CN113897321B - Marseillella and preparation method and application thereof - Google Patents

Marseillella and preparation method and application thereof Download PDF

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CN113897321B
CN113897321B CN202111505225.9A CN202111505225A CN113897321B CN 113897321 B CN113897321 B CN 113897321B CN 202111505225 A CN202111505225 A CN 202111505225A CN 113897321 B CN113897321 B CN 113897321B
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王宇
高文勇
陈琳
李建军
陈育新
舒磊晶
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Beijing Darwin Cell Biotechnology Co ltd
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Abstract

The invention relates to a Marseilles DRWMS001 with the classification name ofMassilia sp.(preservation number is CGMCC No. 23545) and a preparation method and application thereof. The mosaic bacteria extract prepared by the mosaic bacteria contains various growth factors and proteins with the functions of regulating and controlling cell proliferation and differentiation, cell repair and cell nutrition, maintaining tissues and cell growth and development and the like, promotes cell repair and organ injury repair, delays skin aging, reduces or even eliminates melanin deposition, is used for the aspects of joint repair, hair follicle repair, organ injury repair, skin cell repair, oxidative damage cell repair and the like, and is safe and effective.

Description

Marseillella and preparation method and application thereof
Technical Field
The invention relates to the field of biomedicine, and particularly relates to mosaic bacteria, a preparation method and application thereof.
Background
The hair is one of the important indexes of human health and plays an important role in instrument appearance. Modern people are affected by factors such as busy life, working pressure, environmental pollution and the like, and have the problems of easy breakage of hair, early aging and the like, thereby seriously affecting the personal image. Male and female baldness people are more and show a trend of youthfulness. The results of Chinese alopecia crowd survey show that about 2.5 hundred million alopecia crowds in China have the alopecia incidence rate as high as 12 percent. About 60% of people develop alopecia symptoms in about 25 years old, and the people with alopecia tend to be younger. The research result shows that the number and activity of hair follicle stem cells are directly related to alopecia. The decrease in the number and activity of the hair follicle stem cells leads to the decrease in the differentiation ability of the hair follicle cells and failure to complete the regeneration cycle, thereby causing the hair follicle to close to form permanent hair loss. The activity of the hair follicle stem cells determines the regenerative capacity of the hair follicle. Existing forms of hair loss treatment include hair transplantation, oral medication or topical medication. The hair transplantation method causes pain to patients, is expensive and affects beauty. The anti-hair loss medicine utilizes chemical substances to promote the growth of hair follicle stem cells, but has certain toxic and side effects.
Cells are the basic unit of life activities and the basis for body health. When the redox balance of the body is disrupted, disruption of redox signaling and control can be caused, leading to oxidative stress injury and producing various diseases. Neuronal cells damaged by brain oxidative stress can cause various neurodegenerative diseases, including hypomnesis, Alzheimer's disease, Parkinson's disease, multiple sclerosis and the like. Timely repair of damaged cells requires that the associated lesion be treated. The existing cell repair products comprise active peptides, vitamins, probiotics and the like, and have the defects of insignificant effect, high cost and the like. Therefore, more efficient cell repair products are urgently needed in clinic.
Disclosure of Invention
The invention aims to provide a Marseilles DRWMS001 with the classification name ofMassilia sp.，The preservation number is CGMCC No.23545, and is preserved in China center for culture Collection of microorganisms.
The invention aims to provide a mosaic bacterium extract with a cell repairing effect, and the preparation method of the extract comprises the following steps:
(1) placing the frozen Marseilles DRWMS001 with the preservation number of CGMCC No.23545 in a water bath at 30-40 ℃ for melting, coating on a plate, and then placing the plate under the condition of 25-40 ℃ for culturing until a colony grows out;
(2) picking the single colony prepared in the step (1), inoculating the single colony into a basic culture medium, and culturing for 36-60h under the conditions of 25-40 ℃ and 100-;
(3) inoculating the seed culture solution of the activated strain prepared in the step (2) into an acclimatization culture medium according to the inoculation amount of 5-20%, and culturing at 20-35 ℃ and 100-300rpm for 80-200h to obtain a bacterial solution OD6500.5-5.0 of bacterial liquid;
(4) inoculating the bacterial liquid prepared in the step (3) into an acclimation culture medium according to the inoculation amount of 5-20%, culturing at 5-38 ℃ under 100-300rpm for 2-7 days at a circulating temperature change, and receiving the bacterial liquid with the dosage of 3-10mj/cm every 12h during the culture period2Irradiating with ultraviolet for 20-60min, filtering, collecting strain, cleaning, re-suspending with Phosphate Buffer Solution (PBS), physiological saline, Tris buffer solution or their combination, collecting strain, culturing at 10-25 deg.C for 6-24 hr, centrifuging, collecting supernatant, filtering, collecting filtrate, adding lyophilized protectant, and lyophilizing.
According to the preferable technical scheme of the invention, the freezing temperature of the step (1) is-80 ℃.
In a preferred technical scheme of the invention, in the step (2), the culture conditions of the strain in the basic culture medium are as follows: culturing at 30-37 deg.C and 200rpm for 40-50 h.
In a preferred embodiment of the present invention, in step (3), the culture conditions of the strain in the acclimatization medium are: culturing at 25-30 ℃ and under the conditions of 150-250rpm for 120-160 h.
In a preferred embodiment of the present invention, the amount of the inoculum in step (3) or step (4) is 8 to 18%, preferably 10 to 15%.
According to the preferred technical scheme, in the step (3), the bacterial liquid OD is prepared650Is 0.8-3.0, preferably OD650Is 1.0-2.0.
According to the preferable technical scheme of the invention, in the step (4), the circulating temperature-changing culture conditions in the domestication culture system are as follows: culturing at 10-30 deg.C and 150-.
According to the preferable technical scheme of the invention, the circulating temperature-changing culture conditions in the step (4) are as follows: after the temperature of the culture system is raised from 8 ℃ to 38 ℃, the temperature is lowered from 38 ℃ to 8 ℃; then raising the temperature from 8 ℃ to 38 ℃, then lowering the temperature from 38 ℃ to 8 ℃, and carrying out circulating temperature-changing culture.
According to the preferable technical scheme of the invention, the circulating temperature-changing culture conditions in the step (4) are as follows: after the temperature of the culture system is increased from 10 ℃ to 35 ℃, the temperature is reduced from 35 ℃ to 10 ℃; then raising the temperature from 10 ℃ to 35 ℃, then lowering the temperature from 35 ℃ to 10 ℃, and carrying out circulating temperature-changing culture.
According to the preferable technical scheme of the invention, the circulating temperature-changing culture conditions in the step (4) are as follows: after the temperature of the culture system is increased from 12 ℃ to 31 ℃, the temperature is reduced from 31 ℃ to 11 ℃; then raising the temperature from 12 ℃ to 31 ℃, then lowering the temperature from 31 ℃ to 12 ℃, and carrying out circulating temperature-changing culture.
According to the preferable technical scheme of the invention, the heating and cooling rate in the circulating temperature-changing culture in the step (4) is 0.5-5 ℃/h, and preferably 1-4 ℃/h.
According to the preferable technical scheme of the invention, the ultraviolet irradiation conditions in the step (4) are as follows: the dosage is 5-8mj/cm2Irradiating for 30-40 min.
In a preferred embodiment of the present invention, the washing solution in step (4) is selected from any one of Phosphate Buffered Saline (PBS), physiological saline, TBS (Tris NaCl HCl buffered saline) buffer, tbst (Tris buffered saline) buffer, Tris buffer, or a combination thereof.
According to the preferred technical scheme of the invention, the number of times of washing in the step (4) is 1-5, preferably 2-4.
According to the preferable technical scheme, the filtration is multi-stage filtration, the preferable filtration stage number is 2-8 stages, and the more preferable filtration stage number is 3-6 stages.
According to the preferable technical scheme, the centrifugal clear liquid is filtered by membranes with the pore diameters of 80um, 50um, 30um, 10um, 5um, 0.45um and 0.22 um.
In a preferred embodiment of the present invention, the lyoprotectant is selected from any one of mannitol, glycerol, dextran, sucrose, trehalose, glucose, lactose, maltose, dextran, tricaprylin (HES), polyethylene glycol, ethylene-vinyl-diene, phosphate, acetate, citrate, and sorbitol, or a combination thereof.
In a preferred embodiment of the present invention, the lyophilized formulation has a pH of 6-8, preferably 6.5-7.5.
According to the preferred technical scheme, every 100ml of the basic culture medium comprises the following components:
Figure 669659DEST_PATH_IMAGE001
according to the preferred technical scheme, every 100ml of the basic culture medium comprises the following components:
Figure 117958DEST_PATH_IMAGE002
according to the preferred technical scheme, each 100ml of domestication culture medium comprises the following components:
Figure 428853DEST_PATH_IMAGE003
according to the preferred technical scheme, each 100ml of domestication culture medium comprises the following components:
Figure 355221DEST_PATH_IMAGE004
another object of the present invention is to provide a method for preparing a mosaic bacteria extract having a cell repair function, comprising the steps of:
(1) placing the frozen Marseilles DRWMS001 with the preservation number of CGMCC No.23545 in a water bath at 30-40 ℃ for melting, coating on a plate, and then placing the plate under the condition of 25-40 ℃ for culturing until a colony grows out;
(2) picking the single colony prepared in the step (1), inoculating the single colony into a basic culture medium, and culturing for 36-60h under the conditions of 25-40 ℃ and 100-;
(3) inoculating the seed culture solution of the activated strain prepared in the step (2) into an acclimatization culture medium according to the inoculation amount of 5-20%, and culturing at 20-35 ℃ and 100-300rpm for 80-200h to obtain a bacterial solution OD6500.5-5.0 of bacterial liquid;
(4) inoculating the bacterial liquid prepared in the step (3) into an acclimation culture medium according to the inoculation amount of 5-20%, culturing at 5-38 ℃ under 100-300rpm for 2-7 days at a circulating temperature change, and receiving the bacterial liquid with the dosage of 3-10mj/cm every 12h during the culture period2Irradiating with ultraviolet for 20-60min, filtering, collecting strain, cleaning, re-suspending with Phosphate Buffer Solution (PBS), physiological saline, Tris buffer solution or their combination, collecting strain, culturing at 10-25 deg.C for 6-24 hr, centrifuging, collecting supernatant, filtering, collecting filtrate, adding lyophilized protectant, and lyophilizing.
According to the preferable technical scheme of the invention, the freezing temperature of the step (1) is-80 ℃.
In a preferred technical scheme of the invention, in the step (2), the culture conditions of the strain in the basic culture medium are as follows: culturing at 30-37 deg.C and 200rpm for 40-50 h.
In a preferred embodiment of the present invention, in step (3), the culture conditions of the strain in the acclimatization medium are: culturing at 25-30 ℃ and under the conditions of 150-250rpm for 120-160 h.
In a preferred embodiment of the present invention, the amount of the inoculum in step (3) or step (4) is 8 to 18%, preferably 10 to 15%.
According to the preferred technical scheme, in the step (3), the bacterial liquid OD is prepared650Is 0.8-3.0, preferably OD650Is 1.0-2.0.
According to the preferable technical scheme of the invention, in the step (4), the circulating temperature-changing culture conditions in the domestication culture system are as follows: culturing at 10-30 deg.C and 150-.
According to the preferable technical scheme of the invention, the circulating temperature-changing culture conditions in the step (4) are as follows: after the temperature of the culture system is raised from 8 ℃ to 38 ℃, the temperature is lowered from 38 ℃ to 8 ℃; then raising the temperature from 8 ℃ to 38 ℃, then lowering the temperature from 38 ℃ to 8 ℃, and carrying out circulating temperature-changing culture.
According to the preferable technical scheme of the invention, the circulating temperature-changing culture conditions in the step (4) are as follows: after the temperature of the culture system is increased from 10 ℃ to 35 ℃, the temperature is reduced from 35 ℃ to 10 ℃; then raising the temperature from 10 ℃ to 35 ℃, then lowering the temperature from 35 ℃ to 10 ℃, and carrying out circulating temperature-changing culture.
According to the preferable technical scheme of the invention, the circulating temperature-changing culture conditions in the step (4) are as follows: after the temperature of the culture system is increased from 12 ℃ to 31 ℃, the temperature is reduced from 31 ℃ to 11 ℃; then raising the temperature from 12 ℃ to 31 ℃, then lowering the temperature from 31 ℃ to 12 ℃, and carrying out circulating temperature-changing culture.
According to the preferable technical scheme of the invention, the heating and cooling rate in the circulating temperature-changing culture in the step (4) is 0.5-5 ℃/h, and preferably 1-4 ℃/h.
According to the preferable technical scheme of the invention, the ultraviolet irradiation conditions in the step (4) are as follows: the dosage is 5-8mj/cm2Irradiating for 30-40 min.
In a preferred embodiment of the present invention, the washing solution in step (4) is selected from any one of Phosphate Buffered Saline (PBS), physiological saline, TBPS buffer, TBST buffer, Tris buffer, or a combination thereof.
According to the preferred technical scheme of the invention, the number of times of washing in the step (4) is 1-5, preferably 2-4.
According to the preferable technical scheme, the filtration is multi-stage filtration, the preferable filtration stage number is 2-8 stages, and the more preferable filtration stage number is 3-6 stages.
According to the preferable technical scheme, the centrifugal clear liquid is filtered by membranes with the pore diameters of 80um, 50um, 30um, 10um, 5um, 0.45um and 0.22 um.
In a preferred embodiment of the present invention, the lyoprotectant is selected from any one of mannitol, glycerol, dextran, sucrose, trehalose, glucose, lactose, maltose, dextran, tricaprylin (HES), polyethylene glycol, ethylene-vinyl-diene, phosphate, acetate, citrate, and sorbitol, or a combination thereof.
In a preferred embodiment of the present invention, the lyophilized formulation has a pH of 6-8, preferably 6.5-7.5.
According to the preferred technical scheme, every 100ml of the basic culture medium comprises the following components:
Figure 215599DEST_PATH_IMAGE005
according to the preferred technical scheme, every 100ml of the basic culture medium comprises the following components:
Figure 913427DEST_PATH_IMAGE006
according to the preferred technical scheme, each 100ml of domestication culture medium comprises the following components:
Figure 977198DEST_PATH_IMAGE007
according to the preferred technical scheme, each 100ml of domestication culture medium comprises the following components:
Figure 487683DEST_PATH_IMAGE008
the invention also aims to provide a pharmaceutical composition with cell repairing efficacy, which consists of the Marasmius sporophore extract and a pharmaceutically acceptable carrier.
According to a preferred technical scheme, the pharmaceutical composition is a freeze-dried preparation.
The invention also aims to provide application of the Marasmius androsaceus extract or the pharmaceutical composition thereof in preparing products for cell repair, organ injury repair or complications thereof.
In a preferred embodiment of the present invention, the cell repair is any one selected from the group consisting of hair follicle repair, oxidative damage cell repair, joint repair, and skin repair.
According to a preferred technical scheme of the invention, the hair follicle repair is any one selected from healthy hair follicle cell maintenance, damaged hair follicle cell repair, dormant hair follicle cell activation and new hair follicle cell maintenance after hair transplantation.
Another object of the present invention is to provide the use of the extract of mosaicism or its pharmaceutical composition for the preparation of a product for the treatment of acne.
The invention also aims to provide application of the Marseilles extract or the pharmaceutical composition thereof in preparing a product for delaying senescence or resisting senescence.
According to the preferable technical scheme, the Marseilles mosaic fungus extract or the pharmaceutical composition thereof is dissolved in water or physiological saline to prepare a solution with the concentration of 0.1-20%, and then the prepared solution is applied to a diseased part and is massaged or rolled to be absorbed.
Unless otherwise indicated, when the present invention relates to percentages between liquids, said percentages are volume/volume percentages; the invention relates to the percentage between liquid and solid, said percentage being volume/weight percentage; the invention relates to the percentages between solid and liquid, said percentages being weight/volume percentages; the balance being weight/weight percent.
Compared with the prior art, the invention has the following beneficial technical effects:
1. the mosaic bacterium extract contains various growth factors and proteins with the functions of regulating and controlling cell proliferation and differentiation, cell repair and cell nutrition, maintaining tissue and cell growth and development and the like, promotes cell repair and organ damage repair, delays skin aging, reduces or even eliminates melanin deposition, is used for the aspects of joint repair, hair follicle repair, organ damage repair, skin cell repair, oxidative damage cell repair and the like, and is safe and effective.
2. The preparation method has the advantages of simple operation, environmental protection, better cost, suitability for industrial production and the like.
Biological material preservation
The Marseillella DRWMS001 is named as Marseillella in classificationMassilia sp.. The strain is submitted to be preserved in 2021, 10 months and 9 days, and the preservation unit: the general microbiological center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 Hospital No. 1 Xilu of Beijing, Chaoyang districtThe collection number is CGMCC No. 23545.
Drawings
Figure 1 test example 1 results of immunomic cell identification;
FIG. 2 is a study of the effect of Marseilles extract on repairing hair follicle damaged cells;
FIG. 3 is a study of the effect of Marseilles sp extract on repairing oxidative damage cells;
FIG. 4 shows the effect of Marseilles in repairing melanocytes;
FIG. 5 shows the effect of the complex Marseilles extract on the repair of melanocytes;
FIG. 6 shows the effect of Marseilles extract of the present invention on the treatment of facial inflammation.
Detailed Description
The present invention will be further described with reference to the following examples.
The composition per 100ml of basal medium was: tyrose peptone (10g), yeast extract (0.25g), resazurin (0.1mg), hemin (1mg), biotin (1. mu.g), cobalamin (1. mu.g), p-aminobenzoic acid (3. mu.g), folic acid (5. mu.g), pyridoxamine (15. mu.g), NaHCO3(0.4g), cysteine (0.1g), glucose (0.25g), corn starch (0.25g), glycerol (0.2 g), egg white (0.25g) K2HPO4(0.045g)、KH2PO4(0.045g)、NaCl(0.09g)、(NH4)2SO4(0.09g)、MgSO4·7H2O(0.009g)、CaCl2(0.009g)。
The composition of each 100ml of acclimatization medium is: tyrose peptone (5g), yeast extract (0.25g), resazurin (0.1mg), hemin (0.5mg), biotin (5. mu.g), cobalamin (1. mu.g), p-aminobenzoic acid (5. mu.g), folic acid (5. mu.g), pyridoxamine (15. mu.g), cysteine (0.1g), NaHCO3(0.4g), glucose (0.25g), corn starch (0.25g), glycerol (0.2 g), egg white (0.5 g) K2HPO4(0.045g)、KH2PO4(0.045g)、NaCl(0.09g)、(NH4)2SO4(0.1g)、MgSO4·7H2O(0.009g)、CaCl2(0.009g) Riboflavin (0.2 g) and methanol (0.4 g).
Example 1Preparation of Marseilles extract of the invention
The preparation method of the Marseilles extract comprises the following steps:
(1) taking out a Marseilles frozen at-80 ℃ as a starting strain DRWMS001 with the preservation number of CGMCC No.23545, thawing the Marseilles frozen at 37 ℃ in a constant-temperature water bath, coating a plate, and culturing the Marseilles frozen at-28 ℃ in an incubator for one week until a colony grows out;
(2) selecting single colony, inoculating into 50ml basal medium, shake culturing at 37 deg.C and 200rpm for 48 hr, inoculating into acclimatization medium according to 15% inoculum size, and culturing at 25 deg.C and 250rpm for 120 hr to obtain OD6501.0-2.0 bacteria liquid;
(3) inoculating the bacterial liquid into an acclimatization culture medium according to the inoculation amount of 10%, and culturing for 72h at the temperature of 250prm and 10-31 ℃ in a circulating temperature-changing way, wherein the circulating temperature-changing culture operation of the culture system is to heat from 10 ℃ to 31 ℃, then cool from 31 ℃ to 10 ℃, the heating and cooling rate is 3 ℃/h, and the receiving dose is 10mj/cm every 12h2Ultraviolet irradiation for 60 min;
(4) centrifuging (2000 rpm 10 min), collecting the strain, washing with PBS 3 times, suspending the strain in physiological saline, placing the strain in shaking culture at 10-25 deg.C and 200rpm for 12h, centrifuging (2000 rpm 10 min), collecting the centrifugate, filtering with six-stage membrane with pore diameter of 80um, 50um, 30um, 10um, 0.45um and 0.22um, adding mannitol phosphate solution (pH 7.4) with weight ratio of 0.7% into the membrane clear solution, and lyophilizing to obtain the final product.
Example 2Preparation of Marseilles extract of the invention
The preparation method of the Marseilles extract comprises the following steps:
(1) taking out a Marseilles frozen at-80 ℃ as a starting strain DRWMS001 with the preservation number of CGMCC No.23545, thawing the Marseilles frozen at 37 ℃ in a constant-temperature water bath, coating a plate, and culturing the Marseilles frozen at-28 ℃ in an incubator for one week until a colony grows out;
(2) single colonies were picked and inoculated into 50ml basal medium, shake culturing at 37 deg.C and 200rpm for 48h, inoculating into acclimatization medium according to 15%, and culturing at 25 deg.C and 250rpm for 120h to obtain OD6501.0-2.0 bacteria liquid;
(3) inoculating the bacterial liquid into an acclimatization culture medium according to the inoculation amount of 5%, and culturing at the temperature of 250prm and 10-31 ℃ for 48h in a circulating temperature-changing way, wherein the circulating temperature-changing culture operation of the culture system is to heat from 12 ℃ to 31 ℃, then cool from 31 ℃ to 12 ℃, the heating and cooling rate is 1 ℃/h, and the receiving dose is 3mj/cm every 12h2Ultraviolet irradiation for 20 min;
(4) centrifuging (2000 rpm 10 min), collecting the strain, washing with PBS 3 times, suspending the strain in physiological saline, culturing at 10-25 deg.C under 200rpm for 12h, centrifuging (2000 rpm 10 min), collecting the centrifugate, filtering with six-stage membrane with pore diameter of 80um, 50um, 30um, 10um, 0.45um and 0.22um, adding trehalose solution (pH7.4) with weight ratio of 0.7% into the membrane clear liquid, and lyophilizing.
Example 3Preparation of Marseilles extract of the invention
The preparation method of the Marseilles extract comprises the following steps:
(1) taking out a Marseilles frozen at-80 ℃ as a starting strain DRWMS001 with the preservation number of CGMCC No.23545, thawing the Marseilles frozen at 37 ℃ in a constant-temperature water bath, coating a plate, and culturing the Marseilles frozen at-28 ℃ in an incubator for one week until a colony grows out;
(2) selecting single colony, inoculating into 50ml basal medium, shake culturing at 37 deg.C and 200rpm for 48 hr, inoculating into acclimatization medium according to 15% inoculum size, and culturing at 25 deg.C and 250rpm for 120 hr to obtain OD6501.0-2.0 bacteria liquid;
(3) inoculating the bacterial liquid into an acclimatization culture medium according to the inoculation amount of 15%, and performing circulating temperature-changing culture at 250prm and 8-38 ℃ for 120h, wherein the circulating temperature-changing culture operation of the culture system is to heat from 8 ℃ to 38 ℃, then cool from 38 ℃ to 8 ℃, the heating and cooling rate is 5 ℃/h, and the receiving dose is 10mj/cm every 12h2Ultraviolet irradiation for 60 min;
(4) centrifuging (2000 rpm 10 min), collecting the strain, washing with PBS 3 times, suspending the strain in physiological saline, culturing at 10-25 deg.C under 200rpm for 12h, centrifuging (2000 rpm 10 min), collecting the centrifugate, filtering with six-stage membrane with pore diameter of 80um, 50um, 30um, 10um, 0.45um and 0.22um, adding 0.7 wt% glucose solution (pH7.4) into the membrane clear liquid, and lyophilizing.
Test example 1Research on effect of Marseilles extract on repairing hair follicle damaged cells
1. Culturing hair follicle cells:
selecting healthy volunteers, sterilizing hair follicles with iodophor, deiodinating with alcohol, pulling out several hair roots of volunteers, cutting off hair root parts with sterile scissors, soaking the cut hair root parts in precooled Phosphate Buffer Solution (PBS) with pH7.2-7.4 containing penicillin and gentamicin for 5min, lightly blowing with a gun head for several times, taking out hair root parts, transferring the hair root parts to a polylysine-coated 24-well plate to promote adherence, adding 1ml of culture medium (MEM +15% FBS), placing the hair root parts at 37 ℃ and 5% CO2Culturing in an incubator, replacing culture medium every 3 days for half, removing hairy root tissue after the adherent hairy root part appears paving stone-like cells and climbs out for 3 days under the microscope, discarding the culture medium, washing with Phosphate Buffer Solution (PBS) with pH7.2-7.4, adding appropriate amount of trypsin for digestion, adding serum-containing culture medium to stop digestion after the visible cell mass shrinks under the microscope, centrifuging at 1600rpm/5min to collect cells, adding appropriate amount of culture medium into the collected cells to adjust the cell density to 1 × 104Perml, inoculated into a fresh polylysine-coated flask, transferred to 5% CO at 37 ℃2Culturing in an incubator.
CK15 positive (green) and PI cell nucleus staining (red) are identified through immune group cells, and then the cells are determined to be hair follicle cells, and the results are shown in figure 1.
2. Study of repair of damaged cells of Hair follicle
Blank group: DMEM/F12 complete medium (containing 10% FBS) was taken at 1X 104One/well inoculation of hair follicleCell to 96-well plate, placing in incubator at 37 deg.C and 5% CO2After medium culture for 12 h; fresh DMEM/F12 complete medium (containing 10% FBS) was replaced and placed in the incubator at 37 ℃ with 5% CO2After medium culture for 12 h; fresh DMEM/F12 complete medium (containing 10% FBS) was replaced and placed in the incubator at 37 ℃ with 5% CO2And culturing for 12 h. The relative viability of the cells was determined according to the assay method of the invention.
Model group: lipopolysaccharide (LPS) was added to MEM/F12 complete medium (containing 10% FBS), and the mixture was prepared into MEM/F12 complete medium (containing 10% FBS) containing 50ng/ml Lipopolysaccharide (LPS).
DMEM/F12 complete medium (containing 10% FBS) was taken at 1X 104Inoculating hair follicle cells to 96-well plate, placing at 37 deg.C in incubator with 5% CO2After medium culture for 12 h; the fresh MEM/F12 complete medium (containing 10% FBS) containing 50ng/ml Lipopolysaccharide (LPS) was replaced, and the medium was placed in an incubator at 37 ℃ and 5% CO2After medium culture for 12 h; fresh DMEM/F12 complete medium (containing 10% FBS) was replaced and placed in the incubator at 37 ℃ with 5% CO2Culturing for 12h, and detecting the relative activity of the cells according to the detection method of the invention.
Test groups: lipopolysaccharide (LPS) was added to MEM/F12 complete medium (containing 10% FBS), and it was prepared as MEM/F12 complete medium (containing 10% FBS) containing 50ng/ml Lipopolysaccharide (LPS).
The Marseilles sp extract prepared in example 1 was added to MEM/F12 complete medium (containing 10% FBS) and prepared into MEM/F12 complete medium (containing 10% FBS) containing 100ug/ml of Marseilles sp extract.
DMEM/F12 complete medium (containing 10% FBS) was taken at 1X 104Inoculating hair follicle cells to 96-well plate, placing at 37 deg.C in incubator with 5% CO2After medium culture for 12 h; the fresh MEM/F12 complete medium (containing 10% FBS) containing 50ng/ml Lipopolysaccharide (LPS) was replaced, and the medium was placed in an incubator at 37 ℃ and 5% CO2After medium culture for 12 h; the fresh MEM/F12 complete medium (containing 10% FBS) containing 100ug/ml Marseilles extract was replaced and placed in an incubator at 37 ℃ with 5% CO2Culturing for 12h, and detecting the relative activity of the cells according to the detection method of the invention.
The method for detecting the relative activity of the cells comprises the following steps: discarding supernatant, adding MTT working solution (0.5 mg/mL) into each hole, placing the MTT working solution into the holes at 37 ℃ for 2h of lucifugal incubation, discarding supernatant, adding 150 mu L DMSO into each hole, and reading OD (optical Density) by using an enzyme-linked immunosorbent assay (ELISA) instrument490. The relative cell viability of the model group and the test group was calculated as 100% of the relative cell viability of the blank group, and the results are shown in fig. 2. Compared with the model group, the relative activity of the cells of the test group is obviously improved.
Test example 2Research on effect of Marseilles extract on repairing oxidative damage cells
Human fibroblasts were purchased and cultured in DMEM/F12 complete medium (containing 10% FBS).
Blank group: DMEM/F12 complete medium (containing 10% FBS) was taken at 1X 104Inoculating human fibroblast to 96-well plate, placing in incubator at 37 deg.C and 5% CO2After medium culture for 12 h; fresh DMEM/F12 complete medium (containing 10% FBS) was replaced and placed in the incubator at 37 ℃ with 5% CO2After medium culture for 12 h; fresh DMEM/F12 complete medium (containing 10% FBS) was replaced and placed in the incubator at 37 ℃ with 5% CO2Culturing for 12h, and detecting the relative activity of the cells according to the detection method of the invention.
Model group: addition of H to MEM/F12 complete Medium (containing 10% FBS)2O2It was formulated to contain 400umol of H2O2MEM/F12 complete medium (containing 10% FBS).
DMEM/F12 complete medium (containing 10% FBS) was taken at 1X 104Inoculating human fibroblast to 96-well plate, placing in incubator at 37 deg.C and 5% CO2After medium culture for 12 h; replacement of fresh H containing 400umol2O2The MEM/F12 complete medium (containing 10% FBS) was placed in an incubator at 37 ℃ and 5% CO2After medium culture for 12 h; fresh DMEM/F12 complete medium (containing 10% FBS) was replaced and placed in the incubator at 37 ℃ with 5% CO2Culturing for 12h, and detecting the relative activity of the cells according to the detection method of the invention.
Positive control group: addition of H to MEM/F12 complete Medium (containing 10% FBS)2O2It was formulated to contain 400umol of H2O2MEM/F12 complete medium (containing 10% FBS).
Acetylcysteine was added to MEM/F12 complete medium (containing 10% FBS) to prepare 100umol of acetylcysteine in MEM/F12 complete medium (containing 10% FBS).
DMEM/F12 complete medium (containing 10% FBS) was taken at 1X 104Inoculating human fibroblast to 96-well plate, placing in incubator at 37 deg.C and 5% CO2After medium culture for 12 h; replacement of fresh H containing 400umol2O2The MEM/F12 complete medium (containing 10% FBS) was placed in an incubator at 37 ℃ and 5% CO2After medium culture for 12 h; the medium (10% FBS) was replaced with fresh 100. mu. mol of 100 mol of acetylcysteine-containing MEM/F12 and placed in an incubator at 37 ℃ in 5% CO2Culturing for 12h, and detecting the relative activity of the cells according to the detection method of the invention.
Test groups: addition of H to MEM/F12 complete Medium (containing 10% FBS)2O2It was formulated to contain 400umol of H2O2MEM/F12 complete medium (containing 10% FBS).
The Marseilles sp extract prepared in example 1 was added to MEM/F12 complete medium (containing 10% FBS) to prepare MEM/F12 complete medium (containing 10% FBS) containing 100ug/ml of Marseilles sp extract.
DMEM/F12 complete medium (containing 10% FBS) was taken at 1X 104Inoculating human fibroblast to 96-well plate, placing in incubator at 37 deg.C and 5% CO2After medium culture for 12 h; replacement of fresh H containing 400umol2O2The MEM/F12 complete medium (containing 10% FBS) was placed in an incubator at 37 ℃ and 5% CO2After medium culture for 12 h; the medium was replaced with fresh MEM/F12 complete medium (containing 10% FBS) containing 100ug/ml of Marseilles mosaic fungus extract, and the medium was placed in an incubator at 37 ℃ and 5% CO2Culturing for 12h, and detecting the relative activity of the cells according to the detection method of the invention.
The detection method according to the invention is a method for detecting the relative viability of cells: discarding supernatant, adding MTT working solution (0.5 mg/mL) into each hole, placing the holes at 37 ℃ for 2h of dark incubation, discarding supernatant, adding 150 mu L DMSO into each hole, reading OD values at 490nm by an enzyme-labeling instrument, calculating the relative activity of cells in each group by taking the relative activity of the cells in the blank group as 100%, and obtaining a result shown in figure 3. Compared with the model group, the relative activity of the cells of the test group is obviously improved.
Test example 3Research on effect of Marseilles extract on repairing melanocytes
Human melanocyte MC was purchased commercially and cultured in RPMI-1640+10% FBS medium.
Blank group: taking RPMI-1640+10% FBS culture medium, according to 1 × 104Inoculating melanocyte MC to 96-well plate, placing in incubator at 37 deg.C and 5% CO2After medium culture for 12 h; replacing fresh RPMI-1640+10% FBS medium, placing in incubator at 37 deg.C and 5% CO2After culturing for 24h, the melanin expression rate is detected according to the detection method of the invention.
Test groups: the Marseilles mosaic fungus extract prepared in example 1 was added to RPMI-1640+10% FBS medium, and prepared as RPMI-1640+10% FBS medium containing 100ug/ml of Marseilles mosaic fungus extract.
Taking RPMI-1640+10% FBS culture medium, according to 1 × 104Inoculating melanocyte MC to 96-well plate, placing in incubator at 37 deg.C and 5% CO2After medium culture for 12 h; replacing fresh RPMI-1640+10% FBS culture medium containing 100ug/ml Marseilles extract, placing in incubator at 37 deg.C and 5% CO2Culturing for 24h, and detecting the melanin expression rate according to the detection method of the invention.
The method for detecting the melanin expression rate comprises the following steps: abandoning the supernatant, washing each hole for 3 times by PBS, adding 1mL of 1mol/L NaOH aqueous solution containing 10% (volume ratio) DMSO into each hole, sealing the pore plate by a sealing film, placing the pore plate in a water bath kettle at 90 ℃ for heating for 30min, gently blowing the bottom of the plate, centrifuging (2000 revolutions/5 min), collecting the supernatant, adding the supernatant into a 96 pore plate according to groups, and reading an OD value at 405nm by an enzyme-labeling instrument. The melanin expression rate of the test group was calculated with the melanin expression rate of the blank group as 100%, and the results are shown in fig. 4. Compared with the blank group, the melanin expression rate of the test group is obviously reduced.
The supernatant was discarded and the cells were collected in regular groups, stained as required by the DB Annexin V-FITC/PIApoptosis Detection Kit, examined by flow cytometry, and the results are shown in FIG. 5.
Test examples4 Effect research of Marseilles extract for treating facial inflammation
The Marseillea spectabilis extract prepared in example 1 was mixed with physiological saline to prepare a 100ug/ml composite Marseillea spectabilis extract solution.
The female, 32 years old, had inflammation on the face and had acne for more than 6 months. Cleaning the wound surface with normal saline before sleeping every night, naturally drying, smearing 6ml of 100ug/ml Marseilles fungus extract solution for 30 minutes, cleaning with clear water, and moisturizing and caring skin. After one week, the large pox begins to obviously shrink, does not turn red and is not itchy, and after two weeks, marks except the individual deeper pox parts disappear. A front-to-back comparison of the patient's face is shown in FIG. 6.
The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined in the appended claims.

Claims (30)

1. Marseillella DRWMS001 with classification name ofMassilia sp.，The preservation number is CGMCC No.23545, and is preserved in China center for culture Collection of microorganisms.
2. A preparation method of a mosaic bacteria extract with cell repairing effect is characterized by comprising the following steps:
(1) placing the frozen Marseilles DRWMS001 with the preservation number of CGMCC No.23545 in a water bath at 30-40 ℃ for melting, coating on a plate, and then placing the plate under the condition of 25-40 ℃ for culturing until a colony grows out;
(2) picking the single colony prepared in the step (1), inoculating the single colony into a basic culture medium, and culturing for 36-60h under the conditions of 25-40 ℃ and 100-;
(3) inoculating the seed culture solution of the activated strain prepared in the step (2) into an acclimatization culture medium according to the inoculation amount of 5-20%, and placing the culture medium in the acclimatization culture mediumCulturing at 20-35 deg.C and 100-300rpm for 80-200h to obtain bacterial liquid OD6500.5-5.0 of bacterial liquid;
(4) inoculating the bacterial liquid prepared in the step (3) into an acclimation culture medium according to the inoculation amount of 5-20%, culturing at 5-38 ℃ under 100-300rpm for 2-7 days at a circulating temperature change, and receiving the bacterial liquid with the dosage of 3-10mj/cm every 12h during the culture period2Irradiating with ultraviolet for 20-60min, filtering, collecting strain, cleaning, re-suspending with phosphate buffer solution, physiological saline, Tris buffer solution or their combination, collecting strain, culturing at 10-25 deg.C for 6-24 hr, centrifuging, collecting supernatant, filtering, collecting filtrate, adding lyophilized protectant, and lyophilizing.
3. The method of claim 2, wherein the cryopreservation temperature of step (1) is-80 ℃.
4. The method of claim 2, wherein in step (2), the strain is cultured in the basal medium under the following conditions: culturing at 30-37 deg.C and 200rpm for 40-50 h.
5. The method of claim 2, wherein in step (3), the strain is cultured in the acclimatization medium under the following conditions: culturing at 25-30 ℃ and under the conditions of 150-250rpm for 120-160 h.
6. The method of claim 2, wherein the amount of inoculation in step (3) or step (4) is 8-18%.
7. The method of claim 2, wherein in step (3), the bacterial liquid OD is prepared650Is 0.8-3.0.
8. The method of claim 2, wherein in step (4), the temperature cycling culture conditions in the acclimatized culture system are as follows: culturing at 10-30 deg.C and 150-.
9. The method of claim 8, wherein the thermocycling culture conditions in step (4) are: after the temperature of the culture system is raised from 8 ℃ to 38 ℃, the temperature is lowered from 38 ℃ to 8 ℃; then raising the temperature from 8 ℃ to 38 ℃, and then lowering the temperature from 38 ℃ to 8 ℃, thus carrying out circulating temperature-changing culture; or the circulation temperature change culture conditions in the step (4) are as follows: after the temperature of the culture system is increased from 10 ℃ to 35 ℃, the temperature is reduced from 35 ℃ to 10 ℃; then raising the temperature from 10 ℃ to 35 ℃, then lowering the temperature from 35 ℃ to 10 ℃, and carrying out circulating temperature-changing culture; or the circulation temperature change culture conditions in the step (4) are as follows: after the temperature of the culture system is increased from 12 ℃ to 31 ℃, the temperature is reduced from 31 ℃ to 11 ℃; then raising the temperature from 12 ℃ to 31 ℃, then lowering the temperature from 31 ℃ to 12 ℃, and carrying out circulating temperature-changing culture.
10. The method according to claim 2, wherein the temperature increase and decrease rate in the temperature-cycling culture in the step (4) is 0.5-5 ℃/h.
11. The method of claim 2, wherein the ultraviolet irradiation conditions in step (4) are: the dosage is 5-8mj/cm2Irradiating for 30-40 min.
12. The method of claim 2, wherein the washing solution in step (4) is selected from any one of phosphate buffer, physiological saline, TBPS buffer, TBST buffer, Tris buffer, or a combination thereof.
13. The method according to claim 2, wherein the number of washing in step (4) is 1 to 5.
14. The method of claim 2, wherein the filtering is multi-stage filtering with 2-8 stages.
15. The method of claim 2, wherein the centrifuged supernatant is filtered through a membrane having a pore size of 80um, 50um, 30um, 10um, 5um, 0.45um, 0.22 um.
16. The method of claim 2, wherein the lyoprotectant is selected from the group consisting of mannitol, glycerol, dextran, sucrose, trehalose, glucose, lactose, maltose, dextran, tricaprylin, polyethylene glycol, ethylene diene, phosphate, acetate, citrate, sorbitol, and any combination thereof.
17. The method of claim 2, wherein the lyophilized formulation has a pH of 6-8.
18. The method of claim 2, wherein the composition per 100ml of basal medium is:
serial number Components The amount of the components Serial number Components The amount of the components 1 Tyrose peptone 1-20g 12 Glucose 0.1-1g 2 Yeast extract 0.1-1g 13 Corn starch 0.1-1g 3 Leaf of Resazurin 0.01-1mg 14 Glycerol 0.1-1g 4 Hemin 0.1-10mg 15 Egg white 0.1-1g 5 Biotin 0.1-10μg 16 K2HPO4 0.01-1g 6 Cobalamin 0.1-10μg 17 KH2PO4 0.01-1g 7 Para aminobenzoic acid 1-10μg 18 NaCl 0.01-1g 8 Folic acid 1-10μg 19 （NH4)2SO4 0.01-1g 9 Pyridoxamine 1-20μg 20 MgSO4·7H2O 0.001-0.1g 10 Cysteine 0.01-1g 21 CaCl2 0.001-0.1g 11 NaHCO3 0.1-1g
。
19. The method of claim 18, wherein the composition per 100ml of basal medium is:
serial number Components The amount of the components Serial number Components The amount of the components 1 Tyrose peptone 10-15g 12 Glucose 0.25-0.5g 2 Yeast extract 0.25-0.5g 13 Corn starch 0.25-0.5g 3 Leaf of Resazurin 0.1-0.5mg 14 Glycerol 0.2-0.5g 4 Hemin 1-5mg 15 Egg white 0.25-0.5g 5 Biotin 1-5μg 16 K2HPO4 0.045-0.1g 6 Cobalamin 1-5μg 17 KH2PO4 0.045-0.1g 7 Para aminobenzoic acid 3-5μg 18 NaCl 0.09-0.5g 8 Folic acid 5-8μg 19 （NH4）2SO4 0.09-0.5g 9 Pyridoxamine 15-18μg 20 MgSO4·7H2O 0.009-0.5g 10 Cysteine 0.1-0.5g 21 CaCl2 0.009-0.5g 11 NaHCO3 0.4-0.8g
。
20. The method of claim 2, wherein the composition per 100ml of acclimatizing medium is:
serial number Components The amount of the components Serial number Components The amount of the components 1 Tyrose peptone 1-10g 13 Corn starch 0.1-1g 2 Yeast extract 0.1-1g 14 Glycerol 0.1-1g 3 Leaf of Resazurin 0.01-1mg 15 Egg white 0.1-1g 4 Hemin 0.1-1mg 16 K2HPO4 0.01-1g 5 Biotin 1-10μg 17 KH2PO4 0.01-1g 6 Cobalamin 0.1-10μg 18 NaCl 0.01-1g 7 Para aminobenzoic acid 1-10μg 19 （NH4)2SO4 0.01-1g 8 Folic acid 1-10μg 20 MgSO4·7H2O 0.001-0.1g 9 Pyridoxamine 1-25μg 21 CaCl2 0.001-0.1g 10 Cysteine 0.01-1g 22 Riboflavin 0.1-1g 11 NaHCO3 0.1-1g 23 Methanol 0.1-1g 12 Glucose 0.1-1g
。
21. The method of claim 20, wherein the composition per 100ml of acclimatizing medium is:
serial number Components The amount of the components Serial number Components The amount of the components 1 Tyrose peptone 5-8g 13 Corn starch 0.25-0.5g 2 Yeast extract 0.25-0.5g 14 Glycerol 0.2-0.5g 3 Leaf of Resazurin 0.1-0.5mg 15 Egg white 0.5-0.8g 4 Hemin 0.5-0.8mg 16 K2HPO4 0.045-0.1g 5 Biotin 5-8μg 17 KH2PO4 0.045-0.1g 6 Cobalamin 1-5μg 18 NaCl 0.09-0.5g 7 Para aminobenzoic acid 5-8μg 19 （NH4）2SO4 0.1-0.5g 8 Folic acid 5-8μg 20 MgSO4·7H2O 0.009-0.5g 9 Pyridoxamine 15-20μg 21 CaCl2 0.009-0.0.5g 10 Cysteine 0.1-0.5g 22 Riboflavin 0.2-0.5g 11 NaHCO3 0.4-0.8g 23 Methanol 0.4-0.8g 12 Glucose 0.25-0.5g
。
22. A mosaic bacteria extract with cell repair function prepared by the method of any one of claims 2-21.
23. A pharmaceutical composition with cell repairing effect, which comprises the extract of the mosaic bacteria prepared by the preparation method of any one of claims 2-21 and a pharmaceutically acceptable carrier.
24. The pharmaceutical composition of claim 23, wherein the pharmaceutical composition is a lyophilized formulation.
25. Use of a mosaic bacteria extract prepared by a method according to any one of claims 2 to 21 or a pharmaceutical composition according to any one of claims 23 to 24 for the preparation of a cell repair article.
26. The use of claim 25, wherein the cellular repair is selected from any one of hair follicle repair, oxidative damage cell repair, skin repair.
27. The use of claim 26, wherein said hair follicle repair is selected from any of healthy hair follicle cell maintenance, damaged hair follicle cell repair, resting hair follicle cell activation, and new hair follicle cell maintenance after hair transplantation.
28. Use of a mosaic bacteria extract prepared by a process according to any one of claims 2 to 21 or a pharmaceutical composition according to any one of claims 23 to 24 for the preparation of an article of manufacture for the treatment of acne.
29. Use of a mosaic bacteria extract prepared by the method of any one of claims 2-21 or the pharmaceutical composition of any one of claims 23-24 for the preparation of an article of manufacture for delaying skin aging.
30. The use according to any one of claims 25 to 29, wherein the Marseilles mosaic bacteria extract or the pharmaceutical composition thereof is dissolved in water or physiological saline to prepare a 0.1-20% solution, and the prepared solution is applied to the lesion site and massaged or rolled until absorption.
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