CN115678807B - Bifidobacterium longum and application thereof - Google Patents
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- CN115678807B CN115678807B CN202211404065.3A CN202211404065A CN115678807B CN 115678807 B CN115678807 B CN 115678807B CN 202211404065 A CN202211404065 A CN 202211404065A CN 115678807 B CN115678807 B CN 115678807B
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Abstract
The invention discloses bifidobacterium longum and application thereof, and relates to the technical field of microorganisms. The invention discloses bifidobacterium longum (Bifidobacterium longum), which is bifidobacterium longum ProfMIC-231 and is preserved in China center for type culture collection (China center for type culture collection) on 9 th month 13 of 2022, wherein the preservation number is CCTCCNO: M20221386. Experiments show that ProfMIC-231 has the functions of resisting aging, improving cell antibacterial ability and improving alopecia, and can be used for preparing foods, medicines, cosmetics and the like.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to bifidobacterium longum and application thereof.
Background
Photoaging is the predominant form of extrinsic aging of skin, and the primary cause of photoaging of skin is ultraviolet radiation. During aging of the skin, cell proliferation decreases, apoptosis increases, and extracellular matrix components (collagen, elastin, glycosaminoglycan, etc.) significantly decrease with aging of the skin. In addition, active oxygen generated by various factors such as mitochondrial injury, inflammatory reaction and the like is increased, so that oxidative stress is increased, aged and damaged cells cannot be cleared in time, and skin aging is caused.
In the research of aging mechanism, it is found that cell autophagy can delay organ function degradation, remove damaged proteins and lipids in cells, repair DNA, help cells restore metabolic homeostasis, and provide protection for photoaged damaged skin. In addition, reducing apoptosis, reducing cell inflammation, increasing synthesis of extracellular matrix, reducing degradation thereof, improving antioxidant capacity of cells and the like have all been proved to be capable of slowing down skin cell aging, and searching for multi-level and multi-dimensional anti-aging substances is a current research hotspot.
In recent years, it has been found that skin secretes a number of proteins and polypeptides having an antibacterial effect, which are known as antibacterial peptides (antimicrobial peptides, AMPs), which are a class of effector molecules of the natural immune system, capable of contacting microbial membranes, lysing cells, and having a broad spectrum of antimicrobial effects. In addition, they are involved in reactions that interfere with cell proliferation, immune responses, wound healing, cytokine release, leukocyte chemotaxis, protease anti-protease balance, and the like.
Skin microorganisms play an important role in maturation and homeostasis of skin immunity, and most microorganisms living on the skin appear to be symbiotic or mutualistic under homeostatic conditions, and recent studies have demonstrated that the skin microbiota regulates the expression of various innate factors. Skin microorganisms break down phospholipids, sterols, and keratins, which also allow skin cells to absorb and promote cell growth, delay aging, and reduce wrinkles. When the skin is damaged by external causes such as air pollution, excessive use of cosmetics, or use of cosmetics containing hormones, the skin surface flora may be disturbed, thereby causing various skin problems. Studies have shown that changes in skin micro-ecology are associated with the occurrence and development of a variety of inflammatory and infectious diseases, such as psoriasis, atopic dermatitis, acne and chronic wounds. When probiotics are reduced or even eliminated, the skin can have various problems such as reduced resistance, easy inflammation, infection, allergy, acne growth, water-oil imbalance and the like. Therefore, the probiotic related product developed by utilizing the skin microbiological technology is provided, and has important practical significance.
Disclosure of Invention
In view of this, the present invention provides a bifidobacterium longum and uses thereof.
The invention provides bifidobacterium longum (Bifidobacterium longum), which is bifidobacterium longum ProfMIC-231 and is preserved in China center for type culture collection (CCTCC for short, address: eight paths 299 No. in Wuchang district of Wuhan, university of Wuhan, post code 430072) on day 13 of 2022, and the preservation number is bifidobacterium longum of CCTCC NO: M20221386;
it is another object of the present invention to provide the use of the above bifidobacterium longum for the preparation of a product for improving skin conditions.
In some embodiments, the improving the skin condition is at least one of anti-aging, increasing cell antibacterial ability, improving hair loss.
In some embodiments, the anti-aging is up-regulating expression of an extracellular matrix-related gene; the extracellular matrix related gene is at least one of COL1A1, COL13A1, SPTSSA, TIMP1 and/or SMAD 3;
the anti-aging is the up-regulation and inhibition of the expression of apoptosis-related gene BCL-2;
the anti-aging is the up-regulation of the expression of a cell autophagy related gene LC3B;
the anti-aging is the up-regulation of the expression of an immune regulator related gene MOR;
the anti-aging is the up-regulation of the expression of the oxidation-resistance related gene PTEN.
In some embodiments, the anti-aging is down-regulating the expression of genes associated with degradation of extracellular matrix; the degrading extracellular matrix-related gene is at least one of the MMP family;
the anti-aging is the down-regulation of the expression of apoptosis-related genes; the apoptosis-related gene is the expression of at least one of BAX and/or Caspase family;
the anti-aging is the down regulation of the expression of the genes IL-6 and/or TNF-alpha related to the cytokines.
In some embodiments, the enhancing the antimicrobial ability of the skin is up-regulating the expression of the antimicrobial peptide-related genes S100A7, S100A8, and/or DEFB 4.
In some embodiments, the improving hair loss comprises at least one of the following:
a) Up-regulating the expression of hair follicle growth phase related gene beta-catenin and/or LEF 1;
b) Up-regulating expression of hair follicle cell growth factor related genes VEGF, IGF-1 and FGF-2;
c) Up-regulating expression of hair follicle cell cycle regulating factor related gene c-Myc, PP2A and/or Cyclin D1;
d) Up-regulating the expression of a gene HSP27 related to inhibiting the apoptosis of hair follicle cells;
e) Up-regulating expression of hair follicle extracellular matrix related genes CSPG4 and/or HSPG 2;
f) Down-regulating the expression of the gene BMP4 associated with telogen in hair follicles.
In some embodiments, the product is a food product, a pharmaceutical product, or a cosmetic product.
It is another object of the present invention to provide a product for improving skin conditions, made from raw materials comprising the above bifidobacterium longum.
In some embodiments, the bifidobacterium longum in the product comprises one or both of the following (1) or (2):
(1) The bifidobacterium longum flora and/or agent of claim 1;
(2) A culture, exosome, lysate and/or extract of bifidobacterium longum of claim 1.
The invention discloses bifidobacterium longum ProfMIC-231, which has a preservation number of CCTCC NO: M20221386. Experiments show that ProfMIC-231 has the functions of resisting aging, improving cell antibacterial ability and improving alopecia, and can be used for preparing foods, medicines, cosmetics and the like.
Description of biological preservation
Bifidobacterium longum (Bifidobacterium longum) ProfMIC-231 was deposited at China center for type culture Collection (CCTCC for short, address: eight-way 299 of Wuchang district in Wuhan, university of Wuhan, post code 430072) at 9 and 13 days 2022, and the deposit number was CCTCC NO: M20221386.
Detailed Description
The invention provides bifidobacterium longum and application thereof. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The bifidobacterium longum strain ProfMIC-231 of the invention is derived from feces of healthy girls of eight years old and is identified as bifidobacterium longum by 16SrDNA (Bifidobacterium longum). The strain is gram positive and is rod-shaped under a microscope; growing on the BBL flat plate to form a circular colony with smooth and opaque surface, white and neat edge; the strain grows in BBL liquid culture medium in a uniform turbidity mode, the strain is white in precipitation after long-term placement, and the optimal growth temperature is 37 ℃.
Bifidobacterium longum (Bifidobacterium longum) ProfMIC-231, deposit unit: china center for type culture Collection, address: in the Jiuqiu No. 299 university of Wuhan, hubei province, the date of preservation: and 2022, 9 and 13 days, wherein the preservation number is CCTCC NO: M20221386.
Further, the bifidobacterium longum ProfMIC-231 provided by the present invention is present in the use or product according to the invention in the form of living or dead or batch sterilized, or in the form of a fermented lysate and/or extract, or in the form of a bacterial product or in the form of a fermented filtrate or in the form of a derivative, preferably selected from: metabolites, metabolic biological products, exosomes, probiotics, cell walls and components thereof, extracellular polysaccharides, and compounds containing immunogenic components, preferably selected from the group consisting of: fermenting the filtrate and fermenting lysate.
In vitro cell experiments show that the bifidobacterium longum ProfMIC-231 has the effect of up-regulating the expression of a type I collagen alpha chain gene COL1A1 related to HaCaT extracellular matrix, a serine palmitoyltransferase gene SPTSSA, a tissue metalloproteinase inhibitor I gene TIMP1, a signal transduction protein gene SMAD3 and a cell autophagy related gene microtubule related protein 1 light chain 3 beta gene LC3B, and the relative expression multiple of the genes is 1.12-2.04; has the function of down regulating and degrading the expression of matrix metalloproteinase gene MMP1 related to extracellular matrix and tumor necrosis factor alpha gene TNF-alpha related to cell inflammatory factor, and the relative expression multiple of the genes is 0.44-0.75.
In vitro cell experiments show that the bifidobacterium longum ProfMIC-231 has the functions of up-regulating the expression of a collagen-like membrane protein 13 alpha chain gene COL13A1 related to an HFF extracellular matrix, a serine palmitoyl transferase gene SPTSSA, an immunoregulatory factor related beta-endorphin receptor gene MOR, an antioxidant related No. 10 chromosome deleted phosphatase and a tensin homolog PTEN, and inhibiting the expression of a cell apoptosis related B lymphomatosis-2 gene BCL-2, wherein the relative expression multiple of the genes is 1.10-3.51 times; has the functions of down regulating and degrading matrix metalloproteinase family gene MMP related to extracellular matrix, BCL2-Associated X protein gene BAX related to apoptosis, cysteine proteinase family gene Caspase and interleukin 6 gene IL-6 expression related to cell inflammatory factor, and the relative expression multiple of the genes is 0.33-0.83
In vitro cell experiments show that the bifidobacterium longum ProfMIC-231 has the effect of up-regulating the expression of the related genes S100A7, S100A8 and DEFB4 of the antibacterial peptide, and the relative expression multiple of the genes is 1.20-1.91.
In vitro cell experiments show that the bifidobacterium longum ProfMIC-231 has the functions of up-regulating beta-catenin, lymphocyte binding enhancement factor 1LEF1, vascular endothelial growth factor VEGF, insulin-like growth factor 1IGF-1, fibroblast growth factor 2FGF-2, chondroitin sulfate proteoglycan gene CSPG, heparan sulfate proteoglycan gene HSPG, myc family protein gene c-Myc related to hair follicle cell cycle regulation factor, cyclin D1 and protein phosphatase 2A, which are related to hair follicle cell apoptosis, and inhibiting the expression of heat shock protein 27 gene HSP27 related to hair follicle apoptosis by the relative expression multiple of 1.10-2.04. Down-regulating bone morphogenetic protein 4BMP4 related to telogen of hair follicle development, and expressing the gene in relative multiple of 0.47-0.73.
The test materials adopted in the invention are all common commercial products and can be purchased commercially, and the invention is further described below by combining examples:
example 1
Isolation of ProfMIC-231:
sampling was performed in an eight year old healthy girl. After the sample is properly treated, the sample is evenly mixed by shaking in normal saline, the supernatant is streaked on a BBL solid plate, and after anaerobic culture is carried out for 48 hours at the constant temperature of 37 ℃, white bacterial colonies are picked up for repeated inoculation and screening until uniform single bacterial colonies are obtained, and the bacterial colonies are named as ProfMIC-231.
Gram staining microscopy: the bacterial strain ProfMIC-231 is gram-positive and is rod-shaped under a microscope; growing on BBL flat plate to form white, smooth and round opaque microcolonies with regular edges; the strain grows in BBL liquid culture medium in a uniform turbidity manner, and the strain is white in precipitation after long-term placement.
Example 2
Nucleic acid identification of ProfMIC-231:
1. 16S rDNA gene sequence analysis:
single colony is selected and placed in MRS liquid culture medium, after anaerobic culture is carried out at 37 ℃ overnight, the bacterial cells are collected by centrifugation at 12000 r for 1min, and the operation is carried out according to the step of DNA extraction kit. The primer adopts bacterial universal primers 27F and 1492R, the PCR amplification system is a 50 mu L system, and the primer is pre-denatured for 5min at 95 ℃; 15s at 94 ℃, 15s at 57 ℃, 40s at 72 ℃ and 35 cycles; extending at 72℃for 10min.
2. Results
The PCR product sequencing results were compared with the published standard sequences in GenBank (BLASTN) to give ProfMIC-231 strain as Bifidobacterium longum (Bifidobacterium longum).
Example 3
ProfMIC-231 regulates photoaging HaCaT extracellular matrix/autophagy/degradation of extracellular matrix/inflammatory factor-related Gene expression experiments:
1. preparing a ProfMIC-231 fermentation filtrate and a fermentation lysate:
picking up single colony of bifidobacterium longum ProfMIC-231Anaerobic stationary culture in BBL liquid culture medium at 37 deg.c in incubator for 16-18 hr, enzyme-labeled instrument detection and dilution with PBS to regulate OD 600 =0.2, 121 ℃,30min high pressure inactivation, 12000 rotation centrifugation for 2min, filtration through 0.22 μm filter membrane to fermentation filtrate. And (3) after centrifugation, separating and precipitating thalli, washing twice by using PBS, adding liquid nitrogen, grinding and crushing, collecting crushed bacterial mud, re-suspending the bacterial mud to the volume of the centrifuged bacterial mud by using PBS, crushing by using ultrasonic waves for 20 minutes, and inactivating the bacterial mud at the high pressure of 121 ℃ for 30 minutes to obtain the fermentation lysate.
2. HaCaT cell preparation and ultraviolet injury
HaCaT cells were digested and then subjected to a reaction at 0.5 ml/well (containing 2X 10 cells) 5 Cells) were inoculated into 24-well plates and incubated overnight at 37℃in a 5% carbon dioxide incubator. The total dose of cells in the well was 2J/cm 2 Is damaged by ultraviolet UVB irradiation.
3. ProfMIC-231 addition
The fermentation filtrate 5% (V/V) and fermentation lysate 10% (V/V) were added to the stimulated HaCaT cells, respectively (control group replaced fermentation filtrate/fermentation lysate with equal volume of PBS, respectively). Each group was incubated overnight at 37℃in parallel with 3.
4. qPCR method for detecting relative expression fold of extracellular matrix/autophagy/degradation extracellular matrix/inflammatory factor related genes
After the cells are discarded from the culture medium, lysate is added to extract total RNA of the cells, the concentration and purity of the RNA are detected, then the RNA is reversely transcribed into cDNA, GAPDH is used as an internal reference gene, and real-time qPCR is adopted to detect the expression of extracellular matrix related genes COL1A1, SPTSSA, TIMP1 and SMAD3, cell autophagy related gene LC3B, extracellular matrix related gene MMP1 and inflammatory factor related gene TNF-alpha. Relative expression fold f=1, using 2 for control group genes -ΔΔCT The F value of each sample was calculated by the method.
The formula: f=2 -ΔΔCT Wherein:
△CT experiment ＝CT Experiment -CT Internal reference (experiment) ；
△CT Control ＝CT Control -CT Internal reference (control) ；
△△CT＝△CT Experiment -△CT Control 。
Up-regulating extracellular matrix related gene TIMP1 by fermentation filtrate; down-regulating and degrading extracellular matrix related gene MMP1. The results are shown in the following table:
fermentation lysate up-regulates extracellular matrix genes COL1A1, SPTSSA, TIMP1 and SMAD3, cell autophagy-related gene LC3B; down-regulating inflammatory factor related gene TNF-alpha. The results are shown in the following table:
the result shows that the addition of ProfMIC-231 has the anti-aging effect of promoting the synthesis of HaCaT extracellular matrix, promoting autophagy, reducing the degradation of extracellular matrix and reducing inflammatory factors.
Example 4
ProfMIC-231 regulates oxidative damage HFF extracellular matrix/immune modulator/antioxidant/apoptosis-inhibiting/degradation extracellular matrix/apoptosis/inflammatory factor-related gene expression experiments:
1. preparing a ProfMIC-231 fermentation filtrate and a fermentation lysate:
the preparation is described in example 3.
2. HFF cell preparation and H2O 2-induced oxidative damage
After digestion of HFF cells in DMEM culture, the cells were subjected to digestion at 0.5 ml/well (containing 2X 10 cells) 5 Cells) were inoculated into 24-well plates and incubated overnight at 37℃in a 5% carbon dioxide incubator. H was added to each well at a final concentration of 200. Mu.M 2 O 2 Stimulation was performed and the mixture was allowed to stand at 37℃for 1 hour.
ProfMIC-231 addition
The 5% (V/V) fermentation filtrate and 10% (V/V) fermentation lysate were added to stimulated HFF cells, respectively (control groups replaced the fermentation filtrate/fermentation lysate with equal volumes of PBS, respectively). Each group was incubated overnight at 37℃in parallel with 3.
4. qPCR method for detecting relative expression fold of extracellular matrix/immune regulator/antioxidant/apoptosis-inhibiting/extracellular matrix/apoptosis/inflammatory factor-degrading related genes
Removing culture medium from the above cells, adding lysate, extracting total RNA of the cells, detecting RNA concentration and purity, reverse transcribing into cDNA, using GAPDH as reference gene, detecting extracellular matrix related genes COL13A1 and SPTSSA, immune regulator related gene MOR, antioxidant related gene PTEN and apoptosis inhibiting related gene BCL-2 by real-time qPCR; degradation of extracellular matrix related genes MMP family, apoptosis related genes BAX and Caspase family genes and expression of inflammatory factor related genes IL-6. Relative expression fold f=1, using 2 for control group genes -ΔΔCT The F value of each sample was calculated by the method.
Up-regulating extracellular matrix related gene COL13A1, immune regulator related gene MOR and inhibiting apoptosis related gene BCL-2 in fermentation filtrate; down-regulating and degrading extracellular matrix related genes MMP family, apoptosis related genes BAX and Caspase family and inflammatory factor related genes IL-6. The results are shown in the following table:
up-regulating extracellular matrix related gene SPTSSA, antioxidant related gene PTEN and apoptosis-inhibiting gene BCL-2 by fermenting lysate; down-regulating apoptosis-related genes BAX and Caspase-3. The results are shown in the following table:
the results show that the addition of ProfMIC-231 has the anti-aging effects of promoting the synthesis of HFF extracellular matrix, increasing cell immune regulator, increasing antioxidant, reducing apoptosis, reducing extracellular matrix degradation and reducing inflammatory factors.
Example 5
ProfMIC-231 upregulation of HaCaT antibacterial peptide related gene expression experiments:
1. preparation of ProfMIC-231 fermentation lysate:
the preparation is described in example 3.
2. HaCaT cell preparation
HaCaT cells were digested and then subjected to a reaction at 0.5 ml/well (containing 2X 10 cells) 5 Cells) were inoculated into 24-well plates and incubated overnight at 37℃in a 5% carbon dioxide incubator.
3. ProfMIC-231 addition and LPS stimulation
10% (V/V) of the fermentation lysate was added to the cultured overnight HaCaT cells (control group was replaced with equal volume of PBS), and after 2 hours, 0.5ml of LPS solution at a concentration of 0.2. Mu.g/ml was added to induce cell inflammation, and the cells were cultured in a 5% carbon dioxide incubator at 37℃for 20 hours.
4. qPCR method for detecting relative expression times of related genes of antibacterial peptide
After the cells are discarded from the culture medium, lysate is added to extract total RNA of the cells, the cDNA is reversely transcribed after the concentration and purity of the RNA are detected, GAPDH is used as an internal reference gene, and the real-time qPCR is adopted to detect the expression of S100A7, S100A8 and DEFB4 genes. The control (relative gene expression fold f=1) was equal volume PBS treated group, using 2 -ΔΔCT The F value of each sample was calculated by the method.
The results of up-regulating the antibacterial peptide genes S100A7, S100A8 and DEFB4 by the inactivated cells are shown in the following table:
the results show that ProfMIC-231 has the effect of promoting the expression of antibacterial peptide genes.
Example 6
ProfMIC-231 regulates oxidative damage HDPCs follicular anagen/follicular cell growth factor/follicular extracellular matrix/follicular cell cycle regulatory factor/follicular telogen-related gene expression experiments:
1. preparing a ProfMIC-231 fermentation filtrate and a fermentation lysate:
the preparation is described in example 3.
2. Preparation of HDPCs hair follicle cells and H 2 O 2 Induced oxidative damage
HDPCs hair follicle cells cultured in fibroblast medium were digested and then added at a concentration of 2 ml/well (3×10 contained therein) 5 Cells) were inoculated into 6-well plates and incubated overnight at 37℃in a 5% carbon dioxide incubator. H was added to each well at a final concentration of 200. Mu.M 2 O 2 Stimulation was performed and the mixture was allowed to stand at 37℃for 1 hour.
ProfMIC-231 addition
The fermentation filtrate 5% (V/V) and fermentation lysate 10% (V/V) were added to the stimulated HDPCs cells, respectively (control groups replaced the fermentation filtrate/fermentation lysate with equal volumes of PBS, respectively). Each group was incubated overnight at 37℃in parallel with 3.
4. qPCR detection of relative expression fold of hair follicle anagen/hair follicle cell growth factor/hair follicle extracellular matrix/hair follicle cell cycle regulatory factor/hair follicle telogen related gene
Removing culture medium from the above cells, adding lysate, extracting total RNA of the cells, detecting RNA concentration and purity, reverse transcribing into cDNA, using GAPDH as reference gene, detecting hair follicle growth period related genes beta-catenin and LEF1, hair follicle cell growth factor related genes VEGF, IGF-1 and FGF-2, hair follicle extracellular matrix related genes CSPG4 and HSPG2, inhibiting hair follicle cell apoptosis related genes HSP27, hair follicle cell cycle regulatory factor related genes c-Myc, cyclin D1 and PP2A by adopting real-time qPCR; and expression of the telogen related gene BMP 4. Relative expression fold f=1, using 2 for control group genes -ΔΔCT The F value of each sample was calculated by the method.
Up-regulating hair follicle growth period related genes beta-catenin and LEF1, hair follicle cell growth factor related genes VEGF, IGF-1 and FGF-2, hair follicle extracellular matrix related genes CSPG4 and HSPG2, inhibiting hair follicle apoptosis related genes HSP27, and hair follicle cell cycle regulatory factor related genes c-Myc, cyclin D1 and PP2A; down-regulating the telogen related gene BMP4, the results of which are shown in the following table:
up-regulating hair follicle growth phase related gene LEF1, hair follicle cell growth factor related gene VEGF, FGF-2, inhibiting hair follicle cell apoptosis related gene HSP27, hair follicle cell cycle regulating factor related gene c-Myc by fermenting lysate; down-regulating the telogen related gene BMP4, the results of which are shown in the following table:
the result shows that the addition of ProfMIC-231 has the functions of promoting the growth period of HDPCs hair follicle, hair follicle cell growth factors, hair follicle extracellular matrix, inhibiting hair follicle cell apoptosis and hair follicle cell cycle regulating factor related gene expression; has the effects of reducing the expression of the gene related to the telogen phase of the hair follicle and improving alopecia.
Example 7
Preparation example of bifidobacterium longum ProfMIC-231 fermented filtrate granule:
the preparation of the ProfMIC-231 fermented filtrate granule comprises the following components in the formula:
| raw materials | Mass ratio (%) |
| ProfMIC-231 fermented powder | 50 |
| Recombinant collagen powder | 30 |
| Honey peach juice powder | 18.8 |
| Vitamin C | 0.8 |
| Silica dioxide | 0.4 |
The preparation process comprises the following steps: and (3) taking the ProfMIC-231 fermentation filtrate prepared in the example 3, spray-drying to prepare the ProfMIC-231 fermentation powder, adding the recombinant collagen powder, the vitamin C, the juicy peach juice powder and the silicon dioxide, and stirring and dispersing uniformly to obtain the granule. The obtained ProfMIC-231 granule has sour and sweet taste after being infused with water, good palatability and convenient carrying, and can be used as oral beverage for caring skin.
Example 8
Preparation example of bifidobacterium longum ProfMIC-231 fermented lysate emulsion:
preparation of ProfMIC-231 inactivated bacterial emulsion, the formulation is shown in the following table
| Raw materials | Mass ratio (%) |
| ProfMIC-231 fermentation lysate | 50.00 |
| Cyclopentasiloxane | 15.00 |
| Glycerol | 12.00 |
| Vaseline | 2.00 |
| 1, 3-butanediol | 1.50 |
| Tween-80 | 0.75 |
| Vitamin E acetate | 0.20 |
| Hansheng rubber | 0.20 |
| Carbopol Ultrez 10 | 0.10 |
| Triethanolamine salt | Proper amount of |
| Deionized water | To 100 |
The preparation process comprises the following steps: mixing cyclopentasiloxane, vaseline, tween-80, vitamin E acetate and glycerol, and heating to 75deg.C to obtain phase A; then taking the ProfMIC-231 fermented lysate prepared in example 3, adding xanthan gum, carbopol Ultrez 10, 1, 3-butanediol and deionized water, mixing and dispersing uniformly, and heating to 75 ℃ to obtain phase B; slowly adding the preheated phase A into the preheated phase B, homogenizing completely, adding triethanolamine, adjusting pH to 6-7, stirring, and cooling to 35deg.C. The obtained ProfMIC-231 fermented lysate milk has a smooth and comfortable liquid feel.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (6)
1. Bifidobacterium longum @Bifidobacterium longum) The strain is bifidobacterium longum ProfMIC-231 and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M20221386 in 2022, 9 and 06 days.
2. Use of the fermentation filtrate and fermentation lysate of bifidobacterium longum according to claim 1 for the preparation of a product for improving skin conditions, said product being a cosmetic; the improvement of skin condition is at least one of anti-aging, improving cell antibacterial ability and improving alopecia, wherein the preparation methods of fermentation filtrate and fermentation lysate of bifidobacterium longum are as follows:
selecting a single bacterial colony of bifidobacterium longum ProfMIC-231 in a BBL liquid culture medium, performing anaerobic stationary culture in an incubator at 37 ℃ for 16-18 h, detecting by an enzyme-labeled instrument, and diluting with PBS to adjust OD 600 =0.2, 121 ℃,30min high pressure inactivation, 12000 rotation centrifugation for 2min, filtration through 0.22 μm filter membrane to fermentation filtrate; and (3) after centrifugation, separating and precipitating thalli, washing twice by using PBS, adding liquid nitrogen, grinding and crushing, collecting crushed bacterial mud, re-suspending the bacterial mud to the volume of the centrifuged bacterial mud by using PBS, crushing by using ultrasonic waves for 20 minutes, and inactivating the bacterial mud at the high pressure of 121 ℃ for 30 minutes to obtain the fermentation lysate.
3. The use according to claim 2, wherein said anti-aging is up-regulation of expression of HaCaT extracellular matrix-related genes, up-regulation of apoptosis-inhibiting-related genesBCL-2Up-regulating cell autophagy-related genesLC3BUp-regulating immune regulator related genesMORExpression of (c) and up-regulating antioxidant related genesPTENIs expressed by (a); the extracellular matrix-related gene isCOL1A1、COL13A1、SPTSSA、TIMP1And-Or (b)SMAD3At least one of them.
4. The use according to claim 2, wherein said anti-aging is down-regulating the expression of genes associated with degrading extracellular matrix, down-regulating the expression of genes associated with apoptosis and down-regulating genes associated with cytokinesIL-6And/orTNF-αIs expressed by (a); the apoptosis-related gene isBAXAnd/orCaspaseExpression of at least one of the families, saidCaspaseThe related genes of the family areCaspase-3AndCaspase-8At least one of (a) and (b); the related genes for degrading the extracellular matrix areMMPAt least one of the families, theMMPThe related genes of the family areMMP1、MMP2AndMMP8At least one of them.
5. The use according to claim 2, wherein the improvement in the antibacterial ability of the cells is up-regulation of antibacterial peptide-related genesS100A7、S100A8And/orDEFB4Is expressed by (a).
6. The use according to claim 2, wherein the improvement of hair loss is at least one of the following a) to f):
a) Gene related to up-regulating hair follicle growth periodβ-cateninAnd/orLEF1Is expressed by (a);
b) Upregulation of hair follicle cell growth factor related genesVEGF、IGF-1And/orFGF-2Is expressed by (a);
c) Gene related to up-regulating hair follicle cell cycle regulating factorc-Myc、PP2AAnd/orCyclin D1Is expressed by (a);
d) Gene related to up-regulating and inhibiting hair follicle cell apoptosisHSP27Is expressed by (a);
e) Upregulation of genes associated with extracellular matrix of hair follicleCSPG4And/orHSPG2Is expressed by (a);
f) Down-regulating hair follicle telogen related genesBMP4Is expressed by (a).
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