CN114990021A - Lactobacillus salivarius and application thereof in preparation of skin care products - Google Patents
Lactobacillus salivarius and application thereof in preparation of skin care products Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
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- 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
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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Abstract
The invention mainly relates to the technical field of microorganisms, and particularly relates to lactobacillus salivarius and application thereof in preparation of skin care products. The invention discloses a human lactobacillus salivarius GforU-3 separated from the facial skin of a healthy boy of 1 year old, which has the skin care effect and comprises the following components: promoting cell injury repair, inhibiting growth of skin pathogenic bacteria, relieving inflammation, and promoting skin barrier repair. The lactobacillus salivarius GforU-3 provided by the invention has great application potential in foods, medicines, maintenance products or cosmetics.
Description
Technical Field
The invention mainly relates to the technical field of microorganisms, and particularly relates to lactobacillus salivarius and application thereof in preparation of skin care products.
Background
The skin is the largest organ in the human body, the total weight accounts for about 16% of the body weight of an individual, and the skin is the first defense line for maintaining the stability of the body and resisting the invasion of external adverse factors. The skin barrier is a structural barrier formed by the epidermal keratinocytes of the stratum corneum and the lipids between the cutin, which prevents excessive water release from the body and also protects against harmful factors such as chemicals or microorganisms secreting harmful factors into our body. Skin barrier damage can cause skin dryness, skin aging, atopic dermatitis, eczema, psoriasis, ichthyosis, solar dermatitis, skin sensitivity, irritant dermatitis, hormone dependent dermatitis, seborrheic diseases such as acne, rosacea, and seborrheic dermatitis. The stratum corneum of the skin contains natural moisturizing factors, namely amino acids, lactate, saccharides and the like, and usually contains 10-30% of water, and the environment becomes a cradle for the growth of microbial colonies of the skin. However, the water content of the stratum corneum gradually decreases with age, and various problems of the skin are caused when the water content is less than 10%.
In modern society, people have high living pressure, unhealthy diet and irregular work and rest, and skin is easy to have problems such as skin acne, large pores, damaged skin barrier, skin inflammation and the like, so that the demand of people on skin health is higher and higher.
The more scientific skin care method in the modern skin care field comprises the following steps: the probiotics and the preparation thereof are used on medicines and cosmetics to balance the epidermal flora of the skin and repair the skin barrier. The reasonable external application of the probiotics can inhibit the growth of skin pathogenic bacteria, balance the microbial flora on the surface of the skin and promote the repair of the skin barrier. In addition, researches report that the microecological preparation has important functions in maintaining the steady state of a host organism, activating an immune system, maintaining the immune balance of the organism and the like. The Lactobacillus salivarius is gram-positive bacillus, has no catalase or oxidase, can produce lactic acid, and is commonly used in food production. Lactobacillus salivarius belongs to the genus Lactobacillus of the family Lactobacillaceae and is widely present in the intestinal tract of humans and animals. In recent years, lactobacillus salivarius has been widely used as probiotic bacteria in the production of probiotic preparations for human and animals, and has been the hot spot of research in recent years, but the direct external application of lactobacillus salivarius in skin care has been reported to a lesser extent.
Although research and development personnel have made many efforts on skin health, due to the complex causes of skin problems and various aspects of diet, endocrine, environment, immunity, skin micro-ecology and the like, the existing skin care products cannot completely meet the prevention and treatment of skin diseases, and the research and development needs of skin care and products thereof are continuously promoted.
Disclosure of Invention
In view of this, the technical problem to be solved by the present invention is to provide a lactobacillus salivarius and its application in preparation of skin care products.
The invention provides lactobacillus salivarius GforU-3 with the preservation number of CCTCC NO. M20211440.
Specifically, the strain is gram-positive and rod-shaped under a microscope; white round opaque colonies with smooth surface and neat edges can be formed when the colonies grow on an MRS plate.
The invention also provides application of the lactobacillus salivarius in preparation of skin care products.
The skin care includes: promoting repair of cell damage, inhibiting growth of skin pathogenic bacteria, resisting inflammation and/or promoting skin barrier repair.
The inhibiting of growth of skin pathogenic bacteria includes inhibiting growth of at least one of Staphylococcus aureus (Staphylococcus aureus), Staphylococcus hominis (Staphylococcus hominis), Staphylococcus haemolyticus (Staphylococcus haemolyticus), Corynebacterium xerosis (Corynebacterium xerosis), Pseudomonas aeruginosa (Pseudomonas aeruginosa) and Propionibacterium acnes (Propionibacterium acnes).
In some embodiments, the lactobacillus salivarius GforU-3 provided by the present invention inhibits the growth of common skin pathogens at a rate of 20.43% to 31.70%.
The anti-inflammatory comprises reducing NO release and/or down-regulating the expression level of inflammatory factors.
The inflammatory factor includes at least one of IL-8 and TRPV 1.
In some embodiments, the lactobacillus salivarius GforU-3 provided by the invention can reduce the generation of Nitric Oxide (NO) by Lipopolysaccharide (LPS) -induced mouse macrophage raw264.7 by 25.54% -30.53%.
In some embodiments, lactobacillus salivarius GforU-3 down-regulates the expression level of HaCaT cell inflammatory factor-related genes induced by staphylococcus aureus from 30.41% to 48.78%.
The promoting skin barrier repair includes up-regulating the level of a barrier repair-associated gene.
The barrier repair related gene comprises at least one of IVL and OVOL 1.
In some embodiments, expression level of lactobacillus salivarius GforU-3 up-regulated cell repair-related factor involucrin gene IVL and OVO-like transcription factor 1 gene OVOL1 is up-regulated by 1.31-2.70 times.
The lactobacillus salivarius GforU-3 provided by the invention has the effect of promoting the repair of cell damage.
In some embodiments, the lactobacillus salivarius GforU-3 provided herein enhances survival rates of 111.44% to 162.42% of Sodium Dodecyl Sulfate (SDS) -injured HaCaT cells.
The invention provides a skin care product, which comprises the lactobacillus salivarius provided by the invention as raw materials;
the raw materials of the product comprise at least one of live bacteria, dead bacteria, lysate, extract, culture supernatant and derivatives of the Lactobacillus salivarius GforU-3 strain; the derivatives include: at least one of metabolites, metabolic biologicals, prebiotics, cell walls and components thereof, exopolysaccharides and compounds containing immunogenic components.
Preferably, the raw material of the product is the supernatant or the inactivated thallus of the lactobacillus salivarius GforU-3 strain.
The product comprises medicinal products, food, health products or cosmetics.
The invention discloses a human lactobacillus salivarius GforU-3 separated from the facial skin of a healthy boy of 1 year old, which has the skin care effect and comprises the following components: promoting cell injury repair, inhibiting growth of skin pathogenic bacteria, relieving inflammation, and promoting skin barrier repair. The lactobacillus salivarius GforU-3 provided by the invention has great application potential in foods, medicines, maintenance products or cosmetics.
Proof of biological preservation
Lactobacillus salivarius GforU-3 is preserved in China center for type culture Collection at 11 months and 18 days in 2021, with the address of China, Wuhan university and the preservation number of CCTCC NO: M20211440.
Drawings
FIG. 1 shows GforU-3 promotes HaCaT cell repair;
FIG. 2 shows the inhibition rate of GforU-3 against the growth of skin pathogenic bacteria.
Detailed Description
The invention provides a lactobacillus salivarius and application thereof in preparation of skin care products, and a person skilled in the art can use the contents to refer to the contents and appropriately improve process parameters to realize the purpose. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
example 1 isolation of GforU-3
Samples were taken on the facial skin of 1 year old healthy boys. Properly processing a sample, uniformly mixing the sample in normal saline by shaking, taking a supernatant, streaking the supernatant on an MRS solid plate, culturing the MRS solid plate at a constant temperature of 37 ℃ for 24-48 hours, and selecting a white colony for repeated inoculation and screening until a uniform single colony is obtained, wherein the single colony is named as GforU-3.
Gram staining microscopy: the strain GforU-3 is gram-positive and rod-shaped under a microscope; growing on an MRS plate to form white round microcolonies with smooth, mellow and opaque surfaces and regular edges; the strain grows uniformly turbid in MRS liquid culture medium, and the strain is white precipitate after long-term storage.
Example 2 identification of nucleic acid of GforU-3
1. 16s rRNA Gene sequence analysis:
selecting single colony to be placed in MRS liquid culture medium, after overnight culture at 37 ℃, centrifugally collecting thalli, and operating and amplifying the sequence of the thalli according to the steps of a DNA extraction kit. The primer is bacterial universal primer 27F, 1492R; the PCR amplification system is a 50 mu L system, and the PCR amplification procedure is as follows: pre-denaturation at 95 ℃ for 5 min; 15s at 94 ℃, 15s at 57 ℃, 40s at 72 ℃ and 35 cycles; extension at 72 ℃ for 10 min. After amplification, the nucleotide sequence of the nucleic acid is determined.
2. Results
The result of PCR product sequencing is compared with the standard sequence published in GenBank (BL ASTN) to obtain the strain GforU-3 as Lactobacillus salivarius.
Example 3 GforU-3 promotes SDS-induced HaCaT cell injury repair experiment
1. Preparing GforU-3 bacterial liquid:
culturing the activated lactobacillus salivarius GforU-3 bacterial liquid in an MRS liquid culture medium in an incubator at 37 ℃ for standing culture for 16-18 h, detecting and adjusting to OD 600 Inactivating at 121 deg.c for 30min, centrifuging to obtain precipitate as GforU-3 inactivated thallus, and filtering the inactivated fermented liquid with 0.22 micron filter membrane to obtain GforU-3 inactivated supernatant.
2. Experiment for promoting HaCaT cell operation and repair
Inoculation with HaCaT cells (5X 10) 4 One/well) to 96-well plates and cultured overnight until cells adhere. Preparing SDS solution of 50 mu g/ml, adding 100 mu l of SDS solution into each well, incubating for 8h, adding 5% (V/V) GforU-3 inactivated supernatant or 10% (V/V) GforU-3 inactivated thalli, and incubating for 24 h. 10 μ l of CCK-8 solution was added, incubated for 4h, and the absorbance A of the cell culture solution at 450nm was measured.
Cell viability ═ (a experiment-a blank)/(a negative control-a blank).
The results are shown in Table 1:
TABLE 1 GforU-3 promotion of HaCaT cell repair
As shown in Table 1, both the supernatant of GforU-3 and the inactivated cells had a repairing effect on SDS damage of HaCaT cells. The statistical results of GforU-3 promoting HaCaT cell repair are shown in FIG. 1.
Example 4 GforU-3 promotion of HaCaT Barrier repair-related Gene expression experiment
The preparation method of the GforU-3 inactivated supernatant and the inactivated mycelia is the same as that of step 1 of example 3. Inoculation of human immortalized keratinocytes HaCaT (5X 10) 5 One/well) to 6-well plates and cultured overnight until cells adhere. Respectively adding 5% (V/V) of strain supernatant and 10% (V/V) of inactivated thallus, respectively culturing for 24h, adding lysate, extracting total RNA of cells, detecting RNA concentration and purity, adjusting all samples to 1 mu g, carrying out reverse transcription to cDNA, carrying out qPCR (quantitative polymerase chain reaction) to detect expression levels of IVL and OVOL1 genes, and then calculating expression change multiples according to a formula.
The formula is as follows: f is 2 -ΔΔCT
The influence results of the GforU-3 inactivated supernatant and the inactivated thallus on the barrier repair related genes are respectively shown in tables 2 and 3:
TABLE 2 GforU-3 supernatant upregulation of barrier repair-related gene expression
TABLE 3 expression of genes related to barrier repair up-regulated by GforU-3 inactivated bacteria
As shown in tables 2 and 3, the added GforU-3 inactivated supernatant and the inactivated mycelia had the effect of promoting the skin barrier repair.
Example 5 GforU-3 inhibition of pathogenic bacteria experiment-bacterial liquid concentration Change
1. Preparing a lactobacillus salivarius GforU-3 bacterial liquid:
culturing the activated lactobacillus salivarius GforU-3 bacterial liquid in an MRS liquid culture medium in an incubator at 37 ℃ for standing culture for 16-18 h, detecting and adjusting to OD 600 Inactivating at 121 deg.C for 30min, centrifuging to obtain supernatant, and filtering with 0.22 μm filter membrane to obtain GforU-3 inactivated supernatant sample.
2. Preparing a pathogenic bacterium liquid:
the 6 pathogenic bacteria: respectively culturing Staphylococcus aureus CGMCC 1.8721, human Staphylococcus aureus CGMCC 1.493, Staphylococcus haemolyticus CGMCC 1.540, Corynebacterium siccatum CGMCC 1.1919, Pseudomonas aeruginosa CGMCC 1.1783 and Propionibacterium acnes CGMCC 1.5003 with BHI culture medium at 37 deg.C for 18h, detecting and adjusting to OD 600 ＝0.2。
3. Experiment for inhibiting pathogenic bacteria
Adding the GforU-3 inactivated supernatant into each pathogenic bacterium according to the proportion of 10% (V/V), and culturing at 37 ℃ for 12-16 h according to the bacterial liquid concentration (OD) of the pathogenic bacteria 600 ) The percentage reduction is used as an index to evaluate the inhibition effect of GforU-3 on the growth of pathogenic bacteria.
The results are shown in Table 4:
TABLE 4 inhibitory rate of GforU-3 against skin pathogens
As shown in Table 4, GforU-3 has inhibitory effects on Staphylococcus aureus, Staphylococcus hominis, Staphylococcus haemolyticus, Corynebacterium xerosis, Pseudomonas aeruginosa and Propionibacterium acnes, which are common skin pathogens. The statistical result of the inhibition rate of GforU-3 to skin pathogenic bacteria is shown in figure 2.
Example 6 GforU-3 reduces the amount of NO produced by Raw264.7 cells
1. Preparation of GforU-3 bacterial liquid
Culturing the GforU-3 in an MRS culture medium overnight, detecting OD600, adjusting the concentration of a bacterial liquid until the OD600 is 0.2, centrifuging, sterilizing the thalli for 30min at 121 ℃ under high pressure to obtain GforU-3 inactivated thalli, and filtering the centrifuged supernatant by using a 0.22 mu m filter membrane to obtain a GforU-3 inactivated supernatant.
2. Raw264.7 cell preparation
Raw264.7 cells were digested and then digested at 2X 10 5 The cells were inoculated into 24-well plates at a density per well and incubated overnight at 37 ℃ in a 5% carbon dioxide incubator.
3. GforU-3 addition and LPS stimulation
Respectively adding the supernatant of 5% (V/V) and the inactivated thallus of 10% (V/V) into different groups of Raw264.7 cells which are cultured overnight, adding 0.5ml of LPS solution with the concentration of 0.2 mu g/ml after 2h to induce Raw264.7 cells to be inflamed, taking cell culture supernatant after 20h, and carrying out NO content detection on the culture supernatant by using an NO content detection kit, repeating 3 times for each time, and carrying out three experiments.
The results are shown in Table 5:
TABLE 5 GforU-3 reduction of NO production by Raw264.7 cells
As shown in Table 5, GforU-3 had an anti-inflammatory effect and was able to reduce the NO production by Raw264.7 cells induced by LPS, and the supernatant of GforU-3 had a reduced NO production of 25.54% and the inactivated cells of GforU-3 had a reduced NO production of 30.53% as compared with the control group.
Example 7 GforU-3 Down-Regulation of HaCaT cell inflammatory factor expression
1. GforU-3 sample preparation
GforU-3 was cultured overnight with MRS and OD was detected 600 Adjusting the concentration of the bacterial liquid to OD 600 After centrifugation, the cells were autoclaved at 121 ℃ for 30min to give inactivated GforU-3 cells, and the centrifuged supernatant was filtered through a 0.22 μm filter to give an inactivated GforU-3 supernatant.
2. HaCaT cell preparation
HaCaT cells were digested and then treated at 2X 10 5 The cells were inoculated into 24-well plates at a density per well and incubated overnight at 37 ℃ in a 5% carbon dioxide incubator.
3. Preparation and addition of staphylococcus aureus
Inoculating Staphylococcus aureus to nutrient broth culture medium, shake culturing at 37 deg.C overnight, and adjusting bacterial liquid concentration to OD with MEM serum-free culture medium 600 The staphylococcus aureus solution was added to the HaCaT cells cultured overnight at 100 μ l per well to stimulate the production of inflammatory factors, after 3h the cell culture medium was discarded, washed 5 times with PBS, and 1ml of MEM serum-free medium was added to each well.
4. GforU-3 sample addition
The GforU-3 inactivated bacteria are added into HaCaT cells stimulated by staphylococcus aureus according to the proportion of 10% (V/V), each group has 3 multiple holes, and the cells are cultured overnight.
5. qPCR method for detecting relative expression multiple of cell inflammatory factor mRNA
After the cells are discarded with a culture medium, RNA is extracted by using an RNA extraction kit, the concentration and purity of the RNA are detected, all RNA samples are adjusted to 1 mu g, RT-PCR and qPCR are carried out by using a reverse transcription kit and a SYBRGreen qPCR kit, and then the relative expression multiple F of the inflammatory factor genes IL-8 and TRPV1 is calculated.
The formula: f ═ 2 -ΔΔCT 。
The results are shown in Table 6:
TABLE 6 GforU-3 Down-Regulation of the expression of inflammatory factor genes
The result is shown in Table 6, the GforU-3 can reduce HaCaT cell inflammatory factor related gene expression induced by staphylococcus aureus, and the expression level is reduced by 30.41-48.78%. Therefore, GforU-3 has an anti-inflammatory effect.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (10)
1. Lactobacillus salivarius GforU-3 with the preservation number of CCTCC NO: M20211440.
2. Use of lactobacillus salivarius as claimed in claim 1 in the manufacture of a skin care product.
3. Use according to claim 2, wherein the skin care comprises: promoting repair of cell damage, inhibiting growth of skin pathogenic bacteria, resisting inflammation and/or promoting skin barrier repair.
4. The use of claim 3, wherein said inhibiting the growth of a skin pathogenic bacterium comprises inhibiting the growth of at least one of Staphylococcus aureus, Staphylococcus hominis, Staphylococcus haemolyticus, Corynebacterium xerosis, Pseudomonas aeruginosa, and Propionibacterium acnes.
5. The use according to claim 3, wherein the anti-inflammatory comprises a reduction in NO release and/or a down-regulation of the expression level of an inflammatory factor.
6. The use according to claim 5, wherein said inflammatory factor comprises at least one of IL-8 and TRPV 1.
7. The use of claim 3, wherein the promotion of skin barrier repair comprises upregulating the level of a barrier repair-associated gene.
8. The use of claim 7, wherein the barrier repair-associated gene comprises at least one of IVL and OVOL 1.
9. A skin care product characterized in that its raw material comprises the Lactobacillus salivarius of claim 1.
10. The product of claim 9, comprising a pharmaceutical, food, cosmetic or toiletry product.
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