Bifidobacterium for promoting ceramide synthesis and application thereof

Abstract

The present invention relates to the field of microbial fermentation technology, and in particular to bifidobacteria that promote ceramide synthesis and applications thereof. The present invention screens and obtains a strain of Bifidobacterium bifidum JWA68 with a preservation number of CCTCC NO: M 20221852, and its fermentation filtrate can significantly promote epidermal cells to synthesize ceramide, while inhibiting epidermal cells from secreting vascular endothelial growth factor (VEGF), thereby achieving the effect of repairing the skin barrier function and alleviating the vascular hyperreaction of the skin. The strain is used in daily chemical products for sensitive skin, and has the advantages of low cost, safety, reliability, and convenience for industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, in particular to bifidobacteria for promoting ceramide synthesis and application thereof. Background Art

[0002] Improper skin care processes, unqualified skin care products, as well as life stress, staying up late, overeating or environmental pollution can all lead to sensitive skin problems, thus turning people into sensitive skin people. According to a survey, about 46% of Chinese women have experienced varying degrees of skin sensitivity. Sensitive skin refers to the state of high reactivity of the skin to the external environment under physiological or pathological conditions. Clinically, the skin shows subjective symptoms such as burning, stinging, itching and tightness when stimulated by physical, chemical, mental and other factors. Under the interaction of internal and external factors, the skin of sensitive skin people will cause damage to the skin barrier function, increase the afferent signals of sensory nerves, increase the skin's reactivity to external stimuli and the skin's immune inflammatory response. The skin may look normal or accompanied by erythema, which is characterized by low barrier function or damaged barrier.

[0003] Ceramide, also known as N-acyl sphingosine, is the basic unit of sphingomyelin, a component of cell membrane, and is also one of the important biologically active substances in the body. Among them, intercellular lipids are components of the stratum corneum skin barrier, which are composed of a heterogeneous mixture of ceramide (45% to 55%), free fatty acids (15% to 20%) and cholesterol (20% to 25%). Ceramide plays a very important role in maintaining the physical barrier function of the skin. Ceramide can be directly supplemented from the outside or promote the ceramide production capacity of the skin itself. The direct addition of ceramide functional ingredients in the product formula of a large number of existing daily chemical products is a method of directly supplementing ceramide. Since the high-purity ceramide (purity of more than 98%) used as a commodity can cost up to US$500 / kg, the method of supplementing ceramide from the outside has the same problem of insufficient effect sustainability as the use of other moisturizers. In some states, the skin will not fully absorb the exogenously supplemented ceramide, which will lead to its inability to fully exert its efficacy. To promote the skin's own ceramide production ability, there is no need to directly supplement ceramide from the outside. Since the ceramide is produced by the skin itself, the ceramide secreted and synthesized is also the ceramide of the regular structure possessed by the skin, which can avoid problems such as poor adaptability to the skin itself.

[0004] At present, research on how to produce substances that promote epidermal ceramide synthesis in the stratum corneum of the skin is quite popular, such as using plant extracts, such as ganoderma lucidum, Chuanxiong, etc., but due to the limited availability of natural plant resources, the manufacturing cost is high. Microbial fermentation is also commonly used to prepare ceramide synthesis promoters due to the rich active substances. For example, Japanese patent document JP09115236 discloses a ceramide synthesis promoter, which uses a bacterial culture containing nicotinic acid and / or nicotinamide as an effective ingredient. The bacterial culture can be a lactic acid bacteria culture, a bifidobacterium culture, a shiitake mushroom cell culture or a yeast culture. Part of the data in the specification shows that a bacterial culture without nicotinic acid and / or nicotinamide in the culture medium does not produce a ceramide synthesis promoter. The culture medium used in the preparation process of the promoter substantially uses nicotinic acid and / or nicotinamide as an effective ingredient, which also leads to a high preparation cost of this ceramide synthesis promoter. For example, Chinese patent document CN201110170637.1 discloses a method of culturing a microorganism of the genus Lactobacillus in a culture medium containing dregs of soybean and sugars, wherein the microorganism of the genus Lactobacillus is Lactobacillus brevis, Lactobacillus sausage, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus coryneformis, Lactobacillus curvifolius, or Lactobacillus digestus, and obtaining a ceramide production promoter and / or a glucosylceramide production promoter from the supernatant thereof. Existing microbial resources that can produce substances that promote epidermal ceramide synthesis using microbial fermentation technology are relatively few, and more low-cost, efficient superior microorganisms need to be developed to meet the demand. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide bifidobacteria that promote ceramide synthesis and applications thereof.

[0006] The present invention provides Bifidobacterium bifidum JWA68 (Bifidobacterium bifidum JWA68), and its deposit number is CCTCC NO:M 20221852.

[0007] In the present invention, the Bifidobacterium bifidum was isolated from human feces, and was identified as Bifidobacterium bifidum by MRS solid culture and 16SrDNA molecule, and was preserved in the China Center for Type Culture Collection on November 30, 2022.

[0008] The present invention provides a bacterial flora, which comprises the Bifidobacterium bifidum described in the present invention.

[0009] Furthermore, the bacterial community described in the present invention includes a bacterial community formed by a single or multiple bacteria that do not have a mutual antagonistic or competitive relationship with the Bifidobacterium bifidum, and the present invention is not limited to this.

[0010] The present invention provides a bacterial agent, which comprises the Bifidobacterium bifidum described in the present invention or the bacterial flora described in the present invention.

[0011] Furthermore, the bacterial agent of the present invention may be in the form of a powder, granule or liquid preparation, which is not limited in the present invention.

[0012] Furthermore, the bacterial agent of the present invention also includes the fermentation liquid, bacterial cells, supernatant and active substances contained therein of the Bifidobacterium bifidum, which is not limited in the present invention.

[0013] The present invention provides the use of any one of the following I) to III) in the preparation of a skin-improving product:

[0014] 1), the Bifidobacterium bifidum or its lysate, extract or metabolite according to the present invention;

[0015] II), the bacterial flora of the present invention;

[0016] III), the bacterial agent of the present invention.

[0017] In the present invention, the skin improvement includes at least one of epidermal barrier repair, sensitivity relief and / or inhibition of skin pathogens.

[0018] In the present invention, the Bifidobacterium bifidum has the effect of repairing the epidermal barrier. The repairing of the skin barrier includes repairing the skin barrier damage caused by chemical damage, oxidative damage or light damage. In the present invention, the chemical damage includes any chemical substance that can cause mild, moderate or severe epidermal cell damage to the human body, and the present invention is not limited to this.

[0019] Furthermore, the epidermal barrier repair of the present invention includes but is not limited to promoting the synthesis of cell ceramide. Ceramide is a type of phospholipid with ceramide as the skeleton, which is the main component of the cell membrane and plays an important role in maintaining the moisture balance of the stratum corneum and repairing the skin barrier.

[0020] The efficiency of the Bifidobacterium bifidum in promoting ceramide synthesis is significantly higher than that of other strains. In some embodiments of the present invention, HaCaT cells were used as subjects, and the ceramide secretion and secretion rate of different strains were measured. The test results showed that the Bifidobacterium bifidum JWA68 in the present invention promoted the ceramide secretion of HaCaT cells significantly higher than that of other isolated strains, reaching 50.27 μmol / L, and its ceramide secretion rate was 111.86%. At the same time, the ceramide secretion rate of the fermentation broth of Bifidobacterium bifidum of the present invention showed a certain concentration dependence, and the ceramide secretion rate of the fermentation broth at a concentration of 10% was the most significant.

[0021] In the present invention, the Bifidobacterium bifidum has a soothing effect on sensitivity, and the soothing effect includes but is not limited to inhibiting the expression of vascular endothelial growth factor-related genes, and the vascular endothelial growth factor-related genes are not limited to VEGF. In some embodiments of the present invention, Hacat cells are used as subjects to study the effect of the Bifidobacterium bifidum on skin soothing sensitivity. The test results show that the Bifidobacterium bifidum of the present invention can significantly reduce the expression of the VEGF gene in Hacat cells damaged by SDS, thereby achieving the effect of soothing skin sensitivity.

[0022] In the present invention, the Bifidobacterium bifidum has the effect of inhibiting skin pathogens, including but not limited to inhibiting the growth of Staphylococcus aureus. In some specific embodiments of the present invention, 10% concentration of the Bifidobacterium bifidum can significantly inhibit the growth of Staphylococcus aureus.

[0023] The present invention provides the use of the Bifidobacterium bifidum in preparing a product for promoting ceramide synthesis.

[0024] The invention provides the use of the Bifidobacterium bifidum in promoting the expression of VEGF gene in Hacat cells.

[0025] The present invention provides a skin improvement product, wherein the raw materials thereof include at least one of the following i) to iii):

[0026] i) the Bifidobacterium bifidum or its lysate, extract or metabolite according to the present invention;

[0027] ii), the bacterial flora of the present invention;

[0028] iii) the bacterial agent of the present invention.

[0029] Furthermore, the products described in the present invention include cosmetics, and the cosmetic dosage forms include: at least one of creams, emulsions, water-based agents, gels, oils, powders, loose powders, granules, muds, aerosols, patches, films, and freeze-dried agents.

[0030] The present invention also provides a method for improving skin condition, which comprises using the product of the present invention. The method of using includes applying, external application or injection, which is not limited by the present invention.

[0031] The present invention screened and obtained a strain of Bifidobacterium bifidum JWA68 with a preservation number of CCTCC NO: M 20221852. The fermentation filtrate thereof can significantly promote the synthesis of ceramide by epidermal cells, and inhibit the secretion of vascular endothelial growth factor (VEGF) by epidermal cells, thereby achieving the effects of repairing the skin barrier function and alleviating the vascular hyperreaction of the skin. The strain is used in daily chemical products for sensitive skin, and has the advantages of low cost, safety, reliability, and convenience for industrial production.

[0032] Biological Deposit Description

[0033] Bifidobacterium bifidum JWA68 (Bifidobacterium bifidum JWA68) was deposited in the China Center for Type Culture Collection on November 30, 2022, with the address: Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M20221852. DETAILED DESCRIPTION

[0034] The present invention provides bifidobacteria that promote ceramide synthesis and their applications. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0035] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0036] The present invention will be further described below in conjunction with embodiments:

[0037] Example 1 Isolation, purification and functional study of strains

[0038] 1. Isolation, purification and identification of strains

[0039] It was collected from adult fecal samples. After the collected fecal samples were gradient diluted with sterile anaerobic water, the appropriate gradient dilution was selected and spread on MRS solid culture medium, and cultured anaerobically at 37°C for 48 hours. Typical colonies were picked and streaked on MRS solid culture medium 2 to 3 times to obtain pure colonies. After 16SrDNA molecular identification and strain identification using universal primers 27F / 1492R, nucleotide sequence alignment was performed in NCBI, and the results showed a strain of Bifidobacterium bifidum, named Bifidobacterium bifidum JWA68. The strain was deposited in the China Center for Type Culture Collection on November 30, 2022.

[0040] 2. Preparation of Fermentation Filtrate

[0041] The glycerol cryopreservation tube of the strain was taken out from the -80°C refrigerator, thawed at room temperature, and then the strain was picked up using an inoculation loop under a sterile environment, streaked and inoculated on an MRS solid plate, and cultured at 37°C for 48 hours. After observing the colony morphology on the plate and confirming that it was Bifidobacterium bifidum and was free of contamination, a single bacterium on the plate was picked up and inoculated into the corresponding MRS liquid culture medium at a 10% inoculation rate, and placed in a 37°C incubator for static culture for 24 hours until the bacterial liquid grew turbid to obtain an activated bacterial liquid, and the bacterial liquid was transferred to the corresponding MRS fermentation medium at a 10% inoculation rate for a second time, placed in a 37°C incubator for static culture for 24 hours, and the fermentation was completed to obtain a bacterial suspension, and the bacterial suspension was subjected to bacterial cell disruption, centrifugation, and filtration to obtain a fermentation filtrate.

[0042] 3. Efficacy Evaluation

[0043] 1. Detection of ceramide

[0044] Experimental methods:

[0045] (1) Cultivate HaCaT cells: add 3×10 cells per well of a 96-well plate. 4 Cells were cultured in a volume of 200 μL / well at 37°C and 5% CO2 for 16 to 24 hours (cell confluence reached 70%);

[0046] (2) Grouping and induction of cell models: Normal control group: discard the supernatant and add 180 μL / well of fresh DMEM medium; model group: discard the supernatant and add 180 μL / well of fresh medium containing 3 mg / mL SDS; experimental group: discard the supernatant and add 180 μL / well of fresh medium containing 3 mg / mL SDS.

[0047] (3) Adding samples: negative control: adding 20 μL of PBS; model group: adding 20 μL of PBS; experimental group: adding 20 μL of probiotic fermentation liquid, and culturing at 37°C and 5% CO2 for 24 hours.

[0048] (4) Collect the cell culture supernatant by centrifugation at 7500 rpm for 10 min.

[0049] (5) ELISA kit was used to detect the ceramide content. The steps are as follows:

[0050] Standard sample addition: set standard wells and sample wells, add 50 μL of standard samples of different concentrations to each standard well, and add 50 μL of sample diluent to well 0;

[0051] Add sample: Set up blank wells (blank control wells do not add sample and enzyme-labeled reagent, and the rest of the steps are the same) and test sample wells. Add 50 μL of sample to the test sample well on the enzyme-labeled plate, add it to the bottom of the well, try not to touch the well wall, and gently shake to mix;

[0052] Add enzyme: add 100 μL of enzyme labeling reagent to each well, except for the blank well;

[0053] Incubation: Seal the plate with a sealing film and incubate at 37°C for 60 minutes;

[0054] Solution preparation: dilute the 20-fold concentrated washing solution with 20-fold distilled water for later use;

[0055] Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 60 seconds and then discard, repeat this 5 times, and pat dry;

[0056] Color development: First add 50μL of color developer A to each well, then add 50μL of color developer B, gently shake and mix, and develop color at 37℃ in the dark for 15 minutes;

[0057] Stop: Add 50 μL of stop solution to each well to stop the reaction (the blue color immediately turns yellow);

[0058] Determination: adjust the blank well to zero and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm. The determination should be carried out within 15 minutes after adding the stop solution. Enter the measured OD450 value into the ELISA data processing software. The data are shown in Tables 1 and 2 (the fermentation broth concentration is 10%).

[0059] Table 1. Initial screening of ceramide targets of Bifidobacterium bifidum

[0060]

[0061]

[0062] Table 2. The secretion rate of ceramide promoted by different concentrations of JWA68 fermentation broth

[0063] Group Ceramide secretion (μmol / L) Ceramide secretion rate (%) Normal control group 53.62 100％ Injury model group 36.28 67.66％ 2.5% Experimental group 55.54 103.58％ 5% experimental group 57.1 106.49％ 10% experimental group 59.16 110.33％

[0064] Among the five isolated strains of Bifidobacterium bifidum, strain #3 has the highest rate of promoting ceramide secretion, which is also JWA68 mentioned in this scheme, indicating that the fermentation filtrate of Bifidobacterium bifidum JWA68 added to the cultured HaCaT cells can significantly promote the cell secretion of ceramide, thereby achieving the effect of epidermal barrier repair. By studying the effect of different concentrations of JWA68 fermentation broth on the secretion rate of ceramide, the results in Table 2 show that at a certain concentration, the secretion rate of ceramide of JWA 68 fermentation broth shows a certain concentration dependence, and the secretion rate of ceramide of the fermentation broth with a concentration of 10% is the most significant.

[0065] 2. Inhibition of vascular hyperreactivity test

[0066] VEGF (vascular endothelial growth factor) is mainly secreted by keratinocytes and has the activity of promoting angiogenesis, dilation and increasing vascular permeability. Vascular dilation and increased permeability can cause redness of the skin.

[0067] Method: 3×10 4 Hacat cells were cultured until the cell fusion was more than 70%, the supernatant was discarded, 200 μL of DMEM (2% serum) containing 3 mg / mL SDS was added for treatment for 1 hour, and then 10% volume fraction of probiotic fermentation liquid was added. A blank control group, a model group (SDS treatment) and an experimental group (SDS treatment + fermentation filtrate) were set up, and the cells were treated for 24 hours. The supernatant was collected and centrifuged, and the VEGF content in the sample was detected according to the ceramide ELISA kit, as shown in Table 3.

[0068] Table 3. Effect of JWA 68 on VEGF

[0069] Group VEGF (pg / mL) VEGF content (% Blank) Blank control (pg / ml) 533.842649 100％ Model group (pg / ml) 661.759335 123.96％ Experimental group (pg / ml) 615.547875 115.3％％

[0070] The fermentation filtrate of Bifidobacterium bifidum JWA68 can significantly inhibit the secretion of VEGF by HaCaT cells, thereby reducing the skin's vascular hyperreaction, alleviating skin redness, calming the skin and relieving sensitivity.

[0071] 3. Experiment on inhibiting harmful bacteria

[0072] Take the Staphylococcus aureus culture (1×10 8 cfu / ml~1×10 9cfu / ml), diluted with blank TSB liquid culture medium to obtain a bacterial suspension containing 105 CFU / ml of Staphylococcus aureus; prepare antibacterial system: negative control: 1000μl bacterial suspension + 400μl PBS; 10% concentration fermentation liquid: 1000μl bacterial suspension + 140μl raw material liquid + 260μl PBS; the prepared antibacterial systems of various concentrations were placed in a 37℃ incubator for 24h, and then the number of pathogens in each system was detected. The antibacterial rate of the raw material was calculated according to the antibacterial effect test method (suspension quantitative method) of antibacterial daily chemical products in "QB / T2738-2012" Evaluation method for antibacterial and antibacterial effects of daily chemical products".

[0073] The results in Table 4 show that JWA68 fermentation broth at a concentration of 10% can inhibit the growth of harmful bacteria Staphylococcus aureus and has a strong antibacterial effect on Staphylococcus aureus.

[0074] Table 4. Inhibition of pathogens by JWA 68

[0075]

[0076] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Bifidobacterium bifidum JWA68 ( Bifidobacterium bifidum JWA68), and its deposit number is CCTCC NO: M20221852.

2. A flora, characterized in that It comprises the Bifidobacterium bifidum according to claim 1.

3. A bacterial agent, characterized in that It comprises the Bifidobacterium bifidum according to claim 1 or the bacterial flora according to claim 2.

4. Use of any one of the following I) to III) in the preparation of a skin improvement product: 1), the Bifidobacterium bifidum or its lysate according to claim 1; II), the bacterial colony according to claim 2; III), the bacterial agent according to claim 3; The skin improvement is at least one of epidermal barrier repair, sensitivity relief and / or inhibition of skin pathogens; The inhibition of skin pathogens is the inhibition of the growth of Staphylococcus aureus.

5. The use according to claim 4, characterized in that: The epidermal barrier repair includes promoting the synthesis of cell ceramide.

6. The use according to claim 4, characterized in that: The soothing sensitivity includes inhibiting the expression of vascular endothelial growth factor-related genes, and the vascular endothelial growth factor-related genes include VEGF .

7. A skin-improving product, characterized in that: The raw materials include at least one of the following i) to iii): i), the Bifidobacterium bifidum or its lysate according to claim 1; ii) The bacterial flora according to claim 2; iii) The bacterial agent according to claim 3.

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