CN115094011A - Lactobacillus gasseri and application thereof - Google Patents
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- CN115094011A CN115094011A CN202210947360.7A CN202210947360A CN115094011A CN 115094011 A CN115094011 A CN 115094011A CN 202210947360 A CN202210947360 A CN 202210947360A CN 115094011 A CN115094011 A CN 115094011A
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Abstract
The invention discloses lactobacillus gasseri and application thereof, and relates to the technical field of microorganisms. The strain of the lactobacillus gasseri disclosed by the invention is named as ProfMIC-213, and is preserved in China Center for Type Culture Collection (CCTCC) at 23.5.2022, and the preservation number of the lactobacillus gasseri is CCTCC No. M2022691. Experiments show that ProfMIC-213 has the functions of repairing skin barrier, resisting aging, promoting cell proliferation, resisting inflammation, resisting free radical and improving sensitive muscle, and can be used for preparing food, medicines, cosmetics and the like.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus gasseri and application thereof.
Background
The skin is the largest organ in the human body, the total weight accounts for about 16% of the body weight of an individual, and the skin is the first defense line for maintaining the stability of the body and resisting the invasion of external adverse factors. Studies have shown that skin diseases are induced if the external environment causes abnormalities in the relevant genes in the skin barrier.
The skin barrier is a structural barrier formed by the epidermal keratinocytes of the stratum corneum and the lipids between the cutin. The skin barrier prevents the release of excess water from the human body and prevents harmful substances such as chemicals or microorganisms from entering our body. The corneocyte cortex, which constitutes the surface of dead keratinocytes, plays an important role in the stability of intercellular lipids. Skin barrier damage can cause skin dryness, skin aging, atopic dermatitis, eczema, psoriasis, ichthyosis, skin sensitivity such as solar optical dermatitis, irritant dermatitis, and hormone dependent dermatitis, and seborrheic diseases such as acne, rosacea, and seborrheic dermatitis.
The content of the keratinaceous structure lipid ceramide gradually increases in the process of differentiation from the basal layer to the cutin, and the stratum corneum is discharged to the intercellular space, so that a barrier for preventing water loss is formed. The water content in the keratinocytes is high, the shape of the keratinocytes gradually becomes flat as the cells are metabolically differentiated upwards, and the cell nucleus and the organelles begin to degenerate and shrink, so that dead cells without the cell nucleus and the organelles are formed in the horny layer. The stratum corneum generally contains 10 to 30 percent of water due to the hydrophilic and barrier functions of the stratum corneum and the functions of natural moisturizing factors, namely amino acids, lactate, saccharides and the like, contained in the stratum corneum, and the environment becomes a cradle for the growth of microbial colonies of the skin. However, the water content of the stratum corneum gradually decreases with age, and various problems of the skin are caused when the water content is less than 10%.
Skin aging, including extrinsic aging caused by environmental factors such as air pollution, smoking, malnutrition, and Ultraviolet (UV) rays, and intrinsic aging caused by time variation. It is typically characterized by thinning of the skin, fine lines, which may be caused by decreased cell proliferation and significant changes in dermal composition with age. Extracellular matrix components (collagen, elastin, glycosaminoglycans, etc.) are significantly reduced with aging skin. In addition, active oxygen generated by various factors such as mitochondrial damage, inflammatory reaction, etc. is increased with aging, and at the same time, age-related cell repair ability is decreased, so that oxidative stress is increased and aging-damaged cells cannot be removed in time, thereby causing skin aging.
The probiotic is used in cosmetics, and can remarkably inhibit proliferation of skin pathogenic bacteria, balance skin epidermal flora, and repair skin barrier. Meanwhile, the expression of the moisturizing gene is up-regulated, skin aging is resisted, the absorption of the skin to nutrient substances is effectively increased, and the immunity is enhanced.
Acne is a common chronic inflammatory skin disease, and is closely related to factors such as excessive sebum secretion, blockage of pilosebaceous ducts, bacterial infection and inflammatory reaction. Research has shown that Propionibacterium acnes (Propionibacterium acnes), considered as the main pathogenic bacterium causing acne, can induce and activate the initial loop of acne inflammation and convert glycerol into fatty acids, leading to an inflammatory response; simultaneously, protease, hyaluronidase and chemotactic factor are produced, so that the hair follicle is hyperkeratotic and acne is formed. On the other hand, the skin resident staphylococcus epidermidis and propionibacterium acnes can compete with each other antagonistically, and increasing the amount of staphylococcus epidermidis can inhibit the proliferation of propionibacterium acnes. Therefore, reducing the flora ratio of propionibacterium acnes/staphylococcus epidermidis can effectively relieve acne inflammation, thereby maintaining skin health by regulating the skin micro-ecological balance.
Sensitive muscles generally cause skin immunity to be reduced due to skin cell damage, skin moistening degree is insufficient due to the fact that horny layers are thinned, and finally barrier function of the skin is too weak to resist external stimulation, so that discomfort phenomena such as redness, fever, pruritus and stabbing pain are prone to being generated. The damage of the skin barrier further causes the colonization and proliferation of Staphylococcus aureus (Staphylococcus aureus), resulting in inflammation and red swelling. Staphylococcus epidermidis (Staphylococcus epidermidis) resident in the skin and Staphylococcus aureus are mutually antagonistic, and the proliferation of the Staphylococcus epidermidis can be reduced, so that the flora ratio of the Staphylococcus aureus to the Staphylococcus epidermidis is reduced, the skin flora balance distribution is favorably established, and the sensitive muscle flora is improved. Micrococcus luteus (Micrococcus luteus) proliferates to generate pigment, and skin problems such as chloasma, stain or pigmentation and the like easily occur to the skin during overgrowth, so that the flora proportion of the Micrococcus luteus/staphylococcus epidermidis is reduced, the balanced distribution of skin flora is facilitated to be established, and the skin problem flora such as the chloasma, the stain or the pigmentation and the like is improved. Therefore, the probiotic related product developed by utilizing the microecological technology has important practical significance.
Disclosure of Invention
In view of this, the present invention provides a strain of lactobacillus gasseri and its application.
The invention provides lactobacillus gasseri (Lactobacillus. gasseri), which is named as ProfMIC-213 and has been preserved in China center for type culture Collection (CCTCC for short, with the address of No. 299, eight routes in Wuhan district, Wuhan university, zip code 430072) in 5 months and 23 days in 2022, and the preservation number of the lactobacillus gasseri is CCTCC No. M2022691.
Preferably, the lactobacillus gasseri is prepared by adopting the following separation method:
sampling feces of five-year-old healthy girls, properly treating the feces, uniformly mixing the feces in normal saline by shaking, taking the supernatant, streaking the supernatant on an MRS solid plate, culturing the mixture at the constant temperature of 37 ℃ for 48 hours, and selecting white colonies to repeatedly inoculate and screen the white colonies until uniform single colonies are obtained, wherein the single colonies are named as ProfMIC-213.
Preferably, the lactobacillus gasseri is gram-positive under gram staining microscopy, and the strain ProfMIC-213 is rod-shaped under a microscope; growing on an MRS plate to form white round microcolonies with smooth, mellow and opaque surfaces and regular edges; the strain grows in MRS liquid culture medium in a uniform turbid way, and the strain is white and precipitated after being placed for a long time.
The invention also aims to provide the application of the lactobacillus gasseri in preparing products for improving skin conditions.
Preferably, the improving skin condition comprises at least one of repairing skin barrier, moisturizing, anti-aging, inhibiting pathogenic bacteria of skin, regulating the ratio of microbial flora of skin, anti-inflammatory, anti-free radical, and anti-oxidant.
In some embodiments, the repairing a skin barrier is repairing a skin cell and/or up-regulating the expression of a barrier repair-associated gene; the barrier repair-associated genes include FLG, IVL, OVOL1, and/or LOR.
In some embodiments, the moisturizing is up-regulation of the expression of the moisturizing-associated gene AQP 3.
In some embodiments, the anti-aging is upregulating expression of extracellular matrix-associated genes; the extracellular matrix related gene comprises at least one of SMAD3, TIMP1, SPTSSA, Col-I, Col-3A1 and/or MKX.
In some embodiments, the anti-aging is upregulating expression of cellular antioxidant-related genes NRF2 and/or SIRT-1.
In some embodiments, the anti-aging is modulating expression of an apoptosis-related gene; the apoptosis-related gene includes expression of BAX, BCL-2 and/or Caspase families.
In some embodiments, the anti-aging is down-regulation of expression of a gene associated with degrading extracellular matrix; the extracellular matrix-degrading related gene comprises at least one of the P38MAPK and/or MMP families.
In some embodiments, the skin pathogen inhibitor is at least one of staphylococcus hominis, staphylococcus haemolyticus, and/or corynebacterium xerosis.
In some embodiments, the modulating the ratio of the skin microbial flora is inhibiting the growth of staphylococcus aureus, propionibacterium acnes, and/or micrococcus luteus, promoting and/or not affecting the growth of staphylococcus epidermidis.
In some embodiments, the anti-inflammatory is downregulating expression of a cellular inflammation-associated factor gene; the genes of the relevant factors of the cell inflammation comprise at least one of TNF-alpha, IL-6, IL-8 and/or TRPV 1.
In some embodiments, the anti-radical is a scavenger of hydroxyl radicals and/or ABTS radicals.
In some embodiments, the antioxidant is a total antioxidant capacity composed of various antioxidants and antioxidant enzymes, and the like.
In some embodiments, the product is a food product, a pharmaceutical product, or a cosmetic product.
In some embodiments, the lactobacillus gasseri in the product comprises one or both of:
(1) live and/or inactivated bacteria of lactobacillus gasseri;
(2) a culture, lysate and/or extract of lactobacillus gasseri.
The invention discloses a Lactobacillus gasseri ProfMIC-213 with the preservation number of CCTCC No: M2022691. Experiments show that the ProfMIC-213 has the functions of repairing skin barriers, moisturizing, resisting aging, inhibiting skin pathogenic bacteria, regulating the proportion of skin microbial flora, resisting inflammation, resisting free radicals and resisting oxidation, and can be used for preparing foods, medicines, cosmetics and the like.
Biological preservation Instructions
Lactobacillus gasseri ProfMIC-213 is preserved in China center for type culture Collection (CCTCC for short, address: Wuhan university, postcode 430072, eight routes 299 number in Wuchang district, Wuhan city) in 2022, 5 months and 23 days, and the preservation number is CCTCC No: M2022691.
Detailed Description
The invention provides lactobacillus gasseri and application thereof. Those skilled in the art can modify the process parameters appropriately in view of the disclosure herein. 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 methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The Lactobacillus gasseri strain ProfMIC-213 is derived from feces of five-year-old healthy girls and is identified as Lactobacillus gasseri (Lactobacillus) by 16S rDNA. The strain is gram-positive and rod-shaped under a microscope; the bacterial colony grows on the MRS plate, and a round bacterial colony with a smooth and opaque surface can be formed, is white and has a neat edge; the strain grows uniformly and turbulently in MRS liquid culture medium, and the strain is white precipitate after long-term storage, and the optimal growth temperature is 37 ℃.
Lactobacillus gasseri (lactobacillus. gasseri) ProfMIC-213, depository: china center for type culture Collection, Address: wuhan city Wuchang district eight-way 299 # with preservation date of Wuhan university: 23/5/2022, with the preservation number of CCTCC No. M2022691.
Further, the invention provides the lactobacillus gasseri ProfMIC-213 in the product for use according to the invention, in a form that is live or dead or sterilized at intervals, or in the form of a bacterial product or in the form of a supernatant or in the form of a derivative, preferably selected from: metabolites, metabolic biological products, prebiotics, cell walls and components thereof, exopolysaccharides, and compounds containing immunogenic components, preferably selected from: supernatant and inactivated bacteria.
In vitro cell experiments show that the lactobacillus gasseri ProfMIC-213 has the function of up-regulating the expression of skin barrier repair related factors including filaggrin FLG, Involucrin IVL, loricrin LOR and an Ovo-Like transcription factor 1(Ovo Like Transcriptional Reress 1) OVOL1, and the relative expression amount of genes is 1.41-6.12.
In-vitro cell experiments show that the lactobacillus gasseri ProfMIC-213 has the effect of up-regulating the expression of a water channel protein 3 gene AQP3 related to moisture preservation, and the relative expression quantity of the gene is 2.22-3.58.
In vitro cell experiments show that the Lactobacillus gasseri ProfMIC-213 has the functions of up-regulating expression of a tissue metalloproteinase inhibitor 1 gene TIMP1 and a Smad protein 3 gene SMAD3 related to HaCaT keratinocyte extracellular matrix, and a nuclear factor E2 related to antioxidation related gene, namely a 2NRF2 gene, and the relative expression quantity of the genes is 1.20-1.46; the protein has the function of down-regulating and degrading matrix metalloproteinase family genes MMP1 and serine/threonine protein kinase gene P38MAPK related to extracellular matrix, and the relative expression quantity of the genes is 0.15-0.68; has the function of down regulating the tumor necrosis factor-alpha gene TNF-alpha and interleukin 6 gene IL-6, and the relative expression amount of the genes is 0.27-0.75.
In vitro cell experiments show that the Lactobacillus gasseri ProfMIC-213 has the functions of up-regulating the expression of serine palmitoyltransferase gene SPTSSA, collagen type I gene Col-I, collagen type III alpha chain gene Col-3A1 and Mohokk protein gene MKX related to HFF human fibroblast extracellular matrix and antioxidant deacetylated protein 1 gene SIRT-1, and the relative expression amount of the genes is 1.10-3.37; the gene has the function of up-regulating B-lymphocytoma-2 gene (B-cell lymphoma-2) BCL-2 related to apoptosis, and the relative expression quantity of the gene is 1.35-2.01; the protein has the function of down-regulating and degrading matrix metalloproteinase family genes MMP family and serine/threonine protein kinase gene P38MAPK related to extracellular matrix, and the relative expression quantity of the genes is 0.23-0.95; has the function of down-regulating IL-6 of interleukin 6 gene of inflammatory factor, and the relative expression quantity of the gene is 0.51-0.69. The expression of BCL2-Associated X protein gene BAX related to apoptosis and Caspase family gene Caspase is reduced, and the relative expression amount of the genes is 0.28-0.88.
In vitro cell experiments show that the Lactobacillus gasseri ProfMIC-213 has the function of reducing the expression of interleukin 8 gene IL-8, interleukin 6 gene IL-6 and vanillic acid transient receptor subtype 1 gene TRPV1 of HACaT cell inflammation related factors induced by LPS, and the relative expression amount of the genes is 0.33-0.94.
In vitro cell experiments show that the lactobacillus gasseri ProfMIC-213 has the function of removing hydroxyl radicals and ABTS radicals, and the free radical removal rate is 7.87-18.52%.
In vitro experiments show that the Lactobacillus gasseri ProfMIC-213 has the function of inhibiting the proliferation of human staphylococcus, staphylococcus haemolyticus and corynebacterium sicans which are skin pathogenic bacteria, and the inhibition rate is 17.78-58.44%.
In vitro experiments show that the lactobacillus gasseri ProfMIC-213 has the function of reducing the flora proportion of propionibacterium acnes/staphylococcus epidermidis so as to treat acne.
In vitro experiments show that the lactobacillus gasseri ProfMIC-213 has the function of reducing the flora proportion of staphylococcus aureus/staphylococcus epidermidis and/or micrococcus luteus/staphylococcus epidermidis, thereby improving the flora of sensitive muscles and chloasma.
The test materials adopted by the invention are all common commercial products and can be purchased commercially, and the invention is further explained by combining the following embodiments:
EXAMPLE 1 isolation of ProfMIC-213
Collected from feces of five-year-old healthy girls. Properly processing the sample, uniformly mixing the sample in normal saline by shaking, taking the supernatant, streaking the supernatant on an MRS solid plate, culturing the MRS solid plate at the constant temperature of 37 ℃ for 48 hours, and then selecting a white colony to repeatedly inoculate and screen until a uniform single colony is obtained, wherein the colony is named as ProfMIC-213.
Gram staining microscopy: the strain ProfMIC-213 is gram-positive and rod-shaped under a microscope; growing on an MRS plate to form white round microcolonies with smooth, mellow and opaque surfaces and regular edges; the strain grows in MRS liquid culture medium in a uniform turbid way, and the strain is white and precipitated after being placed for a long time.
Example 2 nucleic acid identification of ProfMIC-213
1. 16S rDNA Gene sequence analysis
Selecting a single colony, placing the single colony in an MRS liquid culture medium, culturing overnight at 37 ℃, centrifuging at 12000 rpm for 1min, and collecting thalli, and operating according to the steps of a DNA extraction kit. The primers adopt bacterial universal primers 27F and 1492R, a PCR amplification system is a 50 mu L system, and the pre-denaturation is carried out for 5min at 95 ℃; 15s at 94 ℃, 15s at 57 ℃, 40s at 72 ℃ and 35 cycles; extension at 72 ℃ for 10 min.
2. Results
The homology comparison (BLASTN) of the PCR product sequencing results with the published standard sequences in GenBank gave that the ProfMIC-213 strain was Lactobacillus gasseri.
Example 3 ProfMIC-213 promotion of HaCaT Barrier repair-related Gene expression experiment
1. ProfMIC-213 supernatant and inactivated thallus preparation
Selecting a single bacterial colony of the Lactobacillus gasseri ProfMIC-213 in an MRS liquid culture medium, carrying out static culture in an incubator at 37 ℃ for 16-18 h, detecting by an enzyme-linked immunosorbent assay (ELISA) instrument, and diluting by PBS to adjust OD 600 The cells were inactivated at 121 ℃ for 30min under high pressure, centrifuged at 12000 rpm for 2min, and filtered through a 0.22 μm filter to give a supernatant. Resuspending the pellet with PBS, diluting and adjusting OD 600 0.2, which is an inactivated cell.
2. Experiment for promoting HaCaT barrier repair related gene expression
Inoculation of human immortalized keratinocytes HaCaT (2 ml/well, 5X 10% in it) 5 Cells) to a 6-well plate, and cultured overnight at 37 ℃ in a 5% carbon dioxide incubator until the cells adhere to the wall. 5% (V/V) of the supernatant and 10% (V/V) of the inactivated bacteria were added, and the supernatant/inactivated bacteria were replaced with PBS of the same volume as the control group. After 24h of culture, adding a lysate, extracting total RNA of cells, detecting the concentration and purity of the RNA, performing reverse transcription to obtain cDNA, taking GAPDH as an internal reference gene, and detecting the expression of FLG, IVL, OVOL1 and LOR genes by adopting real-time qPCR. The volume-equivalent PBS-treated group was used as a control (relative gene expression fold F: 1), and 2 was used -ΔΔCT F value was calculated for each sample.
The formula is as follows: f is 2 -ΔΔCT Wherein:
△CT experiment of ＝CT Experiment of -CT Internal reference (experiment) ；
△CT Control ＝CT Control -CT Internal reference (contrast) ；
△△CT＝△CT Experiment of -△CT Control of 。
The results are shown in the following table:
the result shows that the ProfMIC-213 supernatant can up-regulate the repair gene, the expression quantity of the related gene is 1.41-6.12, and the effect of promoting skin barrier repair is achieved.
Example 4 ProfMIC-213 Up-regulating HaCaT moisturizing-related Gene expression experiment
1. ProfMIC-213 supernatant preparation
The preparation method refers to example 3.
2. Experiment for up-regulating HaCaT moisturizing related gene expression
Inoculation of human immortalized keratinocytes HaCaT (2 ml/well, 5X 10 content) 5 Cells) to 6-well plate, 5% carbon dioxide incubator 37 ℃ overnight until cells adhere. The supernatant was added at 5% (V/V) and the control group was replaced with an equal volume of PBS. After 24h of culture, adding a lysate, extracting total RNA of cells, detecting the concentration and purity of the RNA, performing reverse transcription to obtain cDNA, taking GAPDH as an internal reference gene, and detecting the expression of AQP3 by adopting real-time qPCR. The volume-equivalent PBS-treated group was used as a control (relative gene expression fold F: 1) and 2 was used -ΔΔCT F value was calculated for each sample.
The results are shown in the following table:
the results show that the ProfMIC-213 has the effect of promoting skin moisturizing.
Example 5: ProfMIC-213 experiment for regulating light aging HaCaT keratinocyte extracellular matrix/antioxidant/inflammatory factor related gene expression
1. ProfMIC-213 supernatant and inactivated thallus preparation
The preparation method refers to example 3.
2. HaCaT cell preparation and ultraviolet ray damage
HaCaT cells were digested and then digested at 0.5 ml/well (2X 10 contained therein) 5 Cells) were seeded into 24-well plates and cultured overnight at 37 ℃ in a 5% carbon dioxide incubator. Total dose of 2J/cm was applied to cells in the wells 2 Ultraviolet UVB radiation damage.
3. ProfMIC-213 addition
5% (V/V) of the supernatant and 10% (V/V) of the inactivated bacteria were added to the stimulated HaCaT cells (in the control group, the supernatant/inactivated bacteria were replaced with an equal volume of PBS, respectively). Each group 3 was incubated overnight at 37 ℃.
4. qPCR method for detecting relative expression multiple of extracellular matrix/antioxidant/inflammatory factor related genes
Removing culture medium from the above cells, adding lysis solution, extracting total RNA from the cells, detecting RNA concentration and purity, reverse-transcribing to cDNA, and taking GAPDH as internal reference mediumTherefore, the expression of extracellular matrix related genes, extracellular matrix degradation related genes and antioxidant related genes is detected by adopting real-time qPCR. Relative expression fold F of control gene was 1, and 2 was used -ΔΔCT The F value of each sample was calculated.
The supernatant regulation HaCaT keratinocyte-associated gene results are given in the following table:
the results of the gene related to the regulation of HaCaT keratinocyte by the inactivated thallus are shown in the following table:
the result shows that the ProfMIC-213 is added to up-regulate extracellular matrix and oxidation resistance related genes, the expression quantity of the related genes is 1.20-1.46, the expression quantity of the related genes is 0.15-0.75, and the anti-aging effect of promoting synthesis of HaCaT keratinocyte extracellular matrix, reducing degradation of the extracellular matrix, increasing oxidation resistance and reducing inflammatory factors is achieved.
Example 6: ProfMIC-213 experiment for regulating oxidation injury human fibroblast extracellular matrix/antioxidation/apoptosis/inflammatory factor related gene expression
1. ProfMIC-213 supernatant and inactivated thallus preparation
The preparation method is referred to example 3.
2. HFF human fibroblast preparation and H 2 O 2 Inducing oxidative damage
HFF cells cultured in DMEM were digested at a volume of 0.5 ml/well (2X 10 cells contained therein) 5 Cells) were inoculated into 24-well plates and cultured overnight at 37 ℃ in a 5% carbon dioxide incubator. H was added to each well to a final concentration of 200. mu.M 2 O 2 Stimulating, and standing at 37 ℃ for 1 h.
3. ProfMIC-213 addition
The supernatant (5% (V/V) and the inactivated bacteria (10% (V/V)) were added to the stimulated HFF cells (control groups replaced the supernatant/inactivated bacteria with an equal volume of PBS, respectively). Each group 3 was incubated overnight at 37 ℃.
4. qPCR method for detecting relative expression multiple of extracellular matrix/antioxidation/apoptosis/inflammatory factor related genes
Removing the culture medium of the cells, adding a lysis solution, extracting total RNA of the cells, detecting the concentration and purity of the RNA, performing reverse transcription to obtain cDNA, taking GAPDH as an internal reference gene, and detecting the expression of genes related to extracellular matrix, antioxidation, extracellular matrix degradation, apoptosis and apoptosis by adopting real-time qPCR. Relative expression fold F of control gene was 1, and 2 was used -ΔΔCT The F value of each sample was calculated.
The results of the supernatant regulation of HFF human fibroblast-associated genes are shown in the following table:
the results of the genes related to the HFF human fibroblast regulated by the inactivated thallus are shown in the following table:
the result shows that the addition of ProfMIC-213 can up-regulate extracellular matrix, oxidation resistance and apoptosis related genes, the expression quantity of the related genes is 1.10-3.37, the down-regulation degradation of extracellular matrix, inflammatory factors and apoptosis related genes is 0.28-0.94, and the anti-aging effect of promoting the synthesis of HFF human fibroblast extracellular matrix, reducing the degradation of extracellular matrix, reducing the apoptosis, reducing the inflammation of cells and increasing the oxidation resistance is achieved.
Example 7: ProfMIC-213 Down-Regulation of HaCaT cell inflammatory factor-related Gene expression
1. ProfMIC-213 supernatant preparation
The preparation method refers to example 3.
2. HaCaT cell preparation
After digesting Raw264.7 cellsAt a concentration of 0.5 ml/well (2X 10 contained therein) 5 Cells) were inoculated into 24-well plates and cultured overnight at 37 ℃ in a 5% carbon dioxide incubator.
3. LPS preparation and addition
Preparing LPS with the concentration of 200ng/ml, adding 500 μ l of LPS into HaCaT cells cultured overnight, stimulating the cells to produce inflammatory factors, discarding the cell culture medium after 20h, washing with PBS 5 times, and adding 1ml of MEM serum-free medium into each well.
4. ProfMIC-213 supernatant addition
ProfMIC-213 supernatant was added at 5% (V/V) to LPS-stimulated HaCaT cell culture broth, 3 replicates per group, and incubated overnight at 37 ℃.
5. qPCR method for detecting relative expression multiple of cell inflammatory factor gene
Removing the culture medium of the cells, adding a lysate, extracting total RNA of the cells, detecting the concentration and purity of the RNA, performing reverse transcription to obtain cDNA, and detecting the expression of IL-6, IL-8 and TRPV1 genes by using real-time qPCR (quantitative polymerase chain reaction) by using GAPDH as an internal reference gene. The volume-equivalent PBS-treated group was used as a control (relative gene expression fold F: 1) and 2 was used -ΔΔCT The F value of each sample was calculated.
The results are shown in the following table:
the result shows that ProfMIC-213 can down-regulate LPS to induce HaCaT cell inflammatory factors, and the expression quantity of related genes is 0.33-0.94. Thus, ProfMIC-213 has an anti-inflammatory effect.
Example 8 Effect of ProfMIC-213 on scavenging free radicals
1. ProfMIC-213 supernatant preparation
Selecting a single bacterial colony of the Lactobacillus gasseri ProfMIC-213 in an MRS liquid culture medium, statically culturing for 16-18 h in an incubator at 37 ℃, detecting by an enzyme labeling instrument, and diluting with the MRS liquid culture medium to adjust OD 600 The cells were inactivated at 121 ℃ for 30min under high pressure, centrifuged at 12000 rpm for 2min, and the supernatant was filtered through a 0.22 μm filter to give a supernatant.
2. ProfMIC-213 supernatant hydroxyl radical scavenging capacity detection
The reagent preparation and detection method are carried out according to the instruction of the Solebao hydroxyl radical scavenging capability detection kit. The 536nm absorbance of each sample was measured, averaged and the clearance of each sample calculated.
The calculation formula and the result are shown in the following table:
3. ProfMIC-213 supernatant ABTS free radical scavenging ability test
The reagent preparation and detection method are carried out according to the instruction of the detection kit for the free radical scavenging ability of Solebao ABTS. The 405nm absorbance of each sample was measured, averaged and the clearance of each sample calculated.
The calculation formula and the result are shown in the following table:
4. ProfMIC-213 supernatant total antioxidant capacity detection
The reagent preparation and detection method is used for detecting various antioxidant substances, antioxidant enzymes and the like of ProfMIC-213 to form the total antioxidant level according to the specification of the Solebao total antioxidant capacity detection kit. The absorbance at 405nm of each sample was measured, averaged and the total antioxidant capacity of each sample was calculated.
Calculating the formula: total antioxidant capacity (. mu. mol/ml) × V anti-total/V-like
Measurement A-A blank 11.232x +0.0197
V, reverse total: total volume of reaction, 0.204ml
And V sample: sample volume in the reaction, 0.006ml
The results are given in the following table:
the result shows that ProfMIC-213 has the function of eliminating hydroxyl free radicals and ABTS free radicals, the free radical elimination rate is 7.87-18.52 percent, and the total antioxidant capacity is 1.22-1.26 mu mol/ml.
Example 9 Effect of ProfMIC-213 inhibition of skin pathogenic bacteria
1. ProfMIC-213 supernatant preparation
The preparation process is referred to example 8.
2. Preparation of pathogenic bacteria liquid
The 4 pathogenic bacteria: selecting single colony of human staphylococcus CGMCC 1.493, staphylococcus haemolyticus CGMCC 1.540 and corynebacterium siccatum CGMCC 1.1919, placing in BHI liquid culture medium, standing and culturing overnight in 37 deg.C incubator, detecting, and diluting with BHI liquid culture medium to adjust OD 600 ＝0.2。
3. Experiment for inhibiting pathogenic bacteria
Adding the inactivated supernatant into pathogenic bacteria liquid at an addition amount of 10% (V/V), culturing at 37 deg.C for 3 hr with the added equal volume of MRS liquid culture medium as control, and detecting bacterial liquid concentration (OD) 600 ) And calculating the pathogenic bacteria inhibition rate.
The calculation formula and the result are shown in the following table:
the result shows that ProfMIC-213 has the function of inhibiting the proliferation of skin pathogenic bacteria, and the inhibition rate is 17.78-58.44%.
Example 10 ProfMIC-213 Effect of altering the proportion of flora on treating acne
1. ProfMIC-213 supernatant preparation
The preparation process is referred to example 8.
2. Preparation of bacterial liquid of acne-related flora
Selecting single colony of Propionibacterium acnes CGMCC 1.5003 and Staphylococcus epidermidis CGMCC 1.4260 respectively in BHI liquid culture medium, standing and culturing overnight in a 37 ℃ incubator, detecting, and diluting with BHI liquid culture medium to adjust OD 600 ＝0.2。
3. Experiment for influencing growth of acne-related flora by adding supernatant
Adding the supernatant into two skin flora bacterial liquids respectively in an addition amount of 10% (V/V), taking an added equal volume MRS liquid culture medium as a control, culturing for 16h at 37 ℃, and evaluating the influence on the growth of the acne related flora according to the relative concentration ratio of the two bacterial liquids.
The calculation formula and the result are shown in the following table:
the results show that ProfMIC-213 has obvious inhibition effect on Propionibacterium acnes and promotes the growth of Staphylococcus epidermidis. ProfMIC-213 can change the proportion of flora related to acne, thereby achieving the purpose of treating acne.
Example 11: ProfMIC-213 function of changing flora proportion and improving sensitive muscle
1. ProfMIC-213 supernatant preparation
The preparation process is referred to example 8.
2. Preparation of bacterial liquid of sensitive muscle related flora
Respectively picking single colony of Staphylococcus aureus CGMCC 1.8721 and Staphylococcus epidermidis CGMCC 1.4260 to BHI liquid culture medium, standing and culturing at 37 deg.C for overnight, picking single colony of Micrococcus luteus CICC 10209 to nutrient broth liquid culture medium, standing and culturing at 30 deg.C for overnight, detecting, and diluting with BHI/nutrient broth liquid culture medium to adjust OD 600 ＝0.2。
3. Experiment for influencing growth of sensitive muscle related flora by adding supernatant
Adding the supernatant into two kinds of bacteria liquid of skin flora respectively at 10% addition amount, culturing at 37 deg.C for 16 hr and micrococcus luteus at 30 deg.C for 5 hr with the addition of equal volume MRS liquid culture medium as control, and taking the relative concentrations (OD) of the two kinds of bacteria liquid 600 ) The influence of the ratio evaluation on the growth of sensitive muscle-associated flora.
The calculation formula and the result are shown in the following table:
the result shows that the ProfMIC-213 has obvious inhibiting effect on staphylococcus aureus and promotes the growth of staphylococcus epidermidis. ProfMIC-213 can change the flora ratio, thereby effectively improving the sensitive muscle flora.
Example 12: ProfMIC-213 function of changing flora ratio to improve chloasma
1. ProfMIC-213 supernatant preparation
The preparation process is referred to example 8.
2. Preparation of chloasma-related flora bacterial liquid
Selecting single colony of Staphylococcus epidermidis CGMCC 1.4260 in BHI liquid culture medium, static culturing at 37 deg.C in incubator overnight, selecting single colony of Micrococcus luteus CICC 10209 in nutrient broth liquid culture medium, static culturing at 30 deg.C in incubator overnight, detecting, diluting with BHI/nutrient broth liquid culture medium, and adjusting OD 600 ＝0.2。
3. Experiment for influencing chloasma-related flora growth by adding clear liquid
Adding the supernatant into two kinds of bacteria liquid of skin flora respectively at 10% addition amount, culturing at 37 deg.C for 16 hr and micrococcus luteus at 30 deg.C for 5 hr with the addition of equal volume MRS liquid culture medium as control, and taking the relative concentrations (OD) of the two kinds of bacteria liquid 600 ) The ratio evaluation has the influence on the growth of the chloasma-related flora.
The calculation formula and the result are shown in the following table:
the result shows that the ProfMIC-213 has obvious inhibition effect on staphylococcus aureus and micrococcus luteus and promotes the growth of staphylococcus epidermidis. ProfMIC-213 can change the flora proportion, thereby effectively improving the flora related to sensitive muscle and chloasma.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (18)
1. Lactobacillus gasseri (Lactobacillus. gasseri), named as ProfMIC-213, has been preserved in China Center for Type Culture Collection (CCTCC) No. M2022691 in 23.5.2022.
2. The lactobacillus gasseri according to claim 1, characterized in that it is obtained by the following isolation method:
sampling feces of a five-year-old healthy girl, properly treating the feces, shaking and uniformly mixing the feces in normal saline, taking a supernatant, streaking the supernatant on an MRS solid plate, culturing the MRS solid plate at the constant temperature of 37 ℃ for 48 hours, and selecting a white colony to repeatedly inoculate and screen until a uniform single colony is obtained, wherein the colony is named as ProfMIC-213.
3. Lactobacillus gasseri according to claim 1, wherein the strain ProfMIC-213 is gram positive under gram-staphyloscopic examination and rod-shaped under microscope; growing on an MRS plate to form white round microcolonies with smooth, mellow and opaque surfaces and regular edges; the strain grows in MRS liquid culture medium in a uniform turbid way, and the strain is white and precipitated after being placed for a long time.
4. Use of a Lactobacillus gasseri according to any of the claims 1 to 3 for the preparation of a product for improving skin condition.
5. The use of claim 4, wherein said improving skin condition comprises at least one of repairing skin barrier, moisturizing, anti-aging, inhibiting skin pathogens, modulating skin microflora ratio, anti-inflammatory, anti-radical, anti-oxidant.
6. The use according to claim 5, wherein the repairing of the skin barrier is repairing skin cells and/or upregulating the expression of barrier repair-related genes; the barrier repair-associated genes include FLG, IVL, OVOL1, and/or LOR.
7. The use according to claim 5, wherein the moisturizing is up-regulation of the expression of the moisturizing-associated gene AQP 3.
8. The use according to claim 5, wherein the anti-aging is up-regulation of the expression of extracellular matrix-related genes; the extracellular matrix related gene comprises at least one of SMAD3, TIMP1, SPTSSA, Col-I, Col-3A1 and/or MKX.
9. The use of claim 5, wherein the anti-aging is up-regulation of the expression of cellular antioxidant-related genes NRF2 and/or SIRT-1.
10. The use according to claim 5, wherein the anti-aging is modulating expression of apoptosis-related genes; the apoptosis-related genes include expression of BAX, BCL-2 and/or Caspase families.
11. The use according to claim 5, wherein the anti-aging is down-regulation of expression of extracellular matrix-associated genes; the extracellular matrix-degrading related gene includes at least one of the P38MAPK and/or MMP families.
12. Use according to claim 5, wherein the dermatopathogenic inhibitor is at least one of Staphylococcus hominis, Staphylococcus haemolyticus and/or Corynebacterium xerosis.
13. Use according to claim 5, wherein the skin microflora is regulated in a ratio such as to inhibit the growth of Staphylococcus aureus, Propionibacterium acnes and/or Micrococcus luteus, to promote and/or not affect the growth of Staphylococcus epidermidis.
14. The use of claim 5, wherein the anti-inflammatory is down-regulation of the expression of a gene of a cellular inflammation-related factor; the genes of the relevant factors of the cell inflammation comprise at least one of TNF-alpha, IL-6, IL-8 and/or TRPV 1.
15. Use according to claim 5, wherein the anti-radical is a scavenging effect on hydroxyl and/or ABTS radicals.
16. The use according to claim 5, wherein the antioxidant is a total antioxidant capacity of various antioxidants and antioxidant enzymes.
17. Use according to any one of claims 4 to 16, wherein the product is a food product, a pharmaceutical product or a cosmetic product.
18. The use according to claim 17, wherein the lactobacillus gasseri in the product comprises one or both of:
(1) live and/or inactivated bacteria of lactobacillus gasseri;
(2) a culture, lysate and/or extract of lactobacillus gasseri.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115404189A (en) * | 2022-11-01 | 2022-11-29 | 山东锦鲤生物工程有限公司 | Lactobacillus corynebacterium and application thereof |
| CN116948922A (en) * | 2023-09-20 | 2023-10-27 | 杭州微致生物科技有限公司 | Lactobacillus gasseri VB247 and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115404189A (en) * | 2022-11-01 | 2022-11-29 | 山东锦鲤生物工程有限公司 | Lactobacillus corynebacterium and application thereof |
| CN115404189B (en) * | 2022-11-01 | 2023-01-13 | 山东锦鲤生物工程有限公司 | Lactobacillus corynebacterium and application thereof |
| CN116948922A (en) * | 2023-09-20 | 2023-10-27 | 杭州微致生物科技有限公司 | Lactobacillus gasseri VB247 and application thereof |
| CN116948922B (en) * | 2023-09-20 | 2024-03-01 | 杭州微致生物科技有限公司 | Lactobacillus gasseri VB247 and application thereof |
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