Lactobacillus crispatus JYLU-888 strain promoting EGF and EGFR expression, bacterial powder and application

Abstract

The present invention relates to the technical field of probiotics, and in particular to a strain of Lactobacillus crispatus JYLU‑888, bacterial powder and application thereof, which promotes the expression of EGF and EGFR. Lactobacillus curly JYLU‑888 was deposited with the General Microbiology Center of the China Culture Collection Administration on July 4, 2024, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 31177. The Lactobacillus crispatus JYLU‑888 strain provided by the present invention can promote the expression of epidermal growth factor and epidermal growth factor receptor. Compared with traditional methods for promoting the expression of epidermal growth factor and epidermal growth factor receptor, such as the use of chemically synthesized drugs, this strain has no drug side effects, providing a new green, safe, and long-term approach to improving human health and has broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of probiotics technology, specifically to a strain of Lactobacillus curvature JYLU-888 that promotes EGF and EGFR expression, its powder, and its applications. Background Art

[0002] Epidermal growth factor (EGF) is a peptide with strong mitogenic activity for epithelial cells from various tissues. EGF is composed of 50-60 amino acid molecules, with six cysteine ​​molecules in the chain. These cysteine ​​molecules form stable disulfide bonds, making the entire peptide chain an active substance consisting of three linked segments. EGF promotes cell proliferation and maturation, accelerates the replacement of senescent cells and collagen synthesis, and promotes the growth of granulation tissue, including the migration and proliferation of tissue cells, the formation of granulation tissue, and the deposition of extracellular matrix. It also promotes cell migration to damaged areas, accelerating wound healing.

[0003] The epidermal growth factor receptor (EGFR) is a large transmembrane receptor protein with a molecular weight of approximately 180 kDa, possessing ligand-induced tyrosine protein kinase activity. EGFR is responsible for binding with epidermal growth factor, thereby promoting cell division and proliferation.

[0004] Research has shown that the signaling network composed of epidermal growth factor and epidermal growth factor receptor (EGFR) plays a crucial role in maintaining bone homeostasis. EGFR promotes the differentiation of osteoclasts, thereby maintaining a healthy and stable bone state. Furthermore, when bones are damaged or fractured, osteoclast differentiation is regulated, causing them to aggregate to the damaged area, where they absorb and destroy bone tissue, providing the necessary space and conditions for new bone growth.

[0005] Probiotics are a class of live microorganisms that benefit the host by colonizing the human body and altering the composition of the gut microbiota in a specific part of the host. *Lactobacillus curvatureii* (…) Lactobacillus crispatus Lactobacillus curvature is a facultative anaerobic, Gram-positive bacterium, characterized by its slender, curved, and delicate shape. It belongs to the phylum Firmicutes, class Bacillus, order Lactobacilli, family Lactobacillus, and genus Lactobacillus. It is non-flagellated and non-spore-forming. Due to its strong adhesive ability and tolerance to acids and bile salts, Lactobacillus curvature is widely used in various probiotic products.

[0006] However, no *Lactobacillus curvatureensis* strains have yet been found to promote the expression levels of EGF and EGFR proteins. Summary of the Invention

[0007] In view of the current technical problem of lack of Lactobacillus crispatus that can promote the expression level of EGF protein and EGFR protein, the present invention provides a Lactobacillus crispatus JYLU-888 that promotes the expression of EGF and EGFR, bacterial powder and application.

[0008] In a first aspect, the present invention provides a strain of *Lactobacillus curvaturei* that promotes the expression of EGF and EGFR (…). Lactobacillus curly ) JYLU-888 was deposited in the General Microbiology Center of China Culture Collection Administration on July 4, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31177.

[0009] Secondly, the present invention provides a bacterial powder containing the above-mentioned Lactobacillus curvature JYLU-888.

[0010] Furthermore, the preparation method of the bacterial powder is as follows: wash the Lactobacillus curvature JYLU-888 cells with sterile physiological saline, resuspend them in reconstituted skim milk, freeze-dry, and pulverize them to obtain the powder.

[0011] Furthermore, the activated Lactobacillus curvature JYLU-888 was inoculated into MRS liquid medium and cultured at 37°C for 24 hours to obtain bacterial solution. After centrifugation, bacterial cells were obtained.

[0012] In a third aspect, the present invention further provides a use of the above-mentioned Lactobacillus crispatus JYLU-888 in preparing a bacterial agent for promoting the expression of EGF and EGFR, wherein the bacterial agent comprises bacterial powder of Lactobacillus crispatus JYLU-888.

[0013] Furthermore, the microbial agent also contains maltodextrin.

[0014] Furthermore, the bacterial count of Lactobacillus curlis JYLU-888 in the bacterial agent was 5.0 × 10⁻⁶. 9 cfu / g.

[0015] The beneficial effects of the present invention are:

[0016] The Lactobacillus crispatus JYLU-888 strain provided by the present invention can promote the expression levels of epidermal growth factor and epidermal growth factor receptor. Compared with traditional methods for promoting the expression of epidermal growth factor and epidermal growth factor receptor, such as using chemically synthesized drugs, the strain has no drug side effects, provides a new green, safe and long-lasting approach to improving human health, and has broad application prospects. DETAILED DESCRIPTION

[0017] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0018] Example 1: Isolation, screening, and identification of bacterial strains

[0019] 1. Strains screening and purification

[0020] (1) Sampling: In May 2023, samples were collected from Nanchang City, Jiangxi Province and transported back to the laboratory via cold chain for sample preparation;

[0021] (2) Sample preparation: ① Add 1 g of the sample from step (1) into a conical flask containing 9 mL of sterile saline, stir and shake at 4°C for 30 min, and set aside;

[0022] ② Dilute the solution from step ① to prepare samples with different concentration gradients, each at 10... -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , numbered 1#, 2#, 3#, 4#, 5#, 6#, 7# in sequence, for standby use;

[0023] (3) Prepare MRS plate culture medium containing 0.5% CaCO3: weigh 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of potassium hydrogen phosphate heptahydrate, 1mL of Tween 80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate, 5g of calcium carbonate, 15g of agar, and 1000mL of distilled water; mix the above ingredients, adjust the pH to 6.8, heat and mix thoroughly, and sterilize at 121℃ and 0.1MPa for 20min; pour the sterilized culture medium into a plate and let it cool for later use;

[0024] (4) Cultivation: Using a spreading stick, the solutions 1#~7# in step (2) were spread onto MRS plates containing 0.5% CaCO3 and cultured at 37℃ under anaerobic conditions for 48h.

[0025] (5) Select colonies according to the following characteristics: diameter 1-2 mm, round colonies, neat edges, slightly white color with a bulge in the middle, and a large calcium dissolution zone;

[0026] (6) Isolation and purification: Based on the appearance and morphology, six single colonies were selected from the colony characteristics of step (5), and inoculated onto the culture medium of step (3) by the streak method. The culture was cultured at 37°C under anaerobic conditions for 48 h. The above operation was repeated 2-3 times. A single colony was picked and stored in a glycerol tube at -70°C as an alternative strain.

[0027] 2. Identification and preservation

[0028] The isolated and purified single colonies were sent for identification by Sangon Biotech (Shanghai) Co., Ltd. The primers used in the identification process are as follows:

[0029] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 1);

[0030] 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 2).

[0031] The strain was identified as Lactobacillus crispatus and named JYLU-888.

[0032] The curly lactobacillus ( Lactobacillus crispatus The 16S rDNA gene sequence (SEQ ID NO: 3) of JYLU-888 is as follows:

[0033]

[0034] Lactobacillus crispatus JYLU-888 was deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date was July 4, 2024, with the deposit number CGMCC No. 31177, and the classification name was Lactobacillus crispatus. Lactobacillus crispatus .

[0035] Example 2 Preparation of bacterial agent

[0036] Weigh 10 g of peptone, 5 g of beef powder, 5 g of sodium acetate trihydrate, 2 g of dipotassium hydrogen phosphate heptahydrate, 1 mL of Tween 80, 0.05 g of manganese sulfate tetrahydrate, 2 g of triammonium citrate, 20 g of glucose, 0.2 g of magnesium sulfate heptahydrate, 5 g of calcium carbonate, 15 g of agar, and 1000 mL of distilled water; mix the above ingredients, adjust the pH to 6.8, heat to mix, and sterilize at 121°C, 0.1 MPa for 20 min to prepare MRS solid medium;

[0037] Weigh 10 g of peptone, 5 g of beef powder, 5 g of sodium acetate trihydrate, 2 g of dipotassium hydrogen phosphate heptahydrate, 1 mL of Tween 80, 0.05 g of manganese sulfate tetrahydrate, 2 g of triammonium citrate, 20 g of glucose, 0.2 g of magnesium sulfate heptahydrate, and 1000 mL of distilled water; mix the above ingredients, adjust the pH to 6.8, heat and mix thoroughly, and sterilize at 121°C, 0.1 MPa for 20 min to prepare MRS liquid medium;

[0038] JYLU-888, screened in Example 1, was activated on MRS solid medium. The activated bacteria were then inoculated into MRS liquid medium at an inoculation rate of 1% (w / w) and cultured at 37°C for 24 hours to obtain the bacterial culture.

[0039] The bacterial suspension was centrifuged, the cells were collected, washed with sterile saline, and resuspended in 15% (w / w) reconstituted skim milk to obtain a bacterial suspension. The bacterial concentration of the bacterial suspension was controlled at 1.0-2.0×10 10 cfu / mL range, the bacterial suspension was freeze-dried and then pulverized to obtain bacterial powder of Lactobacillus crispatus JYLU-888.

[0040] The powder of Lactobacillus crispatus JYLU-888 was mixed with maltodextrin (purchased from Baolingbao Biotechnology Co., Ltd.) to prepare a bacterial cell count of 1.0×10 9 cfu / g, 2.0×10 9 cfu / g and 5.0×10 9 CFU / g bacterial agent.

[0041] Example 3 Lactobacillus crispatus JYLU-888 promotes the expression of EGF and EGFR in the skin

[0042] 1. Rat modeling and grouping

[0043] Female BALB / c mice weighing 20 ± 2 g were purchased from Changsha Tianqin Biotechnology Co., Ltd. After one week of adaptive feeding, all mice underwent hair removal on their backs, with the hair removal area approximately 2 cm × 2 cm. The mice were randomly divided into a control group, a model group, and a JYLU-888 group, with 8 mice in each group.

[0044] Atopic dermatitis models were established in mice in the model group and JYLU-888 group. The specific methods are as follows:

[0045] On days 1, 4, and 7 of the experiment, sensitization was achieved by applying 200 μL of a 1.0% 1-chloro-2,4-dinitrobenzene solution (a mixture of acetone and olive oil in a 3:1 volume ratio) to the skin tissue on the back of mice, once daily for a total of 3 applications. On days 14, 17, 19, 22, 24, 27, and 29 of the experiment, challenge was achieved by repeatedly applying 20 μL of a 0.5% 1-chloro-2,4-dinitrobenzene solution to the skin tissue on the back of the left ear of mice, once daily for a total of 7 applications. Starting from the day of the experiment, mice in the JYLU-888 group were given 5.0 × 10⁻⁶ bacterial cells prepared in Example 2 daily. 9 Lactobacillus curvaturei JYLU-888 bacterial agent (cfu / g), dosage 0.1 mg, once daily.

[0046] Control group mice were treated with the same volume of solvent at the same time point.

[0047] On day 30 of the experiment, mice were euthanized by cervical dislocation, and half of the lesion tissue was excised, fixed in 4% paraformaldehyde, and then dehydrated, embedded in paraffin, and sectioned. After dewaxing, the tissue was incubated with hydrogen peroxide to quench endogenous peroxidase activity and heated to inactivate endogenous enzymes and biotin. The tissue was incubated with anti-epidermal growth factor antibody and anti-epidermal growth factor receptor protein antibody at 37°C for 60 minutes. A reaction enhancer was added, and the tissue was incubated with secondary antibody for 20 minutes, followed by incubation with 3,3-diaminobenzidine (DAB) for 5 minutes, and finally reverse stained with hematoxylin. The tissue was observed under a microscope, and three fields of view were randomly selected for photography under a light microscope. Cells showing brown signals were defined as immunohistochemically positive. Image J (1.48, National Institutes of Health, Bethesda, MD, USA) was used for analysis, and the cells were scored according to staining intensity and staining area.

[0048] Staining intensity scoring criteria:

[0049] 0 points: Negative (no visible staining);

[0050] 1 point: low positive (light yellow);

[0051] 2 points: moderately positive (brown-yellow);

[0052] 3 points: Highly positive (brown).

[0053] Scoring criteria for the percentage of stained area:

[0054] 0 points: ≤5% of the visual field area was stained;

[0055] 1 point: 5% to 25% of the visual field area is stained (inclusive of 25%);

[0056] 2 points: 25% to 50% of the visual field area is stained (inclusive of 50%);

[0057] 3 points: >50% of the visual field area is stained.

[0058] The total protein expression score is calculated using the formula: Protein Expression Score = Staining Intensity Score × Staining Area Percentage Score. 0 points indicates negative expression, 1-3 points indicate low expression, 4-6 points indicate moderate expression, and 6-9 points indicate high expression.

[0059] The results of EGF and EGFR protein expression scores for each group are shown in Table 1 below.

[0060] Table 1. EGF and EGFR protein expression scores on the back skin of mice in each group.

[0061]

[0062] Note: *p<0.01, compared with the model group.

[0063] As shown in Table 1, the expression scores of EGF and EGFR proteins in the back skin of the control group and the JYLU-888 group were significantly higher than those in the model group (p<0.01). These results indicate that *Lactobacillus curvatureii* JYLU-888 promotes the expression levels of EFG and EGFR proteins in the back skin of rats.

[0064] Example 4: Lactobacillus curvature JYLU-888 promotes the expression of EGF and EGFR in vaginal tissue.

[0065] Adult female SPF rats, purchased from Changsha Tianqin Biotechnology Co., Ltd., weighing 250g±35g, were selected. After two weeks of acclimatization feeding, 8 rats were randomly selected as the sham-operated group; the rest underwent bilateral ovariectomy to establish the model. When the rats' vaginas were congested, swollen, and had thick secretions, it indicated that the ovaries had been completely removed, and the model was successfully established.

[0066] Rats that successfully modeled the bacteria were randomly divided into a model group and a JYLU-888 group, with 8 rats in each group; the JYLU-888 group received 5.0 × 10⁻⁶ bacterial cells prepared in Example 2 daily. 9 cfu / g of Lactobacillus crispatus JYLU-888 bacterial agent, with a bacterial dosage of 0.2 mg, 3 times a day, for three consecutive weeks.

[0067] Rat vaginal tissue was soaked in wax, embedded, and sectioned. After dewaxing, the tissue was incubated with hydrogen peroxide to quench endogenous peroxidase activity and heated to inactivate endogenous enzymes and biotin. The tissue was incubated with anti-epidermal growth factor and anti-epidermal growth factor receptor antibodies, respectively, for 60 minutes at 37°C. A reaction enhancer was added, followed by incubation with secondary antibodies for 20 minutes, 3,3-diaminobenzidine (DAB) for 5 minutes, and finally counterstained with hematoxylin. The tissue was observed microscopically, and three randomly selected fields were photographed under a light microscope. Cells showing a brown signal were defined as immunohistochemically positive. Analysis was performed using Image J (1.48, National Institutes of Health, Bethesda, MD, USA), and cells were scored based on staining intensity and staining area. The scoring criteria were the same as in Example 3. The EGF and EGFR protein expression scores for each group are shown in Table 2 below.

[0068] Table 2 EGF and EGFR protein expression scores in vaginal tissues of rats in each group

[0069]

[0070] Note: *p<0.01, compared with the model group.

[0071] As can be seen from Table 2, there were significant differences in the expression of EGF and EGFR proteins in the JYLU-888 group compared with the model group. The above results indicate that Lactobacillus crispatus JYLU-888 can increase the expression levels of EFG and EGFR proteins in the rat vagina.

[0072] Furthermore, to better determine the effect of *Lactobacillus curvature* JYLU-888 on the mRNA expression of EGF and EGFR in rat vaginal tissue, total RNA was collected from the vaginal tissue of rats in each group and extracted using TRIzol reagent (Thermo Fisher Scientific, Shanghai, China). RNA concentration was detected using a multifunctional enzyme-linked immunosorbent assay (ELISA) reader, and then the total RNA was reverse transcribed into cDNA according to the reverse transcription kit. Amplification conditions were: 95℃ for 10 min, 95℃ for 10 s, 60℃ for 30 s, 72℃ for 30 s, for 40 cycles; lysis curve analysis was performed at 95℃ for 10 s, with a step size of 0.5℃. Using RN-ACTB as an internal control, the relative mRNA expression levels of EGF and EGFR proteins in the vaginal tissue of rats in each group were calculated. The results are shown in Table 3 below.

[0073] Table 3. Relative expression levels of EGF and EGFR protein mRNA

[0074]

[0075] Note: *p<0.01, compared with the model group.

[0076] As can be seen from Table 3, the mRNA expression of EGF and EGFR in the sham operation group and JYLU-888 group was significantly higher than that in the model group (p < 0.01), and the mRNA expression of the Lactobacillus crispatus JYLU-888 group even exceeded that of the sham operation group, indicating that Lactobacillus crispatus JYLU-888 has the effect of promoting the transcriptional expression of EGF and EGFR mRNA.

[0077] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A strain of Lactobacillus crispatus JYLU-888 that promotes the expression of EGF and EGFR, characterized in that: Lactobacillus crispatus ( Lactobacillus crispatus ) JYLU-888 was deposited in the General Microbiology Center of the China Culture Collection Administration on July 4, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31177.

2. A bacterial powder, characterized in that: Contains the Lactobacillus crispatus JYLU-888 as claimed in claim 1.

3. The bacterial powder according to claim 2, wherein The preparation method comprises the following steps: washing Lactobacillus crispatus JYLU-888 bacterial bodies with sterile physiological saline, resuspending the cells in reconstituted skim milk to obtain a bacterial suspension, and freeze-drying and pulverizing the suspension.

4. The bacterial powder according to claim 3, wherein The activated Lactobacillus crispatus JYLU-888 was inoculated into MRS liquid culture medium and cultured at 37° C. for 24 hours to obtain a bacterial solution, which was then centrifuged to obtain bacterial cells.

5. A use of the Lactobacillus crispatus JYLU-888 according to claim 1 in preparing a bacterial agent for promoting the expression of EGF and EGFR, characterized in that: The bacterial agent contains bacterial powder of Lactobacillus crispatus JYLU-888.

6. The use according to claim 5, characterized in that The inoculum also contains maltodextrin.

7. The use according to claim 5, characterized in that The number of Lactobacillus crispatus JYLU-888 in the inoculum was 5.0×10 9 cfu / g.

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