CN115044519A - Lactobacillus amyloliquefaciens and application thereof - Google Patents

Lactobacillus amyloliquefaciens and application thereof Download PDF

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CN115044519A
CN115044519A CN202210965527.2A CN202210965527A CN115044519A CN 115044519 A CN115044519 A CN 115044519A CN 202210965527 A CN202210965527 A CN 202210965527A CN 115044519 A CN115044519 A CN 115044519A
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skin
lactobacillus
cells
amyloliquefaciens
seuneu
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靖培培
孙夏慧
陈奕兴
王熠
李霄
郭青青
张玉
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Shandong Jinli Bioengineering Co ltd
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Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus amyloliquefaciens and application thereof. The invention relates to a Lactobacillus amyloliquefaciens (A)Lactobacillus amylovorus) Stored in China center for type culture Collection with the preservation number of CCTCC NO: M2022368. Experiments show that the invention providesThe provided lactobacillus amyloliquefaciens has the functions of maintaining and repairing skin barriers, moisturizing, resisting aging, promoting cell proliferation, resisting inflammation, inhibiting skin pathogenic bacteria and improving sensitive muscles, and can be used for preparing food, medicines, cosmetics and other fields.

Description

Lactobacillus amyloliquefaciens and application thereof
Technical Field
The invention relates to the technical field of microorganisms, in particular to lactobacillus amyloliquefaciens 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, solar dermatitis, skin sensitivity, irritant dermatitis, hormone dependent dermatitis, 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, and dead cells without the cell nucleus and the organelles are formed in the stratum corneum. The stratum corneum usually contains 10-30% of water due to the hydrophilic and barrier functions of the stratum corneum and the effects 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 the 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.
The probiotics is used in cosmetics, and can obviously inhibit the proliferation of skin pathogenic bacteria, balance skin epidermal flora and repair skin barriers. Meanwhile, the expression of the moisture-keeping, anti-aging and anti-inflammatory genes is up-regulated, the absorption of the skin to nutrient substances is effectively increased, and the immunity of the skin is enhanced.
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 functions of the skin are too weak to resist external stimulation, so that the phenomena of redness, fever, pruritus, stabbing pain and the like are prone to being generated. While an impaired skin barrier may further lead to Staphylococcus aureus (S.aureus) ((R))Staphylococcus aureus) The colonization of (A) and (B) causes inflammation and red swelling. Staphylococcus epidermidis (resident in the skin: (A)Staphylococcus epidermidis) Antagonizes Staphylococcus aureus and reduces the proliferation of the latter. Thus reducing the flora ratio of staphylococcus aureus/staphylococcus epidermidis and helping to establish a balanced distribution of skin flora and thus improve sensitive muscles. Probiotic as a microecological preparation can be used for balancing skin flora and repairing skinThe skin barrier improves the skin state, so the probiotic related product developed by utilizing the microecology technology has important practical significance.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide lactobacillus amyloliquefaciens and applications thereof.
The invention provides a lactobacillus amyloliquefaciens (A)Lactobacillus amylovorus ) The preservation number is CCTCC NO: M2022368.
The lactobacillus amyloliquefaciens is separated from pickled vegetable pulp water, and is identified as lactobacillus amyloliquefaciens (lactobacillus amyloliquefaciens) through 16S rDNA gene sequence analysis and NCBI database comparisonLactobacillus amylovorus ）。
The invention provides a flora comprising the lactobacillus amyloliquefaciens.
Furthermore, the flora of the present invention includes a flora formed by one or more bacteria that do not have a mutual antagonistic or competitive relationship with the lactobacillus amyloliquefaciens, and the present invention is not limited thereto.
The invention provides a microbial inoculum, the raw materials of which comprise the lactobacillus amyloliquefaciens and/or the flora.
Furthermore, the preparation type of the microbial inoculum can be powder, granule or liquid, which is not limited in the invention.
Furthermore, the microbial inoculum of the present invention further includes a fermentation broth, a thallus, a supernatant and active substances contained therein of the strain, which is not limited in the present invention.
The invention provides an application of any one of the following I) -III) in preparing a product for improving skin state;
I) the lactobacillus amyloliquefaciens or lysate, extract and metabolite thereof;
II) the bacterial flora according to the invention;
III) the microbial inoculum.
In the invention, the improvement of the skin state comprises at least one of repairing skin barrier, moisturizing, resisting aging, promoting cell proliferation, resisting inflammation, inhibiting skin pathogenic bacteria or regulating the proportion of skin microbial flora.
In the invention, the lactobacillus amyloliquefaciens has the function of repairing skin barriers. The repairing skin barrier comprises at least one of repairing chemical damage, repairing oxidative damage or repairing light damage, and/or up-regulating barrier repair related genes.
In the present invention, the chemical injury includes any chemical substance that can cause mild, moderate or severe epidermal cell injury to a human body, and the present invention is not limited thereto. Furthermore, the HaCaT cell subject is used for researching the repair effect of the lactobacillus amyloliquefaciens on the HaCaT cell chemical damage caused by SDS (sodium dodecyl sulfate), and the result shows that the lactobacillus amyloliquefaciens can relieve the chemical damage caused by the SDS and improve the cell survival rate, and the cell survival rate is 109.57-124.19%; the lactobacillus amyloliquefaciens has the function of promoting epidermal cell repair.
In the present invention, the oxidative damage includes peroxide or active oxygen damage, which is not limited by the present invention. The repair of oxidative damage includes, but is not limited to, increasing the expression of antioxidant-related genes. The antioxidant related gene comprisesPTEN、SIRT-1。The invention takes HFF cells as subjects to study the pair H of the lactobacillus amyloliquefaciens 2 O 2 The repair effect of the HFF cell oxidative damage is caused, and the result shows that the lactobacillus amyloliquefaciens can up-regulate H 2 O 2 Induced antioxidant related gene of HFF cellPTEN、SIRT-1Thereby playing the role of antioxidation and achieving the aim of enhancing the repair capability of cell oxidative damage.
Further, the repair of the skin barrier according to the present invention includes, but is not limited to, up-regulating the expression of a skin barrier repair-related gene. According to the invention, HaCaT cells are taken as a subject, the influence of the lactobacillus amyloliquefaciens on the expression of HaCaT cell skin barrier repair related genes is researched, and the result shows that the lactobacillus amyloliquefaciens can improve the HaCaT cell skin barrier repair related genesFLG、IVL、OVO1AndLORthe expression in (2) indicates that the lactobacillus amyloliquefaciens has the function of promoting epidermal cell repair.
In the invention, the lactobacillus amyloliquefaciens has the function of moisturizing skin. The moisturizing includes but is not limited to up-regulation of moisturizing related genes.
Further, the genes related to moisture retention include, but are not limited toAQP3、GBA. The invention takes HaCaT cells as a subject and takes aquaporin 3 geneAQP3And glucocerebrosidase geneGBAIs characterized by determining the HaCaT cells of said Lactobacillus amyloliquefaciensAQP3AndGBAthe expression effect shows that the lactobacillus amyloliquefaciens has up-regulationAQP3AndGBAthe expression effect is that the relative expression multiple of the gene is 1.22-6.85, which shows that the lactobacillus amyloliquefaciens can promote skin moisture retention。
In the invention, the lactobacillus amyloliquefaciens has the function of resisting cell aging. The anti-aging comprises at least one of up-regulation of photoaging extracellular matrix synthesis related genes, down-regulation of photoaging extracellular matrix degradation related genes, reduction of apoptosis, increase of cellular immune regulatory factors, increase of cellular beta-endorphin production or reduction of cellular protein carbonyl content.
In the invention, the extracellular matrix synthesis related gene and the extracellular matrix degradation related gene both belong to extracellular matrix related genes. Further, the extracellular matrix synthesis related gene includesLN、MKX、COL1A1、COL13A1、TIMP1AndSMAD3(ii) a The extracellular matrix degradation related gene comprisesMMPOf familyMMP1、MMP2、MMP3、MMP7、MMP8. According to the invention, HaCaT cells and HFF cells are taken as subjects, the influence of the lactobacillus amyloliquefaciens on the expression of HaCaT cells and HFF extracellular matrix related genes is researched, and tests show that the lactobacillus amyloliquefaciens can up-regulate extracellular matrix synthesis genes and down-regulate extracellular matrix degradation genes, and has the function of resisting cell aging.
In the present invention, said reduction of apoptosis comprises reduction of cells caused by photoagingApoptosis, stimulus-induced apoptosis, or damage-induced apoptosis, which is not a limitation of the present invention. Further, the apoptosis-related gene of the present invention includes, but is not limited toBCL-2、Caspase-3、Caspase-9、BAXWhereinBCL-2Is a gene related to the anti-apoptosis of the cell,Caspase-3、Caspase-9 、BAXis a cell apoptosis related gene. Further, the invention takes HaCaT cells and HFF cells as the subjects, researches the influence of the lactobacillus amyloliquefaciens on apoptosis, and shows that: the lactobacillus amyloliquefaciens can reduce the expression of apoptosis related genes and inhibit apoptosis, thereby playing the role of anti-aging.
In the present invention, the genes related to cellular immune regulatory factors include, but are not limited toMORThe present invention is not limited thereto. Further, the present invention uses HFF cells as subjects to treatMORThe gene expression is characterized, the influence of the lactobacillus amyloliquefaciens on the cellular immune regulatory factor is researched, and the result shows that the lactobacillus amyloliquefaciens can up-regulate the related gene of the immune regulatory factor, thereby playing the role of anti-aging.
In the invention, the cells secrete beta-endorphin, and the effects of resisting aging and delaying sexual aging can be achieved. Furthermore, the invention takes HFF cells as a subject, and researches the influence of the lactobacillus amyloliquefaciens on the secretion of the beta-endorphin of the HFF cells, and the results show that the lactobacillus amyloliquefaciens can improve the secretion amount of the beta-endorphin of the cells and has the effects of resisting cell senescence or delaying senescence.
In the present invention, the content of cellular protein carbonyl is an important index of cell aging. Furthermore, the invention takes the HFF cells as the subjects, and researches the influence of the lactobacillus amyloliquefaciens on the content of the protein carbonyl of the HFF cells, and the results show that the lactobacillus amyloliquefaciens can reduce the content of the protein carbonyl of the HFF cells, thereby achieving the aim of resisting cell aging.
In the invention, the lactobacillus amyloliquefaciens has the function of promoting cell proliferation. Further, the cell proliferation includes physiological phenomena in a biological sense such as cell division, development, and propagation, which are within the scope of the definition of cell proliferation, and the present invention is not limited thereto. Furthermore, in some specific embodiments, the human fibroblast HFF is used as the subject, and the effect of lactobacillus amyloliquefaciens on HFF cell proliferation is studied, so that lactobacillus amyloliquefaciens can promote HFF proliferation with a proliferation promoting rate of 26.36-45.74%.
In the invention, the lactobacillus amyloliquefaciens has an anti-inflammatory effect. The anti-inflammatory includes reducing cellular NO production or down-regulating inflammatory factor related genesIL-8、TRPV1、IL-6AndIL-22at least one of (1).
In the invention, cell Nitric Oxide (NO) has double characteristics of promoting inflammation and resisting inflammation, endothelial source NO has the function of inhibiting inflammatory reaction under normal condition, and a large amount of NO synthesized by inducible NO synthetase has cytotoxic effect under pathological condition, thus aggravating inflammatory reaction; the expression of genes related to inflammatory factors, including but not limited to, genes related to inflammatory factors, is also closely related to cell anti-inflammationIL-8、TRPV1、IL-6OrIL-22. Furthermore, the invention takes the cell Raw264.7 as a subject, and researches the influence of the lactobacillus amyloliquefaciens on the NO production amount of the Raw264.7 cell and the expression of the genes related to the inflammatory factors, and the results show that the lactobacillus amyloliquefaciens can reduce the NO production amount of the Raw264.7 cell and down regulate the expression of the genes related to the inflammatory factors, thereby having the anti-inflammatory effect.
In the present invention, the anti-inflammatory agents include, but are not limited to, skin sensitivity, irritant dermatitis, and hormone-dependent dermatitis, such as anti-pigmentation atopic dermatitis and solar dermatitis, and the present invention is not limited thereto.
In the invention, the lactobacillus amyloliquefaciens has the effect of inhibiting skin pathogenic bacteria. Human staphylococcus, hemolytic staphylococcus and corynebacterium crenatum are main pathogenic bacteria of skin, and the fixed values of the human staphylococcus, hemolytic staphylococcus and corynebacterium crenatum can destroy skin cutin to a certain extent and increase skin sensitivity. The invention uses staphylococcus hominis (Staphylococcus hominisCGMCC 1.493), and Staphylococcus hemolyticus (Staphylococcus haemolyticusCGMCC 1.540), and corynebacterium crenatum (Corynebacterium xerosis CGMCC 1.1919) is a subject, and the research on the capability of the lactobacillus amyloliquefaciens to inhibit skin pathogenic bacteria shows that the lactobacillus amyloliquefaciens can inhibit the growth of the skin pathogenic bacteria and achieve the aim of protecting the health of the skin.
In the invention, the lactobacillus amyloliquefaciens has the function of regulating the microbial flora of skin. Staphylococcus aureus and Staphylococcus epidermidis are main skin colonization flora, the proportion of the staphylococcus aureus and the Staphylococcus epidermidis affects the health of the skin flora, and the proportion of the staphylococcus aureus is reduced due to the construction of good skin flora. The invention is provided with
Staphylococcus aureus golden yellow subspecies (Staphylococcus aureus subsp.aureusCGMCC 1.8721 and Staphylococcus epidermidis ((S. epidermidis))Staphylococcus epidermidisCGMCC 1.4260) is a tested object, the regulation effect of the lactobacillus amyloliquefaciens on skin flora is researched, and the result shows that the lactobacillus amyloliquefaciens can reduce the proportion of staphylococcus aureus in the skin flora to achieve the aim of maintaining healthy skin flora.
The invention provides a product for improving skin condition, which comprises any one of the following items I) to III):
I) the lactobacillus amyloliquefaciens or lysate, extract and metabolite thereof;
II) the bacterial flora according to the invention;
III) the microbial inoculum provided by the invention.
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, granules, etc., 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 obtains the following beneficial effects:
the present invention provides a method for understanding Lactobacillus amylovorusLactobacillus amylovorus ) Which isStored in China center for type culture Collection with the preservation number of CCTCC NO: M2022368. Experiments show that the lactobacillus amyloliquefaciens SEUNEU-111 has the functions of maintaining and repairing skin barrier, moisturizing, resisting aging, promoting cell proliferation, resisting inflammation, inhibiting skin pathogenic bacteria and improving sensitive muscles, and can be used for preparing the fields of food, medicines, cosmetics and the like.
Biological preservation Instructions
Lactobacillus amyloliquefaciens SEUNEU-111 (Lactobacillus amylovorusSEUNEU-111) deposited at the China center for type culture Collection on 1/4/2022 at the address: china, Wuhan and Wuhan university, the preservation number is CCTCC NO: M2022368.
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 SEUNEU-111
Sampling in pickled Chinese cabbage slurry water. Properly processing the sample, uniformly mixing the processed 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 selecting a white colony for repeated inoculation and screening until a uniform single colony is obtained, wherein the uniform single colony is named as SEUNEU-111.
Gram staining microscopy: the bacterial strain SEUNEU-111 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 SEUNEU-111
1. 16S rDNA gene sequence analysis:
selecting a single colony to be placed 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 SEUNEU-111 strain is lactobacillus amyloliquefaciens (BLASTN) obtained by homology comparison of the sequencing result of the PCR product and a published standard sequence in GenBankLactobacillus amylovorus）。
Example 3 SEUNEU-111 promotion of SDS-induced HaCaT Damage repair assay in human immortalized keratinocytes
1. Preparation of SEUNEU-111 supernatant and inactivated thallus:
selecting a single colony of lactobacillus amyloliquefaciens SEUNEU-111 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 cell repair
Inoculation of HaCaT cells (5X 10) 4 cells/well) to 96-well plates and cultured overnight until cells adhere. SDS was prepared at 50. mu.g/ml, and 100. mu.l of SDS was added to each well, and the mixture was incubated at 37 ℃ for 8 hours in a 5% carbon dioxide incubator. 5% (V/V) of the supernatant and 10% (V/V) of the inactivated bacteria were added to each well (in the control group, the supernatant/inactivated bacteria were replaced with PBS of the same volume, respectively) and cultured for 24 hours. Mu.l of CCK-8 solution was added to each well, incubated for 4h, and the absorbance A at 450nm was measured. The results are shown in table 1:
Figure 945095DEST_PATH_IMAGE001
the SEUNEU-111 supernatant and the inactivated thallus have a repairing effect on SDS damage of HaCaT cells, and the SEUNEU-111 supernatant and the inactivated thallus are added into human immortalized keratinocyte HaCaT damaged by Sodium Dodecyl Sulfate (SDS), so that the cell survival rate is increased compared with a control, and is 109.57-124.19%. The experimental results show that the lactobacillus amyloliquefaciens SEUNEU-111 has the effect of promoting epidermal cell repair.
Example 4 experiment of promoting HaCaT Barrier repair related Gene expression by SEUNEU-111
1. Preparation of SEUNEU-111 supernatant and inactivated thallus:
the preparation method refers to example 3.
2. Experiment for promoting HaCaT barrier repair related gene expression
Inoculation of HaCaT cells (2 ml/well, 5X 10 content) 5 Cells) to 6-well plate, 5% carbon dioxide incubator 37 ℃ overnight until cells adhere. Respectively adding supernatant 5% (V/V) and inactivated thallus 10% (V/V), culturing for 24 hr, adding lysate, extracting total RNA from cells, detecting RNA concentration and purity, and reverse transcribing to cDNA to obtainGAPDHFor reference gene, real-time qPCR detection is adoptedFLG, IVL, OVOL1 and LORExpression of the gene. The group treated with the supernatant/inactivated cells without SEUNEU-111 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 formula: f =2 -ΔΔCT Wherein:
△CT experiment of =CT Experiment of -CT Internal reference (experiment);
△CT control =CT Control -CT Internal control (control);
△△CT=△CT experiment of -△CT And (6) comparison.
The results are shown in tables 2 and 3:
Figure 393394DEST_PATH_IMAGE002
Figure 969869DEST_PATH_IMAGE003
in vitro cell experiments show that the supernatant and the inactivated thallus of the lactobacillus amyloliquefaciens SEUNEU-111 have the function of up-regulating the filaggrin gene of a relevant factor for skin barrier repairFLGInvolucrin geneIVLOVO-like transcription factor 1 geneOVOL1And loricrine protein geneLORThe relative expression multiple of the gene is 1.55-4.33. The above results show that SEUNEU-111 has the effect of promoting skin barrier repair.
Example 5 SEUNEU-111 experiments to upregulate HaCaT moisturizing-related Gene expression
1. Preparation of SEUNEU-111 supernatant and inactivated thallus:
the preparation method is referred 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 a 6-well plate, and cultured overnight at 37 ℃ in a 5% carbon dioxide incubator until the cells adhere to the wall. Adding supernatant 5% (V/V), inactivating thallus 10% (V/V), culturing for 24 hr, adding lysate, extracting total RNA from cells, detecting RNA concentration and purity, and reverse transcribing to cDNA to obtainGAPDHFor reference gene, real-time qPCR detection is adoptedAQP3AndGBAexpression of the gene. The group treated with the supernatant/inactivated cells without SEUNEU-111 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 tables 4 and 5:
Figure 161816DEST_PATH_IMAGE004
Figure 772926DEST_PATH_IMAGE005
in vitro cell experiments show that the supernatant and the inactivated thallus of the lactobacillus amyloliquefaciens SEUNEU-111 have aquaporin 3 genes related to up-regulation and moisture retentionAQP3And glucocerebrosidase geneGBAExpression ofThe relative expression multiple of the gene is 1.22-6.85. The above results show that SEUNEU-111 has the effect of promoting skin moisturization.
Example 6 SEUNEU-111 experiments to modulate photoaging HaCaT extracellular matrix/inflammatory factor-related Gene expression
1. Preparation of SEUNEU-111 supernatant and inactivated thallus
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 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. SEUNEU-111 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 culture medium from the above cells, adding lysis solution, extracting total RNA from cells, detecting RNA concentration and purity, and reverse transcribing into cDNAGAPDHDetecting related genes of extracellular matrix by adopting real-time qPCR (quantitative polymerase chain reaction) as internal reference genesCOL1A1、TIMP1AndSMAD3and degrading extracellular matrix-related genesMMP1，Genes related to inflammatory factorsTNF-αExpression of (2). Relative expression multiple F =1 of control group gene, using 2 -ΔΔCT The F value of each sample was calculated.
Supernatant up-regulation of extracellular matrix-related genesCOL1A1AndTIMP1(ii) a Down-regulation of extracellular matrix-degrading related genesMMP1，Genes related to inflammatory factorsTNF-α. The results are shown in Table 6:
Figure 392126DEST_PATH_IMAGE006
inactivation of bacteria up-regulation of extracellular matrix genesCOL1A1、SMAD3AndTIMP1(ii) a Down-regulation of inflammatory factor-related genesTNF-α. The results are shown in Table 7.
Figure 452967DEST_PATH_IMAGE007
In vitro cell experiments show that the lactobacillus amyloliquefaciens SEUNEU-111 has the function of up-regulating tissue metalloproteinase inhibitor 1 gene related to HaCaT extracellular matrixTIMP1Type I collagen alpha 1 chain geneCOL1A1AndSMAD3the relative expression multiple of the gene is 1.23-1.84 under the action of gene expression; has down-regulation of matrix metalloprotease family gene associated with degradation of extracellular matrixMMP1And tumor necrosis factor alpha gene related to cytokineTNF-αThe relative expression multiple of the gene is 0.36-0.82. The results show that SEUNEU-111 has the anti-aging effects of promoting synthesis of HaCaT extracellular matrix, reducing degradation of extracellular matrix and reducing inflammatory factors.
Example 7 proliferation of human fibroblast HFF by SEUNEU-111
1. Preparation of SEUNEU-111 supernatant and inactivated thallus:
the preparation method refers to example 3.
2. HFF cell preparation and SEUNEU-111 addition
HFF cells cultured with DMEM were digested at 0.5 ml/well (1.5X 10 per well) 5 Cells) were inoculated into 24-well plates and cultured overnight at 37 ℃ in a 5% carbon dioxide incubator. The supernatant and the inactivated cells were added to HFF cells at 10% (V/V) (control group replaced supernatant/inactivated cells with 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 table 8:
Figure 183026DEST_PATH_IMAGE008
in vitro cell experiments show that the lactobacillus amyloliquefaciens SEUNEU-111 has the effect of promoting the proliferation of human fibroblast HFF, and the proliferation rate is 26.36-45.74%. Shows that SEUNEU-111 has the function of promoting the proliferation of HFF cells.
Example 8 SEUNEU-111 Regulation of oxidative damage to HFF extracellular matrix/apoptosis/antioxidant/immunoregulatory/inflammatory factor-related Gene expression assays
1. Preparation of SEUNEU-111 supernatant and inactivated thallus:
the preparation method refers to example 3.
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 1 h.
3. SEUNEU-111 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 culture medium from the above cells, adding lysis solution, extracting total RNA from the cells, detecting RNA concentration and purity, and reverse transcribing to cDNA to obtainGAPDHDetecting related genes of extracellular matrix by adopting real-time qPCR (quantitative polymerase chain reaction) as internal reference genesLN、MKXAndCOL13A1apoptosis-related genesBCL-2Antioxidant-related genesPTENAndSIRT-1genes related to immune regulatory factorMOR(ii) a And degrading extracellular matrix-related genesMMPFamily, apoptosis-related genesCaspaseFamily andBAXinflammatory factor-relatedGeneIL-6Expression of (2). Relative expression multiple F =1 of control group gene, using 2 -ΔΔCT The F value of each sample was calculated.
Supernatant up-regulation of extracellular matrix-related genesLN、MKXAndCOL13A1，genes inhibiting apoptosisBCL-2Antioxidant-related genesPTENAndSIRT-1genes related to immune regulatory factorMOR(ii) a Down-regulation of extracellular matrix-degrading related genesMMPFamily, apoptosis-related genesCaspaseFamily andBAXinflammatory factor-related genesIL-6The results are shown in Table 9:
Figure 648642DEST_PATH_IMAGE009
inactivation of thallus to up-regulate and inhibit relevant gene of extracellular matrixLNAntioxidant-related genesSIRT-1Genes related to immune regulatory factorMOR(ii) a Down-regulation of apoptosis-related genesCaspase-3AndBAX. The results are shown in Table 10:
Figure 173164DEST_PATH_IMAGE010
in vitro cell experiments show that the lactobacillus amyloliquefaciens SEUNEU-111 of the invention has the function of up-regulating laminin related to HFF extracellular matrixLNMohoke protein geneMKXAnd type XIII collagen alpha 1 chain geneCOL13A1Immunomodulatory factor-related beta-endorphin receptor genesMORPhosphatase and tensin homolog genes resistant to oxidation-related chromosome 10 deletionsPTENSirtuins protein family genesSIRT-1And B-lymphocytoma-2 gene involved in the inhibition of apoptosisBCL-2The relative expression multiple of the gene is 1.08-3.62 times under the action of expression; has down-regulation of matrix metalloprotease family gene associated with degradation of extracellular matrixMMPApoptosis-related BCL2-Associated X protein geneBAXAnd cysteine protease family genesCaspaseAnd interleukin 6 gene related to cytokineIL-6The relative expression multiple of the gene is 0.32-0.95. Indicating SEUNEU-111 has anti-aging effects of promoting synthesis of HFF extracellular matrix, reducing apoptosis, increasing antioxidant activity, increasing cytokine-mediated immunity, reducing degradation of extracellular matrix, and reducing inflammatory factors.
Example 9 SEUNEU-111 increasing the amount of β -endorphin production by HFF cells
1. Preparing SEUNEU-111 supernatant and inactivated bacteria:
selecting a single colony of lactobacillus amyloliquefaciens SEUNEU-111 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 =1.0, which is an inactivated cell.
2. HFF cell preparation and H 2 O 2 Inducing oxidative damage
HFF cells cultured with DMEM were digested at 2 ml/well (5X 10 contained therein) 5 Cells) were inoculated into 6-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. SEUNEU-111 addition
The supernatant and inactivated cells were added to stimulated HFF cells at 5% (V/V) (control group replaced equal volume of PBS for supernatant/inactivated cells). Each group 3 was incubated overnight at 37 ℃.
4. Determination of beta-endorphin expression
Collecting cell culture solution, establishing a standard curve according to the method of a Jianglai biological human beta-endorphin (beta-EP) enzyme-linked immunosorbent assay kit, and calculating the generation amount and the improvement rate of the beta-endorphin in the sample.
The standard curve formula and the measurement results are shown in Table 11:
Figure 724232DEST_PATH_IMAGE011
the result shows that SEUNEU-111 has the anti-aging effect of increasing the generation amount of the beta-endorphin of the HFF cells.
Example 10 SEUNEU-111 experiments to reduce the carbonyl content of HFF cellular proteins
1. Preparation of SEUNEU-111 supernatant:
the preparation method refers to example 3.
2. HFF cell preparation and UV damage
HFF cells were digested and seeded at 0.5 ml/well (1.5X 105 cells in) into 24-well plates and incubated overnight at 37 ℃ in a 5% carbon dioxide incubator. The cells in the wells were subjected to UV UVB irradiation damage at a total dose of 2J/cm 2.
3. SEUNEU-111 addition
The supernatant was added at 5% (V/V) to the stimulated HFF cells (controls replaced supernatant with equal volume of PBS, respectively). Each group 3 was incubated overnight at 37 ℃.
4. Determination of cellular protein content and protein carbonyl content
And removing the culture medium of the cells, adding RIPA lysate to extract cell protein, respectively measuring the protein carbonyl content in the sample according to the methods of a Nanjing constructed total protein measurement (Coomassie brilliant blue method) kit and a protein carbonyl content measurement kit, and calculating the reduction rate. The results are shown in Table 12:
Figure 257981DEST_PATH_IMAGE012
the results show that SEUNEU-111 has an anti-aging effect of reducing the carbonyl content of HFF cellular protein.
Example 11 reduction of NO production by Raw264.7 cells by SEUNEU-111
1. Preparation of SEUNEU-111 supernatant and inactivated thallus:
the preparation method refers to example 3.
2. Raw264.7 cell preparation
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. SEUNEU-111 addition and LPS stimulation
Adding 5% (V/V) of the supernatant and 10% (V/V) of inactivated bacteria into Raw264.7 cells cultured overnight, adding 0.5ml of LPS solution with the concentration of 0.2 mu g/ml after 2h to induce Raw264.7 cells to be inflamed, taking the cell culture supernatant after 20h, drawing a standard curve according to the method of a Biyunyan NO detection kit, and calculating the concentration and the inhibition rate of NO in the sample.
The calculation formula and the result are shown in Table 13:
Figure 312525DEST_PATH_IMAGE013
the result shows that SEUNEU-111 has anti-inflammatory effect, can reduce LPS-induced NO production of Raw264.7 cells, and reduces 14.08% -31.88% compared with an LPS control group.
Example 12 SeUNEU-111 Down-Regulation of HaCaT cell inflammatory factor-related Gene expression
1. Preparation of SEUNEU-111 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. SEUNEU-111 addition and LPS stimulation
The supernatant 5% (V/V) was added to HaCaT cells cultured overnight, and after 2 hours, 0.5ml of LPS solution with 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 culture medium from the above cells, adding lysis solution, extracting total RNA from the cells, detecting RNA concentration and purity, and reverse transcribing to cDNA to obtainGAPDHFor reference gene, real-time qPCR detection is adoptedIL-8、IL-22AndTRPV1expression of the gene. The group treated with the supernatant without the addition of SEUNEU-111 was used as a control (relative gene expression fold F = 1), and the F value of each sample was calculated by the 2-. DELTA.CT method. The results are shown in Table 14:
Figure 273528DEST_PATH_IMAGE014
in vitro cell experiments show that the lactobacillus amyloliquefaciens SEUNEU-111 has the function of down-regulating HaCaT cell inflammation related factors induced by LPSIL-8、IL-22AndTRPV1the relative expression multiple of the gene is 0.32-0.87. Shows that SEUNEU-111 has anti-inflammatory effect.
Example 13 inhibition of skin pathogenic bacteria by SEUNEU-111
1. Preparation of SEUNEU-111 supernatant:
selecting a single colony of lactobacillus amyloliquefaciens SEUNEU-111 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 with 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-micron filter membrane to obtain the supernatant.
2. Preparing a pathogenic bacterium liquid:
the three types of pathogenic bacteria: staphylococcus hominis (Staphylococcus hominisCGMCC 1.493), and Staphylococcus hemolyticus (Staphylococcus haemolyticusCGMCC 1.540), and corynebacterium crenatum (Corynebacterium xerosisCGMCC 1.1919), respectively picking single colony 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 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:
Figure 311891DEST_PATH_IMAGE015
in vitro experiments show that the lactobacillus amyloliquefaciens SEUNEU-111 has the effect of inhibiting the proliferation of skin pathogenic bacteria staphylococcus hominis, staphylococcus haemolyticus and corynebacterium crenatum, and the inhibition rate is 31.58-80.77%.
Example 14 Effect of SEUNEU-111 altering the flora ratio on improving sensitive muscle
1. Preparation of SEUNEU-111 supernatant:
the preparation method refers to example 12.
2. Preparing sensitive muscle related flora bacterial liquid:
staphylococcus aureus golden yellow subspecies (A), (B), (C)Staphylococcus aureus subsp.aureusCGMCC 1.8721 and Staphylococcus epidermidis ((S. epidermidis))Staphylococcus epidermidisCGMCC 1.4260), respectively selecting single colony 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 influencing growth of sensitive muscle related flora by adding supernatant
Adding the supernatant into two kinds of skin flora bacterial liquid respectively at 10% (V/V) addition amount, culturing at 37 deg.C for 16 hr with the addition of equal volume MRS liquid culture medium as control, and taking the relative concentrations (OD) of the two kinds of bacterial liquid 600 ) The influence of the ratio evaluation on the growth of sensitive muscle-associated flora. The calculation formula and the results are shown in Table 16:
Figure 383752DEST_PATH_IMAGE016
the result shows that SEUNEU-111 has obvious inhibition effect on staphylococcus aureus, but has no inhibition effect on staphylococcus epidermidis. Shows that SEUNEU-111 can change the flora proportion, thereby effectively improving sensitive muscles.
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 (11)

1. Lactobacillus amyloliquefaciens (A)Lactobacillus amylovorus ) The preservation number is CCTCC NO: M2022368.
2. A population of bacteria comprising the lactobacillus amyloliquefaciens of claim 1.
3. A microbial inoculum, characterized in that its raw material comprises the Lactobacillus amyloliquefaciens strain according to claim 1 and/or the flora according to claim 2.
4. The application of any one of the following I) -III) in preparing a product for improving the skin state;
I) the lactobacillus amyloliquefaciens of claim 1;
II) the population of claim 2;
III) the microbial agent according to claim 3.
5. The use of claim 4, wherein the improvement in skin condition comprises at least one of promoting cell proliferation, repairing skin barrier, moisturizing, anti-aging, anti-inflammatory, inhibiting skin pathogens, regulating skin microflora.
6. The use according to claim 5, wherein the repair of skin barrier comprises repair of chemical damage, repair of oxidative damage, and/or up-regulation of barrier repair-related genesFLG、IVL、OVO1AndLORat least one of (1).
7. The use of claim 5, wherein said moisturizing comprises up-regulation of a moisturizing-associated geneAQP3And/orGBAExpression of (2).
8. The use of claim 5, wherein said anti-aging comprises promoting extracellular matrix synthesis; inhibiting degradation of extracellular matrix; reducing apoptosis; increasing at least one of cellular immune regulatory factors, increasing the production of cellular beta-endorphins, or decreasing the carbonyl content of cellular proteins.
9. The use of claim 5, wherein said anti-inflammatory comprises reducing cellular NO production or down-regulating an inflammatory factor-related geneIL-8、IL-22、TRPV1AndIL-6at least one of the expressions.
10. The use of claim 5, wherein said inhibition of skin pathogens comprises inhibition of staphylococcus hominis, staphylococcus haemolyticus, corynebacterium parvum; the regulating of the skin microbial flora comprises reducing the ratio of staphylococcus aureus to staphylococcus epidermidis.
11. The product for improving the skin state is characterized by comprising any one of the following I) -III):
I) the lactobacillus amyloliquefaciens of claim 1;
II) the population of claim 2;
III) the microbial preparation according to claim 3.
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