CN115044519B - Lactobacillus amyloliquefaciens and application thereof - Google Patents

Lactobacillus amyloliquefaciens and application thereof Download PDF

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CN115044519B
CN115044519B CN202210965527.2A CN202210965527A CN115044519B CN 115044519 B CN115044519 B CN 115044519B CN 202210965527 A CN202210965527 A CN 202210965527A CN 115044519 B CN115044519 B CN 115044519B
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lactobacillus
cells
seuneu
amyloliquefaciens
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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 lactobacillus amyloliquefaciens provided by the invention 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 the fields of food, medicines, cosmetics and the like.

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 keratinocyte structural lipid ceramide gradually increases in the process of differentiation from a basal layer to a cutin, so that the keratinocyte is discharged to intercellular spaces, and the keratinocyte structural lipid ceramide forms a barrier for preventing water loss. 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 to 30% of water due to its hydrophilicity and barrier function, and natural moisturizing factors contained in the stratum corneum, such as amino acids, lactate and saccharides, and this environment becomes a cradle for the growth of the microbial colonies of the skin itself. However, the water content of the stratum corneum gradually decreases with age, and various problems of the skin are caused when the water content is less than 10%.
Skin aging, including extrinsic aging caused by environmental factors such as air pollution, smoking, malnutrition, and Ultraviolet (UV) rays, and intrinsic aging caused by time variation. It is typically characterized by thinning of the skin, fine lines, which may be caused by decreased cell proliferation and significant changes in dermal composition with age. Extracellular matrix components (collagen, elastin, glycosaminoglycans, etc.) are significantly reduced with 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 moisturizing, anti-aging and anti-inflammatory genes is up-regulated, so that the absorption of the skin on nutrient substances is effectively increased, and the immunity of the skin is enhanced.
Sensitive muscles generally cause skin immunity reduction due to skin cell damage, skin moistening is insufficient due to the fact that horny layers are thinned, and finally the barrier function of the skin is too weak to resist external stimulation, so that the phenomena of redness, fever, pruritus, stabbing pain and the like are easy to generate. While an impaired skin barrier may further lead to Staphylococcus aureus (S.aureus) ((R))Staphylococcus aureus) Leading to inflammation and red swelling. Staphylococcus epidermidis (resident in the skin) ((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. The probiotics serving as a microecological preparation can be used for balancing skin flora, repairing skin barriers and improving skin states, so that the probiotics related product developed by utilizing the microecological 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) (by 16S rDNA gene sequence analysis and NCBI database comparisonLactobacillus amylovorus ）。
The invention provides a flora comprising the lactobacillus amyloliquefaciens.
Further, 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 preparation, which is not limited by the invention.
Furthermore, the microbial inoculum of the invention also comprises fermentation liquor, thallus, supernatant and active substances contained in the strain, which is not limited by the 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 provided by the invention.
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 repair of the skin barrier comprises at least one of repair of chemical damage, repair of oxidative damage or repair of light damage, and/or up-regulation of a barrier repair-related gene.
In the present invention, the chemical injury includes any chemical substance that can cause mild, moderate or severe epidermal cell injury to human body, and the present invention is not limited thereto. Furthermore, the invention researches the repair effect of the lactobacillus amyloliquefaciens on HaCaT cell chemical damage caused by SDS (sodium dodecyl sulfate) by using a HaCaT cell tested object, and the result shows that the lactobacillus amyloliquefaciens can relieve the chemical damage caused by the SDS and improve the cell survival rate, wherein the cell survival rate is 109.57-124.19%; the lactobacillus amyloliquefaciens has the function of promoting the repair of epidermal cells.
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 the increase of 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 induced HFF cell oxidative damage 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 genes related to HaCaT cell skin barrier repair is researched, and the result shows that the lactobacillus amyloliquefaciens can improve the genes related to HaCaT cell skin barrier repairFLG、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 to 6.85, which shows that the lactobacillus amyloliquefaciens can promote the 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. The invention takes HaCaT cells and HFF cells as subjects, and researches the influence of the lactobacillus amyloliquefaciens on the expression of genes related to HaCaT cells and HFF extracellular matrix.
In the present invention, the reduction of apoptosis includes reduction of apoptosis caused by photoaging, apoptosis caused by stimuli or apoptosis caused by injury, which is not limited in 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, toMORThe gene expression is characterized, the influence of the lactobacillus amyloliquefaciens on the cell immune regulatory factor is researched, and the result shows that the lactobacillus amyloliquefaciens is subjected to the starch degradationThe lactobacillus plantarum can up-regulate related genes of the immune regulatory factor, thereby playing a role in resisting aging.
In the invention, the cell secretes 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 the lactobacillus amyloliquefaciens on the proliferation of the HFF cells is studied, so that the lactobacillus amyloliquefaciens can promote the proliferation of the HFF, and the proliferation promoting rate is 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, the cellular Nitric Oxide (NO) has the dual characteristics of promoting inflammation and resisting inflammation, the endothelial source NO has the function of inhibiting inflammatory reaction under the normal condition, and the inducible NO synthetase synthesizes a large amount of NO under the pathological condition, so that the cellular toxicity is realized, and the inflammatory reaction is aggravated; 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 xerostomia (C. Xerostomia)Corynebacterium xerosisCGMCC 1.1919) is a tested subject, the research on the capability of the lactobacillus amyloliquefaciens for inhibiting 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 subspecies aureobasidium: (Staphylococcus aureus subsp.aureusCGMCC 1.8721 and Staphylococcus epidermidis (CGMCC)Staphylococcus epidermidisCGMCC 1.4260) is a subject, 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 floraThe purpose is.
The invention provides a product for improving skin condition, which comprises any one of the following I) -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 for using the composition comprises smearing, external application or injection, and the invention is not limited to the method.
The invention obtains the following beneficial effects:
the present invention provides a method for understanding Lactobacillus amylovorusLactobacillus amylovorus ) It is preserved 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 barriers, 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 (A)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 skill in the art that variations and modifications in the methods and applications disclosed herein, or appropriate variations and combinations thereof, 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 vegetable slurry water. Properly processing a sample, uniformly mixing the sample in normal saline by shaking, taking a supernatant, streaking the supernatant on an MRS solid plate, culturing the MRS solid plate at a 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 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:
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 ℃; 15s at 94 ℃, 15s at 57 ℃, 40s at 72 ℃,35 cycles; extension at 72 ℃ for 10min.
2. As a result, the
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 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 16-18h, 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 5% carbon dioxide was added to each well and incubated at 37 ℃ for 8 hours. 5% (V/V) of the supernatant and 10% (V/V) of the inactivated bacteria (PBS equal to the volume of the control group is used for replacing the supernatant/the inactivated bacteria) are added into each well respectively for culturing for 24h. 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 bacteria:
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 a 6-well plate, and cultured overnight at 37 ℃ in a 5% carbon dioxide incubator until the cells adhere to the wall. Respectively adding 5% (V/V) of the supernatantInactivating thallus by 10% (V/V), culturing for 24h, adding lysate, extracting total RNA of 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 of -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 fold of the gene is 1.55 to 4.33. The above results show that SEUNEU-111 has the effect of promoting skin barrier repair.
Example 5 SEUNEU-111 experiment for Up-regulating HaCaT moisturizing related Gene expression
1. Preparation of SEUNEU-111 supernatant and inactivated thallus:
the preparation method refers to example 3.
2. Experiment for up-regulating HaCaT moisturizing related gene expression
Inoculation of human immortalized keratinocytes HaCaT (2 ml/well, 5X 10 therein) 5 Cells) to 6-well plate, 5% carbon dioxide incubator 37 ℃ overnight until cells adhere. Adding 5% (V/V) of supernatant, inactivating 10% (V/V) of thallus, culturing for 24h, adding lysate, extracting total RNA of cells, detecting RNA concentration and purity, and reverse transcribing into 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 multiple F = 1) and 2 was used -ΔΔCT F value was calculated for each sample.
The results are shown in tables 4 and 5:
Figure 161816DEST_PATH_IMAGE004
Figure 772926DEST_PATH_IMAGE005
in vitro cell experiments show that the supernate and the inactivated thallus of the lactobacillus amyloliquefaciens SEUNEU-111 have aquaporin 3 genes related to up-regulation and moisture retentionAQP3And glucocerebrosidase geneGBARelative expression fold of the gene is 1.22 to 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 dispensed at 0.5 ml/well (2X 10 contents) 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, respectively (in the control group, the supernatant/inactivated bacteria were replaced with PBS of the same volume). 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 to 1.84; 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 fold of the gene is 0.36 to 0.82. The above results show thatSEUNEU-111 has effects of promoting synthesis of HaCaT extracellular matrix, reducing degradation of extracellular matrix and anti-aging effect of inflammatory factor.
Example 7 proliferation of human fibroblast HFF by SEUNEU-111
1. Preparing SEUNEU-111 supernatant and inactivated bacteria:
the preparation method is referred 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 to 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%. The SEUNEU-111 is shown to have 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. Preparing SEUNEU-111 supernatant and inactivated bacteria:
the preparation method is referred 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 1h.
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-related genesIL-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
inactivatingBacterial body up-regulation inhibition of extracellular matrix related geneLNAntioxidant-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 associated with the inhibition of apoptosisBCL-2The relative expression multiple of the gene is 1.08 to 3.62 times; has the function of down regulating matrix metalloproteinase family genes related to the degradation of extracellular matrixMMPApoptosis-related BCL2-Associated X protein GeneBAXAnd cysteine protease family genesCaspaseAnd interleukin 6 gene associated with a cytokineIL-6Relative expression fold of the gene is 0.32 to 0.95. The SEUNEU-111 has the anti-aging effects of promoting the synthesis of HFF extracellular matrix, reducing apoptosis, increasing antioxidation, increasing cell immune regulatory factor, reducing extracellular matrix degradation and reducing inflammatory factor.
Example 9 SEUNEU-111 increasing the amount of β -endorphin production by HFF cells
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 16-18h, 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 in DMEM were digested at 2 ml/well (5X 10 cells 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 1h.
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 the 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 (3) removing a culture medium from the cells, adding RIPA lysate, extracting cell protein, measuring the protein carbonyl content in the sample according to the methods of a Nanjing-built total protein measurement (Coomassie brilliant blue method) kit and a protein carbonyl content measurement kit respectively, and calculating the reduction rate. The results are shown in Table 12:
Figure 257981DEST_PATH_IMAGE012
the results show that SEUNEU-111 has the anti-aging effect of reducing the content of carbonyl of HFF cell 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 is referred 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 seeded 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 downregulation 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 seeded 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 into HaCaT cells cultured overnight, adding 0.5ml of LPS solution with concentration of 0.2 μ g/ml after 2h to induce cell inflammation, and culturing at 37 deg.C in a 5% carbon dioxide incubator for 20h.
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 cells, detecting RNA concentration and purity, and reverse transcribing into cDNAGAPDHFor reference gene, real-time qPCR detection is adoptedIL-8、IL-22AndTRPV1expression of the gene. F values of the samples were calculated by the 2- Δ Δ CT method using a group treated with supernatant without SEUNEU-111 as a control (gene relative expression fold F = 1). The results are shown in Table 14:
Figure 273528DEST_PATH_IMAGE014
in vitro cell experiments show that the lactobacillus amyloliquefaciens SEUNEU-111 can reduce the LPS-induced HaCaT cell inflammation related factorIL-8、IL-22AndTRPV1the relative expression fold of the gene is 0.32 to 0.87. The 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 16-18h, detecting by an enzyme-linked immunosorbent assay, and diluting with the MRS liquid culture mediumRelease adjustment of 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. 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 and improving sensitive muscles
1. Preparation of SEUNEU-111 supernatant:
the preparation method refers to example 12.
2. Preparing sensitive muscle related flora bacterial liquid:
mixing golden yellow grape subspecies aureococcus (golden yellow)Staphylococcus aureus subsp.aureusCGMCC 1.8721 and Staphylococcus epidermidis (CGMCC) Staphylococcus epidermidisCGMCC 1.4260) are respectively picked into a BHI liquid culture medium, the culture box is kept still for overnight culture at 37 ℃, and the OD is adjusted by diluting the BHI liquid culture medium 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. The SEUNEU-111 is shown to change the flora ratio, thereby effectively improving the sensitive muscle.
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 (8)

1. The lactobacillus amyloliquefaciens has a preservation number of CCTCC NO: M2022368.
2. A microbial inoculum, characterized in that the raw material thereof comprises the Lactobacillus amyloliquefaciens strain as set forth in claim 1.
3. Use of the lactobacillus amyloliquefaciens of claim 1 or the microbial inoculum of claim 2 for preparing a product for improving skin conditions; the skin improving state is at least one of cell proliferation promotion, skin barrier repair, moisture retention, aging resistance, anti-inflammation, skin pathogenic bacteria inhibition and skin microbial flora regulation;
the promotion of cell proliferation is the promotion of HFF cell proliferation;
the skin pathogenic bacteria are staphylococcus hominis, staphylococcus haemolyticus or corynebacterium parvum;
the regulating of the skin microbial flora is to reduce the ratio of staphylococcus aureus to staphylococcus epidermidis.
4. Use according to claim 3, wherein the repair of skin barrier is the repair of chemical damage, the repair of oxidative damage, and/or the up-regulation of a gene involved in barrier repairFLG、IVL、OVO1AndLORat least one of (a).
5. The use of claim 3, wherein the moisturizing is up-regulation of moisturizing-associated genesAQP3And/orGBAExpression of (2).
6. The use according to claim 3, wherein the anti-aging is 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-endorphin, or reducing the content of cellular protein carbonyl.
7. The use of claim 3, wherein the anti-inflammatory is reducing cellular NO production or down-regulating an inflammatory factor-related geneIL-8、IL-22、TRPV1AndIL-6at least one of the expressions.
8. A skin condition improving product comprising the Lactobacillus amyloliquefaciens according to claim 1 or the microbial agent according to claim 2.
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