CN115786208B - A kind of Lactobacillus brevis and application thereof - Google Patents
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
本发明公开了一种短乳杆菌及其应用，涉及微生物技术领域。本发明公开的短乳杆菌(Lactobacillus brevis)，该菌株为短乳杆菌ProfMIC‑217，已于2022年7月18日保藏在中国典型培养物保藏中心，其保藏编号为CCTCC NO:M20221130。实验表明，ProfMIC‑217具有促进细胞增殖、抗衰老能力的功能，可用于制备食品、药品、化妆品等。The invention discloses a Lactobacillus brevis and its application, and relates to the field of microbial technology. The Lactobacillus brevis disclosed in the invention is a strain of Lactobacillus brevis ProfMIC-217, which has been deposited in the China Center for Type Culture Collection on July 18, 2022, and its deposit number is CCTCC NO:M20221130. Experiments show that ProfMIC-217 has the function of promoting cell proliferation and anti-aging ability, and can be used to prepare food, medicine, cosmetics, etc.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to lactobacillus brevis and application thereof.
Background
Photoaging is the predominant form of extrinsic aging of skin, and the primary cause of photoaging of skin is ultraviolet radiation. During aging of the skin, cell proliferation decreases, apoptosis increases, and extracellular matrix components (collagen, elastin, glycosaminoglycan, etc.) significantly decrease with aging of the skin. In addition, active oxygen generated by various factors such as mitochondrial injury, inflammatory reaction and the like is increased, so that oxidative stress is increased, aged and damaged cells cannot be cleared in time, and skin aging is caused.
In the research of aging mechanism, it is found that cell autophagy can delay organ function degradation, remove damaged proteins and lipids in cells, repair DNA, help cells restore metabolic homeostasis, and provide protection for photoaged damaged skin. In addition, reducing apoptosis, reducing cell inflammation, increasing synthesis of extracellular matrix, reducing degradation thereof, improving antioxidant capacity of cells and the like have all been proved to be capable of slowing down skin cell aging, and searching for multi-level and multi-dimensional anti-aging substances is a current research hotspot.
Skin microorganisms play an important role in maturation and homeostasis of skin immunity, and most microorganisms living on the skin appear to be symbiotic or mutualistic under homeostatic conditions, and recent studies have demonstrated that the skin microbiota regulates the expression of various innate factors. Skin microorganisms break down phospholipids, sterols, and keratins, which also allow skin cells to absorb and promote cell growth, delay aging, and reduce wrinkles. Therefore, the probiotic related product developed by utilizing the skin microbiological technology is provided, and has important practical significance.
Disclosure of Invention
In view of the above, the present invention provides a Lactobacillus brevis and its application.
The invention provides lactobacillus brevis (Lactobacillus brevis), which is lactobacillus brevis ProfMIC-217 and is preserved in China center for type culture collection (CCTCC for short, address: eight paths 299 in Wuchang district of Wuhan, university of Wuhan, post code 430072) for 7 months and 18 days in 2022, wherein the preservation number is lactobacillus brevis of CCTCC NO: M20221130;
it is another object of the present invention to provide the use of the above Lactobacillus brevis for the preparation of a product for improving skin cell conditions.
In some embodiments, the improving the skin condition is at least one of promoting cell proliferation, anti-aging ability.
In some embodiments, the promoting cell proliferation comprises promoting the proliferation of skin keratinocytes and/or fibroblasts.
In some embodiments, the anti-aging is up-regulating the expression of an extracellular matrix-related gene, wherein the extracellular matrix-related gene is at least one of COL1A1, TIMP1, SPTSSA, SMAD3, LN, and/or FN;
The anti-aging is the up-regulation of the expression of cell anti-oxidation related genes SIRT-1, SIRT-3 and/or PTEN.
In some embodiments, the anti-aging is down-regulating the expression of the extracellular matrix-associated gene MMP 1;
The anti-aging is to down regulate the expression of cell inflammatory factor related gene TNF-alpha and up regulate the expression of immune regulator related gene MOR.
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, made from raw materials 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) Live bacteria and/or inactivated bacteria of the above Lactobacillus brevis;
(2) Culture, exosomes, lysates and/or extracts of lactobacillus brevis as described above.
The invention discloses Lactobacillus brevis ProfMIC-217, which has 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 foods, medicines, cosmetics, etc.
Description of biological preservation
Lactobacillus brevis (Lactobacillus brevis) ProfMIC-217 is preserved in China center for type culture collection (CCTCC for short, address: eight-path 299 No. of Wuchang district of Wuhan, university of Wuhan, post code 430072) at the year of 2022 and 7 and 18, and the preservation number is CCTCC NO: M20221130.
Detailed Description
The invention provides lactobacillus brevis and application thereof. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The Lactobacillus brevis strain ProfMIC-217 is derived from Korean fermentation of Oriental cherry, and is identified as Lactobacillus brevis (Lactobacillus brevis) by 16S rDNA. The strain is gram positive, rod-shaped under a microscope, grows on an MRS flat plate, can form round colonies with smooth and opaque surfaces, is white and neat in edge, grows in uniform turbidity in an MRS liquid culture medium, and has white precipitate after long-term placing, and the optimal growth temperature is 37 ℃.
Lactobacillus brevis (Lactobacillus brevis) ProfMIC-217, wherein the preservation number is CCTCC NO: M20221130, and the preservation number is 2022, 7 and 18 in the China center for type culture Collection, eight-path 299 university of Wuhan in Wuhan, hubei province.
Further, the present invention provides Lactobacillus brevis ProfMIC-217 in the use or product according to the invention, in the form of living or dead or batch sterilized, 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 derivative, preferably selected from the group consisting of metabolites, metabolic biological products, exosomes, probiotics, cell walls and components thereof, extracellular polysaccharides, and compounds containing immunogenic components, preferably selected from the group consisting of supernatants, inactivated bacteria.
In vitro cell experiments show that the Lactobacillus brevis ProfMIC-217 has the effect of promoting the repair of epidermal cells HaCaT, profMIC-217 promotes the proliferation rate of SDS-damaged HaCaT cells, and the proliferation rate is 112.43% -117.68%
In vitro cell experiments show that the Lactobacillus brevis ProfMIC-217 has the functions of up-regulating the expression of a type I collagen alpha chain gene COL1A1 related to HaCaT extracellular matrix, a tissue metalloproteinase inhibitor I gene TIMP1, a serine palmitoyl transferase gene SPTSSA, a signal transduction protein gene SMAD3 and a cell autophagy related gene microtubule related protein 1 light chain 3 beta gene LC3B, the relative expression multiple of the genes is 1.03-1.77, the function of down-regulating the expression of a matrix metalloproteinase family gene MMP1 related to extracellular matrix and a tumor necrosis factor alpha gene TNF-alpha related to cell inflammatory factors, and the relative expression multiple of the genes is 0.46-0.81.
In vitro cell experiments show that the lactobacillus brevis ProfMIC-217 has the effect of promoting proliferation of human fibroblast HFF, and the proliferation rate is 109.18% -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, resisting oxidation related genes Sirtuins protein family genes SIRT-1 and SIRT-3, and phosphoenzyme and tensin homolog gene PTEN deleted from chromosome 10, and the beta-endorphin receptor gene MOR related to immune regulation factor, wherein the relative expression multiple of the genes is 1.11-3.56 times.
The test materials adopted in the invention are all common commercial products and can be purchased commercially, and the invention is further described below by combining examples:
examples 1ProfMIC-217 isolation
Sampling in Korean family fermented Oriental cherry. After the sample is properly treated, the sample is evenly mixed in normal saline in a vibrating way, the supernatant is streaked on an MRS solid plate, and after the sample is cultured for 48 hours at the constant temperature of 37 ℃, white colonies are picked up for repeated inoculation and screening until uniform single colonies are obtained, and the colonies are named ProfMIC-217.
Gram staining microscopic examination shows that strain ProfMIC-217 is gram positive and rod-shaped under microscope, and can form white, smooth and round opaque microcolonies with clean surface and regular edges on MRS flat plate, and can form uniform turbid growth in MRS liquid culture medium and white precipitate after long-term bacteria.
Nucleic acid identification of examples 2ProfMIC-217
1. 16S rDNA gene sequence analysis:
Single colony is selected and placed in MRS liquid culture medium, after being cultured overnight at 37 ℃, the single colony is centrifuged for 1min at 12000 revolutions to collect thalli, and the operation is carried out according to the step of DNA extraction kit. The primers were bacterial universal primers 27F,1492R, PCR amplification system was 50. Mu.L system, pre-denatured at 95℃for 5min, 94℃for 15s,57℃for 15s,72℃for 40s,35 cycles, 72℃for 10min.
2. Results
The result of sequencing the PCR product was compared with the published standard sequence in GenBank (BLASTN) to give ProfMIC-217 strain as Lactobacillus brevis (Lactobacillus brevis).
EXAMPLE 3 ProfMIC-217 promotes SDS-induced repair of HaCaT damage to human immortalized keratinocytes
1. ProfMIC-217 inactivated bacteria preparation:
And (3) picking single colony of lactobacillus brevis ProfMIC-217 in an MRS liquid culture medium, standing and culturing for 16-18 h in a 37 ℃ incubator, inactivating at high pressure for 30min at 121 ℃, centrifuging for 2min at 12000 r, re-suspending the centrifugal precipitate with a proper amount of PBS, and diluting and adjusting OD 600 =0.2 to obtain the inactivated thallus.
2. Promoting HaCaT cell repair experiments
HaCaT cells (5×10 4 cells/well) were seeded into 96-well plates and cultured overnight to cell attachment. 50. Mu.g/ml SDS was prepared, 100. Mu.l of each well was added, and incubated at 37℃in a 5% carbon dioxide incubator for 8 hours. 10% inactivated cells (control group 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, and incubated for 4 hours, and the absorbance at 450nm was measured for brightness A.
Cell viability calculation formula and results are shown in the following table:
As shown in the above table, profMIC-217 inactivated cells have a repair effect on SDS damage of HaCaT cells.
EXAMPLE 4 ProfMIC-217 regulates light aging HaCaT extracellular matrix/autophagy/degradation of extracellular matrix/inflammatory factor-related Gene expression experiments
1. ProfMIC-217 inactivated bacteria preparation:
Preparation method reference example 3
2. HaCaT cell preparation and ultraviolet injury
HaCaT cells were digested and inoculated at 0.5 ml/well (containing 2×10 5 cells) into 24-well plates and incubated overnight at 37 ℃ in a 5% carbon dioxide incubator. Cells in the wells were subjected to UV UVB irradiation injury at a total dose of 2J/cm 2.
3. ProfMIC-217 addition of
10% (V/V) of the inactivated cells were added to the stimulated HaCaT cells (control was replaced with equal volume of PBS). Each group was run in parallel at 37 ℃ overnight.
4. QPCR method for detecting relative expression fold of extracellular matrix/autophagy/degradation extracellular matrix/inflammatory factor related genes
After the cells are discarded from the culture medium, lysate is added to extract total RNA of the cells, the concentration and purity of the RNA are detected, then the RNA is reversely transcribed into cDNA, GAPDH is used as an internal reference gene, real-time qPCR is adopted to detect the expression of extracellular matrix related genes COL1A1, TIMP1, SPTSSA and SMAD3, the autophagy related gene LC3B is used to degrade the expression of extracellular matrix related genes MMP1 and inflammatory factor related genes TNF-alpha. The F value of each sample was calculated using the 2 -ΔΔCT method with the relative expression fold f=1 for the control group genes.
Formula f=2 -ΔΔCT, wherein:
△CT Experiment ＝CT Experiment -CT Internal reference ( Experiment );
△CT Control ＝CT Control -CT Internal reference ( Control );
△△CT=△CT Experiment -△CT Control .
the method comprises the steps of inactivating thalli to up-regulate extracellular matrix genes COL1A1 TIMP1, SPTSSA and SMAD3, up-regulate autophagy gene LC3B, down-regulate degradation extracellular matrix gene MMP1, and down-regulate inflammatory factor related gene TNF-alpha. The results are shown in the following table:
The results show that ProfMIC-217 has the anti-aging effects of promoting the synthesis of the extracellular matrix of HaCaT, promoting autophagy of cells, reducing degradation of the extracellular matrix and inflammatory factors.
EXAMPLE 5 ProfMIC-217 promotes proliferation of human fibroblast HFF
1. ProfMIC-217 supernatant and inactivated bacteria preparation:
And (3) picking single colony of lactobacillus brevis ProfMIC-217 in an MRS liquid culture medium, standing and culturing for 16-18 h in a 37 ℃ incubator, detecting by using an enzyme-labeling instrument, diluting and adjusting OD 600 = 0.2,121 ℃ by using PBS, inactivating at high pressure for 30min, centrifuging for 2min at 12000 r, and filtering to obtain a supernatant through a 0.22 mu m filter membrane. The pellet was resuspended in an appropriate amount of PBS and diluted to adjust OD 600 =0.2 to inactivate the cells.
2. HFF cell preparation and ProfMIC-217 addition
HFF cells cultured in DMEM were seeded, transferred to 96-well plates at 100 ul/well (3X 10 4 cells per well), and cultured overnight until the cells attached. 200 mu M H 2O2 was prepared, 100. Mu.l of each well was added and incubated at 37℃in a 5% carbon dioxide incubator for 1 hour. The original medium was discarded, PBS was washed twice, 100. Mu.l of fresh medium was added, 5% (V/V) of supernatant and 10% (V/V) of inactivated cells were added to each well, respectively, (the control group replaced supernatant/inactivated cells with equal volume of PBS). Culturing for 24h. Mu.l of CCK-8 solution was added to each well, and incubated for 4 hours, and the absorbance at 450nm was measured for brightness A.
The calculation formula and the results are shown in the following table:
the results show that adding ProfMIC-217 has the effect of promoting proliferation of HFF cells.
EXAMPLE 6 ProfMIC-217 mediated oxidative damage HFF extracellular matrix/antioxidant/immunomodulatory factor-related Gene expression experiments
1. ProfMIC-217 supernatant and inactivated bacteria preparation:
The preparation is described in example 5.
2. HFF cell preparation and H 2O2 induced oxidative damage
After digestion of DMEM-cultured HFF cells, 0.5 ml/well (containing 2X 10 5 cells) was inoculated into 24-well plates and incubated overnight in a 5% carbon dioxide incubator at 37 ℃. The wells were stimulated with H 2O2 at a final concentration of 200. Mu.M and allowed to stand at 37℃for 1H.
3. ProfMIC-217 addition of
5% (V/V) of the supernatant and 10% (V/V) of the inactivated cells were added to the stimulated HFF cells, respectively (the control group replaced the supernatant/the inactivated cells with equal volumes of PBS, respectively). Each group was run in parallel at 37 ℃ overnight.
4. QPCR method for detecting relative expression fold of extracellular matrix/antioxidant/immune regulator related genes
After the cells are discarded from the culture medium, lysate is added to extract total RNA of the cells, the concentration and purity of the RNA are detected, then the RNA is reversely transcribed into cDNA, GAPDH is used as an internal reference gene, and real-time qPCR is adopted to detect the expression of extracellular matrix related genes LN and FN, antioxidant related genes SIRT-1, SIRT-3 and PTEN and an immune regulator related gene MOR. The F value of each sample was calculated using the 2 -ΔΔCT method with the relative expression fold f=1 for the control group genes.
The supernatant up-regulates extracellular matrix related gene FN and antioxidant related gene SIRT-3. The results are shown in the following table:
The cell up-regulating extracellular matrix related gene LN, antioxidant related genes SIRT-1, SIRT-3 and PTEN and the immune regulator related gene MOR are inactivated. The results are shown in the following table:
The results show that the addition of ProfMIC-217 has anti-aging effects of promoting synthesis of HFF extracellular matrix, increasing antioxidant and increasing cellular immune regulator.
EXAMPLE 7 ProfMIC-217 preparation example of supernatant infusion
ProfMIC-217 supernatant granule is prepared, and the formula is shown in the following table
| Raw materials | Mass ratio (%) |
| ProfMIC-217 fermented powder | 40.0 |
| Red bean powder | 39.0 |
| Cocoa powder | 20.6 |
| Silica dioxide | 0.4 |
The preparation method comprises collecting ProfMIC-217 supernatant prepared in example 5, spray drying to obtain ProfMIC-217 fermented powder, adding semen Phaseoli powder, cocoa powder, and silicon dioxide, stirring, and dispersing to obtain granule. The ProfMIC-217 granule is convenient to carry, has natural taste and good palatability after being infused with water, and can be used as oral beverage for caring skin.
EXAMPLE 8 ProfMIC-217 preparation example of inactivated bacterial essence
ProfMIC-217 preparation of inactivated bacteria essence water, the composition is shown in the following table
The preparation method comprises heating ProfMIC-217 inactivated bacteria liquid prepared in example 5 to 50deg.C, adding glycerol, 1, 3-butanediol, phenoxyethanol and vanillin, stirring and dispersing until clear, and cooling to 35deg.C. The obtained ProfMIC-217 inactivated thallus essence water has fresh smell, can be used for suspending thallus by shaking before use, and has the effects of lubricating and non-sticky feeling when being smeared on skin.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
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