CN113897300B - Bifidobacterium animalis for improving skin barrier function injury and skin sensitivity - Google Patents
Bifidobacterium animalis for improving skin barrier function injury and skin sensitivity Download PDFInfo
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- A61K8/00—Cosmetics or similar toiletry preparations
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- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
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Abstract
The invention discloses bifidobacterium animalis for improving skin barrier function injury and skin sensitivity, belonging to the technical field of microorganisms and the technical field of medicines. The invention provides a bifidobacterium animalis CCFM1160, which can relieve the damage of a human cell barrier, and is specifically characterized in that: (1) significantly improving the survival rate of SDS-induced HaCaT cells; (2) The barrier damage caused by SDS induced HaCaT cells is remarkably improved; (3) the ability to significantly promote cell migration; (4) Can effectively promote the expression of natural moisturizing factors and barrier integrity factors AQP-3, so that the bifidobacterium animalis CCFM1160 has great application prospect in preparing products for preventing or treating skin barrier injury.
Description
Technical Field
The invention relates to an animal bifidobacterium strain capable of improving skin barrier function injury and skin sensitivity, belonging to the technical field of microorganisms and the technical field of medicines.
Background
In recent years, the incidence of sensitive skin is gradually increased, the living quality of people is seriously affected, and the people are paid attention to the sensitivity. According to studies, skin barrier dysfunction is a major cause of sensitive skin development. Skin barrier dysfunction easily causes skin irritation to cause symptoms such as redness, tingling, itching and the like, and the symptoms frequently recur, which very puzzles the life of patients.
The definition of sensitive skin is not unified at present, and in general, sensitive skin is more susceptible to external stimulus than normal skin to generate high sensitivity and high response, and is poor in skin tolerance against external changes, so sensitive skin can be summarized as a highly sensitive skin state. However, the cause of the sensitive skin is unknown, and the subjective feeling of the patient is mainly the cause, and the clinical signs are unknown, but in summary, the sensitive skin has three major characteristics: high sensitivity, high reactivity and poor tolerance.
In general, factors causing sensitive skin are roughly classified into two types: endogenous factors and exogenous factors. Endogenous factors include sex, age, race skin conditions, etc. Exogenous factors are varied, such as environmental factors, use of chemical products, unhealthy lifestyles, etc. Environmental factors include contaminated air, intense ultraviolet radiation or prolonged exposure to the sun, which can cause sensitive skin. In addition, irritants contained in some fresheners and the like used in people's daily lives may also cause sensitive skin. Also, sensitive skin is easily induced by long-term eating of some irritating foods in life.
Skin barrier damage mechanism current research considers that the occurrence of sensitive skin is caused by the combined action of internal and external factors, so that the skin barrier function is damaged, sensory nerve afferent signals are increased, and the responsiveness of the skin to external stimulus is enhanced. The skin barrier function is damaged, the structural integrity of the epidermis horny layer of the skin is damaged, the lipid content among epidermis cells is unbalanced, and the water content of the horny layer is reduced, so that the sensitivity of the skin to external stimulus is enhanced. In addition, ambient temperature is also an important factor in initiating or aggravating sensitive skin, since too low or too high a temperature of the skin surface delays the rate of repair of the skin barrier.
In addition to medication and surgery, skin barrier repair therapy is currently becoming a new research direction for sensitive skin, and more scholars try to research the appearance and progress of skin diseases from the aspect of skin barrier pathology and molecules. Although there are few studies on drugs for treating various kinds of skin diseases, dependence and side effects are brought about due to the complex mechanism of action of the drugs.
Therefore, there is still a need to develop a natural non-irritating product to enhance skin barrier function, improve skin defenses, reduce the occurrence of sensitive skin disorders, promote the efficacy of skin external drugs and the efficacy of skin care products.
Disclosure of Invention
The technical scheme of the invention is as follows:
in order to solve the problems, the invention provides a bifidobacterium animalis (Bifidobacterium animalis) CCFM1160 which is preserved in the Guangdong province microorganism strain preservation center, the preservation number is GDMCC No.61500, the preservation date is 2021, 2 months and 4 days, and the preservation address is Guangzhou Miao 100 institute of China, no. 59 building 5.
The bifidobacterium animalis is derived from human excrement samples, the strain is subjected to sequencing analysis, the 16S rDNA sequence of the strain is shown as SEQ ID NO.1, the sequence obtained by sequencing is subjected to nucleic acid sequence comparison in GeneBank, and the result shows that the strain is bifidobacterium animalis (Bifidobacterium animalis) and is named as bifidobacterium animalis GDMCC No.61500.
The microbial morphological characteristics of the bifidobacterium animalis CCFM1160 are: the polymorphic bacillus is Y-shaped, V-shaped, bent and spatula-shaped, and has the morphological characteristics of bifurcated bacillus. The colony is smooth, convex and round, the edge is complete, the cream is white, and the cream is flashing and has soft texture.
The invention also provides a composition, which is characterized by comprising one or more of cells, fermentation supernatant and intracellular matters of the bifidobacterium animalis.
In one embodiment, the intracellular material is the supernatant of the disrupted bifidobacterium animalis.
In one embodiment of the invention, the method of preparing the cell is: inoculating the bifidobacterium animalis CCFM1160 into a proliferation culture medium for fermentation, centrifuging the obtained bifidobacterium animalis fermentation liquid under the conditions of 8000-10000g and 15-20 min, and collecting cells of the bifidobacterium animalis.
In one embodiment of the invention, the preparation method of the fermentation supernatant of the bifidobacterium animalis comprises the following steps: inoculating the bifidobacterium animalis CCFM1160 into a proliferation culture medium for fermentation, centrifuging the obtained bifidobacterium animalis fermentation liquid under the conditions of 8000-10000g and 15-20 min, filtering and sterilizing by a 0.22-0.45 mu m filter membrane, and collecting fermentation supernatant of the bifidobacterium animalis.
In one embodiment of the present invention, the method for preparing the intracellular material of bifidobacterium animalis comprises the steps of: inoculating the bifidobacterium animalis CCFM1160 into a proliferation culture medium for fermentation, centrifuging the obtained bifidobacterium animalis fermentation liquid under the conditions of 8000-10000g and 15-20 min, adding a proper amount of PBS, repeatedly grinding by using liquid nitrogen until powder is formed, adding a proper amount of PBS, performing ultrasonic treatment with 100-500W for 5-15 min, centrifuging under the conditions of 8000-10000g and 15-20 min, filtering and sterilizing by a 0.22-0.45 mu m filter membrane, and collecting supernatant to obtain the intracellular matters of the bifidobacterium animalis.
In one embodiment, the viable count of the fermentation liquid is 4 to 6×10 9 cfu/mL。
In one embodiment of the invention, the carbon source in the proliferation culture medium is 10-40 g/L glucose, the nitrogen source is 5-25. 25g/L yeast extract powder, tryptone or soytone, the inorganic salt is 0.1-0.5 g/L magnesium sulfate, 0.1-0.5 g/L manganese sulfate, the trace elements are 1-5 g/L anhydrous sodium acetate, 1-5 g/L diammonium hydrogen citrate, 1-5 g/L potassium dihydrogen phosphate, 0.25-0.75 g L-cysteine hydrochloride and tween 80 0.5-1.5 mL/L.
The invention also provides application of the bifidobacterium animalis CCFM1160 in preventing or treating skin barrier injury.
The invention also provides the application of the composition in preventing or treating the injury of skin barrier.
The beneficial effects are that:
the invention provides a bifidobacterium animalis (Bifidobacterium animalis) CCFM1160, which can relieve and alleviate skin cell barrier damage, and is specifically characterized in that:
(1) The survival rate of SDS-induced HaCaT cells is remarkably improved;
(2) The barrier damage caused by SDS induced HaCaT cells is remarkably improved;
(3) The ability to significantly promote cell migration;
(4) Can effectively promote the expression of natural moisturizing factors and barrier integrity factors AQP-3,
therefore, the bifidobacterium animalis (Bifidobacterium animalis) has great application prospect in preparing products for preventing or treating skin barrier injury.
Preservation of biological materials
A bifidobacterium animalis (Bifidobacterium animalis) CCFM1160 was deposited at the microorganism strain collection in Guangdong province on 2 nd month 4 of 2021 under the accession number GDMCC No.61500 and deposited at floor 5 of the national institute of Mitrex 100 in Guangzhou City.
Drawings
Fig. 1: effect comparison of different groups to improve SDS-induced HaCaT cell survival.
Fig. 2: comparison of the morphological effects of different groups on HaCaT cells.
Fig. 3: the effects of different groups on promoting cell migration capacity are compared.
Fig. 4: gene expression levels of AQP-3 in different groups.
Detailed Description
The skin is covered on the surface of the human body and is one of the largest organs of the human body. The skin is composed of two parts, namely epidermis and dermis, wherein the key function of the epidermis is to form a barrier between an organism and the outside, and the skin has an important protective effect on a human body. Skin Keratinocytes (KCs) are the major components of the epidermis, whereas human immortalized cortical forming cells (HaCaT cells) belong to a cell line spontaneously transformed by adult epidermal cells and have the same proliferation, differentiation characteristics and genetic stability as normal keratinocytes of the human body, and are commonly used in the biomedical field as cell lines for in vitro studies such as skin repair healing, sun protection and anti-aging. Also, keratinocytes are an important component in the study of oxidative damage to skin. Thus, cell experiments were performed using SDS-induced human immortalized cortical forming cells as a barrier damaging cell model in the following examples.
Human immortalized cortical forming cells (HaCaT cells) referred to in the examples below were purchased from the chinese collection of typical cultures; DMEM medium referred to in the examples below was purchased from sameimer femto instruments limited; fetal Bovine Serum (FBS), penicillin, streptomycin and trypsin referred to in the examples below were purchased from sigma aldrich (Shanghai) trade company, inc.; the MTT referred to in the examples below was purchased from beijing solibao technologies limited; the RNA kit (product number: DP 419) referred to in the following examples was purchased from Shanghai Tiangen Biotechnology Co., ltd; PCR kit (cat# q 711-02) was purchased from Northenan Biotechnology Co., ltd.
The following examples relate to the following media:
MRS liquid medium: 10g/L peptone, 10g/L beef extract, 20g/L glucose, 2g/L sodium acetate, 5g/L yeast powder, 2g/L, K diammonium hydrogen citrate 2 PO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 0 0.1g/L、MnSO 4 0.05g/L Tween 80:1ml/L, pH 7.0.
The formula of the fermentation medium of bifidobacterium animalis No.1 comprises the following components: glucose: 20g; yeast soaking powder: 10g; na (Na) 2 HPO 4 ：3.5g；MgSO 4 -7H 2 O：0.25g；MnSO 4 -H 2 O:0.1g; diammonium hydrogen citrate: 2g; KH (KH) 2 PO 4 :3.5g; l-cysteine hydrochloride: 0.5g; tween 80:1mL; the pH was adjusted to 6.8.
The formula of the fermentation medium of bifidobacterium animalis No. 2 comprises the following components: glucose: 20g; tryptone (tryptone): 15g; na (Na) 2 HPO 4 ： 3g；MgSO 4 -7H 2 O：0.25g；MnSO 4 -H 2 O:0.1g; diammonium hydrogen citrate: 2g; KH (KH) 2 PO 4 :3g; l-cysteine hydrochloride: 0.5g; tween 80:1mL; the pH was adjusted to 6.8.
The preparation methods of the bifidobacterium animalis culture broth and intracellular material involved in the following examples were as follows:
1. activation and culture of strains
All containers and tools were sterilized. And (3) after the strain preservation tube is uniformly vibrated, a small amount of bacterial liquid is dipped in a sterile inoculating loop, and the bacterial liquid is streaked and purified in an MRS solid culture medium. After standing for a period of time, the plates were placed in an anaerobic incubator at 37℃for 48h. After the culture is finished, single colony is selected and inoculated into 5mL of MRS liquid culture medium, and the culture is kept in a constant temperature incubator at 37 ℃ for 12-18 hours. Obtaining the seed liquid of the strain. Two successive generations of activation were performed with an inoculum size of 2%. Inoculating the seed solution into MRS liquid culture medium with 2% inoculum size, and culturing in incubator at 37deg.C for 12-18 hr. The procedure was repeated 1 time (note: bifidobacteria were cultured in anaerobic conditions).
The MRS culture medium can be replaced by a bifidobacterium animalis No.1 fermentation culture medium or a bifidobacterium animalis No. 2 fermentation culture medium, and the culture conditions are the same as those of the method.
2. Preparation of external probiotic samples
(1) Probiotic fermented supernatant: centrifuging 8000r of cultured probiotics for 15min to obtain supernatant; adjusting the pH to 7.2-7.4; filtering with 0.22 μm disposable filter, sterilizing, packaging, and storing in refrigerator at-20deg.C.
(2) Probiotic intracellular: taking 10mL of cultured probiotics, centrifugally collecting bacterial sludge, and adding 1mL of PBS solution for dissolution; adding liquid nitrogen, grinding and crushing, and collecting bacterial mud suspension; adding 1mL of PBS solution for dissolution and ultrasonic crushing; centrifuging at 8000r for 10min, and collecting supernatant; a disposable filter of 0.22 μm was used for sterilization, and the resulting mixture was stored in a refrigerator at-20℃after packaging (0.1 g of wet bacterial sludge gave 1mL of intracellular supernatant).
Complete medium: 5% Fetal Bovine Serum (FBS), 100U/mL penicillin, 100mg/mL streptomycin, 95% DMEM medium.
Example 1: screening and identification of strains
The method comprises the following specific steps:
1. screening
Taking human body excrement from Jiangsu tin-free areas as a sample, pretreating the sample, storing the sample in a refrigerator at the temperature of minus 80 ℃ in 20% glycerol, taking out the sample for thawing, uniformly mixing the sample, absorbing 0.5mL of the sample, adding the sample into 4.5mL of the sample, carrying out gradient dilution by using 9g/L of physiological saline containing 0.05g/L of cysteine, selecting proper gradient diluent to be coated on an MRS solid culture medium, culturing the sample at the temperature of 37 ℃ for 48 hours, picking a typical colony of bifidobacterium animalis, streaking and purifying the typical colony on the MRS solid culture medium, picking a single colony, transferring the single colony to the MRS liquid culture medium (containing 0.05g/L of cysteine) for enrichment, and storing 30% glycerol to obtain a strain; wherein, the typical colony of the bifidobacterium animalis is milky white, irregular, round convex and smooth.
2. Authentication
The genome of the strain CCFM1160 was extracted, the 16S rDNA of the strain CCFM1160 was amplified and sequenced (the nucleotide sequence of the 16S rDNA of the strain CCFM1160 is shown as SEQ ID NO.1 by Jin Weizhi Biotechnology Co., ltd., suzhou) and the sequence was subjected to nucleic acid sequence alignment in NCBI, which revealed that the strain CCFM1160 was bifidobacterium animalis and was named as bifidobacterium animalis (Bifidobacterium animalis) CCFM1160.
Example 2: effect of Bifidobacterium animalis on SDS-induced HaCaT cell survival
The method comprises the following specific steps:
after the frozen HaCaT cells are rewarmed at 37 ℃, the cells are centrifuged at 1000rpmTaking out precipitate after 5 min; after washing the sediment once with the complete medium, re-suspending the cells with the complete medium and counting to obtain a cell re-suspension; inoculating the cell heavy suspension into 10cm culture dish, and gas phase containing 5% (v/v) CO at 37deg.C 2 The culture in the cell incubator of (2) was continued by replacing the complete medium the next day at 37℃with a gas phase containing 5% (v/v) CO 2 Is cultured in a cell culture incubator. Cells were passaged when grown to 70% -80% density of the dishes.
Selecting HaCaT cells with good growth state, digesting the HaCaT cells by trypsin with the concentration of 2.5g/L, centrifuging, and re-suspending by using a complete culture medium, and performing cell counting to obtain a cell re-suspension; the cell resuspension was followed by 5X 10 3 Inoculating each well into a 96-well culture plate, wherein each well is 100 mu L; the 96-well culture plate was placed at 37℃and the gas phase contained 5% (v/v) CO 2 Culturing for 24 hours in a cell culture box; cells were treated with a complete medium containing 50. Mu.M SDS for 24h.
Setting a normal control group, an animal bifidobacterium MRS culture medium fermentation liquor experimental group D1, an animal bifidobacterium No.1 fermentation culture medium fermentation liquor experimental group D2, an animal bifidobacterium No. 2 fermentation culture medium fermentation liquor experimental group D3, an animal bifidobacterium live bacteria experimental group D4, an animal bifidobacterium intracellular matter experimental group D5, a short bifidobacterium R1 experimental group and a long bifidobacterium R2 experimental group.
Culturing Bifidobacterium animalis with MRS culture medium, bifidobacterium animalis No.1 fermentation culture medium or Bifidobacterium animalis No. 2 fermentation culture medium until the bacterial concentration reaches 6×10 9 CFU/mL, centrifuged and the supernatant collected, recorded as incubation supernatants of test groups D1, D2 and D3, respectively; selecting fermentation liquor of bifidobacterium animalis cultured by MRS culture medium, centrifuging to obtain viable bacteria, and resuspending the viable bacteria with complete culture medium until the bacterial concentration reaches 6×10 9 CFU/mL, designated as incubation supernatant for experimental group D4; selecting fermentation liquor of lactobacillus plantarum cultured by using an MRS culture medium, centrifuging to obtain living bacteria, preparing an intracellular matter, and recording the intracellular matter as an incubation supernatant of an experimental group D5; culturing Bifidobacterium breve experimental group R1 and Bifidobacterium longum experimental group R2 with MRS culture medium until the bacterial concentration reaches 6X10 9 CFU/mL, centrifuged and the supernatant collected, recorded as incubation supernatants for test groups R1 and R2, respectively.
The culture supernatants of D1, D2, D3, D4, D5 and R1, R2 were mixed with complete medium (5%: 95%), respectively, and 100. Mu.L of each was aspirated and cultured for 24h, respectively, of SDS-treated HaCaT cells. Control groups were supplemented with 100 μl of complete medium, each group was provided with 5 duplicate wells, and the MTT assay was used to measure cell viability.
As can be seen from fig. 1, the proliferation survival rate of the normal control group was 62.30%, the proliferation of cells was significantly promoted by bifidobacterium animalis (P < 0.001), the survival rate of cells in bifidobacterium animalis MRS medium broth experimental group D1 was 83.53%, the survival rate of cells in bifidobacterium animalis No.1 fermentation medium broth experimental group D2 was 80.48%, the survival rate of cells in bifidobacterium animalis No. 2 fermentation medium broth experimental group D3 was 78.32%, the survival rate of cells in bifidobacterium animalis live bacteria experimental group D4 was 78.90%, the survival rate of cells in bifidobacterium animalis intracellular experimental group D5 was 69.48%, and the proliferation effect was better than that of bifidobacterium breve R1 (cell survival rate= 47.23%), bifidobacterium longum (cell survival rate=48.56%).
Compared with bifidobacterium breve R1 and bifidobacterium longum R2, the bifidobacterium animalis can remarkably promote the growth of HaCaT cells and relieve the damage of cell barriers.
Example 3: influence of bifidobacterium animalis on HaCaT cell morphology
The method comprises the following specific steps:
re-warming the frozen HaCaT cells at 37 ℃, centrifuging at 1000rpm for 5min, and taking a precipitate; after washing the sediment once with the complete medium, re-suspending the cells with the complete medium and counting to obtain a cell re-suspension; inoculating the cell heavy suspension into 10cm culture dish, and gas phase containing 5% (v/v) CO at 37deg.C 2 The culture in the cell incubator of (2) was continued by replacing the complete medium the next day at 37℃with a gas phase containing 5% (v/v) CO 2 Is cultured in a cell culture incubator. Cells were passaged when grown to 70% -80% density of the dishes.
Selecting HaCaT cells with good growth state, digesting the HaCaT cells with trypsin with the concentration of 2.5g/L, centrifuging, re-suspending with DMEM culture medium, and performing cell counting to obtain cell re-suspension; the cell resuspension was followed by 5X 10 6 Inoculating the culture medium into 6 holesIn the plates, 1mL per well, 6-well plates were placed at 37℃in a gas phase containing 5% (v/v) CO 2 Culturing for 24 hours in a cell culture box; cells were treated with a complete medium containing 50. Mu.M SDS for 24h.
A normal control group, an animal bifidobacterium D1, a bifidobacterium breve R1 experimental group, and a bifidobacterium longum R2 experimental group were set (fig. 2). The incubation supernatants of bifidobacterium animalis D1, R1 and R2 were each mixed with complete medium (5%: 95%) using the same concentration of incubation supernatant as in example 2, and 2mL each aspirated and cultured separately SDS-treated HaCaT cells for 24h. The normal control group was supplemented with 2mL of complete medium.
As can be seen from FIG. 2, after the HaCaT cells of the normal control group are cultured for 24 hours, the cells grow vigorously on the wall under a microscope, adjacent cells grow and fuse into sheets, the cytoplasm is full, the cell outline is clear, the cell shape is fusiform or polygonal, and the cell is well spread; after the bifidobacterium animalis CCFM1160 is added, the HaCaT cells are observed to grow well on the wall after 24 hours, and the cells are in a fusiform shape or a polygonal shape, can grow in a clustered shape, have large nuclei and high nuclear-mass ratio, slightly increase the suspension of the HaCaT cells in the bifidobacterium breve R1 experimental group and the bifidobacterium longum R2 experimental group, and have the phenomena of cell shrinkage and rounding.
Therefore, the bifidobacterium animalis can maintain the normal form of the HaCaT cells, increase the cell number and enhance the cell activity, and has the promotion effect on the growth of the HaCaT cells.
Example 4: effect of Bifidobacterium animalis on HaCaT cell migration ability
The method comprises the following specific steps:
re-warming the frozen HaCaT cells at 37 ℃, centrifuging at 1000rpm for 5min, and taking a precipitate; after washing the sediment once with the complete medium, re-suspending the cells with the complete medium and counting to obtain a cell re-suspension; inoculating the cell heavy suspension into 10cm culture dish, and gas phase containing 5% (v/v) CO at 37deg.C 2 The culture in the cell incubator of (2) was continued by replacing the complete medium the next day at 37℃with a gas phase containing 5% (v/v) CO 2 Is cultured in a cell culture incubator. Cells were passaged when grown to 70% -80% density of the dishes.
Firstly, a ruler is marked on the back surface of a 24-hole plate, two transverse parallel straight lines are marked along the ruler by a marker pen, and the distance between the straight lines is 0.5-1 cm. Selecting HaCaT cells with good growth state, digesting the HaCaT cells with trypsin with the concentration of 2.5g/L, centrifuging, re-suspending with DMEM culture medium, and performing cell counting to obtain cell re-suspension; haCaT cells were plated at 6X 10 cells per well 5 Inoculating the individual cells into a 24-well plate, and culturing for 48 hours to enable the cells to achieve 100% fusion; after 24h of treatment of cells with complete medium containing 50. Mu.M SDS, calibration was performed with a ruler, and a 200. Mu.L tip was used to scratch the bottom of the plate (using the same tip, keeping the tip perpendicular to the bottom surface, one pass) along the pre-marked parallel lines at the bottom of the 24-well plate, after light washing with PBS, fresh serum-free medium containing the sample was replaced, and photographs were observed under a microscope (quick completion, reduction of cell stress) according to the marker pen marked position, and a normal control group, a bifidobacterium animalis experimental group D1, a bifidobacterium breve R1 experimental group, and a bifidobacterium longum R2 experimental group were set (FIG. 2). Normal control group was added with 200 μl of complete medium, the experimental group used the same concentration of incubation supernatant as in example 2, the incubation supernatants of bifidobacterium animalis experimental groups D1, R1 and R2 were mixed with complete medium (5%: 95%), and 200 μl of SDS-treated HaCaT cells were each aspirated and cultured for 48h, the cells were washed with serum-free medium, and the same visual field observations as before were selected and photographed according to marker pen-labeled positions.
As shown in fig. 3, compared with the normal control group, the relative width of the scratch after the treatment of bifidobacterium animalis D1 is reduced, the cell migration rate is increased, the scratch healing rate is (49.57 ±0.85)%, and the cell migration capability is obviously improved; the relative width of scratches after the treatment of the bifidobacterium breve R1 and the bifidobacterium longum R2 is wider, the cell migration rate is reduced, the cell scratch healing rate is reduced to (35.41 +/-0.96)%, and (38.23+/-1.04)%, respectively, and the cell migration is inhibited.
Example 5: effect of Bifidobacterium animalis on HaCaT cell AQP-3mRNA levels
The method comprises the following specific steps:
re-warming the frozen HaCaT cells at 37 ℃, centrifuging at 1000rpm for 5min, and taking a precipitate; the sediment is used in complete culture mediumAfter one washing, the cells are resuspended in complete medium and counted to obtain a cell resuspension; inoculating the cell heavy suspension into 10cm culture dish, and gas phase containing 5% (v/v) CO at 37deg.C 2 The culture in the cell incubator of (2) was continued by replacing the complete medium the next day at 37℃with a gas phase containing 5% (v/v) CO 2 Is cultured in a cell culture incubator. Cells were passaged when grown to 70% -80% density of the dishes.
Selecting HaCaT cells with good growth state, digesting the HaCaT cells with trypsin with the concentration of 2.5g/L, centrifuging, re-suspending with DMEM culture medium, and performing cell counting to obtain cell re-suspension; haCaT cells were plated at 5X 10 cells per well 6 Inoculating the individual cells into a 6cm cell culture dish for culturing for 24 hours; cells were treated with a complete medium containing 50. Mu.M SDS for 24h. .
A normal control group, an animal bifidobacterium experimental group, a bifidobacterium breve R1 experimental group, and a bifidobacterium longum R2 experimental group were set (fig. 2). Using the same concentration of incubation supernatants as in example 2, the incubation supernatants of D1, D2, D3, D4, R1 and R2 were each mixed with complete medium (5%: 95%) and 7mL of each aspirated, each of which was used to culture SDS-treated HaCaT cells for 24h. Normal control groups were supplemented with 7mL of complete medium.
Collecting cells, extracting total RNA, measuring RNA concentration, reverse transcribing, and storing in a refrigerator at-20deg.C for use. The Q-PCR method detects the expression level of the AQP-3 gene.
Primer name
AQP-3-F：5'-AGGTGGACCCAGAAGTGAGT-3'；
AQP-3-R：5'-CCCCAGTCAAGGGTCATAGC-3'。
human-Actin-F：5'-GTGACGTTGACATCCGTAAAGA-3'；
human-Actin-R：5'-GCCGGACTCATCGTACTCC-3'。
Q-PCR: for detailed steps, refer to the PCR kit instructions.
As can be seen from FIG. 4, the relative expression level of AQP-3mRNA in HaCaT cells cultured by adding bifidobacterium animalis is significantly increased by about 1.6-2 times as compared with the normal control group under the same culture time. The bifidobacterium animalis can obviously improve the relative expression quantity of the AQP-3 genes of the HaCaT human keratinocytes, which indicates that the bifidobacterium animalis can promote the expression of the AQP-3 of the skin and improve the barrier function and the moisturizing capability of the skin.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of Jiangnan
<120> A bifidobacterium animalis strain with improved skin barrier function damage and skin sensitivity
<130> BAA210511A
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 434
<212> DNA
<213> artificial sequence
<400> 1
cgaacgggtc ccttgaccgg gtcagtcaca ccggcggcca gcaggtcctc gtaggtgtcg 60
gtggcggcgt tgaagccttc cccatcgggc agagaacgaa ccttgttgat caccacgtcg 120
ccggacacgc tgcgttctcg gcaatctgct tgatcggggc ttcgatggcg cggaacacga 180
tggcggcacc ggtagcctct tcgccggtca gggaggtgac ggcctcaccc ttctcggcct 240
tggcagcagc ctgaacgagg gccacgccac cgccgggcag caggccttcc tcaatggcgg 300
ccttggcgtt acgcacggca tcttcgatgc ggtgcttgcg ctccttggcc tcgacctcgg 360
tgacagcgcc gaccttgatg acagccacgc cgccggccag cttggccaga cgctcctgca 420
gttccttcac ggaa 434
Claims (9)
1. Bifidobacterium animalis @Bifidobacteriumanimalis) CCFM1160, deposited on 2.4.2021 with the microorganism strain of Guangdong provinceThe center, accession number is GDMCC No.61500.
2. A composition comprising one or more of the cells, intracellular material or fermentation supernatant of bifidobacterium animalis CCFM1160 of claim 1.
3. The composition of claim 2, wherein the intracellular material is the disrupted supernatant of bifidobacterium animalis of claim 1.
4. The composition of claim 2, wherein the cells are prepared by a method comprising: inoculating the bifidobacterium animalis of claim 1 into a culture medium for fermentation, centrifuging the obtained fermentation liquid, and collecting bifidobacterium animalis cells.
5. The composition of claim 2, wherein the fermentation supernatant is prepared by a process comprising: inoculating the bifidobacterium animalis of claim 1 into a culture medium for fermentation, centrifuging the obtained fermentation liquor, removing solids, and collecting fermentation supernatant of the bifidobacterium animalis.
6. The composition of claim 2, wherein the intracellular material is prepared by a process comprising: inoculating the bifidobacterium animalis of claim 1 into a culture medium for fermentation, centrifuging the obtained fermentation liquid, collecting and breaking cells, centrifuging to remove solids, and collecting the intracellular matters of the bifidobacterium animalis.
7. The composition of any one of claims 2 to 6, wherein the viable count of the fermentation broth is up to 4 to 6 x 10 9 cfu/mL。
8. The composition of any one of claims 4 to 6, wherein the carbon source in the culture medium is 10 to 40g/L glucose, the nitrogen source is 5 to 25g/L yeast extract, tryptone or soy peptone, the inorganic salt is 0.1 to 0.5g/L magnesium sulfate, 0.1 to 0.5g/L manganese sulfate, the trace elements are 1 to 5g/L anhydrous sodium acetate, 1 to 5g/L diammonium hydrogen citrate, 1 to 5g/L potassium dihydrogen phosphate and 800.5 to 1.5mL/L tween.
9. Use of bifidobacterium animalis as claimed in claim 1 in the manufacture of a medicament for the prophylaxis or treatment of skin barrier damage.
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