CN111019857B - Lactobacillus plantarum HG20 strain and application thereof - Google Patents
Lactobacillus plantarum HG20 strain and application thereof Download PDFInfo
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- CN111019857B CN111019857B CN201911292365.5A CN201911292365A CN111019857B CN 111019857 B CN111019857 B CN 111019857B CN 201911292365 A CN201911292365 A CN 201911292365A CN 111019857 B CN111019857 B CN 111019857B
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Abstract
Lactobacillus plantarum HG20 strain and application thereof, belonging to the field of functional food microorganisms. The lactobacillus plantarum HG20 strain disclosed by the invention is preserved in the China center for type culture Collection in 11 and 21 months in 2019, and the preservation numbers are as follows: CCTCC NO: M2019964. According to the invention, a collagen type rheumatoid arthritis rat model is adopted, and lactobacillus plantarum HG20 strain is administrated by intragastric administration for 21 days continuously, and the result shows that lactobacillus plantarum HG20 strain can reduce the diameter of ankle joint of rat; reducing inflammatory reaction, reducing the level of inflammatory factors IL-1 beta and TNF-alpha, and improving the level of anti-inflammatory factors IL-10; reducing levels of C-reactive protein, immunoglobulin IgG, IgM during the onset of rheumatoid arthritis; can effectively exert the anti-inflammatory effect and improve the symptoms of red, swollen and painful joints.
Description
Technical Field
The invention belongs to the technical field of functional food microorganisms, and particularly relates to a lactobacillus plantarum HG20 strain and application thereof.
Background
Rheumatoid Arthritis (RA) is a chronic autoimmune disease mainly manifested by joint pain, joint capsule swelling, lesions, and the like. The occurrence and development of RA are related to the release of a large number of inflammatory cytokines and inflammatory mediators, the main pathological change is chronic synovitis, cartilage and bone destruction is gradually caused, and finally, joint deformity and even joint function loss of patients are caused, so that RA is one of the main causes of labor loss and disability in China.
At present, no medicine for radically treating RA exists in clinic. Current drug therapy is mainly aimed at relieving pain, controlling the rate of progression of arthritis, and preventing articular cartilage damage. In modern clinic, non-steroidal anti-inflammatory drugs, glucocorticoids, methotrexate, novel biological agents and the like are mainly applied to control symptomatic treatment of diseases. Although these drugs can improve the symptoms of RA to some extent and inhibit bone destruction, they have strong toxic and side effects, such as inhibiting the immune function of the body and inducing severe adverse reactions such as infection of RA patients, etc., when used for a long time. Therefore, the search for safe and effective drugs for treating RA is of great significance.
Disclosure of Invention
The invention aims to provide a lactobacillus plantarum HG20 strain and application thereof, and the lactobacillus plantarum HG20 strain is used for preventing or treating rheumatoid arthritis.
The technical scheme adopted by the invention for solving the technical problem is as follows:
the lactobacillus plantarum (L.plantarum) HG20 strain is preserved in the China center for type culture Collection in 11-21.2019 with the preservation numbers as follows: CCTCC NO: M2019964.
The microbial inoculum takes the lactobacillus plantarum HG20 strain as an active component.
In a preferred embodiment, the concentration of the Lactobacillus plantarum (L.plantarum) HG20 strain is 109～1010CFU/g。
The preparation method of the microbial inoculum mainly comprises the following steps:
culturing Lactobacillus plantarum HG20 strain to 2L, centrifuging at 6000r/min for 5min, resuspending with physiological saline, cleaning, centrifuging at 6000r/min for 5min, collecting bacterial sludge, dissolving in 1L lyophilized protectant, and lyophilizing to obtain Lactobacillus plantarum HG20 leaven with bacterial count of 1.0 × 1010CFU/g。
In a preferred embodiment, the lyoprotectant is prepared by dissolving 40g of glucose, 5g of sodium ascorbate, 80g of fructo-oligosaccharide and 30g of trehalose in tertiary water, and making the volume of the solution equal to 750 mL.
The lactobacillus plantarum HG20 strain can be applied to preparation of products for preventing and treating rheumatoid arthritis.
The invention has the beneficial effects that:
the invention takes lactobacillus separated and identified from the surface of cucumber peel as a research object, and obtains a new lactobacillus strain named as lactobacillus plantarum HG20 strain through a large amount of experimental screening. The invention adopts a collagen type rheumatoid arthritis rat model, lactobacillus plantarum (L.plantarum) HG20 strain is administered by gastric lavage for 21 days continuously, and the treatment effect of the lactobacillus plantarum (L.plantarum) HG20 strain on the collagen type rheumatoid arthritis rat model is discussed. Research results show that lactobacillus plantarum (l.plantarum) HG20 strain can reduce the diameter of the ankle joint of rats; reducing inflammatory reaction, reducing the level of inflammatory factors IL-1 beta and TNF-alpha, and improving the level of anti-inflammatory factors IL-10; during the onset of rheumatoid arthritis, levels of C-reactive protein, immunoglobulin IgG, IgM were reduced.
Orally ingesting Lactobacillus plantarum (L.plantarum) HG20 strain or its bacterial preparation, and can reduce ankle joint diameter of rat; reducing inflammatory reaction, reducing the level of inflammatory factors IL-1 beta and TNF-alpha, and improving the level of anti-inflammatory factors IL-10; during the attack period of rheumatoid arthritis, the level of C-reactive protein, immunoglobulin IgG and IgM is reduced, and the anti-rheumatoid arthritis drug has the effects of preventing and treating the rheumatoid arthritis and relieving the inflammatory reaction. Therefore, the lactobacillus plantarum (L.plantarum) HG20 strain provided by the invention can be used for preparing medicines, medicines and the like for resisting rheumatoid arthritis, and meanwhile, the lactobacillus plantarum (L.plantarum) HG20 strain provided by the invention can also be used as functional probiotics to be applied to the fields of foods, health products and medicines, so that the application prospect is very wide.
Drawings
FIG. 1 is a diagram showing changes in the appearance of a collagen-type arthritis rat joint. In the figure, A is a control group; b is a model group; c is Lactobacillus plantarum HG20 group.
Detailed Description
The lactobacillus plantarum (L.plantarum) HG20 strain is preserved in China Center for Type Culture Collection (CCTCC) in 11-21.2019, and the address is as follows: eight-path Wuhan university school (Wuhan university collection center) 299 in Wuchang area, Wuhan city, Hubei province has the collection number: CCTCC NO: M2019964.
The active ingredient of the microbial inoculum is a lactobacillus plantarum (L.plantarum) HG20 strain. Wherein the concentration of Lactobacillus plantarum (L.plantarum) HG20 strain is 109～1010CFU/g。
The microbial inoculum provided by the invention has at least one of the following functions:
(a1) reducing the diameter of the ankle joint of a rat
(a2) Reducing serum levels of inflammatory factors (TNF-alpha, IL-1 beta)
(a3) Increasing the level of anti-inflammatory factor (IL-10) in serum;
(a4) reducing C-reactive protein (CRP), immunoglobulin (IgG, IgM) levels
The microbial inoculum is powder prepared by using a bacterial liquid containing a lactobacillus plantarum HG20 strain, and the method for preparing the powder is a freeze drying method.
The preparation method of the microbial inoculum comprises the following steps:
culturing Lactobacillus plantarum HG20 strain to 2L, centrifuging at 6000r/min for 5min, resuspending with physiological saline, cleaning, centrifuging at 6000r/min for 5min, collecting bacterial sludge, dissolving in 1L lyophilized protectant, and lyophilizing to obtain Lactobacillus plantarum HG20 leaven with bacterial count of 1.0 × 1010CFU/g。
Wherein the freeze-drying protective agent is prepared by dissolving 40g of glucose, 5g of sodium ascorbate, 80g of fructo-oligosaccharide and 30g of trehalose in tertiary water and fixing the volume to 750 mL.
The lactobacillus plantarum HG20 strain can be applied to preparation of products with functions of preventing or treating rheumatoid arthritis. The product can be food, health product or medicine. The lactobacillus plantarum HG20 strain or its microbial inoculum taken daily is used for replacing drugs for treating rheumatoid arthritis, which can obviously promote the absorption of bone joint cartilage inflammatory substances, thereby inhibiting the level of inflammatory factors (IL-1 beta, TNF-alpha) excessively produced in joint fluid, improving the level of anti-inflammatory factors IL-10 and relieving the pathological process of joint cartilage; during the attack period of rheumatoid arthritis, the level of C-reactive protein (CRP) and immunoglobulin (IgG and IgM) is reduced, and the pharmaceutical composition has the effects of preventing and treating the rheumatoid arthritis, and is safe and free from toxic and side effects.
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the 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.
The test materials used in the following examples were purchased from conventional biochemicals, unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Experimental materials:
solid MRS medium: the solvent is water, and contains peptone 10g/L, beef extract 10g/L, and yeast extract 5g/L, KH2PO42g/L, sodium acetate 5g/L, sodium citrate 5g/L, MgSO4·7H2O 0.2g/L、MnSO4·4H2O0.05 g/L, Tween-801 mL/L, agar 15g/L and glucose 20 g/L; the pH was 6.6.
Solid MRS 5.2 medium: MRS medium at pH 5.2.
Liquid MRS medium differs from solid MRS medium only in that no agar is added.
SD rats (body weight 180-220 g): cincha Changchun city Yinshi laboratory animal technology, Inc.
Basic feed: cincha Changchun city Yinshi laboratory animal technology, Inc.
Detecting the content of TNF-alpha in serum by adopting a Jiangsu enzyme-labeled rat tumor necrosis factor (TNF-alpha) ELISA kit; detecting the content of IL-1 beta in serum by adopting a Jiangsu enzyme-labeled rat interleukin 1 beta (IL-1 beta) ELISA kit; detecting the content of IL-10 in serum by adopting a Jiangsu enzyme-labeled rat interleukin 10(IL-10) ELISA kit; detecting the content of CRP in serum by adopting a Jiangsu enzyme-labeled rat C Reactive Protein (CRP) ELISA kit; adopting a Jiangsu enzyme-labeled rat immunoglobulin IgG (IgG) ELISA kit to detect the content of IgG in serum; and (3) detecting the IgM content in the serum by adopting a Jiangsu enzyme-labeled rat interleukin IgM (IgM) ELISA kit.
Among them, Freund's complete adjuvant was purchased from Sigma, USA, and type II collagen was purchased from Beijing Boleigdeko technology development Co.
Example 1 preparation of Lactobacillus plantarum (L.plantarum) HG20 Strain
Separation of Lactobacillus plantarum strains
1. Taking a sample: the surface of cucumber peel.
Adding 45mL of normal saline (10 times diluted) into 5g of cucumber peel, mixing, taking 0.5mL of diluted solution, diluting with normal saline again to 10 times-3Taking 100 μ L of liquid (10)-1、10-2、10-3) And coating the mixture on a solid MRS 5.2 culture medium for culturing (the culture temperature is 37 ℃, and the culture time is 48-72 h). And selecting a single colony, inoculating the single colony on a solid MRS medium plate, continuously culturing (the culture temperature is 37 ℃ and the culture time is 48h), and repeatedly performing streak culture and purification (the culture temperature is 37 ℃ and the culture time is 48h) by the solid MRS medium plate to obtain multiple pure culture strains.
2. Respectively inoculating the obtained multiple pure culture strains into a liquid MRS culture medium for culture (the culture temperature is 37 ℃, the culture time is 14-18 h), then adding 60% of glycerol, and storing in a refrigerator at-80 ℃.
3. Gram staining positive, catalase test negative and 16s rDNA sequence homology comparison identification are carried out, a plurality of lactobacillus plantarum strains are screened from a plurality of pure culture strains, and 1 strain is selected and named as lactobacillus plantarum (L.plantarum) HG20 strain.
II, identification of the strains
1. The physiological and biochemical identification results of lactobacillus plantarum (l.plantarum) HG20 strain of the present invention are as follows:
gram positive, catalase negative.
The strain grows in a uniform turbid way in a liquid MRS culture medium, and the strain is white and precipitated after being placed for a long time.
2. 16S rDNA sequence homology analysis
(1) Extraction of strain genome DNA by CTAB method
Inoculating the separated multiple strains of lactobacillus plantarum in a liquid MRS culture medium, culturing at 37 ℃ for 16h, taking 10mL of culture solution, centrifuging at 10000r/min for 10min, discarding supernatant after centrifugation, and cleaning the thalli for 1 time by using TE buffer solution; resuspending the washed thallus in 1.9mL of TE buffer solution, adding 0.1mL of SDS solution with the mass fraction of 10%, 10 mu L of proteinase K with the concentration of 20mg/mL and 5 mu L of lysozyme with the concentration of 50 mg/mL, uniformly mixing, and carrying out water bath for 1h in a water bath kettle at 37 ℃; after water bath, adding 300 mu L of 5M NaCl solution and 300 mu L of CTAB-NaCl solution with the mass fraction of 10 percent into the system, uniformly mixing, and then carrying out water bath for 20min in a water bath kettle at 65 ℃; after water bath, adding a chloroform-isoamyl alcohol solution with the same volume (the volume ratio of the chloroform solution to the isoamyl alcohol solution is 24:1) into the system, fully and uniformly mixing, and centrifuging at 10000r/min for 30 min; sucking the supernatant into a new centrifugal tube, adding an equal volume of phenol-chloroform-isoamyl alcohol solution (the volume ratio of the phenol solution to the chloroform solution to the isoamyl alcohol solution is 25:24:1), fully and uniformly mixing, and centrifuging at 10000r/min for 30 min; sucking the supernatant into a new centrifugal tube, adding 0.6 times of isoamylol solution, standing at-20 deg.C for 2h, centrifuging at 10000r/min for 20min, and discarding the supernatant; washing the precipitate with 70% ethanol solution, centrifuging at 10000r/min for 20min, and standing at room temperature until ethanol is completely dried; dissolving the precipitate in 20 μ LTE buffer solution, adding 1 μ L RNase, and water-bathing in 37 deg.C water bath for 1h to obtain strain genome DNA, and storing at-20 deg.C.
(2) PCR amplification
Amplification primers: according to the conserved region of the 16S rDNA sequence of lactobacillus, a bacterial universal primer 27F/1492R is adopted, and the sequence is as follows:
27F：5'-AGAGTTTGATCCTGGCTCAG-3'；
1492R：5'-GGTTACCTTGTTACGACTT-3'。
the PCR reaction system is shown in Table 1:
TABLE 1
| Components of the System | Reaction system |
| TaKaRa Ex Taq(5U/μL) | 0.25μL |
| 10×Ex Taq Buffer(Mg2+Plus)5μL | 5μL |
| dNTP mix (2.5 mmol/L each) | 4μL |
| Genomic DNA | 1μL |
| Forward primer (10. mu. mol/L) | 1μL |
| Reverse primer (10. mu. mol/L) | 1μL |
| Sterilization double distilled water | 37.75μL |
| Total volume | 50μL |
PCR amplification procedure as follows:
detecting PCR amplification product
Agarose gel with mass fraction of 1% is prepared in advance (10 mg/mL ethidium bromide EB is added), after PCR amplification is finished, 5 mu L of PCR amplification product and 1 mu L of 6 × Loading buffer are sucked and mixed evenly, and a pipettor is added into a sample application hole of the agarose gel plate for electrophoresis. The length of the target fragment is about 1500bp, a DL2000 DNA Marker is used, 1 × TAE is used as an electrophoretic solution, the electrophoretic voltage is 100V, and the electrophoretic time is 40 min.
(3)16S rDNA Gene sequence analysis
Sending the purified product to Changchun Kumei for sequencing; obtaining the 16S rDNA sequence of each strain, and carrying out homology comparison analysis on the sequence information of known strains in a GenBank library through a BLAST program to identify the strain to be detected; and (3) classifying the species by taking the 16S rDNA sequence homology of more than 99 percent as a standard, and then constructing a phylogenetic tree by utilizing the 16S rDNA sequences of the strain to be detected and the model strain by utilizing a Neighbor-Joining method in MEGA4.0 software. The 16s rDNA sequence is shown as SEQ ID NO 1 in the sequence table.
Based on the above analysis results, it was confirmed that the strain HG20 of the present invention is Lactobacillus plantarum (L.
Third, preservation of the Strain
The Lactobacillus plantarum HG20 strain of the invention has been preserved in China Center for Type Culture Collection (CCTCC) in 11 months and 21 days in 2019, and the addresses are as follows: eight-path Wuhan university school (Wuhan university collection center) 299 in Wuchang area, Wuhan city, Hubei province has the collection number: CCTCC NO: M2019964.
Example 2 Effect of Lactobacillus plantarum (Lactobacillus plantarum) HG20 Strain according to the present invention on collagen type arthritis model rats
First, establishment and grouping of animal models
Fully emulsifying the calf cartilage type II collagen with Freund's complete adjuvant to obtain a modeling agent, dissolving 20mg of the calf cartilage type II collagen in 10mL of acetic acid with the concentration of 0.1mol/L, and fully emulsifying with the same amount of complete Freund's adjuvant after overnight at 4 ℃. After the rats were sterilized by wiping the left and right plantar areas with alcohol, 100. mu.L/rat was injected intradermally into the flesh pads of the left and right plantar areas. After 7d, the injection was boosted. And 30 male SD rats, randomly divided into 3 groups: control, model and lactobacillus plantarum HG20 groups, 10 per group. The preparation is administered by gavage 7 days after intensive injection, and the control group and the model group are administered with 2mL/d of gastric physiological saline, the lactobacillus plantarum HG20 group is administered with 202mL/d of gastric bacterial strain, and the preparation is administered continuously for 21 days. After the test is finished, measuring the diameter of the ankle joint and the volume of toes; blood is taken from the heart of each group of rats, centrifuged at 3000r/min for 15min, and serum is collected.
Secondly, the influence of the lactobacillus plantarum HG20 on the overall condition of the collagen type arthritis rats
Control group: the rat had smooth and colored dorsal hair. Diet and stool were normal. The ankle joints on both sides have no abnormal conditions such as swelling and the like. The rat moves freely, prefers to move.
Model group: the rat had unsmooth dorsal hair and dull color. Less food intake and partially unformed stools. The ankle joints on both sides have obvious swelling, difficult movement and no pleasure.
Lactobacillus plantarum HG20 group: the rat has smooth dorsal hair and good color. Good diet and good stool formation. The ankle joints on both sides are slightly swollen, and the movement is almost not difficult and is favored.
Third, degree of ankle swelling in rats
TABLE 2 Effect of Lactobacillus plantarum HG20 strain on the degree of swelling of ankle joints in rats with collagen arthritis
| Group of | Degree of swelling (cm) | Toe volume (mL) |
| Control group | 0.90±0.19** | 1.77±0.31** |
| Model set | 3.80±0.48 | 6.07±0.33 |
| Lactobacillus plantarum HG20 group | 1.31±0.30** | 2.35±0.25** |
Note: differences were very significant compared to model groups (p < 0.01).
Before molding, 3 groups have no obvious difference, and no symptoms of swelling and inconvenient movement are observed. On the 7 th day of molding, except for the normal group, the other 2 groups showed symptoms of swelling, enlarged ankle diameter, mobility disorder, etc., indicating successful molding. After the administration day 21, the diameters of ankle joints of lactobacillus plantarum HG20 groups were decreased, and toe volumes were decreased, indicating that it had the effects of relieving joint swelling and mobility impairment, as shown in fig. 1.
Fourth, related index detection in serum
TABLE 3 Effect of Lactobacillus plantarum HG20 strain on inflammatory factors in serum of collagen-type arthritis rats
| Group of | TNF-α(pg/mL) | IL-lβ(pg/mL) | IL-10(pg/mL) |
| Control group | 41.81±7.44** | 43.49±10.15** | 70.32±3.95** |
| Model set | 122.58±16.09 | 120.20±13.33 | 36.98±3.44 |
| HG20 | 58.13±2.29** | 74.21±9.79** | 57.46±7.04** |
Note: differences were very significant compared to model groups (p < 0.01).
During the onset of RA, the levels of inflammatory cytokines such as TNF- α, IL-1 β, etc., increase and the levels of the anti-inflammatory cytokine IL-10 decrease, which aggravate the inflammatory response and cause damage to the synovial membrane and articular cartilage. The content change of three cytokines which have key effects in the pathogenesis of RA is detected, and the lactobacillus plantarum HG20 has certain anti-inflammatory effect, can reduce the levels of TNF-alpha and IL-1 beta of collagen arthritis rats, and increase the level of pathologically reduced IL-10, thereby relieving the illness state and reducing the morbidity.
TABLE 4 Effect of Lactobacillus plantarum HG20 Strain on immunoglobulin levels in collagen type arthritic rat serum
Note: differences were very significant compared to model groups (p < 0.01).
The initiation factor inducing RA can make the organism produce IgG antibody and change its molecular structure, so as to stimulate the formation of rheumatoid factor. IgM is mainly involved in the acute immune response of the body and is an important component of the body's defense system. C-reactive protein (CRP) is an acute phase reactive protein, and during the onset of RA, the body is in an acute inflammatory phase, and the concentration of the C-reactive protein is increased. The result of the invention shows that the immune indexes IgG, IgM and CRP of the model group rat are obviously increased, and after the rat passes through the lactobacillus plantarum HG20, the immune indexes in the serum are obviously reduced.
The invention discloses a lactobacillus plantarum HG20 strain and application thereof, and can be realized by appropriately improving process parameters by taking the contents of the strain as reference by a person skilled in the art. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the technology can be practiced and applied by modifying or appropriately combining the products described herein without departing from the spirit and scope of the invention.
Sequence listing
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cctgccctga agcgggggat aacacctgga aacagatgct aataccgcat aacaacttgg 60
accgcatggt ccgagtttga aagatggctt cggctatcac ttttggatgg tcccgcggcg 120
tattagctag atggtggggt aacggctcac catggcaatg atacgtagcc gacctgagag 180
ggtaatcggc cacattggga ctgagacacg gcccaaactc ctacgggagg cagcagtagg 240
gaatcttcca caatggacga aagtctgatg gagcaacgcc gcgtgagtga agaagggttt 300
cggctcgtaa aactctgttg ttaaagaaga acatatctga gagtaactgt tcaggtattg 360
acggtattta accagaaagc cacggctaac tacgtgccag cagccgcggt aatacgtagg 420
tggcaagcgt tgtccggatt tattgggcgt aaagcgagcg caggcggttt tttaagtctg 480
atgtgaaagc cttcggctca accgaagaag tgcatcggaa actgggaaac ttgagtgcag 540
aagaggacag tggaactcca tgtgtagcgg tgaaatgcgt agatatatgg aagaacacca 600
gtggcgaagg cggctgtctg gtctgtaact gacgctgagg ctcgaaagta tgggtagcaa 660
acaggattag ataccctggt agtccatacc gtaaacgatg aatgctaagt gttggagggt 720
ttccgccctt cagtgctgca gctaacgcat taagcattcc gcctggggag tacggccgca 780
aggctgaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat 840
tcgaagctac gcgaagaacc ttaccaggtc ttgacatact atgcaaatct aagagattag 900
acgttccctt cggggacatg gatacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga 960
gatgttgggt taagtcccgc aacgagcgca acccttatta tcagttgcca gcattaagtt 1020
gggcactctg gtgagactgc cggtgacaaa ccggaggaag gtggggatga cgtcaaatca 1080
tcatgcccct tatgacctgg gctacacacg tgctacaatg gatggtacaa cgagttgcga 1140
actcgcgaga gtaagctaat ctcttaaagc cattctcagt tcggattgta ggctgcaact 1200
cgcctacatg aagtcggaat cgctagtaat cgtggat 1237
Claims (3)
1. Lactobacillus plantarum (II)L. plantarum) HG20 strain, characterized in thatThe strain is preserved in China center for type culture Collection in 2019, 11 and 21 months, and the preservation numbers are: CCTCC NO: M2019964.
2. The Lactobacillus plantarum (F) (L) as defined in claim 1L. plantarum) HG20 strain as active component, and the lactobacillus plantarum (A) (B)L. plantarum) The concentration of HG20 strain was 109～1010CFU/g, the method for preparing the microbial inoculum comprises the following steps:
culturing Lactobacillus plantarum HG20 strain to 2L, centrifuging at 6000r/min for 5min, resuspending with physiological saline, cleaning, centrifuging at 6000r/min for 5min, collecting bacterial sludge, dissolving in 1L lyophilized protectant, and lyophilizing to obtain Lactobacillus plantarum HG20 leaven with bacterial count of 1.0 × 1010 CFU/g; the freeze-drying protective agent is prepared by dissolving 40g of glucose, 5g of sodium ascorbate, 80g of fructo-oligosaccharide and 30g of trehalose in tertiary water and fixing the volume to 750 mL.
3. The Lactobacillus plantarum (F) (as defined in claim 1)L. plantarum) The HG20 strain is used in preparing medicine for preventing and treating rheumatoid arthritis.
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| CN111019857A (en) | 2020-04-17 |
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