CN115317431A - Preparation of bifidobacterium infantis YLGB-1496 and new application thereof in skin health - Google Patents
Preparation of bifidobacterium infantis YLGB-1496 and new application thereof in skin health Download PDFInfo
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- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- 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 provides a preparation of bifidobacterium infantis YLGB-1496 and a new application thereof in skin health, in particular to an application of bifidobacterium infantis in preparing a skin care product with skin care effect, wherein the bifidobacterium infantis comprises bifidobacterium infantis with the preservation number of CGMCC No. 21109. The bifidobacterium infantis has the effects of moisturizing, enhancing skin barrier, promoting skin antibacterial peptide expression and the like, and can be applied to preparing skin care products.
Description
Technical Field
The invention relates to a skin care application and a skin care product of bifidobacterium infantis, in particular to an application of bifidobacterium infantis with the preservation number of CGMCC No.21109 or fermentation liquor thereof in preparing a skin care product with skin care effect.
Background
The conventional skin care products generally use chemical preparations as main ingredients, but the chemical preparations often cause various skin problems, and the skin is finally damaged by using the skin care products with the chemical preparations as main ingredients for a long time. With the increasing demand for natural skin-friendly skin care products, there are skin care products containing plant extracts as main ingredients. However, the plant extract is usually extracted by using chemical agents as auxiliary agents, so that chemical agent residues are inevitable, and even a trace amount of chemical agent residues can cause skin problems and even skin seeps into blood to cause diseases.
The barrier function of the skin is an important structure for maintaining the normal physiological function of the skin, and the structure is figuratively likened to a "brick wall structure". Wherein the "bricks" of the brick wall structure refer to keratinocytes, and the "mortar" filling the spaces between the bricks and tightly connecting the bricks refers to intercellular lipids. The skin barrier function has two aspects: preventing the invasion of external chemical, physical or biological harmful factors; preventing the loss of nutrient substances and water in the body. The main cause of the formation of sensitive skin is the impairment of the skin barrier function. The stratum corneum barrier function of sensitive skin is relatively weak compared to normal skin. The reduction of keratinocytes leads to a thinning of the stratum corneum of the skin, an increase of the transdermal penetration rate and a higher skin sensitivity.
Disclosure of Invention
An object of the present invention is to provide a bifidobacterium infantis and skin care applications of its preparation.
Another object of the present invention is to provide a skin care product prepared from Bifidobacterium infantis and/or its fermentation product.
The invention provides Bifidobacterium longum subsp. The Bifidobacterium longum baby subspecies with the preservation number of CGMCC No.21109 is preserved in China general microbiological culture Collection center (CGMCC) in 12 months and 05 days in 2020, and the preservation unit address is as follows: west road No. 1, north west of the morning area, beijing, institute of microbiology, china academy of sciences, date of deposit: year 2020, 12 months and 05 days; the preservation number is: CGMCC No.21109; and (3) classification and naming: bifidobacterium longum subsp. The strain was also preserved in China Center for Type Culture Collection (CCTCC) at 10.04.2011, and the preservation unit address is as follows: wuhan university in Wuhan, china, 430072; the preservation date is as follows: 2011, 04 month 10 days; the preservation number is: CCTCC NO: m2011122, category naming: bifidobacterium longum subsp. The strain is also called YLGB-1496 or GB-1496 in the invention.
The inventor of the invention discovers in research that the fermentation broth of Bifidobacterium infantis (Bifidobacterium longum subsp. Infanis) with the preservation number of CGMCC No.21109 has a skin-care function, contains rich organic acid, amino acid and polysaccharide compounds, can enhance the expression of barrier-related genes in skin cells, improves the expression level of an antibacterial peptide gene CAMP and has the effect of improving skin barriers.
Thus, in one aspect, the invention provides an application of bifidobacterium infantis in preparing a skin care product with skin care efficacy, wherein the bifidobacterium infantis comprises bifidobacterium infantis with the preservation number of CGMCC No. 21109. The invention also provides a preparation of the bifidobacterium infantis with the preservation number of CGMCC No.21109 and skin care application thereof. In the present invention, the preparation of bifidobacterium infantis refers to an article prepared from bifidobacterium infantis, for example, in some specific embodiments, the preparation of bifidobacterium infantis is a fermentation product, such as a fermentation broth, obtained by fermenting bifidobacterium infantis; in other specific embodiments, the preparation of bifidobacterium infantis is a skin care product further prepared from bifidobacterium infantis or a fermentation broth thereof.
According to a specific embodiment of the invention, the bifidobacterium infantis with the preservation number of CGMCC No.21109 is fermented liquid used for preparing skin care products.
According to the specific embodiment of the invention, in the fermentation liquor, the content of lactic acid is 1-3%, the content of acetic acid is 1-4%, the content of total polysaccharide is 8-12 mg/mL, and the content of total amino acid is 2-6 mg/mL.
According to a particular embodiment of the invention, the skin care effect comprises an improvement in skin barrier function.
Preferably, the improving skin barrier function comprises: promote the expression of barrier-associated genes such as FLG, LOR, IVL and/or AQP-3, promote the expression of skin antibacterial peptides such as CAMP, hBD-2 and/or hBD-3, and/or reduce skin water loss.
On the other hand, the invention also provides a skin care product, which comprises the bifidobacterium infantis with the preservation number of CGMCC No.21109 and/or a fermentation product thereof.
According to a specific embodiment of the present invention, in the skin care product of the present invention, the fermentation product is a fermentation broth.
Preferably, the fermentation liquor is prepared according to the following method:
inoculating the bifidobacterium infantis with the preservation number of CGMCC No.21109 into a culture solution containing lactalbumin, and fermenting and culturing to obtain a fermentation solution.
According to a specific embodiment of the present invention, the fermentation broth of bifidobacterium infantis of the present invention is prepared such that the whey protein content of the culture broth containing whey protein is 2% to 10%. The culture solution may also contain 3% -12% white sugar. The remaining component of the culture medium may be water. The inoculation amount of the bifidobacterium infantis (seed solution) with the preservation number of CGMCC No.21109 can be 2-5%. After culturing at about 37 ℃ for 18h to 72h, the resulting preparation (which can be centrifuged to remove the cells as needed to obtain a supernatant) can be used for skin care applications of the invention.
According to the specific embodiment of the invention, in the skin care product, the fermentation liquor contains 1-3% of lactic acid, 1-4% of acetic acid, 8-12 mg/mL of total polysaccharide and 2-6 mg/mL of total amino acid. Specifically, the amino acid comprises one or more of aspartic acid, glutamic acid, serine, glycine, histidine, arginine, threonine, alanine, proline, tyrosine, valine, methionine (methionine), cystine, isoleucine, leucine, phenylalanine, lysine and tryptophan.
According to a specific embodiment of the invention, the fermentation liquor is added into the cosmetic in an amount of 0.1-30%.
According to a specific embodiment of the present invention, the skin care product of the present invention may be a skin lotion or a skin cream.
According to a particular embodiment of the invention, the skin care product of the invention may be a lotion or a mask.
The skin care product has good effect of improving skin barrier.
Drawings
FIG. 1 shows the results of the fermentation broth of Bifidobacterium infantis of the present invention promoting the expression level of the barrier-associated gene FLG in HaCaT cells.
FIG. 2 shows the results of promoting the expression level of barrier-associated gene LOR in HaCaT cells by using the fermentation broth of Bifidobacterium infantis of the present invention.
FIG. 3 shows the results of promoting the expression level of barrier-associated gene IVL in HaCaT cells by using fermentation broth of Bifidobacterium infantis of the present invention.
FIG. 4 shows the results of the fermentation broth of Bifidobacterium infantis of the present invention promoting the expression level of the barrier-associated gene AQP3 in HaCaT cells.
FIG. 5 shows the results of the fermentation broth of Bifidobacterium infantis of the present invention promoting the expression level of the antimicrobial peptide gene CAMP in HaCaT cells.
FIG. 6 shows the results of the fermentation broth of Bifidobacterium infantis of the present invention promoting the expression level of antimicrobial peptide gene hBD-2 in HaCaT cells.
FIG. 7 shows the results of the fermentation broth of Bifidobacterium infantis of the present invention promoting the expression level of antimicrobial peptide gene hBD-3 in HaCaT cells.
Fig. 8 shows the results of the lactic acid sting test after using the skin care product of the present invention.
In the figure: "+" indicates significant difference, p is more than or equal to 0.01 and less than 0.05, "+" indicates significant difference, p is more than or equal to 0.001 and less than 0.01; "x" indicates significant difference, p <0.001.
Microbial deposits for patent procedures:
bifidobacterium infantis YLGB-1496 (or GB-1496) strain:
the preservation date is as follows: year 2020, 12 months and 05 days;
the preservation unit: china general microbiological culture Collection center (CGMCC);
the address of the depository: western road No. 1, north west city of township, beijing, institute of microbiology, china academy of sciences;
the preservation number is as follows: CGMCC No.21109;
and (3) classification and naming: bifidobacterium longum subsp.
Detailed Description
For a more clear understanding of the technical features, objects and advantages of the present invention, reference is now made to the following detailed description taken in conjunction with the accompanying specific embodiments, and the technical solutions of the present invention are described, it being understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.
Example 1: preparation of probiotic fermentation liquor
A Bifidobacterium infantis YLGB-1496 (originated from the mother's milk of China health mother, which has been preserved in China general microbiological culture Collection center (CGMCC) at 12/05/2020 for 12/05, wherein the preservation unit address is No. 3 of Beijing's republic of south China West institute of microbiology, the preservation date is No. 05/2020/12/09, the preservation number is CGMCC No.21109, and the classification name is Bifidobacterium longum subsp.
Example 2: content detection of organic acid, amino acid and polysaccharide
Determining the content of organic acid in the probiotic fermentation broth obtained in example 1 by using high performance liquid chromatography; measuring the polysaccharide content by a phenol-sulfuric acid method; and (4) determining the content of the amino acid by an automatic amino acid analyzer method. The results of measuring the content of organic acids are shown in table 1.
TABLE 1 detection of organic acid content in probiotic fermentation broth
Total polysaccharide test results: 9.792mg/mL.
The amino acid test results include: aspartic acid 0.194mg/mL; glutamic acid 0.201mg/mL; serine 0.064mg/mL; glycine 0.049mg/mL; histidine 0.080mg/mL; arginine 0.157mg/mL; threonine 0.154mg/mL; alanine 0.222mg/mL; proline 0.213mg/mL; tyrosine 0.168mg/mL; valine 0.190mg/mL; methionine (methionine) cystine 0.110mg/mL; isoleucine 0.202mg/mL; leucine 0.295mg/mL; phenylalanine 0.129mg/mL; lysine 0.168mg/mL; tryptophan 0.083mg/mL.
Example 3: enhancement of skin barrier function by probiotic fermentation broth
The experimental grouping was specifically set up as shown in table 2.
TABLE 2 Experimental groups
Cell culture:
1. plate preparation: cells were digested, counted and 3X 10 cells per well 5 2mL of cell culture solution is put in a six-hole plate; wherein the cell culture fluid comprises MEM culture medium (89%) + FBS (serum 10%) + double antibody (1%); culture medium: national experimental cell resource sharing platform MEM, serum: gibco cat # s: 10099-141C, double antibody: hyClone cargo number: SV30010;
2. culturing in an incubator at 37 ℃ for 24h, allowing the cells to adhere to the wall, discarding the supernatant, adding 2mL of fermentation broth samples and competitive products with different concentrations (5%, 3% and 1%) into each well, and adding cell culture solution into a blank control;
3. after 24h of culture, removing the supernatant, adding 1ml of a lysis solution into each hole, blowing by using a pipette and transferring into an EP (EP) tube;
4. extracting sample RNA according to the RNA kit;
5. performing reverse transcription on the extracted RNA according to the kit instruction to obtain cDNA; the primer sequences are shown in Table 3.
TABLE 3 primers for semi-quantitative RT-PCR detection of barrier-associated genes
Statistical method
The results are expressed as Mean ± SD using GraphPad Prism mapping. The statistical analysis of t-test is adopted for comparison among groups. All statistical analyses were two-tailed. p <0.05 was considered to have a significant difference, and p <0.01 was considered to have a very significant difference.
And (3) test results:
filaggrin (FLG) mainly functions to connect keratinocyte fibers, and maintains the shape of keratinocytes to form a cornified envelope. Compared with a control group, the fermentation liquid sample has obvious effect on the expression quantity of the barrier related gene FLG in the HaCaT cell when the concentration is 3 percent and 5 percentInduction of (p)<0.05). Compared with the control group, the fermentation liquor of the Schizosaccharomyces cerevisiae (Renair Complex CLR PF) (for short, the German CLR company)
) The competitive product has obvious induction effect on the expression quantity of the barrier related gene FLG in HaCaT cells when the concentration is 5 percent (p)<0.05 (FIG. 1).
Compared with a control group, the fermentation liquid sample has obvious induction effect (p) on the expression level of the barrier related gene LOR in the HaCaT cells when the concentration is 1%, 3% and 5%<0.05). Compared with the control group, the compound of the formula,
the competitive products have obvious induction effect on the expression quantity of the barrier related gene LOR in HaCaT cells when the concentration is 1%, 3% and 5% (p)<0.05 And the effect is dose-dependent (figure 2).
Compared with a control group, the fermentation liquid sample has obvious induction effect (p) on the expression quantity of the barrier related gene IVL in HaCaT cells when the concentration is 3% and 5%<0.05). Compared with the control group, the compound of the formula,
the competitive product has obvious induction effect on the expression level of the barrier related gene IVL in HaCaT cells when the concentration is 5 percent (p)<0.05 (FIG. 3).
Compared with a control group, the fermentation liquor sample has obvious induction effect (p) on the expression quantity of the barrier related gene AQP-3 in HaCaT cells when the concentration is 3% and 5%<0.01). Compared with the control group, the compound had better effect,
the competitive product has obvious induction effect on the expression quantity of the barrier related gene AQP3 in HaCaT cells when the concentration is 3% and 5% (p<0.01). Aquaporin-3 (AQP-3, aquaporia-3) is primarily involved in the processes of absorption and secretion of cellular substances in keratinocytes, helping to maintain skin elasticity and repair damage (FIG. 4).
Example 4: evaluation of the Effect of probiotic fermentates on the expression of skin antimicrobial peptides at the cellular level
The experimental grouping was specifically set up as shown in table 4.
TABLE 4 Experimental groups
Cell level antibacterial peptide experimental method
1. Blank plate: the cells were digested, counted and 3X 10 cells per well 5 2mL of cell culture medium per well (same as example 3) was placed in six-well plates;
2. culturing in an incubator at 37 ℃ for 24h, allowing the cells to adhere to the wall, discarding the supernatant, adding 2mL of fermentation broth samples and competitive products with different concentrations (5%, 3% and 1%) into each well, and adding cell culture solution into a blank control;
3. after 24h of culture, discarding the supernatant, adding 1mL of Trizol lysate into each hole, blowing by using a pipette and transferring into an EP tube;
4. extracting sample RNA according to the RNA kit;
5. performing reverse transcription on the extracted RNA according to the kit instruction to obtain cDNA;
6. combining the cDNA and the primer, and operating according to the instruction of the real-time fluorescent quantitative PCR kit.
Statistical method
The results are expressed as Mean ± SD using GraphPad Prism mapping. The comparison between groups was performed by statistical analysis of t-test. All statistical analyses were two-tailed. p <0.05 was considered to have a significant difference, and p <0.01 was considered to have a very significant difference.
And (3) test results:
compared with a control group, the fermentation liquid sample has obvious induction effect (p is less than 0.05) on the expression quantity of the antimicrobial peptide gene CAMP in the HaCaT cells when the concentration is 3% and 5%, and the effect has dose dependence. Compared with a control group, the CLR competitive product has a remarkable induction effect (p is less than 0.01) on the expression level of the antimicrobial peptide gene CAMP in HaCaT cells at the concentration of 3% and 5% (figure 5).
Human beta-defensin (hBD-2) dominates the innate immunity of the skinAnd the major role of the antibacterial agent is to be considered as a hotspot in anti-infection clinical research due to the broad-spectrum antibacterial activity of the antibacterial agent. Compared with a control group, the fermentation liquor sample has obvious induction effect (p) on the expression quantity of the antibacterial peptide gene hBD-2 in HaCaT cells when the concentration is 1%, 3% and 5%<0.05 And the effect is dose-dependent. Compared with the control group
The competitive products have obvious induction effect on the expression quantity of the antimicrobial peptide gene hBD-2 in HaCaT cells when the concentration is 3% and 5% (p)<0.05 And the effect is dose-dependent (figure 6). Compared with a control group, the fermentation liquor sample has obvious induction effect (p) on the expression quantity of the antibacterial peptide gene hBD-3 in HaCaT cells when the concentration is 1%, 3% and 5%<0.01 And the effect is dose-dependent. Compared with the control group, the compound of the formula,
no significant induction effect is seen on the expression quantity of the antibacterial peptide gene hBD-3 in HaCaT cells when the concentration of the competitive products is 1%, 3% and 5% (p)>0.05 (FIG. 7).
Example 5: preparation of mask containing probiotic fermentation product and function evaluation
The formula of the mask product comprises: water, butylene glycol, bifidobacterium infantis YLGB-1496 broth (3%) prepared in example 1, oat β -glucan, stem extract of dendrobium stem, aloe barbadensis leaf extract, root extract of sophora flavescens ait, lycium barbarum fruit extract, echinacea purpurea extract, hydroxyethylpiperazineethanesulfonic acid, p-hydroxyacetophenone, hydrolyzed sclerotium rolfsii gum, sodium hyaluronate, 1, 2-ethanediol, carnosine, disodium EDTA. The product information is shown in table 5.
TABLE 5 detection of product information
| Name (R) | Product batch number | Colour and state of matter | Specification of | Number of | Package (I) |
| Fermentation mask | BR0105087 | Surface-mounted mask | Is not marked | 300 pieces | Bag for containing liquid medicine |
The test method comprises the following steps: the test result is obtained by using the test product for 28 days by 30 healthy female testees in the age range of 25-40 years by the skin being dry and rough, the face having desquamation phenomenon, the eye periphery having fine lines, the skin being sensitive, the lactic acid reaction being positive (the total score is more than or equal to 3), and the test result shows that the product has the following effects:
the experimental results are as follows:
the experimental results are shown in table 6, after 14 days of using the test product, the moisture content of the horny layer of the cheek of the subject significantly increases by 20.34%, the erythema index EI value of the cheek skin significantly decreases by 10.84%, the moisture loss amount of the cheek skin significantly decreases by 8.21%, the elasticity R2 value of the cheek skin significantly increases by 3.01%, the elasticity Q1 value of the cheek skin significantly increases by 3.09%, the roughness sor of the cheek skin significantly decreases by 12.22%, the smoothness of the cheek skin significantly decreases by 8.50%, the fine line area of the skin under the eye significantly decreases by 15.89%, and the fine line area of the fishtail significantly decreases by 12.67%. The results show that the test product has remarkable effects of moisturizing, repairing skin barriers, relieving skin, improving skin elasticity, smoothing skin, reducing fine wrinkles and reducing fishtail wrinkles 14 days after being used.
After the test product is used for 28 days, the moisture content of the horny layer of the cheek of the subject is remarkably increased by 24.11%, the erythema index EI value of the cheek skin is remarkably reduced by 15.60%, the moisture loss of the cheek skin is remarkably reduced by 12.80%, the elasticity R2 value of the cheek skin is remarkably increased by 6.38%, the elasticity Q1 value of the cheek skin is remarkably increased by 6.22%, the roughness SEr of the cheek skin is remarkably reduced by 22.06%, the smoothness SEsm of the cheek skin is remarkably reduced by 14.91%, the fine line area of the skin under the eyes is remarkably reduced by 19.27%, and the area of the crow's feet is remarkably reduced by 12.48%. The results show that the test product has remarkable effects of moisturizing, repairing skin barriers, relieving skin, improving skin elasticity, smoothing skin, reducing fine wrinkles and reducing fishtail wrinkles after being used for 28 days.
Table 6 fermented mask use 28-day efficacy data
Table 6 (continue)
In the table, p <0.05, p <0.001.
Lactic acid sting test:
the results show that 14 days after using the test product, the nasolabial fold lactic acid sting score was significantly reduced by 51.28% at 2.5min, 52.54% at 5min, and 52.04% at the total score of 2.5min and 5 min. The above results indicate that the test product has the effect of remarkably enhancing the skin tolerance after 14 days.
After the test product is used for 28 days, the nasolabial sulcus lactic acid sting score is remarkably reduced by 56.41% at 2.5min, is remarkably reduced by 55.93% at 5min, and is remarkably reduced by 56.12% at the total score of 2.5min and 5min, and the results show that the test product has the effect of remarkably enhancing the skin tolerance after the test product is used for 28 days (figure 8).
The product safety evaluation result shows that:
all subjects did not experience skin erythema, infiltration, edema, papules, blisters, and other adverse skin reactions, acne, enlarged pores, dulling skin color, symptoms associated with impaired barrier (including but not limited to skin itching, stinging, fever, tightness, redness and swelling, redness and redness, etc.) and allergic symptoms during the test period.
SEQUENCE LISTING
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Claims (10)
1. An application of Bifidobacterium infantis in preparing skin care products with skin care effect is disclosed, wherein the Bifidobacterium infantis comprises Bifidobacterium infantis with preservation number of CGMCC No. 21109.
2. The use according to claim 1, wherein the bifidobacterium infantis with the collection number of CGMCC No.21109 is fermented broth for use in the preparation of skin care products.
3. The use of claim 2, wherein the fermentation broth comprises 1-3% lactic acid, 1-4% acetic acid, 8-12 mg/mL total polysaccharide, and 2-6 mg/mL total amino acids.
4. Use according to claim 1, wherein the skin care efficacy comprises improving skin barrier function;
preferably, the improving skin barrier function comprises: promote the expression of barrier related genes FLG, LOR, IVL and/or AQP-3, promote the expression of skin antibacterial peptide, and/or reduce the water loss of skin.
5. A skin care product comprises Bifidobacterium infantis with preservation number of CGMCC No.21109 and/or its fermentation product.
6. The skin care product of claim 5, wherein the fermentation product is a fermentation broth;
preferably, the fermentation liquor is prepared according to the following method:
inoculating Bifidobacterium infantis with the preservation number of CGMCC No.21109 into a culture solution containing lactalbumin, and fermenting and culturing to obtain a fermentation solution.
7. The skin care product of claim 6, wherein the fermentation broth contains 1-3% lactic acid, 1-4% acetic acid, 8-12 mg/mL total polysaccharide and 2-6 mg/mL total amino acids.
8. The skin care product of claim 6 or 7, wherein the fermentation liquid is added in an amount of 0.1-30% in the skin care product.
9. The skin care product according to claim 5, which is a skin lotion or a skin cream.
10. The skin care preparation according to claim 5, which is a cosmetic water or a pack.
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| CN117551590A (en) * | 2024-01-10 | 2024-02-13 | 广州集妍化妆品科技有限公司 | Bifidobacterium longum subspecies infantis, microbial agent and application thereof |
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| 金鑫等: "益生菌调节防御素表达的研究进展", 《中国兽医杂志》, vol. 54, no. 5, pages 66 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117551590A (en) * | 2024-01-10 | 2024-02-13 | 广州集妍化妆品科技有限公司 | Bifidobacterium longum subspecies infantis, microbial agent and application thereof |
| CN117551590B (en) * | 2024-01-10 | 2024-04-05 | 广州集妍化妆品科技有限公司 | Bifidobacterium longum subspecies infantis, microbial agent and application thereof |
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