CN115786208A

CN115786208A - Lactobacillus brevis and application thereof

Info

Publication number: CN115786208A
Application number: CN202211598936.XA
Authority: CN
Inventors: 廖梅香
Original assignee: Individual
Current assignee: Individual
Priority date: 2022-12-12
Filing date: 2022-12-12
Publication date: 2023-03-14

Abstract

The invention discloses lactobacillus brevis and application thereof, and relates to the technical field of microorganisms. The strain of the Lactobacillus brevis disclosed by the invention is Lactobacillus brevis ProfMIC-217 which is preserved in China center for type culture collection at 7-18 months in 2022, and the preservation number is CCTCC NO: M20221130. Experiments show that ProfMIC-217 has the functions of promoting cell proliferation and resisting aging, and can be used for preparing food, medicines, cosmetics and the like.

Description

Lactobacillus brevis and application thereof

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to lactobacillus brevis and application thereof.

Background

Photoaging is the predominant form of extrinsic aging of the skin, and the primary cause of photoaging of the skin is ultraviolet radiation. In the aging process of skin, cell proliferation is reduced, apoptosis is increased, and extracellular matrix components (collagen, elastin, glycosaminoglycan, etc.) are significantly reduced with skin aging. In addition, oxidative stress is increased due to increased reactive oxygen species generated by various factors such as mitochondrial damage, inflammatory reaction, etc., and aging-damaged cells cannot be removed in time, thereby causing skin aging.

In the research of aging mechanism, the autophagy of cells is found to delay the function degradation of organs, clear damaged proteins and lipids in cells, repair DNA, help cells to restore metabolic homeostasis and provide protection for photoaging damaged skin. In addition, reduction of apoptosis, reduction of cell inflammation, increase of synthesis and degradation of extracellular matrix, improvement of oxidation resistance of cells and the like are proved to be capable of slowing down skin cell aging, and the search for multi-layer and multi-dimensional anti-aging substances is a research hotspot at present.

Skin microorganisms play an important role in the maturation and homeostasis of skin immunity, most of the microorganisms living on the skin behave symbiotically or synergetically under steady-state conditions, and recent studies have demonstrated that skin microbiota regulates the expression of various innate factors. Skin microbial decomposition of phospholipids, sterols and keratin also allows skin cells to absorb and promote cell growth, delay aging and reduce wrinkle production. Therefore, the probiotic related product developed by utilizing the skin microbial technology has important practical significance.

Disclosure of Invention

In view of the above, the present invention provides a lactobacillus brevis and applications thereof.

The invention provides Lactobacillus brevis (Lactobacillus brevis), which is a strain of Lactobacillus brevis ProfMIC-217 and has been preserved in China center for type culture Collection (CCTCC for short, with the address of No. 299 in Wuchang district, university of Wuhan, zip code 430072) in 7 months and 18 days in 2022, and the preservation number of the Lactobacillus brevis is M20221130;

another object of the present invention is to provide the use of the above Lactobacillus brevis for preparing a product for improving skin cell conditions.

In some embodiments, the improvement in skin condition is at least one of promotion of cell proliferation, anti-aging ability.

In some embodiments, the pro-cell proliferation includes an effect of promoting proliferation of skin keratinocytes and/or fibroblasts.

In some embodiments, the anti-aging is upregulating expression of extracellular matrix-associated genes; the extracellular matrix-associated gene is at least one of COL1A1, TIMP1, SPTSSA, SMAD3, LN and/or FN;

the anti-aging is the expression of up-regulated cell antioxidant related genes SIRT-1, SIRT-3 and/or PTEN.

In some embodiments, the anti-aging is down-regulation of expression of a degradation extracellular matrix-associated gene, MMP1; up-regulating the expression of the autophagy-related gene LC3B;

the anti-aging is the expression of down-regulation of a gene TNF-alpha related to a cell inflammatory factor; up-regulating the expression of the gene MOR related to the immune regulatory factor.

In some embodiments, the product is a food product, a pharmaceutical product, or a cosmetic product.

It is another object of the present invention to provide a product for improving skin conditions, which is made from a raw material comprising lactobacillus brevis as described above.

In some embodiments, the lactobacillus brevis in the product comprises one or both of the following (1) or (2):

(1) Viable and/or inactivated bacteria of the aforementioned Lactobacillus brevis;

(2) Cultures, exosomes, lysates and/or extracts of the aforementioned lactobacillus brevis.

The invention discloses Lactobacillus brevis ProfMIC-217 with a preservation number of CCTCC NO: M20221130. Experiments show that ProfMIC-217 has the functions of promoting cell proliferation and resisting aging, and can be used for preparing food, medicines, cosmetics and the like.

Biological preservation Instructions

Lactobacillus brevis (Lactobacillus brevis) ProfMIC-217 is preserved in China center for type culture Collection (CCTCC for short, address: wuhan university, wuhan's Wuchang district No. eight-way 299) at 7 months and 18 days in 2022, and the preservation number is CCTCC NO: M20221130.

Detailed Description

The invention provides lactobacillus brevis and application thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The Lactobacillus brevis strain ProfMIC-217 is derived from Korean fermented oriental cherry, and is identified as Lactobacillus brevis (Lactobacillus brevis) by 16S rDNA. The strain is gram-positive and rod-shaped under a microscope; the bacterial colony grows on an MRS plate, can form a round bacterial colony with a smooth and opaque surface, is white and has a regular edge; the strain grows uniformly and turbulently in MRS liquid culture medium, and the strain is white precipitate after long-term storage, and the optimal growth temperature is 37 ℃.

Lactobacillus brevis (Lactobacillus brevis) ProfMIC-217, depository: china center for type culture Collection, address: in the Wuhan university school of Wuhan 299 in the Wuchang area of Wuhan city, hubei province, the preservation date is as follows: 18 months 7 in 2022, the preservation number is CCTCC NO: M20221130.

Further, the invention provides lactobacillus brevis ProfMIC-217 in the use or product according to the invention in a form that is live or dead or that is sterilized at intervals, or in the form of a lysate and/or extract, or in the form of a bacterial product or in the form of a supernatant or in the form of a derivative, preferably selected from: metabolites, metabolic biological products, exosomes, prebiotics, cell walls and components thereof, exopolysaccharides, and compounds containing immunogenic components, preferably selected from: supernatant and inactivated bacteria.

In vitro cell experiments show that the Lactobacillus brevis ProfMIC-217 has the function of promoting epidermal cell HaCaT repair, and the ProfMIC-217 promotes the proliferation rate of the HaCaT cells damaged by SDS (sodium dodecyl sulfate) and has the proliferation rate of 112.43-117.68%

In vitro cell experiments show that the lactobacillus brevis ProfMIC-217 has the functions of up-regulating expression of a collagen type I alpha chain gene COL1A1, a tissue metalloproteinases inhibitor I gene TIMP1, a serine palmitoyltransferase gene SPTSSA, a signal transduction protein gene SMAD3 and a microtubule-associated protein 1 light chain 3 beta gene LC3B related to a cell autophagy-associated gene, wherein the relative expression multiple of the genes is 1.03-1.77; has the function of down-regulating the expression of matrix metalloproteinase family gene MMP1 related to degrading extracellular matrix and tumor necrosis factor alpha gene TNF-alpha related to cell inflammatory factors, and the relative expression multiple of the gene is 0.46-0.81.

In vitro cell experiments show that the Lactobacillus brevis ProfMIC-217 has the effect of promoting human fibroblast HFF proliferation, and the proliferation rate is 3238-113.59%.

In vitro cell experiments show that the Lactobacillus brevis ProfMIC-217 has the functions of up-regulating laminin LN and fibronectin FN related to HFF extracellular matrix, antioxidant related genes Sirtuins protein family genes SIRT-1 and SIRT-3, phosphatase deleted from No. 10 chromosome and tensin homolog gene PTEN, and immune regulatory factor related beta-endorphin receptor gene MOR with the relative expression multiple of 1.11-3.56.

The test materials adopted by the invention are all common commercial products and can be purchased commercially, and the invention is further explained by combining the following embodiments:

EXAMPLE 1 isolation of ProfMIC-217

Sampling in Korean fermented cherry vegetable. Properly processing the sample, uniformly mixing the sample in normal saline by shaking, taking the supernatant, streaking the supernatant on an MRS solid plate, culturing the MRS solid plate at the constant temperature of 37 ℃ for 48 hours, and then selecting a white colony to repeatedly inoculate and screen until a uniform single colony is obtained, wherein the colony is named as ProfMIC-217.

Gram staining microscopic examination: the strain ProfMIC-217 is gram-positive and rod-shaped under a microscope; growing on an MRS plate to form white round microcolonies with smooth, mellow and opaque surfaces and regular edges; the strain grows in MRS liquid culture medium in a uniform turbid way, and the strain is white and precipitated after being placed for a long time.

Example 2 nucleic acid identification of ProfMIC-217

1. 16S rDNA gene sequence analysis:

picking single colony in MRS liquid culture medium, culturing overnight at 37 deg.C, centrifuging at 12000 deg.C for 1min, collecting thallus, and performing operation according to DNA extraction kit. The primers adopt bacterial universal primers 27F and 1492R, a PCR amplification system is a 50 mu L system, and the pre-denaturation is carried out for 5min at 95 ℃;94 ℃ 15s,57 ℃ 15s,72 ℃ 40s,35 cycles; extension at 72 ℃ for 10min.

2. Results

The homology comparison (BLASTN) of the PCR product sequencing result with the published standard sequence in GenBank indicates that the strain ProfMIC-217 is Lactobacillus brevis (Lactobacillus brevis).

Example 3: profMIC-217 experiment for promoting SDS-induced HaCaT injury repair of human immortalized keratinocytes

1. Preparation of ProfMIC-217 inactivated bacteria:

selecting single bacterial colony of Lactobacillus brevis ProfMIC-217 in MRS liquid culture medium, static culturing at 37 deg.c in culture box for 16-18h, deactivating at 121 deg.c for 30min, centrifuging for 2min at 12000 deg.c, re-suspending the centrifugal precipitate with proper amount of PBS, diluting and regulating 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. 10% inactivated cells (control was replaced with equal volume of PBS) were added to each well and incubated for 24h. Mu.l of CCK-8 solution was added to each well, incubated for 4h, and absorbance A at 450nm was measured.

The formula for calculating the cell viability and the results are shown in the following table:

the results in the table show that the inactivated thallus of ProfMIC-217 has a repairing effect on SDS damage of HaCaT cells.

Example 4: profMIC-217 experiment for regulating light-aging HaCaT extracellular matrix/cell autophagy/degradation extracellular matrix/inflammatory factor related gene expression

1. Preparation of ProfMIC-217 inactivated bacteria:

preparation method reference 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. The total dose of the cells in the wells is 2J/cm ² Ultraviolet UVB radiation damage.

3. ProfMIC-217 addition

10% (V/V) of the inactivated cells were added to the stimulated HaCaT cells (control group replaced by an equal volume of PBS). Each group 3 was incubated overnight at 37 ℃.

4. qPCR method for detecting relative expression multiple of extracellular matrix/cell autophagy/extracellular matrix degradation/inflammatory factor related genes

Removing a culture medium from the cells, adding a lysis solution, extracting total RNA of the cells, detecting the concentration and purity of the RNA, performing reverse transcription to obtain cDNA, detecting expressions of extracellular matrix related genes COL1A1, TIMP1, SPTSSA and SMAD3, extracellular autophagy related gene LC3B, degraded extracellular matrix related gene MMP1 and inflammatory factor related gene TNF-alpha by using GAPDH as an internal reference gene and adopting real-time qPCR. Relative expression multiple F =1 of control group gene, using 2 ^-ΔΔCT The F value of each sample was calculated.

The formula: f =2 ^-ΔΔCT Wherein:

△CT _{experiment of the invention} ＝CT _{Experiment of} -CT _{Internal reference (experiment);}

△CT _control ＝CT _Control -CT _{Internal control (control);}

△△CT＝△CT _{experiment of} -△CT _{And (6) comparison.}

The inactivated thallus can up-regulate extracellular matrix genes COL1A1 TIMP1, SPTSSA and SMAD3; up-regulating autophagy gene LC3B; down-regulating and degrading extracellular matrix gene MMP1; down-regulating the inflammatory factor-related gene TNF-alpha. The results are given in the following table:

the result shows that the addition of ProfMIC-217 has the anti-aging effects of promoting the synthesis of HaCaT extracellular matrix, promoting the autophagy of cells, reducing the degradation of the extracellular matrix and reducing inflammatory factors.

Example 5: profMIC-217 promotes proliferation of human fibroblast HFF

1. Preparing ProfMIC-217 supernatant and inactivated thallus:

selecting single bacterial colony of Lactobacillus brevis ProfMIC-217 in MRS liquid culture medium, standing and culturing at 37 deg.C for 16-18 h, detecting with microplate reader, diluting with PBS to adjust OD ₆₀₀ Deactivation at 121 ℃ for 30min under high pressure, centrifugation at 12000 rpm for 2min, and filtration through a 0.22 μm filter membrane to give a supernatant. Resuspending the pellet with PBS, diluting and adjusting OD ₆₀₀ =0.2, is inactivated bacterial cell.

2. HFF cell preparation and ProfMIC-217 addition

HFF cells cultured with DMEM were seeded at 100 ul/well (3X 10 cells per well) ⁴ Cells) were transferred to 96-well plates and cultured overnight until cells attached. Formulation 200. Mu. M H ₂ O ₂ Mu.l of the culture medium was added to each well, and the culture was carried out at 37 ℃ for 1 hour in a 5% carbon dioxide incubator. The original culture medium was discarded, washed twice with PBS, 100. Mu.l of fresh culture medium was added, 5% (V/V) of supernatant and 10% (V/V) of inactivated bacteria were added to each well, respectively (control group replaced supernatant/inactivated bacteria with equal volume of PBS). And culturing for 24h. Mu.l of CCK-8 solution was added to each well, incubated for 4h, and absorbance A at 450nm was measured.

The calculation formula and the result are shown in the following table:

the results show that the addition of ProfMIC-217 has the effect of promoting the proliferation of HFF cells.

Example 6: profMIC-217 experiment for regulating oxidative damage HFF extracellular matrix/antioxidant/immune regulatory factor related gene expression

1. Preparing ProfMIC-217 supernatant and inactivated thallus:

the preparation method refers to example 5.

2. HFF cell preparation and H ₂ O ₂ Inducing oxidative damage

The HFF cells cultured with DMEM were digested at 0.5 ml/well (2X 10 contained therein) ⁵ Cells) were inoculated into 24-well plates and cultured overnight at 37 ℃ in a 5% carbon dioxide incubator. H was added to each well to a final concentration of 200. Mu.M ₂ O ₂ Stimulating, and standing at 37 ℃ for 1h.

3. ProfMIC-217 addition

5% (V/V) of the supernatant and 10% (V/V) of the inactivated bacteria were added to the stimulated HFF 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/antioxidant/immune regulatory factor related genes

Removing the culture medium of the cells, adding a lysis solution, extracting total RNA of the cells, detecting the concentration and purity of the RNA, performing reverse transcription to obtain cDNA, and detecting the expression of extracellular matrix related genes LN and FN, antioxidant related genes SIRT-1, SIRT-3 and PTEN and an immunoregulatory factor related gene MOR by using GAPDH as an internal reference gene and adopting real-time qPCR. Relative expression multiple F =1 of control group gene, using 2 ^-ΔΔCT The F value of each sample was calculated.

The supernatant fluid up-regulates extracellular matrix related gene FN and antioxidant related gene SIRT-3. The results are shown in the following table:

the inactivated thallus can up-regulate extracellular matrix related gene LN, antioxidant related genes SIRT-1, SIRT-3 and PTEN, and immunoregulation factor related gene MOR. The results are given in the following table:

the result shows that the addition of ProfMIC-217 has the anti-aging effects of promoting the synthesis of HFF extracellular matrix, increasing the oxidation resistance and increasing the cell immune regulatory factor.

Example 7 preparation of supernatant infusion of ProfMIC-217

The preparation of the supernatant granule of ProfMIC-217 is shown in the following table

Raw materials	Mass ratio (%)
		ProfMIC-217 fermentation powder	40.0
Red bean powder	39.0
		Cocoa powder	20.6
Silicon dioxide	0.4

The preparation process comprises the following steps: and (3) taking the supernatant of ProfMIC-217 prepared in the example 5, preparing ProfMIC-217 fermentation powder after spray drying, adding red bean powder, cocoa powder and silicon dioxide, and stirring and dispersing uniformly to obtain the medicinal granules. The obtained instant granules of ProfMIC-217 are convenient to carry, have natural taste after being soaked in water, have good palatability, and can be used as oral beverage for caring skin.

Example 8 preparation of serum containing inactivated bacteria of ProfMIC-217

Preparation of ProfMIC-217 inactivated thallus essence water, formula is shown in the following table

The preparation process comprises the following steps: heating the sterilized bacterial body fluid of ProfMIC-217 prepared in example 5 to 50 ℃, adding glycerol, 1,3-butanediol, phenoxyethanol and vanillin, stirring and dispersing uniformly until the mixture is clear, and cooling to 35 ℃ to obtain the product. The obtained ProfMIC-217 inactivated thallus essence has fresh water smell, and is shaken before use to resuspend thallus, so that the thallus can be applied to skin to achieve smooth and non-greasy feeling.

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

1. Lactobacillus brevis (Lactobacillus brevis) is Lactobacillus brevis ProfMIC-217, which has been preserved in China center for type culture Collection in 7 months and 18 days 2022 with the preservation number of CCTCCNO: M20221130.

2. Use of a lactobacillus brevis according to claim 1 for the preparation of a product for improving the condition of skin cells.

3. The use of claim 2, wherein the improvement in skin condition is at least one of promotion of cell proliferation, anti-aging capacity.

4. Use according to claim 3, wherein said pro-cell proliferation comprises an effect of promoting the proliferation of skin keratinocytes and/or fibroblasts.

5. The use according to claim 3, wherein the anti-aging is up-regulation of the expression of extracellular matrix-related genes; the extracellular matrix-related gene is at least one of COL1A1, TIMP1, SPTSSA, SMAD3, LN and/or FN;

6. The use according to claim 3, wherein the anti-aging is down-regulation of the expression of an extracellular matrix-associated gene MMP1; up-regulating the expression of autophagy-related gene LC3B;

7. Use according to any one of claims 1 to 6, wherein the product is a food product, a pharmaceutical product or a cosmetic product.

8. A product for improving skin conditions, made from a material comprising the lactobacillus brevis of claim 1.

9. The product according to claim 8, wherein the Lactobacillus brevis in the product comprises one or both of the following (1) or (2):

(1) Viable and/or inactivated bacteria of Lactobacillus brevis according to claim 1;

(2) A culture, exosome, lysate and/or extract of Lactobacillus brevis according to claim 1.