WO2022155881A1

WO2022155881A1 - Skin source bacteria kbfs1720 having skin ultraviolet burn repair function, and application thereof

Info

Publication number: WO2022155881A1
Application number: PCT/CN2021/073227
Authority: WO
Inventors: 李英; 冉海霞; 张涛; 张兴银; 郑文潇
Original assignee: 佛山科学技术学院
Priority date: 2021-01-22
Filing date: 2021-01-22
Publication date: 2022-07-28

Abstract

Provided is a skin source bacteria KBFS1720 having a skin ultraviolet burn repair function, and an application thereof. Also provided is a method for culturing the skin source bacteria.

Description

A kind of skin-derived bacteria KBFS1720 with skin ultraviolet burn repair function and its application

technical field

The present invention relates to the field of human microecology. Specifically, it relates to a skin-derived bacterium KBFS1720 with the function of repairing skin ultraviolet burns and its application.

Background technique

UV is an electromagnetic wave with a wavelength of 200nm to 400nm. According to its wavelength, it is divided into long-wave ultraviolet UVA, medium-wave ultraviolet UVB and short-wave ultraviolet UVC. Among them, UVC is absorbed by the ozone layer, and the main ones that can reach the ground are 95% of UVA and 5% of UVA. UVB, excess ultraviolet radiation poses a danger to human health, especially exposed skin. Long-term exposure of the skin to ultraviolet radiation can cause skin melanin deposition, oxidative stress in skin tissue, DNA, RNA, and protein damage, skin structural damage, and skin immunosuppression. As the first barrier of the body, the skin protects the body from physical and chemical damage, while skin bacteria colonize the surface of the human body, forming a unique ecological structure called skin microecology with human skin in the common historical evolution process . Bacteria that colonize the skin play an important role in the formation and homeostasis of skin immunity, including inhibition of colonization resistance to pathogen invasion, regulation of spontaneous inflammation and immune responses, and promotion of host immune tolerance against commensal microorganisms. In addition, studies have shown that skin commensal microbiota can induce an adaptive immunity that combines anti-infection and tissue repair functions, and can also interfere with UV-induced skin photodamage. However, the existing common flora colonized on human skin has poor resistance to ultraviolet radiation, and when the flora is applied in a product, the damage to the skin by ultraviolet radiation cannot be effectively slowed down. In addition, at present, most of the skin damage repair products induced by ultraviolet radiation are plant extracts, and there are relatively few products that apply microorganisms to skin damage repaired by ultraviolet radiation. have great research significance.

To sum up, in the field of microecology, there are still technical problems that need to be solved urgently.

SUMMARY OF THE INVENTION

Based on this, in order to solve the problem that the common flora has poor resistance to ultraviolet radiation, it cannot effectively slow down the damage to the skin caused by ultraviolet radiation and the loss of genetic material, which is unstable. The present invention provides a skin-derived bacterium KBFS170 with the function of repairing skin ultraviolet burns. The strain was deposited in the Guangdong Provincial Microbial Culture Collection Center on December 23, 2020. Building 59 of the compound, No. 100, Xianlie Middle Road, City.

The invention also provides the application of the skin-derived bacteria KBFS1720 with the skin ultraviolet burn repair function in resisting ultraviolet radiation and repairing light damage.

The invention also provides the application of the skin-derived bacteria KBFS1720 with skin ultraviolet burn repair function in the preparation of products for resisting ultraviolet radiation and repairing light damage.

Preferably, the product acts on the skin.

In addition, the present invention also provides a method for culturing skin-derived bacteria KBFS1720 with skin ultraviolet burn repairing function, comprising the following steps:

isolate the bacterial strain to obtain the first bacterial suspension;

The first bacterial suspension is placed in the first culture medium for cultivation;

After several times of purification and cultivation, 16S full-length sequencing was performed to identify the bacterial species, and the strains enriched in the skin were screened out;

The screened strains are activated in the second medium, and cultured overnight at a constant temperature to obtain activated strains;

The activated bacterial strain is inoculated in the third medium, the constant temperature air bath, the shaking overnight culture, the centrifugation, and the collection of the bacterial precipitation;

The bacterial cell precipitate is washed with sterile PBS buffer, and after washing is completed, the bacterial cell precipitate is suspended in sterile PBS buffer to obtain a bacterial suspension.

Preferably, the strain is isolated from the skin of healthy adults who have been exposed to sunlight for a long time.

Preferably, the first medium comprises one or more of peptone nutrient agar medium, yeast extract powder agar medium, brain heart extract agar medium, sucrose medium, tryptone soybean agar medium.

Preferably, the second medium is a nutrient agar medium.

Preferably, the third medium is a nutrient broth.

Preferably, the bacterial concentration of the bacterial suspension is 1×10 ⁶ CFU/mL to 1×10 ⁸ CFU/mL.

The genetic properties of the skin-derived bacterium KBFS1720 with the skin ultraviolet burn repair function in the above scheme are not easily lost, and the obtained strain has significant stability. At the same time, the skin-derived bacterium KBFS1720 is applied in products that resist ultraviolet radiation and repair light damage It can effectively slow down the damage of ultraviolet radiation to the skin, and can significantly repair and improve the skin damage after receiving solar ultraviolet radiation.

Description of drawings

Fig. 1 is the bacterial colony morphology schematic diagram of skin-derived bacteria KBFS1720 of the present invention;

Fig. 2 is the schematic diagram of Gram staining 10×60 microscopic examination of skin-derived bacteria KBFS1720 of the present invention;

Fig. 3 is the growth curve schematic diagram of skin-derived bacteria KBFS1720 of the present invention;

Fig. 4 is the schematic diagram of chromosome group circle of skin-derived bacteria KBFS1720 of the present invention;

Fig. 5 is the schematic diagram of the plasmid circle of skin-derived bacteria KBFS1720 of the present invention, wherein ABCD represents the circle schematic diagram of 4 plasmids;

Fig. 6 is the schematic flow chart of constructing subacute light damage model mice in Example 5 of the present invention;

7 is a schematic diagram of processing model mice in Example 5 of the present invention;

8 is a schematic diagram of H&E staining sections of mouse skin with different degrees of injury in Example 5 of the present invention;

FIG. 9 is a schematic diagram of the statistical results of skin morphology and tissue scores of different test groups in Example 5 of the present invention.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to its embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, and do not limit the protection scope of the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used herein in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The skin-derived bacterium KBFS1720 with skin ultraviolet burn repair function in an embodiment of the present invention was deposited in the Guangdong Provincial Microorganism Culture Collection Center on December 23, 2020, and the culture collection number is GDMCC: 61382, and the deposit address is Building 59, No. 100 Xianlie Middle Road, Guangzhou City. The appearance of the skin-derived bacteria KBFS1720 with skin ultraviolet burn repair function shows that the surface is viscous, smooth and easy to be picked, the edges are neat and complete, and there is light transmittance and glossy round shape, the outer periphery of the colony is raised, and the middle is depressed; The color of the front and back, as well as the colony edge and center are cream yellow, as shown in Figure 1.

In one embodiment, the present invention also provides the application of the skin-derived bacteria KBFS1720 with the function of repairing skin ultraviolet burns in resisting ultraviolet radiation and repairing light damage.

In one of the embodiments, the present invention also provides the application of the skin-derived bacteria KBFS1720 with the function of repairing skin ultraviolet burns in the preparation of products for resisting ultraviolet radiation and repairing light damage.

The genetic properties of the skin-derived bacteria KBFS1720 with skin ultraviolet burn repair function in the above scheme are not easily lost, and the obtained strain has significant stability. At the same time, the skin-derived bacteria KBFS1720 with skin ultraviolet burn repair function is applied to resist ultraviolet radiation. Among the irradiated products, it can effectively slow down the damage to the skin caused by ultraviolet radiation, and has a significant effect of repairing and improving the skin damage after receiving solar ultraviolet radiation.

In one embodiment, the product acts on the skin.

In one of the embodiments, the present invention also provides a method for culturing skin-derived bacteria KBFS1720 with skin ultraviolet burn repairing function, comprising the following steps:

isolate the bacterial strain to obtain the first bacterial suspension;

The bacterial cell precipitate is washed with sterile PBS buffer, and after the washing is completed, the bacterial cell precipitate is suspended in sterile PBS buffer to obtain a second bacterial suspension.

In one embodiment, the strain is isolated from the skin of healthy adults who have been exposed to sunlight for a long period of time.

In one embodiment, the specific steps of isolating the strain are: using SCF-1 buffer to wet the cotton swab head, pressing the cotton swab head on the surface of the human forehead skin, and wiping it repeatedly for more than 30 times; placing the collected cotton swab head on the Sealed in a 2mL EP tube; sterile phosphate buffer was added to the 2mL EP tube, and the first bacterial suspension was obtained after sufficient shaking.

In one embodiment, the SCF-1 buffer comprises 50 mM Tris buffer, 1 mM EDTA, 0.5% Tween-20, and the pH of the Tris buffer is 7.6 and the pH of the EDTA is 8.0.

In one embodiment, the first medium comprises one or more of peptone nutrient agar medium, yeast extract powder agar medium, brain heart extract agar medium, sucrose medium, tryptone soy agar medium kind.

In one embodiment, the second medium is a nutrient agar medium.

In one embodiment, the temperature of the constant temperature overnight culture is 25°C-30°C.

In one embodiment, the third medium is a nutrient broth.

In one embodiment, the temperature of the constant temperature gas bath is 25°C-30°C.

In one embodiment, the conditions of the centrifugation are: the rotating speed is 5000r/min, and the time is 5min

In one embodiment, the bacterial cell concentration of the second bacterial suspension is 1×10 ⁶ CFU/mL to 1×10 ⁸ CFU/mL.

In one embodiment, the specific step of performing 16S full-length sequencing to identify the bacterial species to which it belongs is: performing Gram staining on the bacterial strain after multiple purification and culturing, and then confirming its purity through microscopic examination, using the mass percentage concentration as 15% glycerol was used to store the strain at -80°C. The 16S rDNA sequence was amplified and sequenced by primers 17F/1429R, and the sequence homology was compared with the sequence in the database, and the sequence homology with Pantoea eucrina strain LMG 2781 was obtained. 99.7% and numbered it KBFS1720. Microscopic examination by Gram staining showed that the bacterial cells were short rod-shaped, and the Gram staining was red, as shown in Figure 2.

The embodiments of the present invention will be described in detail below with reference to specific examples.

Example 1:

Isolated strains: Choose healthy adults in Daocheng County, Ganzi Prefecture, Sichuan Province at an altitude of 3750 meters, who have long-term outdoor activities and whose skin is often exposed to sunlight. Wet the cotton swab head with SCF-1 buffer, and then press the cotton swab head on the forehead. On the skin, wipe it repeatedly for more than 30 times; put the cotton swab into the 2mL EP tube to seal, and then fully shake with sterile phosphate buffer solution to obtain the first bacterial suspension; take 200 mL of the first bacterial suspension and spread it on peptone nutrient agar for culture base, yeast extract powder agar medium, brain heart extract agar medium, sucrose medium and tryptone soybean agar medium; 420 single colonies were selected from each medium, and 420 single colonies were selected for multiple purification and culture; 6 strains of skin were screened. The enriched bacteria are kept in glycerol;

Obtain the second bacterial suspension: streak the strains and the first bacterial suspension stored in glycerol in a nutrient agar medium for activation, and cultivate at a constant temperature of 28 °C overnight; select a single colony and inoculate it in 50 mL of nutrient broth, and place it in 50 mL of nutrient broth. 28 °C constant temperature air bath, after shaking overnight, centrifuge at 5000 r/min for 5 min to collect the cell pellet; wash the cell pellet three times with the same amount of sterilized PBS buffer, and then resuspend the cell pellet in In sterilized PBS buffer, the second bacterial suspension was obtained; through the count detection of viable bacteria on the plate, it was found that the concentration of the second bacterial suspension was between 1×10 ⁶ CFU/mL and 1×10 ⁸ CFU/mL; the first bacterial suspension Without dilution, the first bacterial suspension obtained from the culture was directly used for strain colonization, and each strain colonization was reactivated and cultured overnight with shaking.

Example 2:

16S full-length sequencing to identify the species: Gram-stain the strains purified and cultivated for several times, and confirm their purity by microscopy, and then store the strains at -80°C with 15% glycerol by mass. . The results showed that the strain of the present invention was isolated from the NA medium, and the 16S rDNA sequence was amplified and sequenced with the universal primer 17F/1429R, and homologous comparison was performed with the sequence in the database to obtain the same strain as Pantoea eucrina strain LMG 2781. The sequence homology reaches 99.7%, and it is numbered as KBFS1720, that is, the skin-derived bacterium KBFS1720 with skin ultraviolet burn repair function according to the present invention is obtained; the appearance of the bacteria is directly observed by culturing, and the colony is wet and the surface is sticky , smooth and easy to be picked, with neat and complete edges, transparent and shiny round, the colony is uplifted at the periphery, and the middle is concave; Microscopic examination by Gram staining showed that the bacterial cells were short rod-shaped, and the Gram staining was red, as shown in Figure 2.

Example 3:

Strain growth characteristics:

Take the second bacterial suspension obtained in Example 1, pick a ring of bacterial liquid with aseptic operation and streak culture until a single bacterial plaque appears, pick a ring of bacterial plaque aseptically, and insert it into the liquid culture solution at 150 r. /min, after culturing at 28°C for 18h, respectively take 1000μL of seed solution into a pre-prepared centrifuge tube containing 30mL of NB liquid medium, culture under the same conditions, take out the centrifuge tube on time and take 200μL of homogeneous culture solution in enzyme Three parallel experiments were set up to measure OD ₆₀₀ on a standard instrument, and the results were averaged. The results are shown in Figure 3.

Example 4:

Strain whole genome sequence determination:

The genomic DNA of the strain in the second bacterial suspension in Example 1 was extracted, and the whole genome sequence was determined. The results show: as shown in Figure 4.

The whole genome sequence is 4,053,112bp in length, contains 3781 independent coding regions (CDSs), contains 1 circular chromosome, and 4 plasmids. The chromosomal genome is circular, the average content of the sum of guanine (G) and thymine (C) is 55.91%, the length is 3 365 778 bp, and it contains 3100 CDSs with an average length of 1086 bp; Figure A) has a sequence length of 388 872 bp and contains 361 CDSs; plasmid 2 (Figure B in Figure 5) has a sequence length of 160 430 bp and contains 110 CDSs; plasmid 3 (Figure C in Figure 5) has a sequence length of 91 185 bp , contains 103 CDSs; plasmid 4 (panel D in Figure 5) has a sequence length of 100 847 bp and contains 107 CDSs. The total length of the coding region is 3 532 818 bp, accounting for 87.2% of the whole genome sequence. Among all 3 781 CDSs, 2 957 genes can be classified into the orthologous (Cluster of Orthologous Group, COG) functional classification database, of which 2 549 genes are distributed on chromosomes, and each distribution on plasmids 1-4 with 273, 81, 9, and 45 genes. Among them, 304 are involved in carbohydrate metabolism, 390 are involved in amino acid metabolism, 117 are involved in signal transfer mechanism, 235 are involved in the transport and metabolism of inorganic ions, 258 genes whose function is unknown, and 824 encoding genes related to COG There are no matches for genes in the database.

Example 5:

Construction of subacute light damage model mice, the specific process is shown in Figure 6; the model mice were treated with the 6 strains of bacteria enriched in the skin, as shown in Figure 7; the skin H&E of mice with different degrees of damage Stained sections are shown in Figure 8.

Test results and data analysis: After the test, the mice were sacrificed by cervical dislocation, and their back skin was removed, and the skin was immersed in a 4% by mass formaldehyde solution in time after ex vivo, and stored at 4°C; then paraffin-embedded, Sectioning, HE staining, and microscopic observation, a representative diagram of histopathological sections of mouse skin as shown in Figure 8 was obtained. Among them, the bare skin on the back of the mice in the normal group was pink flesh-colored, smooth and plump, and no obvious histopathological damage was found (Panel A in Figure 8); mild skin damage showed a small amount of inflammatory cell infiltration (Panel B in Figure 8). ); moderate skin lesions showed more inflammatory cell infiltration, hyperemia (panel C in Figure 8); severe skin lesions showed inflammatory cell infiltration, hyperemia, and inflammatory crusting (panel D in Figure 8). The observation results under the optical microscope showed that, except for the back skin of the normal group of ICR mice, the other groups of ICR mice had different degrees of histopathological changes, mainly manifested in the formation of inflammatory cell crusts on the skin surface attached to the epidermis. At the same time, the epidermis is obviously thickened; the fibrous tissue structure of the dermis is disordered, the capillaries are congested and the inflammatory cells infiltrate in different numbers; the subcutaneous is relatively more fat cells, and inflammatory cell infiltration can be seen there. According to the degree of skin epidermal damage and the severity of inflammatory cell infiltration, a scoring standard for histopathological damage was established. 1, 2, and 3 points were used to indicate mild, moderate and severe damage, and the cumulative score was calculated. The results are shown in Table 1 below. .

Table 1:

It should be noted that: test groups A-F respectively represent treatment groups using bacterial suspensions of Arthrobacter gandavensis, Bacillus psychrosaccharolyticus, the skin-derived bacteria KBFS1720 of the present invention, Paenibacillus amylolyticus, Paenibacillus terrae, and Psychrobacter psychrophilus.

Compared with the UV-irradiated control group, the experimental group C had a statistically significant difference (p<0.01), and other experimental groups had no statistical significance compared with the blank control group (p>0.05). Compared with the normal group, the blank control group and the test group had a statistically significant difference (p<0.01), as shown in Figure 9. In conclusion, the strain of the present invention plays a significant positive role in the repair of UV-induced skin damage.

The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity, all possible combinations of the technical features in the above-described embodiments are not described. However, as long as there is no contradiction between the combinations of these technical features, All should be regarded as the scope described in this specification.

The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims

A skin-derived bacterium KBFS1720 with a skin ultraviolet burn repair function, characterized in that the strain was preserved in the Guangdong Provincial Microorganism Culture Collection Center on December 23, 2020, and the strain preservation number is GDMCC: 61382, and the preservation address is Guangzhou Building 59 of the compound, No. 100, Xianlie Middle Road, City.
The application of the skin-derived bacterium KBFS1720 with skin ultraviolet burn repair function of claim 1 in resisting ultraviolet radiation and repairing light damage.
The application of the skin-derived bacterium KBFS1720 with skin ultraviolet burn repair function of claim 1 in the preparation of a product that resists ultraviolet radiation and repairs light damage.
The use according to claim 3, wherein the product is capable of acting on the skin.
A method for culturing skin-derived bacteria KBFS1720 with a skin ultraviolet burn repair function, characterized in that the method comprises the following steps:

isolate the bacterial strain to obtain the first bacterial suspension;

The bacterial suspension is placed in the first medium for cultivation;

After several times of purification and cultivation, 16S full-length sequencing was performed to identify the bacterial species, and the strains enriched in the skin were screened out;

The screened strains are activated in the second medium, and cultured overnight at a constant temperature to obtain activated strains;

The activated bacterial strain is inoculated in the third medium, the constant temperature air bath, the shaking overnight culture, the centrifugation, and the collection of the bacterial precipitation;

The bacterial cell precipitate is washed with sterile PBS buffer, and after washing is completed, the bacterial cell precipitate is suspended in sterile PBS buffer to obtain a bacterial suspension.
The method for culturing skin-derived bacteria KBFS1720 with skin ultraviolet burn repair function according to claim 5, wherein the bacterial strain is isolated from the skin of healthy adults exposed to sunlight for a long time.
The method for culturing skin-derived bacteria KBFS1720 with skin ultraviolet burn repair function according to claim 5, wherein the first culture medium comprises peptone nutrient agar medium, yeast extract powder agar medium, brain heart extract agar One or more of medium, sucrose medium, tryptone soy agar medium.
The method for culturing skin-derived bacteria KBFS1720 with skin ultraviolet burn repair function according to claim 5, wherein the second medium is a nutrient agar medium.
The method for culturing skin-derived bacteria KBFS1720 with skin ultraviolet burn repair function according to claim 5, wherein the third culture medium is a nutrient broth.
The method for culturing skin-derived bacteria KBFS1720 with skin ultraviolet burn repair function according to claim 5, wherein the bacterial suspension has a bacterial concentration of 1×10 6 CFU/mL to 1×10 8 CFU/mL mL.