CN115197883A - Lactobacillus amyloliquefaciens and skin repair and anti-aging application thereof - Google Patents
Lactobacillus amyloliquefaciens and skin repair and anti-aging application thereof Download PDFInfo
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- CN115197883A CN115197883A CN202210968688.7A CN202210968688A CN115197883A CN 115197883 A CN115197883 A CN 115197883A CN 202210968688 A CN202210968688 A CN 202210968688A CN 115197883 A CN115197883 A CN 115197883A
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Abstract
The invention relates to the technical field of microorganisms, in particular to lactobacillus amyloliquefaciens and application thereof. The invention discloses a Lactobacillus amyloliquefaciens (Lactobacillus amylovorus) which is preserved in China center for type culture collection with the preservation number of CCTCC NO: M2022370. Experiments show that the lactobacillus amyloliquefaciens has the functions of maintaining and repairing skin barriers, enhancing the antibacterial capacity of skin cells, resisting aging, promoting cell proliferation, whitening, resisting inflammation and free radicals, inhibiting skin pathogenic bacteria and treating nasosinusitis, 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 lactobacillus amyloliquefaciens and application thereof in skin repair and anti-aging.
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 dry skin, skin aging, atopic dermatitis, eczema, psoriasis, ichthyosis, and dermatitis with symptoms of sensitive skin, irritant dermatitis, and hormone-dependent dermatitis, and seborrheic diseases such as acne, rosacea, and seborrheic dermatitis.
The antibacterial peptide is mainly secreted by epithelial cells on the surface layer of the mucous membrane, and is expressed in cells of eyes, skin, oral mucosa, urogenital system and respiratory system. Antimicrobial peptides have a direct effect on most bacteria, certain fungi and viruses. The common mode of action is that positive charges carried by the bacteria react with negatively charged components (such as lipopolysaccharide) on the bacterial cell membrane, so that the permeability of the bacterial cell membrane is increased, the bacteria are cracked, and the bacteria die.
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 skin aging. 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.
Tyrosinase is also called polyphenol oxidase and widely exists in animals, plants, microorganisms and human bodies. Tyrosinase has important physiological functions in organisms, is a rate-limiting enzyme for melanin synthesis, directly influences the synthesis of melanin, and is also related to the occurrence of diseases such as excessive melanin deposition of freckles, chloasma and the like of a human body. Tyrosine generates dopa under the action of tyrosinase, and melanin is finally generated through a series of steps. Therefore, inhibition of tyrosinase activity is of great significance for skin whitening.
The probiotic is used in cosmetics, and has effects in resisting inflammation, scavenging free radicals, inhibiting proliferation of skin pathogenic bacteria, balancing skin epidermis flora, and repairing skin barrier. And meanwhile, the tyrosinase activity is inhibited, the absorption of skin to nutrient substances is effectively increased, the skin whitening is improved, and the immunity is enhanced.
Nasosinusitis is a common disease in otorhinolaryngology, and is mainly caused by respiratory tract allergy, nasal and sinus mucosa cleaning dysfunction, respiratory tract microbial infection, abnormal local anatomical structure of nose and the like. Research shows that Haemophilus influenzae (Haemophilus influenzae) is an important pathogenic bacterium of nasosinusitis, and Lactobacillus sake (Lactobacillus sakei) has a positive effect on maintaining normal functions of nasal cavities. Therefore, the proportion of the flora of the haemophilus influenzae/the sake lactobacillus is reduced, and the treatment of the nasosinusitis is facilitated. The probiotics can be used as a microecological preparation for adjusting the proportion of respiratory tract flora and maintaining the respiratory tract flora balance for a long time. Therefore, it is important to develop probiotics for maintaining the balance of respiratory flora and explore the potential properties and utilization value of the strains.
Disclosure of Invention
In view of the above, the technical problem to be solved by the invention is lactobacillus amyloliquefaciens and its application in skin repair and anti-aging.
The invention provides a Lactobacillus amyloliquefaciens (Lactobacillus amylovorus) with the preservation number of CCTCC NO: M2022370. The Lactobacillus amyloliquefaciens is separated from Szechwan pickled vegetable pulp water and named as ProfMIC-212, and the strain is determined to be Lactobacillus amyloliquefaciens (Lactobacillus amylovorus) after 16S sequencing and sequence comparison.
Further, the invention provides a flora comprising the lactobacillus amyloliquefaciens.
Further, the present invention provides a material comprising the bacterial agent of the lactobacillus amyloliquefaciens or the flora of the present invention. The formulation of the microbial inoculum comprises particles, liquid and dry powder, which is not limited in the invention.
The invention also provides application of the lactobacillus amyloliquefaciens and/or the flora and/or the microbial inoculum in preparing products for improving skin conditions.
Further, the improvement of the skin condition according to the present invention includes at least one of repairing skin barrier, enhancing skin cell antibacterial ability, anti-aging, promoting cell proliferation, anti-inflammatory, whitening, anti-free radical, inhibiting skin pathogenic bacteria, and regulating microbial flora ratio.
In the invention, the repair of the skin barrier of the lactobacillus amyloliquefaciens includes but is not limited to repair of epidermal cell damage, improvement of epidermal cell survival capability or up-regulation of the expression of barrier repair related genes. Further, the barrier repair-related gene comprises at least one of FLG, IVL, OVO1 and LOR. Furthermore, the HaCaT cell is taken as a subject, and the HaCaT cell is treated by using the inactivated supernatant and the thallus of the lactobacillus amyloliquefaciens, and the result shows that the supernatant and the thallus of the lactobacillus amyloliquefaciens can both up-regulate the related genes for cell barrier repair, and have the effect of promoting the cell barrier repair.
In the present invention, the lactobacillus amyloliquefaciens can enhance the antibacterial ability of skin cells, including but not limited to, increasing the expression of antibacterial related genes or expressing antibacterial proteins or polypeptides, which is not limited in the present invention. Furthermore, the HaCaT cell is a subject, and the cell is treated by using the inactivated supernatant and the thallus of the lactobacillus amyloliquefaciens, and the result shows that the supernatant and the thallus of the lactobacillus amyloliquefaciens can up-regulate the expression of beta-defensin genes DEFB1, S100A7 and S100A8 of the HaCaT cell and have the effect of enhancing the antibacterial capacity of epidermal cells.
In the invention, the lactobacillus amyloliquefaciens has the function of resisting cell aging. The anti-aging comprises any one of the following A) to D):
a) Up-regulating the expression of extracellular matrix related genes and down-regulating extracellular matrix degradation related genes; the extracellular matrix synthesis-related gene comprises at least one of TIMP1, MKX, SMAD3, LN, COL1A1 and COL13A 1; the extracellular matrix degradation related gene comprises at least one of P38MAPK and/or MMP families.
B) Up-regulating the expression of the apoptosis-related gene BCL-2; downregulating the expression of at least one gene of the apoptosis-related gene BAX and/or Caspase family.
C) Up-regulating the expression of the gene MOR related to the cell immune regulatory factor.
D) Up-regulating the expression of the cell antioxidant related gene; the antioxidant-related gene comprises at least one of PTEN, NRF2 and SIRT families.
In the invention, haCaT cells and HFF cells are taken as subjects, and extracellular matrix related gene expression is taken as a representation to study the influence of the lactobacillus amyloliquefaciens on cell anti-aging. The results show that the inactivated supernatant and thalli of the lactobacillus amyloliquefaciens can both up-regulate at least one of extracellular matrix synthesis related genes TIMP1, MKX, SMAD3, LN, COL1A1 and COL13A1 and down-regulate at least one of extracellular matrix degradation related genes P38MAPK and/or MMP families; the lactobacillus amyloliquefaciens can promote the synthesis of extracellular matrixes and inhibit the degradation of the extracellular matrixes so as to delay cell aging.
In the invention, the HFF cell is a subject, and the effect of the lactobacillus amyloliquefaciens on cell anti-aging is researched by taking the expression of apoptosis-related genes as characteristics. The result shows that the inactivated supernatant and thalli of the lactobacillus amyloliquefaciens can both up-regulate and inhibit the expression of apoptosis related genes BCL-2 and down-regulate the expression of at least one gene in apoptosis related genes BAX and/or Caspase families.
According to the invention, the HFF cells are taken as a subject, the expression of a cell immune regulatory factor related gene MOR is taken as a representation, the influence of the lactobacillus amyloliquefaciens on the anti-aging of the HFF cells is researched, and the result shows that the sterilized supernatant of the lactobacillus amyloliquefaciens can up-regulate the expression of the MOR gene of the HFF cells, improve the cell immune regulatory capacity and further play a role in resisting cell aging.
The lactobacillus amyloliquefaciens has the effect of promoting cell proliferation. Further such cells include, but are not limited to, HFF cells, haCaT cells, or other biologically commonly used tool cells, as the present invention is not limited in this respect. Furthermore, the subject of the present invention was tested for HFF, and the effect of Lactobacillus amyloliquefaciens on cell proliferation was investigated. The results show that the sterilized supernatant and the thallus of the lactobacillus amyloliquefaciens can promote the proliferation of HFF cells, and that the lactobacillus amyloliquefaciens can promote the proliferation of the cells.
The lactobacillus amyloliquefaciens has the function of improving the anti-inflammatory effect of cells. The anti-inflammation includes, but is not limited to, down-regulating the expression of a cellular inflammation-related factor gene, and/or reducing the release level of cellular NO. Further, the genes related to the cytokines comprise at least one of IL-8, COX-2, TNF-alpha or IL-6. Furthermore, the invention takes the HFF cells as a subject, researches the influence of the sterilized supernatant and the thallus of the lactobacillus amyloliquefaciens on the expression of the genes related to the HFF cell inflammation, and shows that the lactobacillus amyloliquefaciens can up-regulate the expression of the genes related to the inflammatory factors and has the function of improving the anti-inflammatory capability of the cells. Furthermore, the invention takes Raw264.7 cells as a subject, researches the influence of the lactobacillus amyloliquefaciens on the NO production of LPS stimulated cells, and shows that the sterilized supernatant and the precipitate of the lactobacillus amyloliquefaciens can reduce the NO production of the LPS stimulated cells and increase the anti-inflammatory effect of the cells.
The lactobacillus amyloliquefaciens has the function of resisting free radicals. Further, the radical includes, but is not limited to, hydroxyl radical and/or ABTS radical, which is not limited in the present invention. The results show that the lactobacillus amyloliquefaciens can eliminate cellular hydroxyl radicals and/or ABTS radicals.
The lactobacillus amyloliquefaciens has a whitening effect. Further, the whitening includes any one of reducing cytochrome precipitation, inhibiting related gene of pigmentation, inhibiting related enzyme activity of pigmentation, and increasing skin cell brightness, which is not limited in the present invention. Furthermore, the invention takes tyrosinase as a subject, and researches the inhibition effect of the lactobacillus amyloliquefaciens on the activity of the lactobacillus amyloliquefaciens, and the result shows that the sterilized supernatant and the precipitate of the lactobacillus amyloliquefaciens can inhibit the activity of the tyrosinase, so that the skin pigmentation is reduced, and the whitening effect is achieved.
The lactobacillus amyloliquefaciens has the function of inhibiting skin pathogenic bacteria. The skin pathogenic bacteria comprise: at least one of staphylococcus hominis, staphylococcus haemolyticus and corynebacterium sicans.
Furthermore, the invention takes human Staphylococcus (Staphylococcus hominis CGMCC 1.493), staphylococcus haemolyticus (Staphylococcus haemolyticus CGMCC 1.540) and Corynebacterium parahaemolyticus (Corynebacterium xenosis CGMCC 1.1919) as subjects, and researches the killing effect of the lactobacillus amyloliquefaciens on skin pathogenic bacteria. The result shows that the lactobacillus amyloliquefaciens can inhibit the growth of human staphylococcus, hemolytic staphylococcus and corynebacterium xerosis and has the function of inhibiting the proliferation of skin pathogenic bacteria.
The lactobacillus amyloliquefaciens has the effect of changing the flora ratio and treating nasosinusitis. Further, the adjusting of the flora ratio comprises: inhibiting the growth of Haemophilus influenzae, and/or promoting or not affecting the growth of Lactobacillus sake. Furthermore, the invention takes haemophilus influenzae and lactobacillus sake as the subjects, and the influence of the lactobacillus amyloliquefaciens on the flora ratio is researched. The results show that the lactobacillus amyloliquefaciens has obvious inhibition effect on haemophilus influenzae and no inhibition effect on lactobacillus sake, thereby effectively treating nasosinusitis.
The invention provides a product for improving skin conditions, and raw materials of the product comprise the lactobacillus amyloliquefaciens and a culture, a lysate and/or an extract thereof, and/or a flora and a microbial inoculum thereof.
Further, the product of the present invention includes food, medicine or cosmetics, but the present invention is not limited thereto.
Further, the product of the present invention comprises a cosmetic, and the cosmetic formulation comprises: at least one of creams, emulsions, mists, gels, oils, powders, loose powders, mud, aerosols, patches, films, and lyophilized powders.
The invention also provides a method for improving skin condition, which comprises using the product. The method of application includes smearing, external application or injection, but the invention is not limited thereto.
The invention provides a Lactobacillus amylovorus ProfMIC-212 (Lactobacillus amylovorus) preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2022370. Experiments show that the ProfMIC-212 has the functions of maintaining and repairing skin barrier, enhancing skin cell antibacterial ability, resisting aging, promoting cell proliferation, whitening skin, resisting inflammation and free radicals, inhibiting skin pathogenic bacteria and treating nasosinusitis, and can be used for preparing foods, medicines, cosmetics and the like.
Biological preservation Instructions
Lactobacillus amyloliquefaciens ProfMIC-212 (Lactobacillus amylovorus ProfMIC-212) is preserved in China center for type culture Collection at 1/4 in 2022, with the address of China, wuhan and Wuhan university with the preservation number of CCTCC NO: M2022370.
Detailed Description
The invention provides a method for understanding lactobacillus amyloliquefaciens and application thereof, and a person skilled in the art can realize the method by appropriately improving process parameters by referring to the content. 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 test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
EXAMPLE 1 isolation of ProfMIC-212
Sampling in Sichuan pickle juice. 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-212.
Gram staining microscopy: the strain ProfMIC-212 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-212
1. 16S rDNA gene sequence analysis:
picking single colony in MRS liquid culture medium, culturing overnight at 37 deg.C, centrifuging at 12000 deg.C for 1min, collecting thallus, and performing operation according to 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 ℃;94 ℃ 15s,57 ℃ 15s,72 ℃ 40s,35 cycles; extension at 72 ℃ for 10min.
2. As a result, the
Homology comparison (BLASTN) of the 16S rDNA gene PCR product sequence with the published standard sequence in GenBank results in the strain ProfMIC-212 being Lactobacillus amyloliquefaciens (Lactobacillus amylovorus).
Example 3 ProfMIC-212 Gene expression test for promoting HaCaT Barrier repair in human immortalized keratinocytes
1. Preparation of ProfMIC-212 supernatant and inactivated thallus:
selecting a single colony of the lactobacillus amyloliquefaciens ProfMIC-212 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 And (3) inactivating at 121 ℃ for 30min under high pressure, centrifuging at 12000 rpm for 2min, and filtering with 0.22 μm filter membrane to obtain supernatant. Resuspending the pellet with PBS, diluting and adjusting OD 600 And =0.2, which is an inactivated cell.
2. Experiment for promoting HaCaT barrier repair related gene expression
Inoculation with HaCaT (2 ml/well, 5X 10 content) 5 Cells) to 6-well plate, 5% carbon dioxide incubator 37 ℃ overnight until cells adhere. Respectively adding 5% (V/V) of supernatant, 10% (V/V) of inactivated bacteria, culturing for 24h, adding lysate, extracting total RNA of cells, detecting RNA concentration and purity, performing reverse transcription to obtain cDNA, and detecting the expression of FLG, IVL, OVOL1 and LOR genes by real-time qPCR (quantitative polymerase chain reaction) by taking GAPDH as an internal reference gene. Control groups treated with PBS of equal volume (relative gene expression fold F = 1) were each added with 2 -ΔΔCT The F value of each sample was calculated.
The formula: f =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 the invention -△CT Control 。
The results are shown in tables 1 and 2:
TABLE 1 supernatant upregulation of HaCaT Barrier repair Gene expression by ProfMIC-212
TABLE 2 expression of HaCaT barrier repair gene up-regulated by ProfMIC-212 inactivated bacteria
In vitro cell experiments show that the supernatant and the inactivated thallus of the lactobacillus amyloliquefaciens ProfMIC-212 have the effect of up-regulating the expression of a silk polymerization protein gene FLG, an involucrin gene IVL, an OVO-like transcription factor 1 gene OVOL1 and an loricrin gene LOR which are relevant factors for the skin barrier repair of a human immortalized keratinocyte HaCaT, and the relative expression multiple of the genes is 1.26-4.25, which shows that the ProfMIC-212 has the effect of promoting the skin barrier repair.
Example 4 ProfMIC-212 Up-regulating HaCaT antimicrobial peptide-related Gene expression experiment
1. Preparation of ProfMIC-212 supernatant and inactivated thallus:
the preparation method refers to example 3.
2. HaCaT cell preparation
HaCaT cells were digested and then digested at 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.ProfMIC-212 addition and LPS stimulation
Respectively adding the supernatant of 5% (V/V) and the inactivated thallus of 10% (V/V) into HaCaT cells cultured overnight (equal volume of PBS is used for replacing the supernatant in the control group respectively), adding 0.5ml of LPS solution with the concentration of 0.2 mu g/ml after 2h to induce cell inflammation, and culturing for 20h at 37 ℃ in a 5% carbon dioxide incubator.
4. qPCR method for detecting relative expression multiple of cell inflammatory factor gene
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, and detecting the expression of DEFB1, S100A7 and S100A8 genes by using real-time qPCR (quantitative polymerase chain reaction) by using GAPDH as an internal reference gene. Control with an equal volume of PBS-treated group (gene relative expression fold F = 1) using 2 -ΔΔCT F value was calculated for each sample.
The results are shown in tables 3 and 4:
TABLE 3 expression of HaCaT antimicrobial peptide Gene upregulated by ProfMIC-212 supernatant
TABLE 4 expression of HaCaT antimicrobial peptide gene up-regulated by ProfMIC-212 inactivated bacteria
In vitro cell experiments show that the supernatant and the inactivated thallus of the lactobacillus amyloliquefaciens ProfMIC-212 have the effect of up-regulating the expression of HaCaT cell antibacterial peptide beta-defensin genes DEFB1, S100A7 and S100A8, and the relative expression multiple of the genes is 1.44-2.14, so that the ProfMIC-212 has the effect of promoting the expression of the antibacterial peptide genes.
Example 5 ProfMIC-212 Regulation of photoaging HaCaT extracellular matrix/antioxidant/inflammatory factor-related Gene expression assay
1. Preparing a ProfMIC-212 supernatant and inactivated bacteria:
the preparation method refers to example 4.
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 inoculated 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-212 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/autophagy/antioxidation related genes
Removing a culture medium from 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 extracellular matrix related genes TIMP1, COL1A1 and SMAD3, antioxidant related gene NRF2, degraded extracellular matrix related genes P38MAPK and MMP1, and inflammatory factor related genes TNF-alpha by adopting real-time qPCR. Relative expression multiple F =1 of control group gene, using 2 -ΔΔCT The F value of each sample was calculated.
The supernatant liquid can regulate extracellular matrix related genes COL1A1 and SMAD3 and antioxidant related gene NRF2; down-regulating and degrading extracellular matrix related gene MMP1 and inflammatory factor related gene TNF-alpha. The results are shown in Table 5:
TABLE 5 ProfMIC-212 supernatant regulates expression of extracellular matrix-associated genes
The inactivated bacteria up-regulated the extracellular matrix genes TIMP1, COL1A1 and SMAD3, and the results are shown in table 6:
TABLE 6 expression of genes associated with cellular senescence regulated by inactivated thallus ProfMIC-212
In vitro cell experiments show that the lactobacillus amyloliquefaciens ProfMIC-212 has the function of up-regulating expression of tissue metalloproteinase inhibitor 1 gene TIMP1, type I collagen alpha 1 chain gene COL1A1 and SMAD3 genes related to HaCaT extracellular matrix and anti-oxidation related gene nuclear factor E2 related factor 2 gene NRF2, and the relative expression quantity of the genes is up-regulated by 1.17 to 1.81 times; has the functions of down-regulating matrix metalloproteinase family gene MMP1 related to degrading extracellular matrix and interleukin 6 gene IL-6 related to cell inflammation factors, and the relative expression multiple of the genes is 0.31-0.78. The ProfMIC-212 is shown to have the anti-aging effects of promoting the synthesis of HaCaT extracellular matrix, increasing the antioxidant capacity, reducing the degradation of the extracellular matrix and reducing inflammatory factors.
Example 6 ProfMIC-212 promotes proliferation of human fibroblast HFF
1. Preparing a ProfMIC-212 supernatant and inactivated bacteria:
the preparation method refers to example 4.
2. HFF cell preparation and ProfMIC-212 addition
The HFF cells cultured with DMEM were digested at 0.5 ml/well (per well)The pores contain 1.5X 10 5 Cells) were seeded into 24-well plates and cultured overnight at 37 ℃ in a 5% carbon dioxide incubator. The 10% (V/V) supernatant and the inactivated cells were added to HFF cells, respectively (control with an equal volume of PBS). Each group 3 was incubated overnight at 37 ℃.
3. HFF cell transfer and stain counting
HFF cells in 24-well plates were counted and diluted appropriately at 2 ml/well (2.0X 10 per well) 3 Cells) were transferred to 6-well plates, each group was 3-parallel, and cultured in a 5% carbon dioxide incubator at 37 ℃ for 7-10 days. The cells in the wells were counted after fixation with paraformaldehyde followed by crystal violet staining. And calculating the cell proliferation rate according to a formula.
The calculation formula and the results are shown in the following table 7:
TABLE 7 ProfMIC-212 promotes HFF cell proliferation
The result shows that the addition of ProfMIC-212 has the function of promoting the proliferation of HFF cells, and the proliferation rate is 8.85-106.96%.
Example 7 ProfMIC-212 Regulation of oxidative damage HFF extracellular matrix/apoptosis/antioxidant/immunomodulatory factor/inflammatory factor-related Gene expression assay
1. Preparing a ProfMIC-212 supernatant and inactivated bacteria:
the preparation method refers to example 4.
2. HFF cell preparation and H 2 O 2 Inducing oxidative damage
The HFF cells cultured with DMEM were digested at 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. H was added to each well to a final concentration of 200. Mu.M 2 O 2 Stimulating, and standing at 37 ℃ for 1h.
3.ProfMIC-212 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/apoptosis/antioxidation/immunoregulation factor/inflammatory factor related gene
Removing a culture medium from 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, detecting extracellular matrix related genes MKX, COL13A1 and LN, apoptosis-inhibiting related genes and BCL-2, oxidation-resisting related genes PTEN, SIRT-1 and SIRT-3 and immune regulatory factor related genes MOR by using GAPDH as an internal reference gene and adopting real-time qPCR; and degrading the expression of MMP family and P38MAPK of extracellular matrix related genes, caspase family and BAX of apoptosis related genes and IL-6 of inflammatory factor related genes. Relative expression multiple F =1 of control group gene, using 2 -ΔΔCT F value was calculated for each sample.
The supernatant fluid up-regulates extracellular matrix related genes MKX and COL13A1, inhibits apoptosis gene BCL-2, antioxidant related genes PTEN, SIRT-1 and SIRT-3, and immunoregulation factor related gene MOR; down-regulating and degrading extracellular matrix related genes MMP family, apoptosis related genes BAX and Caspase family and inflammatory factor related genes IL-6, and the results are shown in a table 8:
TABLE 8 ProfMIC-212 supernatant modulating the expression of extracellular matrix/apoptosis/antioxidant/immunomodulatory factor/inflammatory factor-related genes
The inactivated thallus can up-regulate and inhibit extracellular matrix related genes LN and MKX and oxidation resistance related genes SIRT-1; down-regulating and degrading extracellular matrix related genes MMP family and P38MAPK and apoptosis related gene BAX, and finding the results in Table 9:
TABLE 9 expression of ProfMIC-212 inactivated bacteria to modulate degradation of extracellular matrix/antioxidant/apoptosis related genes
The results show that ProfMIC-212 has the anti-aging effects of promoting HFF extracellular matrix synthesis, reducing apoptosis, increasing antioxidation, increasing cellular immune regulatory factors, reducing extracellular matrix degradation and reducing inflammatory factors.
Example 8 inhibition of tyrosinase Activity by ProfMIC-212
1. Preparation of ProfMIC-212 supernatant:
the preparation method refers to example 3.
2. Tyrosinase activity inhibition assay
Tyrosinase, 6.0mM L-dopa and 80mM PBS were prepared at a concentration of 250U/ml, 50. Mu.l PBS + 20. Mu.l supernatant + 50. Mu.l tyrosinase + 50. Mu.l L-dopa were sequentially added to each well of a 96-well plate, and the supernatant was replaced with MRS medium in the control group. After mixing, the reaction is carried out for 15min at room temperature in a dark place, the absorbance A is detected at 475nm, and the tyrosinase activity inhibition rate is calculated.
The calculation formula and the results are shown in table 10:
TABLE 10 inhibition of tyrosinase Activity by ProfMIC-212 supernatant
The result shows that the supernatant of ProfMIC-212 has the function of inhibiting the activity of tyrosinase, and the inhibition rate is 50.77-56.92%
Example 9 ProfMIC-212 reduction of NO production by Raw264.7 cells
1. Preparing a ProfMIC-212 supernatant and inactivated bacteria:
the preparation method refers to example 3.
2. Preparation of Raw264.7 cells
Raw264.7 cells were digested and then digested at 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.ProfMIC-212 addition and LPS stimulation
Adding 5% (V/V) of the supernatant and 10% (V/V) of inactivated bacteria into different groups of Raw264.7 cells which are cultured overnight (equal volume of PBS is respectively used for replacing the supernatant and the inactivated bacteria for a control group), adding 0.5ml of LPS solution with the concentration of 0.2 mu g/ml after 2 hours to induce Raw264.7 cells to be inflamed, taking cell culture supernatant after 20 hours, drawing a standard curve according to the method of a Biyunnan NO detection kit, and calculating the concentration and the inhibition rate of NO in the sample.
The calculation formula and the results are shown in the following table 11:
TABLE 11 ProfMIC-212 reduction of NO production by Raw264.7 cells
The results show that ProfMIC-212 has the effect of reducing the release of inflammatory factors, namely has the anti-inflammatory effect, can reduce the NO production of Raw264.7 cells induced by LPS, and reduces 31.83-58.48% compared with an LPS control group.
Example 10 ProfMIC-212 Down-Regulation of HaCaT cell inflammatory factor-related Gene expression
1. Preparation of ProfMIC-212 supernatant:
the preparation method refers to example 3.
2. HaCaT cell preparation
HaCaT cells were digested and then digested at 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.ProfMIC-212 addition and LPS stimulation
The supernatant was added at 5% (V/V) to HaCaT cells cultured overnight (the control group replaced the supernatant with an 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 multiple of cell inflammatory factor gene
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 IL-8 and COX-2 genes by adopting real-time qPCR. Control group treated with PBS added at equal volume (gene relative expression fold F = 1) using 2 -ΔΔCT The F value of each sample was calculated.
The results are shown in Table 12:
TABLE 12 ProfMIC-212 supernatant downregulating expression of inflammatory factor-related genes
The result shows that ProfMIC-212 can down-regulate the expression of genes related to the LPS induced HaCaT cell inflammatory factors, has an anti-inflammatory effect, and has a relative expression multiple of 0.32-0.83.
Example 11 Effect of ProfMIC-212 on scavenging free radicals
1. Preparation of ProfMIC-212 supernatant:
selecting a single colony of the lactobacillus amyloliquefaciens ProfMIC-212 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 the MRS liquid culture medium to adjust OD 600 And (3) inactivating at 121 ℃ for 30min under high pressure, centrifuging at 12000 rpm for 2min, and filtering the supernatant with a 0.22 μm filter membrane to obtain the supernatant.
2.ProfMIC-212 supernatant hydroxyl radical scavenging capacity test
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 Table 13:
TABLE 13 ProfMIC-212 hydroxyl radical scavenging ratio (%)
3. Testing of ABTS free radical scavenging ability of ProfMIC-212 supernatant
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 Table 14:
TABLE 14 ProfMIC-212ABTS radical scavenging ratio (%)
The result shows that ProfMIC-212 has the function of eliminating hydroxyl radical and ABTS radical, and the radical eliminating rate is 25.93-32.94%.
EXAMPLE 12 Effect of ProfMIC-212 on inhibiting skin pathogenic bacteria
1. Preparation of ProfMIC-212 supernatant:
the preparation process is as in example 11.
2. Preparing a pathogenic bacterium liquid:
the three types of pathogenic bacteria: respectively picking single colony from human Staphylococcus (Staphylococcus hominis CGMCC 1.493), staphylococcus haemolyticus (Staphylococcus haemolyticus CGMCC 1.540) and Corynebacterium (Corynebacterium ferrosis CGMCC 1.1919), placing single colony in 1ml BHI liquid culture medium, static culturing at 37 deg.C culture box overnight, 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 MRS liquid culture medium of the same volume as the control, and detecting the bacterial liquid concentration (OD) 600 ) And calculating the pathogenic bacteria inhibition rate.
The calculation formula and the result are shown in Table 15:
TABLE 15 inhibitory effect of ProfMIC-212 on skin pathogenic bacteria
The result shows that the ProfMIC-212 has the function of inhibiting the proliferation of skin pathogenic bacteria, and the inhibition rate is 37.84-45.24%.
EXAMPLE 13 Effect of ProfMIC-212 altering the flora ratio in the treatment of sinusitis
1. Preparation of ProfMIC-212 supernatant:
the preparation process is as in example 11.
2. Preparing a bacterium liquid of nasosinusitis-related flora:
selecting single colony of Haemophilus influenzae (Haemophilus influenzae ATCC 49766) in BHI liquid culture medium, selecting single colony of Lactobacillus sake in MRS liquid culture medium, standing and culturing at 37 deg.C in incubator overnight, detecting with enzyme labeling instrument, diluting with blank liquid culture medium, adjusting OD 600 ＝0.2。
3. Experiment for influencing growth of nasosinusitis-related flora by adding supernatant
Adding the supernatant into two bacterial liquids of strains related to sinusitis at 10% (V/V) respectively, culturing at 37 deg.C for 16 hr with the same volume of MRS liquid culture medium as control, and taking the relative concentrations (OD) of the two bacterial liquids 600 ) The ratio evaluation has influence on the growth of the flora related to the nasosinusitis. The calculation formula and the results are shown in Table 16:
TABLE 16 influence of ProfMIC-212 on growth of the sinusitis-related flora
The results show that ProfMIC-212 has significant inhibition effect on Haemophilus influenzae, but has no inhibition effect on sake lactobacillus. ProfMIC-212 is able to alter the flora ratio and thus is effective in treating sinusitis.
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 (12)
1. Lactobacillus amyloliquefaciens (Lactobacillus amylovorus) with the preservation number of CCTCC NO: M2022370.
2. A flora comprising Lactobacillus amyloliquefaciens (Lactobacillus amylovorus) according to claim 1.
3. A bacterial agent comprising the bacterium Lactobacillus amyloliquefaciens (Lactobacillus amylovorus) according to claim 1 or the bacterium population according to claim 2.
4. Use of a lactobacillus amyloliquefaciens according to claim 1, and/or a flora according to claim 2, and/or an inoculant according to claim 3 for the preparation of a product for improving skin conditions.
5. The use of claim 4, wherein the improvement in skin condition comprises at least one of promoting cell proliferation, repairing skin barrier, enhancing skin cell antibacterial ability, anti-aging, anti-inflammatory, whitening, anti-free radical, inhibiting skin pathogens, and regulating microbial flora ratio.
6. The use of claim 5, wherein the repair of the skin barrier comprises up-regulating the expression of a barrier repair-associated gene; the barrier repair-related gene comprises at least one of FLG, IVL, OVO1 and LOR.
7. The use of claim 5, wherein said enhancing the antimicrobial capacity of skin cells comprises up-regulating antimicrobial peptide-related gene expression; the antibacterial peptide related gene comprises at least one of DEFB1, S100A7 and S100A 8.
8. The use according to claim 5, wherein the anti-aging agent comprises any one of the following A) to D):
a) Up-regulating the expression of extracellular matrix synthesis related genes and down-regulating the expression of extracellular matrix degradation related genes; the extracellular matrix synthesis-related gene comprises at least one of TIMP1, MKX, SMAD3, LN, COL1A1 and COL13A 1; the extracellular matrix-degrading related gene comprises at least one of P38MAPK and/or MMP families;
b) Up-regulating the expression of the apoptosis-related gene BCL-2; down-regulating the expression of at least one gene of the apoptosis-related gene BAX and/or Caspase family;
c) Up-regulating the expression of the gene MOR related to the cellular immune regulatory factor;
d) Up-regulating the expression of the cell antioxidant related gene; the antioxidant-related genes include at least one of genes in the PTEN, NRF2, and SIRT families.
9. The use according to claim 5, wherein said anti-inflammatory comprises: down-regulating the expression of a cellular inflammation-associated factor gene, and/or reducing the level of cellular NO release; the cell inflammation related factor gene comprises at least one of IL-8, COX-2, TNF-alpha or IL-6.
10. Use according to claim 5, wherein the anti-radical comprises:
scavenging hydroxyl radicals and/or ABTS radicals; the whitening comprises inhibiting tyrosinase activity.
11. The use according to claim 5, wherein said inhibiting of skin pathogens comprises: inhibiting at least one of staphylococcus hominis, staphylococcus haemolyticus and corynebacterium crenatum; the adjusting of the microbial flora ratio comprises: inhibiting the growth of Haemophilus influenzae, and/or promoting or not affecting the growth of Lactobacillus sake.
12. A product for improving skin conditions, the raw material comprising Lactobacillus amyloliquefaciens strain and a culture, lysate and/or extract thereof according to claim 1, and/or a population according to claim 2, and/or a bacterial agent according to claim 3.
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