glycerine cryopreservation tube when activating, melts under condition of ice bath
Change, bacterium solution is inoculated into preprepared MRS culture dishes with aseptic inoculation ring, is placed in anaerobic culture box and grows the left sides 40h
The right side in culture dish until grow complete bacterium colony, you can is passed on.
In addition, lactobacillus plantarum (Lactobacillusplantarum) AR495 bacterial strains are on 04 07th, 2017
Preservation is carried out in China General Microbiological culture presevation administrative center, deposit number is CGMCC No.14004.
Embodiment two：Lactobacillus plantarum AR495 improves osteoporosis function evaluation
1, animal model is established
SPF grades of female C57BL/6 mouse adapted to environment after 1 week, took weight 18g ± 1g mouse 32, and fasting 12 hours is (no
Prohibit water), it is random to be grouped, every group 8, to the intraperitoneal injection 70mg/kg anesthetic yellow Jackets of every group of mouse, and to wherein
8 only operate notch in corresponding site, do not extract ovary as sham-operation group, remaining 24 progress bilateral ovaries enucleation,
Mouse it is postoperative can normal activity, voluntarily drinking-water ingest, postoperative wound healing is normal, without red and swollen, exudation, suppurate phenomena such as.
The method of 1.1 bilateral ovaries enucleation
It carries out back of the body position after anaesthetizing mouse and anaesthetizing successfully to fix, the position of about 1cm is cut under away from most end rib cage
Hair cuts the skin of about 1.5-2cm, by away within the scope of 2cm at left and right sides of incision dorsal muscles and skin detach, it is visible in the visual field
White cellulite below dorsal muscles, ovary embed wherein；Cellulite is gently pressed from both sides with elbow ophthalmic tweezers to move to outside notch, you can see
The cellular ovary of pink；The fallopian tubal that connection ovary is tightened with No. 4 operation suture threads to prevent bleeding, then ovary is completely plucked
It removes, finally uterus is taken advantage of a situation with method excision other side ovary and sends original position back to, sutures dorsal muscles and skin successively.
2, animal packet and administration
Sham-operation group mouse is denoted as SHAM groups, the normal mouse of post-operative recovery is randomly divided into 3 groups, is denoted as OVX groups respectively,
AR495 groups and E2 groups, SHAM groups and OVX groups give 0.9% physiological saline, and it is outstanding that AR495 groups give lactobacillus plantarum AR495 bacterium
Liquid, E2 groups give 0.02%17 beta estradiol solution, then gastric infusion, and administered volume is 100 μ L/10g weight, is given daily
Medicine, continuous 8 weeks, and during the administration, a weight is weighed daily, gavage amount is adjusted according to weight.
Wherein, the preparation method of lactobacillus plantarum AR495 bacteria suspensions is：It it is 4 DEG C in temperature, speed is the item of 6000rpm
Centrifugation reaches the bacterium solution of the third generation under part, is often 2%, growth time 16h for inoculum concentration, abandons supernatant, 0.9% physiological saline weight
It is outstanding, it repeats the above steps 3 times, until the supernatant water white transparency after centrifugation, according to AR495OD600Light absorption value and CFU/mL
Relationship adjusts bacteria concentration with ultraviolet-uisible spectrophotometer so that bacteria suspension ultimate density is 10^9CFU/mL.
3, sample collection
4 groups of mouse in fasting 12 hours (can't help water) after the last administration, then weigh in, and collect urine and excrement,
- 80 DEG C of refrigerators are deposited in, 70mg/kg anesthetic yellow Jackets are then injected intraperitoneally, mouse heart puncture takes blood and puts to death small
Mouse, complete separating mouse bilateral femur and shin bone, it is fixed in 10% formalin solution.
4, femur bone trabecula parametric measurement
Fig. 1 is the femur bone trabecula μ CT images of the SHAM groups in the embodiment of the present invention, and Fig. 2 is the embodiment of the present invention
In OVX groups femur bone trabecula μ CT images, Fig. 3 is the femur bone trabecula μ CT figure of the AR495 groups in the embodiment of the present invention
Picture；Fig. 4 is the femur bone trabecula μ CT images of the E2 groups in the embodiment of the present invention.
Left femur is scanned with micro-CT, it is thick to measure femoral bmd, diaphysis fraction, bone trabecula number, bone trabecula
Degree and bone trabecula separating degree, as a result as shown in table 1 and Fig. 1-Fig. 4.
Table 1：Femur bone trabecula parameter
5, femur mechanics of materials strength detection
Three point bending test is carried out to right side femur using universal testing machine, measures femur maximum stress in bend, most
Macrobending load and elasticity modulus, the results are shown in Table 2.
Table 2：Femur mechanical parameters
The effect of embodiment
In the above embodiment of the present invention, by mouse osteoporosis model, lactobacillus plantarum AR495 patients before and after intervention is measured
Femur bone trabecula parameter：Bone density, diaphysis fraction, bone trabecula number, bone trabecula thickness and bone trabecula separating degree；Femur material
Mechanical strength：Maximum stress in bend, maximum deflection load and elasticity modulus.
By OVX groups compared with SHAM groups, significant difference is all had in terms of bone trabecula parameter and elasticity modulus,
AR495 intervene after compared with OVX groups, significantly improve femoral bmd, diaphysis fraction, bone trabecula number, bone trabecula thickness and
Elasticity modulus, and bone trabecula caliper recovery, to the level of sham-operation group, elasticity modulus is significantly better than SHAM groups, at the same time,
Positive controls compared with model group, are significantly increased in above-mentioned parameter, illustrate lactobacillus plantarum AR495 can be effectively improved by
Osteoporosis symptoms caused by estrogen deficiency.
Therefore, the lactobacillus plantarum AR495 that the embodiment of the present invention is filtered out can be effectively improved to be caused because of removal ovary
Mouse osteoporosis, can dramatically increase its trabecular bone density, bone trabecula number and bone trabecula thickness through statistical analysis, to
Elasticity modulus is dramatically increased to reduce risk of bone fracture, so lactobacillus plantarum AR495 can be used for preparing for improving sclerotin
Loose lactic acid bacteria product, to ensure the health of body.
The above embodiment is the preferred case of the present invention, is not intended to limit protection scope of the present invention.
Claims (2)
1. a kind of lactobacillus plantarum (Lactobacillusplantarum) AR495 bacterial strains improving osteoporosis, feature exist
In：The lactobacillus plantarum AR495 bacterial strains have been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center,
Deposit number is CGMCC No.14004.
2. lactobacillus plantarum AR495 bacterial strains according to claim 1 prepare the lactic acid for improving osteoporosis in fermentation
Application in bacterium product.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810414459.4A CN108467843B (en) | 2018-05-03 | 2018-05-03 | Lactobacillus plantarum for improving osteoporosis |
| PCT/CN2019/081367 WO2019210762A1 (en) | 2018-05-03 | 2019-04-04 | Lactobacillus plantarum for improving osteoporosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810414459.4A CN108467843B (en) | 2018-05-03 | 2018-05-03 | Lactobacillus plantarum for improving osteoporosis |
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| Publication Number | Publication Date |
|---|---|
| CN108467843A true CN108467843A (en) | 2018-08-31 |
| CN108467843B CN108467843B (en) | 2021-01-22 |
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| CN201810414459.4A Active CN108467843B (en) | 2018-05-03 | 2018-05-03 | Lactobacillus plantarum for improving osteoporosis |
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| WO (1) | WO2019210762A1 (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108624520A (en) * | 2017-03-16 | 2018-10-09 | 景岳生物科技股份有限公司 | Promote the lactobacillus plantarum strain GMNL-662 and combinations thereof of inostosis |
| WO2019210762A1 (en) * | 2018-05-03 | 2019-11-07 | 上海理工大学 | Lactobacillus plantarum for improving osteoporosis |
| CN111621449A (en) * | 2020-07-02 | 2020-09-04 | 重庆第二师范学院 | Probiotic and application thereof in secondary osteoporosis |
| CN114196600A (en) * | 2022-01-10 | 2022-03-18 | 上海理工大学 | Lactobacillus plantarum with effect of relieving irritable bowel syndrome and application thereof |
| CN114681494A (en) * | 2021-12-24 | 2022-07-01 | 上海理工大学 | Application of lactobacillus plantarum in preparation of medicament with protective effect on osteoarthritis |
| CN115772483A (en) * | 2022-08-10 | 2023-03-10 | 重庆第二师范学院 | Lactobacillus plantarum HYF15 and application thereof |
| CN115806898A (en) * | 2022-08-10 | 2023-03-17 | 重庆第二师范学院 | Lactobacillus plantarum KSFY04 and application thereof |
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| CN111621449A (en) * | 2020-07-02 | 2020-09-04 | 重庆第二师范学院 | Probiotic and application thereof in secondary osteoporosis |
| CN114681494A (en) * | 2021-12-24 | 2022-07-01 | 上海理工大学 | Application of lactobacillus plantarum in preparation of medicament with protective effect on osteoarthritis |
| CN114196600A (en) * | 2022-01-10 | 2022-03-18 | 上海理工大学 | Lactobacillus plantarum with effect of relieving irritable bowel syndrome and application thereof |
| CN114196600B (en) * | 2022-01-10 | 2023-12-08 | 上海理工大学 | Lactobacillus plantarum with effect of relieving irritable bowel syndrome and application thereof |
| CN115772483A (en) * | 2022-08-10 | 2023-03-10 | 重庆第二师范学院 | Lactobacillus plantarum HYF15 and application thereof |
| CN115806898A (en) * | 2022-08-10 | 2023-03-17 | 重庆第二师范学院 | Lactobacillus plantarum KSFY04 and application thereof |
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| CN108467843B (en) | 2021-01-22 |
| WO2019210762A1 (en) | 2019-11-07 |
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