Lactobacillus amylolyticus and its application in skin repair and anti-aging

Abstract

The present invention relates to the field of microbial technology, and in particular to Lactobacillus amylovorus and its application. The Lactobacillus amylovorus disclosed in the present invention is deposited with the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2022370. Experiments have shown that the Lactobacillus amylovorus has the functions of maintaining and repairing the skin barrier, enhancing the antibacterial ability of skin cells, resisting aging, promoting cell proliferation, whitening, anti-inflammatory and anti-free radical, inhibiting skin pathogens, and treating sinusitis. The Lactobacillus amylovorus can be used to prepare food, medicine, cosmetics, etc.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, in particular to Lactobacillus amyloliquefaciens and its application in skin repair and anti-aging. Background Art

[0002] The skin is the largest organ in the human body, accounting for approximately 16% of body weight. It maintains the body's stability while also serving as the first line of defense against external factors. Studies have shown that abnormalities in genes involved in the skin barrier can lead to skin diseases.

[0003] The skin barrier is a structural barrier formed by the lipids between the epidermal cells of the stratum corneum and the keratin. The skin barrier prevents the body from releasing excess water and prevents harmful substances such as chemicals and microorganisms from entering our bodies. The corneocyte keratinocyte sheath, which forms the surface of dead keratinocytes, plays a key role in stabilizing the lipids between cells. A damaged skin barrier can lead to dry skin, skin aging, skin allergies such as atopic dermatitis, eczema, psoriasis, ichthyosis, and photodermatitis, oily skin such as irritant dermatitis and hormone-dependent dermatitis, and seborrheic disorders such as acne, rosacea, and seborrheic dermatitis.

[0004] Antimicrobial peptides are primarily secreted by epithelial cells lining the mucosa and are expressed in cells of the eyes, skin, oral mucosa, urogenital system, and respiratory system. They have direct effects on most bacteria, certain fungi, and viruses. Their common mode of action is through the interaction of their positive charge with negatively charged components of the bacterial cell membrane (such as lipopolysaccharide), thereby increasing the permeability of the bacterial cell membrane, lysing the bacteria, and causing their death.

[0005] Skin aging includes external aging caused by environmental factors such as air pollution, smoking, malnutrition and ultraviolet (UV) rays, and internal aging caused by time changes. Its typical characteristics are thinning of the skin and the formation of fine lines, which may be caused by reduced cell proliferation and significant changes in the composition of the dermis with aging. Extracellular matrix components (collagen, elastin, glycosaminoglycans, etc.) decrease significantly with skin aging. In addition, with aging, the reactive oxygen species produced by various factors such as mitochondrial damage and inflammatory response increase. At the same time, the age-related cell repair capacity decreases, which increases oxidative stress and makes it impossible to clear aging and damaged cells in time, thus leading to skin aging.

[0006] Tyrosinase, also known as polyphenol oxidase, is widely present in animals, plants, microorganisms, and the human body. Tyrosinase plays an important physiological role in organisms. It is the rate-limiting enzyme in melanin synthesis, directly influencing melanin synthesis and contributing to the development of conditions such as excessive melanin deposition, such as freckles and brown spots. Tyrosinase converts tyrosine into DOPA, which then undergoes a series of steps to ultimately produce melanin. Therefore, inhibiting tyrosinase activity is crucial for skin whitening.

[0007] Probiotics used in cosmetics can fight inflammation and free radicals, significantly inhibiting the proliferation of skin pathogens, balancing the epidermal flora, and repairing the skin barrier. They also inhibit tyrosinase activity, effectively increasing the skin's absorption of nutrients, improving skin whitening, and boosting immunity.

[0008] Sinusitis is a common disease in otorhinolaryngology, primarily caused by respiratory allergies, impaired nasal and sinus mucosal cleansing, respiratory microbial infection, and abnormalities in the local nasal anatomy. Studies have shown that Haemophilus influenzae is a major pathogen of sinusitis, and that Lactobacillus sakei plays a positive role in maintaining normal nasal function. Therefore, reducing the H. influenzae / Lactobacillus sakei bacterial ratio may aid in the treatment of sinusitis. Probiotics can be used as microecological agents to regulate the proportion of respiratory tract flora and maintain long-term balance. Therefore, developing probiotics that maintain respiratory tract microbial balance and exploring the potential properties and utility of these strains is crucial. Summary of the Invention

[0009] In view of this, the technical problem to be solved by the present invention is Lactobacillus amyloliquefaciens and its skin repair and anti-aging application.

[0010] The present invention provides a Lactobacillus amylolyticus with a deposit number of CCTCC NO: M 2022370. The Lactobacillus amylolyticus was isolated from Sichuan pickle pulp and named ProfMIC-212. After 16S sequencing and sequence alignment, the strain was confirmed to be Lactobacillus amylolyticus.

[0011] Furthermore, the present invention provides a bacterial community comprising the Lactobacillus amyloliquefaciens.

[0012] Furthermore, the present invention provides a bacterial agent comprising the Lactobacillus amyloliquefaciens or the bacterial flora of the present invention. The formulation of the bacterial agent includes granules, liquids, and dry powders, which are not limited by the present invention.

[0013] The present invention also provides the use of the Lactobacillus amyloliquefaciens, and / or the bacterial flora, and / or the bacterial agent in preparing a product for improving skin condition.

[0014] Furthermore, the improvement of skin condition described in the present invention includes at least one of repairing the skin barrier, enhancing the antibacterial ability of skin cells, anti-aging, promoting cell proliferation, anti-inflammation, whitening and anti-free radicals, inhibiting skin pathogens, and regulating the proportion of microbial flora.

[0015] In the present invention, the repair of the skin barrier by the Lactobacillus amyloliquefaciens includes but is not limited to repairing epidermal cell damage, improving the viability of epidermal cells or upregulating the expression of barrier repair-related genes. Furthermore, the barrier repair-related genes include at least one of FLG, IVL, OVO1 and LOR. Furthermore, the present invention uses HaCaT cells as the test subject and uses the inactivated supernatant and bacteria of the Lactobacillus amyloliquefaciens to treat the HaCaT cells. The results show that the supernatant and bacteria of the Lactobacillus amyloliquefaciens can upregulate cell barrier repair-related genes and have the effect of promoting cell barrier repair.

[0016] In the present invention, the Lactobacillus amyloliquefaciens enhances 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 are not limited in the present invention. Furthermore, HaCaT cells were used as test subjects, and the cells were treated with the inactivated supernatant and cells of the Lactobacillus amyloliquefaciens. The results showed that both the supernatant and cells of the Lactobacillus amyloliquefaciens could upregulate the expression of the β-defensin genes DEFB1, S100A7, and S100A8 in HaCaT cells, thereby enhancing the antibacterial ability of epidermal cells.

[0017] In the present invention, the Lactobacillus amyloliquefaciens has an anti-aging effect. The anti-aging effect includes any one of the following A) to D):

[0018] A), upregulating the expression of extracellular matrix-related genes and downregulating extracellular matrix degradation-related genes; the extracellular matrix synthesis-related genes include at least one of TIMP1, MKX, SMAD3, LN, COL1A1 and COL13A1; the extracellular matrix degradation-related genes include at least one of P38MAPK and / or MMP family.

[0019] B) Upregulating the expression of BCL-2, a gene related to inhibiting cell apoptosis; downregulating the expression of BAX, a gene related to cell apoptosis, and / or at least one gene in the Caspase family.

[0020] C) Upregulate the expression of MOR, a gene related to cellular immune regulatory factors.

[0021] D) Upregulating the expression of cellular antioxidant-related genes; the antioxidant-related genes include at least one of PTEN, NRF2 and SIRT families.

[0022] In this study, the anti-aging effects of Lactobacillus amyloliquefaciens on HaCaT and HFF cells were investigated, using expression of extracellular matrix-related genes as a metric. The results showed that both the inactivated supernatant and the cells of Lactobacillus amyloliquefaciens upregulated at least one of the extracellular matrix synthesis-related genes TIMP1, MKX, SMAD3, LN, COL1A1, and COL13A1, and downregulated at least one of the extracellular matrix degradation-related genes P38MAPK and / or MMP family members. This suggests that Lactobacillus amyloliquefaciens can promote extracellular matrix synthesis and inhibit extracellular matrix degradation, thereby delaying cellular aging.

[0023] In this study, HFF cells were used as test subjects, and the anti-aging effects of Lactobacillus amyloliquefaciens on cells were investigated, characterized by expression of apoptosis-related genes. The results showed that both the inactivated supernatant and the cells of Lactobacillus amyloliquefaciens upregulated the expression of the apoptosis-inhibiting gene BCL-2 and downregulated the expression of the apoptosis-related gene BAX and / or at least one gene in the caspase family.

[0024] In the present invention, HFF cells were used as test subjects, and the expression of the cell immune regulatory factor-related gene MOR was used as a characterization to study the anti-aging effect of the Lactobacillus amyloliquefaciens on HFF cells. The results showed that the sterilized supernatant of the Lactobacillus amyloliquefaciens can upregulate the expression of the MOR gene in HFF cells, improve the cell immune regulatory ability, and thus play an anti-cell aging role.

[0025] The Lactobacillus amyloliquefaciens of the present invention has the effect of promoting cell proliferation. Further, the cells include but are not limited to HFF cells, HaCaT cells, or other commonly used biological tool cells, which the present invention does not limit. Furthermore, the present invention uses HFF as a test subject to study the effect of the Lactobacillus amyloliquefaciens on cell proliferation. The results show that the sterilized supernatant and bacterial cells of the Lactobacillus amyloliquefaciens can promote the proliferation of HFF cells, indicating that the Lactobacillus amyloliquefaciens can promote cell proliferation.

[0026] The Lactobacillus amyloliquefaciens described in the present invention has the effect of improving cellular anti-inflammatory. The anti-inflammatory includes but is not limited to downregulating the expression of cellular inflammation-related factor genes, and / or reducing the level of cellular NO release. Furthermore, the cellular inflammatory factor-related genes include at least one of IL-8, COX-2, TNF-α or IL-6. Furthermore, the present invention uses HFF cells as test subjects to explore the effects of the sterilized supernatant and bacteria of the Lactobacillus amyloliquefaciens on the expression of HFF cell inflammation-related genes. The results show that the Lactobacillus amyloliquefaciens can upregulate the expression of inflammatory factor-related genes and has the effect of improving the anti-inflammatory ability of cells. Furthermore, the present invention uses Raw264.7 cells as test subjects to explore the effect of the Lactobacillus amyloliquefaciens on the NO production of LPS-stimulated cells. The results show that the sterilized supernatant and precipitate of the Lactobacillus amyloliquefaciens can reduce the NO production of LPS-stimulated cells and increase the anti-inflammatory effect of cells.

[0027] The Lactobacillus amyloliquefaciens strain of the present invention has an anti-free radical effect. Furthermore, the free radicals include, but are not limited to, hydroxyl radicals and / or ABTS free radicals, which are not limited in the present invention. Results indicate that the Lactobacillus amyloliquefaciens strain can scavenge cellular hydroxyl radicals and / or ABTS free radicals.

[0028] The Lactobacillus amyloliquefaciens described in the present invention has a whitening effect. Furthermore, the whitening effect includes any one of reducing cell pigment deposition, inhibiting related pigment deposition-related genes, inhibiting related pigment deposition-related enzyme activity, and increasing skin cell brightness, which is not limited by the present invention. Furthermore, the present invention uses tyrosinase as a test subject to study the inhibitory effect of the Lactobacillus amyloliquefaciens on its activity. The results show that the sterilized supernatant and precipitate of the Lactobacillus amyloliquefaciens can inhibit the activity of tyrosinase, thereby reducing skin pigmentation and achieving a whitening effect.

[0029] The Lactobacillus amyloliquefaciens of the present invention has the effect of inhibiting skin pathogens, which include at least one of Staphylococcus hominis, Staphylococcus haemolyticus and Corynebacterium xeroticum.

[0030] Furthermore, the present invention used Staphylococcus hominis (CGMCC 1.493), Staphylococcus haemolyticus (CGMCC 1.540), and Corynebacterium xerosis (CGMCC 1.1919) as test subjects to study the killing effect of Lactobacillus amyloliquefaciens on skin pathogens. The results showed that Lactobacillus amyloliquefaciens inhibited the growth of Staphylococcus hominis, Staphylococcus haemolyticus, and Corynebacterium xerosis, and had the effect of inhibiting the proliferation of skin pathogens.

[0031] The Lactobacillus amyloliquefaciens described in the present invention has the effect of altering bacterial population proportions to treat sinusitis. Furthermore, regulating bacterial population proportions includes inhibiting the growth of Haemophilus influenzae and / or promoting or not affecting the growth of Lactobacillus sakei. Furthermore, the present invention used Haemophilus influenzae and Lactobacillus sakei as test subjects to study the effect of Lactobacillus amyloliquefaciens on bacterial population proportions. The results showed that Lactobacillus amyloliquefaciens significantly inhibited Haemophilus influenzae but had no inhibitory effect on Lactobacillus sakei, thereby effectively treating sinusitis.

[0032] The present invention provides a product for improving skin condition, the raw materials of which include the Lactobacillus amyloliquefaciens and its culture, lysate and / or extract, and / or the bacterial flora and the bacterial agent of the present invention.

[0033] Furthermore, the products described in the present invention include medicines, which is not limited in the present invention.

[0034] The present invention also provides a method for improving skin condition, which comprises using the product of the present invention. The method of using the product includes applying, external application or injection, which is not limited by the present invention.

[0035] The present invention provides Lactobacillus amylolyticus ProfMIC-212, which has been deposited with the China Center for Type Culture Collection under the accession number CCTCC No. M 2022370. Experiments have shown that ProfMIC-212 has the functions of maintaining and repairing the skin barrier, enhancing the antibacterial capacity of skin cells, resisting aging, promoting cell proliferation, whitening, fighting inflammation and free radicals, inhibiting skin pathogens, and treating sinusitis. It can be used to prepare pharmaceuticals, among other things.

[0036] Biological Deposit Description

[0037] Lactobacillus amylolyticus ProfMIC-212 was deposited in the China Center for Type Culture Collection on April 1, 2022, at Wuhan University, Wuhan, China, with the deposit number being CCTCC NO: M 2022370. DETAILED DESCRIPTION

[0038] The present invention provides Lactobacillus amyloliquefaciens and application thereof, and those skilled in the art can learn from this article content, suitably improve process parameter and realize.It is particularly important to point out that all similar replacements and changes are obvious to those skilled in the art, and they are all considered to be included in the present invention.The method and application of the present invention have been described by preferred embodiment, and relevant personnel obviously can change or suitably change and combine the method and application of this article without departing from the content, spirit and scope of the present invention, to realize and apply the technology of the present invention.

[0039] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0040] The present invention will be further described below in conjunction with the embodiments:

[0041] Example 1 Isolation of ProfMIC-212

[0042] Samples were collected from Sichuan pickle pulp. After appropriate treatment, the samples were shaken and mixed in physiological saline. The supernatant was streaked onto MRS solid plates and incubated at 37°C for 48 hours. White colonies were selected and repeatedly inoculated and screened until a uniform single colony was obtained, designated ProfMIC-212.

[0043] Gram staining microscopy: Strain ProfMIC-212 is Gram-positive and rod-shaped under a microscope; it grows on MRS plates and forms white, smooth, round, opaque small colonies with neat edges; it grows uniformly turbid in MRS liquid culture medium and forms a white precipitate after prolonged exposure.

[0044] Example 2 Nucleic acid identification of ProfMIC-212

[0045] 1. 16S rDNA gene sequence analysis:

[0046] Single colonies were picked and placed in MRS liquid medium. After incubation at 37°C overnight, the cells were centrifuged at 12,000 rpm for 1 minute and the DNA extraction kit was followed. The primers used were bacterial universal primers 27F and 1492R. The PCR amplification system consisted of a 50 μL system with a 95°C initial denaturation for 5 minutes, 35 cycles of 94°C for 15 seconds, 57°C for 15 seconds, and 72°C for 40 seconds, and an extension at 72°C for 10 minutes.

[0047] 2. Results

[0048] The 16S rDNA gene PCR product sequencing sequence was compared with the standard sequence published in GenBank by BLASTN, and it was concluded that the ProfMIC-212 strain was Lactobacillus amylolyticus.

[0049] Example 3 Experiment on ProfMIC-212 Promoting the Expression of Barrier Repair-Related Genes in Human Immortalized Keratinocytes HaCaT

[0050] 1. Preparation of ProfMIC-212 supernatant and inactivated bacteria:

[0051] A single colony of Lactobacillus amyloliquefaciens ProfMIC-212 was picked and placed in MRS liquid medium, incubated at 37°C for 16-18 hours, and detected by microplate reader and diluted with PBS to adjust the OD 600 = 0.2, 121 ° C, 30 min high pressure inactivation, 12000 rpm centrifugation for 2 min, filter through 0.22 μm filter membrane to obtain supernatant. Resuspend the centrifugal precipitate with appropriate amount of PBS and dilute to adjust OD 600 =0.2, which means inactivated bacteria.

[0052] 2. Experiment on promoting HaCaT barrier repair-related gene expression

[0053] Inoculate HaCaT (2 ml / well, containing 5×10 5 Cells) were plated in 6-well plates and cultured overnight at 37°C in a 5% CO2 incubator until the cells adhered. 5% (V / V) supernatant and 10% (V / V) inactivated bacteria were added respectively. After culturing for 24 hours, lysis buffer was added to extract total cellular RNA. The RNA concentration and purity were tested and then reverse transcribed into cDNA. GAPDH was used as the internal reference gene, and real-time qPCR was used to detect the expression of FLG, IVL, OVOL1 and LOR genes. The group treated with equal volume of PBS was used as the control (relative gene expression F=1). 2 -ΔΔCT The F value of each sample was calculated by this method.

[0054] Formula: F = 2 -ΔΔCT ,in:

[0055] △CT 实验 =CT 实验 -CT 内参(实验) ;

[0056] △CT 对照 =CT 对照 -CT 内参(对照) ;

[0057] △△CT=△CT 实验 -△CT 对照 .

[0058] The results are shown in Table 1 and Table 2:

[0059] Table 1. ProfMIC-212 supernatant upregulates expression of HaCaT barrier repair genes

[0060]

[0061] Table 2. ProfMIC-212 inactivated bacteria upregulate the expression of HaCaT barrier repair genes

[0062]

[0063] In vitro cell experiments showed that the supernatant and inactivated bacteria of the Lactobacillus amyloliquefaciens ProfMIC-212 of the present invention have the effect of upregulating the expression of skin barrier repair-related factors such as the filaggrin gene FLG, the involucrin gene IVL, the OVO-like transcription factor 1 gene OVOL1, and the loricrin gene LOR in human immortalized keratinocytes HaCaT, with the relative expression multiples of the genes being 1.26 to 4.25, indicating that ProfMIC-212 has the effect of promoting skin barrier repair.

[0064] Example 4 Experiment on upregulating HaCaT antimicrobial peptide-related gene expression by ProfMIC-212

[0065] 1. Preparation of ProfMIC-212 supernatant and inactivated bacteria:

[0066] The preparation method is as in Example 3.

[0067] 2. Preparation of HaCaT cells

[0068] After digestion, HaCaT cells were diluted with 0.5 ml / well (containing 2×10 5 Cells were seeded into 24-well plates and cultured overnight at 37°C in a 5% carbon dioxide incubator.

[0069] 3. ProfMIC-212 addition and LPS stimulation

[0070] 5% (V / V) of the supernatant and 10% (V / V) of the inactivated bacteria were added to HaCaT cells cultured overnight (the control group used an equal volume of PBS instead of the supernatant). After 2 hours, 0.5 ml of a 0.2 μg / ml LPS solution was added to induce cell inflammation, and the cells were cultured at 37°C in a 5% carbon dioxide incubator for 20 hours.

[0071] 4. qPCR method to detect the relative expression of inflammatory factor genes

[0072] After discarding the culture medium from the above cells, lysis buffer was added to extract total cell RNA, and the RNA concentration and purity were tested before reverse transcription into cDNA. The expression of DEFB1, S100A7 and S100A8 genes was detected by real-time qPCR with GAPDH as the internal reference gene. The group treated with equal volume of PBS was used as the control (relative gene expression F = 1). -ΔΔCT The F value of each sample was calculated by this method.

[0073] The results are shown in Tables 3 and 4:

[0074] Table 3. ProfMIC-212 supernatant upregulates the expression of HaCaT antimicrobial peptide genes

[0075]

[0076] Table 4. ProfMIC-212 inactivated bacteria upregulate the expression of HaCaT antimicrobial peptide genes

[0077]

[0078] In vitro cell experiments showed that the supernatant and inactivated bacteria of the Lactobacillus amyloliquefaciens ProfMIC-212 of the present invention have the effect of upregulating the expression of the antimicrobial peptide β-defensin genes DEFB1, S100A7 and S100A8 in HaCaT cells, with the relative expression multiples of the genes being 1.44 to 2.14, indicating that ProfMIC-212 has the effect of promoting the expression of antimicrobial peptide genes.

[0079] Example 5 Experiment on the regulation of photoaging HaCaT cell extracellular matrix / antioxidant / inflammatory factor related gene expression by ProfMIC-212

[0080] 1. Preparation of ProfMIC-212 supernatant and inactivated bacteria:

[0081] The preparation method is as in Example 4.

[0082] 2. HaCaT cell preparation and UV damage

[0083] After digestion, HaCaT cells were diluted with 0.5 ml / well (containing 2×10 5 Cells were seeded into 24-well plates and cultured overnight in a 5% CO2 incubator at 37°C. The total dose of 2 J / cm 2 UVB radiation damage.

[0084] 3. ProfMIC-212 added

[0085] 5% (V / V) supernatant and 10% (V / V) inactivated bacteria were added to stimulated HaCaT cells (the control group used equal volumes of PBS instead of supernatant / inactivated bacteria), three replicates per group, and cultured at 37°C overnight.

[0086] 4. qPCR detection of relative expression of extracellular matrix / autophagy / antioxidation related genes

[0087] After discarding the culture medium from the above cells, lysis buffer was added to extract total cell RNA, and the RNA concentration and purity were tested before reverse transcription into cDNA. GAPDH was used as the internal reference gene, and real-time qPCR was used to detect the expression of extracellular matrix-related genes TIMP1, COL1A1 and SMAD3, antioxidant-related gene NRF2, and extracellular matrix degradation-related genes P38MAPK and MMP1, and inflammatory factor-related gene TNF-α. The relative expression of the control gene was F = 1, and 2 was used. -ΔΔCT The F value of each sample was calculated by this method.

[0088] The supernatant up-regulated the extracellular matrix-related genes COL1A1 and SMAD3, and the antioxidant-related gene NRF2; and down-regulated the degradation-related gene MMP1 and the inflammatory factor-related gene TNF-α. The results are shown in Table 5:

[0089] Table 5. ProfMIC-212 supernatant regulates the expression of extracellular matrix-related genes

[0090]

[0091] Inactivated bacteria up-regulated the extracellular matrix genes TIMP1, COL1A1 and SMAD3. The results are shown in Table 6:

[0092] Table 6. ProfMIC-212 inactivated bacteria regulates the expression of cellular senescence-related genes

[0093]

[0094] In vitro cell experiments showed that the Lactobacillus amyloliquefaciens ProfMIC-212 of the present invention has the effect of upregulating the expression of the tissue inhibitor of metalloproteinase 1 gene TIMP1, the type I collagen α1 chain gene COL1A1 and the SMAD3 gene, and the antioxidant-related gene nuclear factor E2-related factor 2 gene NRF2, with the relative expression levels increased by 1.17 to 1.81 times. It also has the effect of downregulating the matrix metalloproteinase family gene MMP1, which is involved in extracellular matrix degradation, and the interleukin 6 gene IL-6, which is involved in cellular inflammatory factors, with the relative expression levels of 0.31 to 0.78 times. This shows that ProfMIC-212 has the anti-aging effect of promoting extracellular matrix synthesis in HaCaT cells, increasing antioxidant capacity, and reducing extracellular matrix degradation and inflammatory factors.

[0095] Example 6 ProfMIC-212 promotes the proliferation of human fibroblasts HFF

[0096] 1. Preparation of ProfMIC-212 supernatant and inactivated bacteria:

[0097] The preparation method is as in Example 4.

[0098] 2. HFF cell preparation and ProfMIC-212 addition

[0099] HFF cells cultured in DMEM were digested and diluted with 0.5 ml / well (1.5×10 5 Cells were seeded into 24-well plates and cultured overnight at 37°C in a 5% CO2 incubator. 10% (v / v) supernatant and inactivated cells were added to HFF cells (an equal volume of PBS was added to the control group). Each group was cultured in triplicate at 37°C overnight.

[0100] 3. HFF cell transfer and staining count

[0101] Count the HFF cells in the 24-well plate and dilute them appropriately with 2 ml / well (2.0×10 3 Cells were transferred to 6-well plates, with three replicates per group, and cultured in a 5% CO2 incubator at 37°C for 7–10 days. Cells were fixed with paraformaldehyde and stained with crystal violet, then counted. Cell proliferation rate was calculated according to the formula.

[0102] The calculation formula and results are shown in Table 7 below:

[0103] Table 7. ProfMIC-212 promotes HFF cell proliferation

[0104]

[0105] The results showed that the addition of ProfMIC-212 could promote the proliferation of HFF cells, with the proliferation rate ranging from 8.85% to 106.96%.

[0106] Example 7 Experiment on the regulation of MIC-212 on the expression of genes related to extracellular matrix / apoptosis / antioxidant / immune regulatory factors / inflammatory factors in HFF cells damaged by oxidative stress

[0107] 1. Preparation of ProfMIC-212 supernatant and inactivated bacteria:

[0108] The preparation method is as in Example 4.

[0109] 2. HFF cell preparation and H2O2-induced oxidative damage

[0110] HFF cells cultured in DMEM were digested and then diluted with 0.5 ml / well (containing 2×10 5 Cells were seeded into 24-well plates and cultured overnight at 37°C in a 5% CO2 incubator. H2O2 was added to each well at a final concentration of 200 μM for stimulation and the cells were incubated at 37°C for 1 hour.

[0111] 3. ProfMIC-212 added

[0112] 5% (V / V) supernatant and 10% (V / V) inactivated bacteria were added to stimulated HFF cells (the control group used equal volumes of PBS instead of supernatant / inactivated bacteria), three replicates per group, and cultured at 37°C overnight.

[0113] 4. qPCR detection of relative expression of genes related to extracellular matrix / apoptosis / antioxidation / immune regulatory factors / inflammatory factors

[0114] After discarding the culture medium from the above cells, lysis buffer was added and total RNA was extracted. The RNA concentration and purity were tested and then reverse transcribed into cDNA. GAPDH was used as the internal reference gene. Real-time qPCR was used to detect the expression of extracellular matrix-related genes MKX, COL13A1 and LN, apoptosis-inhibiting genes and BCL-2, antioxidant-related genes PTEN, SIRT-1 and SIRT-3, immune regulatory factor-related gene MOR; as well as extracellular matrix degradation-related genes MMP family and P38MAPK, apoptosis-related genes Caspase family and BAX, and inflammatory factor-related gene IL-6. The relative expression of genes in the control group was calculated as F = 1, and 2 was used. -ΔΔCT The F value of each sample was calculated by this method.

[0115] The supernatant up-regulated the extracellular matrix-related genes MKX and COL13A1, inhibited apoptosis gene BCL-2, antioxidant-related genes PTEN, SIRT-1 and SIRT-3, and immune regulatory factor-related gene MOR; and down-regulated the degradation of extracellular matrix-related genes MMP family, apoptosis-related genes BAX and Caspase family, and inflammatory factor-related gene IL-6. The results are shown in Table 8:

[0116] Table 8. ProfMIC-212 supernatant regulates the expression of genes related to extracellular matrix / apoptosis / antioxidation / immunomodulatory factors / inflammatory factors

[0117]

[0118]

[0119] The inactivated bacteria up-regulated the inhibition of extracellular matrix-related genes LN and MKX, and the antioxidant-related gene SIRT-1; down-regulated the degradation of extracellular matrix-related genes MMP family and P38MAPK, and the apoptosis-related gene BAX. The results are shown in Table 9:

[0120] Table 9. ProfMIC-212 inactivated bacteria regulates the expression of genes related to degradation of extracellular matrix / antioxidation / apoptosis

[0121]

[0122] The results showed that ProfMIC-212 has anti-aging effects by promoting HFF extracellular matrix synthesis, reducing cell apoptosis, increasing antioxidant capacity, increasing cellular immune regulatory factors, reducing extracellular matrix degradation, and reducing inflammatory factors.

[0123] Example 8 ProfMIC-212 inhibits tyrosinase activity

[0124] 1. Preparation of ProfMIC-212 supernatant:

[0125] The preparation method is as in Example 3.

[0126] 2. Tyrosinase activity inhibition rate detection

[0127] Prepare 250 U / ml tyrosinase, 6.0 mM L-DOPA, and 80 mM PBS. Add 50 μl of PBS, 20 μl of supernatant, 50 μl of tyrosinase, and 50 μl of L-DOPA to each well of a 96-well plate. For the control group, replace the supernatant with MRS medium. Mix thoroughly and incubate at room temperature in the dark for 15 minutes. Measure absorbance (A) at 475 nm to calculate the tyrosinase inhibition rate.

[0128] The calculation formula and results are shown in Table 10:

[0129] Table 10. ProfMIC-212 supernatant inhibits tyrosinase activity

[0130]

[0131] The results showed that the supernatant of ProfMIC-212 had the effect of inhibiting tyrosinase activity, with an inhibition rate of 50.77% to 56.92%.

[0132] Example 9 ProfMIC-212 reduces NO production in Raw264.7 cells

[0133] 1. Preparation of ProfMIC-212 supernatant and inactivated bacteria:

[0134] The preparation method is as in Example 3.

[0135] 2. Raw264.7 Cell Preparation

[0136] Raw264.7 cells were digested and then diluted with 0.5 ml / well (containing 2×10 5 Cells were seeded into 24-well plates and cultured overnight at 37°C in a 5% carbon dioxide incubator.

[0137] 3. ProfMIC-212 addition and LPS stimulation

[0138] 5% (V / V) supernatant and 10% (V / V) inactivated bacteria were added to Raw264.7 cells cultured overnight in different groups (the control group used equal volumes of PBS instead of supernatant / inactivated bacteria, respectively). After 2 hours, 0.5 ml of 0.2 μg / ml LPS solution was added to induce inflammation of the Raw264.7 cells. After 20 hours, the cell culture supernatant was collected, and the standard curve was drawn according to the method described in the Biyuntian NO detection kit to calculate the NO concentration and inhibition rate in the sample.

[0139] The calculation formula and results are shown in Table 11 below:

[0140] Table 11. ProfMIC-212 reduces NO production in Raw264.7 cells

[0141]

[0142]

[0143] The results showed that ProfMIC-212 has the effect of reducing the release of inflammatory factors, that is, it has an anti-inflammatory effect and can reduce the NO production in Raw264.7 cells induced by LPS, which is reduced by 31.83% to 58.48% compared with the LPS control group.

[0144] Example 10 ProfMIC-212 downregulates the expression of inflammatory factor-related genes in HaCaT cells

[0145] 1. Preparation of ProfMIC-212 supernatant:

[0146] The preparation method is as in Example 3.

[0147] 2. Preparation of HaCaT cells

[0148] After digestion, HaCaT cells were diluted with 0.5 ml / well (containing 2×10 5 Cells were seeded into 24-well plates and cultured overnight at 37°C in a 5% carbon dioxide incubator.

[0149] 3. ProfMIC-212 addition and LPS stimulation

[0150] 5% (V / V) of the supernatant was added to HaCaT cells cultured overnight (an equal volume of PBS was used instead of supernatant in the control group). After 2 h, 0.5 ml of 0.2 μg / ml LPS solution was added to induce cell inflammation, and the cells were cultured at 37°C in a 5% carbon dioxide incubator for 20 h.

[0151] 4. qPCR method to detect the relative expression of inflammatory factor genes

[0152] After discarding the culture medium from the above cells, lysis buffer was added to extract total RNA from the cells. After testing the RNA concentration and purity, reverse transcription was performed into cDNA. GAPDH was used as the internal reference gene, and real-time qPCR was used to detect the expression of IL-8 and COX-2 genes. The group treated with equal volume of PBS was used as the control (relative gene expression F = 1). -ΔΔCT The F value of each sample was calculated by this method.

[0153] The results are shown in Table 12:

[0154] Table 12. ProfMIC-212 supernatant down-regulates the expression of inflammatory factor-related genes

[0155]

[0156] The results showed that ProfMIC-212 could downregulate the expression of genes related to inflammatory factors in HaCaT cells induced by LPS, and had an anti-inflammatory effect, with the relative gene expression fold increase ranging from 0.32 to 0.83.

[0157] Example 11: Effect of ProfMIC-212 in scavenging free radicals

[0158] 1. Preparation of ProfMIC-212 supernatant:

[0159] A single colony of Lactobacillus amyloliquefaciens ProfMIC-212 was picked and placed in MRS liquid medium, cultured in a 37°C incubator for 16-18 hours, and detected by a microplate reader and diluted with MRS liquid medium to adjust the OD 600 = 2.0, 121 ° C, high pressure inactivation for 30 min, centrifugation at 12000 rpm for 2 min, and the supernatant was filtered through a 0.22 μm filter membrane to obtain the supernatant.

[0160] 2. Detection of hydroxyl radical scavenging ability of ProfMIC-212 supernatant

[0161] Reagent preparation and detection methods were performed according to the instructions for the Solebol Hydroxyl Radical Scavenging Ability Test Kit. The absorbance of each sample at 536 nm was measured, the average was calculated, and the scavenging rate of each sample was calculated.

[0162] The calculation formula and results are shown in Table 13:

[0163] Table 13. ProfMIC-212 hydroxyl radical scavenging rate (%)

[0164]

[0165] 3. ABTS free radical scavenging ability test of ProfMIC-212 supernatant

[0166] Reagent preparation and detection methods were performed according to the instructions for the Solebol ABTS Free Radical Scavenging Ability Test Kit. The absorbance of each sample at 405 nm was measured, and the average was calculated to calculate the scavenging rate of each sample.

[0167] The calculation formula and results are shown in Table 14:

[0168] Table 14. ProfMIC-212ABTS free radical scavenging rate (%)

[0169]

[0170] The results showed that ProfMIC-212 had the effect of scavenging hydroxyl radicals and ABTS free radicals, with a free radical scavenging rate of 25.93% to 32.94%.

[0171] Example 12: Effect of ProfMIC-212 on Inhibition of Skin Pathogens

[0172] 1. Preparation of ProfMIC-212 supernatant:

[0173] The preparation method is similar to that in Example 11.

[0174] 2. Preparation of pathogenic bacteria solution:

[0175] Single colonies of three pathogens, Staphylococcus hominis (CGMCC 1.493), Staphylococcus haemolyticus (CGMCC 1.540), and Corynebacterium xerosis (CGMCC 1.1919), were picked and placed in 1 ml of BHI liquid medium, cultured in a 37°C incubator overnight, and tested and diluted with BHI liquid medium to adjust the OD 600 =0.2.

[0176] 3. Pathogen inhibition experiment

[0177] The inactivated supernatant was added to the pathogenic bacteria solution at a volume of 10% (V / V), and an equal volume of MRS liquid medium was added as a control. The culture was cultured at 37°C for 3 h, and the concentration of the bacterial solution (OD 600 ) and calculated the pathogen inhibition rate.

[0178] The calculation formula and results are shown in Table 15:

[0179] Table 15. Inhibitory effect of ProfMIC-212 on skin pathogens

[0180]

[0181] The results showed that ProfMIC-212 had the effect of inhibiting the proliferation of skin pathogens, with an inhibition rate of 37.84% to 45.24%.

[0182] Example 13 Effect of ProfMIC-212 on Changing the Proportion of Microflora in Treating Sinusitis

[0183] 1. Preparation of ProfMIC-212 supernatant:

[0184] The preparation method is similar to that in Example 11.

[0185] 2. Preparation of Sinusitis-related Bacteria Fluid:

[0186] Single colonies of Haemophilus influenzae ATCC 49766 were picked and placed in BHI liquid medium, and single colonies of Lactobacillus sakei were picked and placed in MRS liquid medium. The cells were cultured in a 37°C incubator overnight, and the OD values were adjusted by using a microplate reader to detect the cells. 600 =0.2.

[0187] 3. Experiment on the effect of adding supernatant on the growth of sinusitis-related bacteria

[0188] The supernatant was added to two sinusitis-related bacterial cultures at a 10% (V / V) addition rate, and an equal volume of MRS liquid culture medium was added as a control. The cultures were cultured at 37°C for 16 h. The relative concentrations of the two bacterial cultures (OD 600 ) ratio to evaluate the effect on the growth of sinusitis-related bacteria. The calculation formula and results are shown in Table 16:

[0189] Table 16. Effect of ProfMIC-212 on the growth of sinusitis-related bacteria

[0190]

[0191] The results showed that ProfMIC-212 had a significant inhibitory effect on Haemophilus influenzae, but no inhibitory effect on Lactobacillus sakei. ProfMIC-212 can alter the bacterial flora, thereby effectively treating sinusitis.

[0192] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Lactobacillus amylolyticus with a deposit number of CCTCC NO: M 2022370.

2. A bacterial population comprising the Lactobacillus amylolyticus according to claim 1.

3. A bacterial agent comprising the Lactobacillus amylolyticus according to claim 1 or the bacterial flora according to claim 2.

4. Use of the Lactobacillus amyloliquefaciens according to claim 1, and / or the bacterial flora according to claim 2, and / or the bacterial agent according to claim 3 in the preparation of a medicament for improving skin condition; The improvement of skin condition is at least one of repairing the skin barrier, anti-aging, anti-inflammation, and inhibiting skin pathogens; The method of inhibiting skin pathogens comprises inhibiting at least one of Staphylococcus aureus, Staphylococcus hemolyticus and Corynebacterium shrivelensis.

5. A medicine for improving skin condition, characterized in that: The raw material of the medicine is the inactivated bacteria of Lactobacillus amyloliquefaciens according to claim 1; The preparation of the inactivated bacteria of Lactobacillus amyloliquefaciens comprises: picking a single colony of the Lactobacillus amyloliquefaciens described in claim 1 and placing it in an MRS liquid culture medium, culturing it in a 37°C incubator for 16-18 hours, diluting it with PBS to adjust the OD600 to 0.2, inactivating it under high pressure at 121°C for 30 minutes, centrifuging it at 12,000 rpm for 2 minutes, and filtering it through a 0.22 μm filter membrane to obtain a supernatant; and resuspending the centrifugal precipitate with an appropriate amount of PBS, diluting it to adjust the OD600 to 0.2, and obtaining an inactivated bacteria.

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