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

Abstract

The invention relates to the technical field of probiotics, in particular to lactobacillus crispatus JYLU-888 capable of promoting EGF (epidermal growth factor) and EGFR (epidermal growth factor receptor) expression, bacterial powder and application. The lactobacillus crispatus JYLU-888 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on July 4, 2024, the preservation address is No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.31177. The lactobacillus crispatus JYLU-888 has the advantages that the lactobacillus crispatus JYLU-888 can be used for preparing the lactobacillus crispatus JYLU-888; the lactobacillus crispatus JYLU-888 strain provided by the invention can promote the expression level of an epidermal growth factor and an epidermal growth factor receptor, and compared with a traditional method for promoting the expression of the epidermal growth factor and the epidermal growth factor receptor, such as using a chemical synthetic drug, the strain does not have side effects of the drug, and can be used for preparing an epidermal growth factor and an epidermal growth factor receptor. The invention provides a green, safe and long-acting new way for improving human health, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotics, and particularly relates to a Lactobacillus crispatus JYLU-888 that promotes the expression of EGF and EGFR, a bacterial powder, and applications thereof. Background Art

[0002] Epidermal growth factor (EGF) is a peptide with strong mitogenic activity for epithelial cells from various tissue sources. The epidermal growth factor is composed of 50-60 amino acid molecules, and the chain contains 6 cysteine molecules. Stable disulfide bonds are formed between the cysteine molecules, making the entire peptide chain an active substance with three loop-connected parts. The epidermal growth factor can promote cell proliferation and maturation, accelerate the replacement of senescent cells and collagen synthesis, and promote the growth of granulation tissue, including the migration and proliferation of tissue cells, the formation of granulation tissue, and the deposition of extracellular matrix. In addition, it can also promote the migration of cells to the damaged area and accelerate wound healing.

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

[0004] Research shows that the signal transduction network composed of epidermal growth factor and epidermal growth factor receptor also has an important impact on the maintenance of bone homeostasis. The epidermal growth factor receptor can promote the differentiation of osteoclasts, thereby maintaining the bone in a healthy and stable state. Further, when the bone is externally damaged or fractured, by regulating the differentiation of osteoclasts, enabling them to aggregate to the damaged area and absorb and destroy the bone tissue in the damaged area, it can provide the necessary space and conditions for the growth of new bone.

[0005] Probiotics are a class of beneficial active microorganisms for the host that colonize the human body and change the flora composition in a certain part of the host. Lactobacillus crispatus ( Lactobacillus crispatus ) is a facultative anaerobe, Gram-positive, a slender, curved, and delicate bacillus, belonging to the phylum Firmicutes, class Bacilli, order Lactobacillales, family Lactobacillaceae, genus Lactobacillus, without flagella and spores. Due to its strong adhesion ability and tolerance to acid and bile salts, Lactobacillus crispatus is widely used in various probiotic products.

[0006] However, there has not been found a Lactobacillus crispatus with the efficacy of promoting the expression levels of EGF protein and EGFR protein. Summary of the Invention

[0007] In view of the current technical problem of the lack of Lactobacillus crispatus with the efficacy of promoting the expression levels of EGF protein and EGFR protein, the present invention provides a strain of Lactobacillus crispatus JYLU-888, a bacterial powder, and applications thereof that promote the expression of EGF and EGFR.

[0008] In a first aspect, the present invention provides a strain of Lactobacillus crispatus ( Lactobacillus crispatus ) JYLU-888, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on July 4, 2024. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31177.

[0009] In a second aspect, the present invention provides a bacterial powder containing the above-mentioned Lactobacillus crispatus JYLU-888.

[0010] Furthermore, the preparation method of the bacterial powder is to wash the cells of Lactobacillus crispatus JYLU-888 with sterile physiological saline, resuspend them in reconstituted skim milk, and then obtain the powder by freeze-drying and pulverizing.

[0011] Furthermore, the activated Lactobacillus crispatus JYLU-888 is inoculated into MRS liquid medium and cultured at 37 °C for 24 h to obtain a bacterial solution, and the cells are obtained after centrifuging the bacterial solution.

[0012] In a third aspect, the present invention also provides an application of the above-mentioned Lactobacillus crispatus JYLU-888 in the preparation of a bacterial agent that promotes the expression of EGF and EGFR. The bacterial agent contains the bacterial powder of Lactobacillus crispatus JYLU-888.

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

[0014] Furthermore, the cell count of Lactobacillus crispatus JYLU-888 in the bacterial agent is 5.0×10 9 cfu / g.

[0015] The beneficial effects of the present invention are as follows: The strain of Lactobacillus crispatus JYLU-888 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, this strain has no drug side effects, providing a new green, safe, and long-acting way to improve human health and having broad application prospects. Detailed implementation manners

[0016] To enable those skilled in the art to better understand the technical solutions in the present invention, the following will clearly and completely describe the technical solutions of the present invention in combination with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0017] Example 1 Isolation, Screening and Identification of Bacterial Strains 1. Strain Screening and Purification (1) Sampling: In May 2023, samples were taken from Nanchang City, Jiangxi Province and transported back to the laboratory by cold chain for sample preparation; (2) Sample preparation: ① Add 1 g of the sample in step (1) to a conical flask containing 9 mL of sterile normal saline, stir and shake at 4°C for 30 min, and set aside; ② Dilute the solution in step ① to prepare samples with different concentration gradients, which are 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 in sequence, labeled as 1#, 2#, 3#, 4#, 5#, 6#, 7# respectively, and set aside; (3) Preparation of MRS plate medium containing 0.5% CaCO3: 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 ammonium 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; After mixing the above raw materials, adjust the pH value to 6.8, heat and mix evenly, and sterilize at 121°C and 0.1 MPa for 20 min; Pour the sterilized medium into petri dishes and let it cool for later use; (4) Cultivation: Use a spreading rod to spread the solutions of 1# - 7# in step (2) on the MRS plate medium containing 0.5% CaCO3 respectively, and cultivate at 37°C under anaerobic conditions for 48 h; (5) Select colonies according to the following colony characteristics: The diameter is 1 - 2 mm, the colonies are round, the edges are neat, slightly white with a bulge in the middle, and the calcium dissolution circle is relatively large; (6) Isolation and purification: According to the appearance and morphology judgment, select 6 single colonies with the colony characteristics in step (5), inoculate them onto the medium in step (3) by the streaking method, cultivate at 37°C under anaerobic conditions for 48 h, repeat the above operation 2 - 3 times, pick the single colonies, place them in glycerol tubes and store at -70°C as alternative strains.

[0018] 2. Identification and preservation The single colonies obtained after separation and purification were sent for identification. The identification unit is Sangon Biotech (Shanghai) Co., Ltd. During the identification process, the following primers were used: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 1); 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 2).

[0019] The strain was identified as Lactobacillus crispatus and named JYLU-888.

[0020] The 16S rDNA gene sequence (SEQ ID NO: 3) of this Lactobacillus crispatus Lactobacillus crispatus (

[0021] Lactobacillus crispatus JYLU-888 was deposited with the General Microbiological Center of the China Committee for Culture Collection of Microorganisms. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is July 4, 2024, and the deposit number is CGMCC No. 31177. The taxonomic name is Lactobacillus crispatus Lactobacillus crispatus .

[0022] Example 2 Preparation of Bacterial Agent 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 ammonium 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; after mixing the above raw materials, adjust the pH value to 6.8, heat and mix evenly, and sterilize at 121 °C and 0.1 MPa for 20 min to prepare MRS solid medium; 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 ammonium citrate, 20 g of glucose, 0.2 g of magnesium sulfate heptahydrate, and 1000 mL of distilled water; after mixing the above raw materials, adjust the pH to 6.8, heat and mix evenly, and sterilize at 121 °C and 0.1 MPa for 20 min to prepare MRS liquid medium; Take the JYLU-888 screened in Example 1 and activate it on MRS solid medium. Inoculate the activated bacteria into MRS liquid medium at an inoculation amount of 1% (w / w), and then culture at 37 °C for 24 h to obtain a bacterial solution; Centrifuge the bacterial solution, collect the bacterial cells, wash them with sterile normal saline, and resuspend them in 15% (w / w) reconstituted skim milk to obtain a bacterial suspension. Control the bacterial concentration of the bacterial suspension within the range of 1.0 - 2.0×10 10 cfu / mL. After freeze-drying and pulverizing the bacterial suspension, obtain the bacterial powder of Lactobacillus crispatus JYLU-888.

[0023] Mix the bacterial powder of Lactobacillus crispatus JYLU-888 with maltodextrin (purchased from Baolingbao Biology Co., Ltd.) to make bacterial agents with bacterial cell numbers of 1.0×10 9 cfu / g, 2.0×10 9 cfu / g, and 5.0×10 9 cfu / g.

[0024] Example 3 Lactobacillus crispatus JYLU-888 Promotes the Expression of Skin EGF and EGFR 1. Rat Modeling and Grouping Female BALB / c mice weighing 20±2 g were selected. The mice were purchased from Changsha Tianqin Biotechnology Co., Ltd. After one week of adaptive feeding, all mice were depilated on the back skin tissue, and the depilated area was about 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.

[0025] Atopic dermatitis models were established in the mice of the model group and the JYLU-888 group. The specific method is as follows: On the 1st, 4th, and 7th days of the experiment, a 200 μL solution of 1-chloro-2,4-dinitrobenzene with a concentration of 1.0% (the solvent is a mixture of acetone and olive oil with a volume ratio of 3:1) was applied to the back skin tissue of the mice for sensitization, once a day, for a total of 3 times. On the 14th, 17th, 19th, 22nd, 24th, 27th, and 29th days of the experiment, a 20 μL solution of 1-chloro-2,4-dinitrobenzene with a concentration of 0.5% was repeatedly applied to the back skin tissue of the left ear of the mice for challenge, once a day, for a total of 7 times. Starting from the day of the experiment, the mice in the JYLU-888 group were given the Lactobacillus crispatus JYLU-888 bacterial agent prepared in Example 2 with a bacterial count of 5.0×10 9 cfu / g, and the dose of the bacterial agent was 0.1 mg, once a day.

[0026] The mice in the control group were smeared with an equal volume of the solvent at the same time points. After administration on the 30th day of the experiment, the mice were decapitated and sacrificed, and half of the skin lesion tissue was cut off and fixed in 4% paraformaldehyde, and then dehydrated, paraffin-embedded, and sectioned. After dewaxing, the tissue was incubated with hydrogen peroxide to quench the activity of endogenous peroxidase 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 dropwise, and the secondary antibody was incubated for 20 minutes, incubated with 3,3-diaminobenzidine (DAB) for 5 minutes, and finally counterstained with hematoxylin. The tissue was observed under a microscope and three visual fields were randomly selected for photographing under a light microscope. Cells showing brown signals were defined as immunohistochemically positive. Analysis was performed using Image J (1.48, National Institutes of Health, Bethesda, MD, USA), and scoring was performed according to the staining intensity and staining area of the cells, respectively.

[0027] Staining intensity scoring criteria: 0 points: negative (no visible staining); 1 point: low positive (light yellow); 2 points: moderately positive (brownish yellow); 3 points: highly positive (brownish black).

[0028] Staining area ratio scoring criteria: 0 points: ≤5% of the visual field area is stained; 1 point: 5% to 25% of the visual field area is stained (including 25%); 2 points: 25% to 50% of the visual field area is stained (including 50%); 3 points: >50% of the visual field area is stained.

[0029] According to the formula: Protein expression score = staining intensity score × staining area proportion score, calculate the total protein expression score. 0 points is negative, 1 - 3 points is low expression, 4 - 6 points is medium expression, and 6 - 9 points is high expression.

[0030] The results of EGF and EGFR protein expression scores in each group are shown in Table 1 below.

[0031] Table 1 EGF and EGFR protein expression scores in the dorsal skin of mice in each group

[0032] Note: *p < 0.01, compared with the model group.

[0033] It can be seen from Table 1 that the EGF and EGFR protein expression scores in the dorsal skin of the control group and JYLU - 888 group are significantly higher than those in the model group (p < 0.01). The above results indicate that Lactobacillus crispatus JYLU - 888 promotes the expression levels of EFG protein and EGFR protein in the dorsal skin of rats.

[0034] Example 4 Lactobacillus crispatus JYLU - 888 promotes the expression of EGF and EGFR in vaginal tissues Adult female SPF rats were selected. The rats were purchased from Changsha Tianqin Biotechnology Co., Ltd., with a body weight of 250g ± 35g. After two weeks of adaptive feeding, 8 rats were randomly selected as the sham - operation group; the rest were subjected to bilateral ovariectomy for modeling. When the vagina of the rats was congested, swollen, and the secretions were thick, it indicated that the ovaries had been completely removed and the model was successfully established.

[0035] The successfully modeled rats were randomly divided into a model group and a JYLU - 888 group, with 8 rats in each group; among them, the JYLU - 888 group was given the Lactobacillus crispatus JYLU - 888 bacterial agent with a cell number of 5.0×10 9 cfu / g prepared in Example 2 every day, with a bacterial dose of 0.2mg, 3 times a day, and continuously administered for three weeks.

[0036] The vaginal tissues of rats were soaked in wax, embedded and sectioned. After dewaxing, the tissues were incubated with hydrogen peroxide to quench the activity of endogenous peroxidase and heated to inactivate endogenous enzymes and biotin. The tissues were incubated with anti-epidermal growth factor antibody and anti-epidermal growth factor receptor protein antibody at 37 °C for 60 minutes respectively. The reaction enhancer was added dropwise, the secondary antibody was incubated for 20 minutes, incubated with 3,3-diaminobenzidine (DAB) for 5 minutes, and finally counterstained with hematoxylin. The tissues were observed under a microscope and three visual fields were randomly selected for photographing under a light microscope. Cells showing brown signals were defined as immunohistochemically positive. Analysis was performed using Image J (1.48, National Institutes of Health, Bethesda, MD, USA), and scoring was performed according to the staining intensity and staining area of the cells respectively. The scoring criteria were the same as in Example 3. The scoring results of EGF and EGFR protein expression in each group are shown in Table 2 below.

[0037] Table 2 Scoring of EGF and EGFR protein expression in vaginal tissues of rats in each group

[0038] Note: *p < 0.01, compared with the model group.

[0039] As can be seen from Table 2, there were significant differences in the expression of EGF protein and EGFR protein between the JYLU-888 group and the model group. The above results indicate that Lactobacillus crispatus JYLU-888 can increase the expression levels of EFG protein and EGFR protein in the vagina of rats.

[0040] Furthermore, in order to better determine the effect of Lactobacillus crispatus JYLU-888 on the mRNA expression of EGF and EGFR in rat vaginal tissues, total RNA of vaginal tissues from each group of rats was collected and extracted using TRIzol reagent (Thermo Fisher Scientific, Shanghai, China). The concentration of RNA was detected using a multifunctional enzyme-labeling instrument, and then the total RNA was reverse transcribed into cDNA according to the steps of the reverse transcription kit. The amplification conditions were: 95 °C for 10 min, 95 °C for 10 s, 60 °C for 30 s, 72 °C for 30 s, 40 cycles; dissociation curve analysis: 95 °C for 10 s, stepwise sampling, step temperature 0.5 °C. Using RN-ACTB as an internal reference, the relative mRNA expression levels of EGF protein and EGFR protein in vaginal tissues of rats in each group were calculated. The results are shown in Table 3 below.

[0041] Table 3 Relative mRNA expression levels of EGF protein and EGFR protein

[0042] Note: *p < 0.01, compared with the model group.

[0043] As can be seen from Table 3, the mRNA expressions of EGF and EGFR in the sham operation group and the JYLU-888 group were significantly higher than those in the model group (p<0.01), and the mRNA expression in the Lactobacillus crispatus JYLU-888 group even exceeded that in the sham operation group, indicating that Lactobacillus crispatus JYLU-888 has the effect of promoting the mRNA transcriptional expression of EGF and EGFR.

[0044] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, those of ordinary skill in the art can make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions should all be within the scope of the present invention. / Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered by 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 China Microbiological Culture Collection Administration on July 4, 2024. The deposit address is No. 3, Yard No. 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, characterized in that The preparation method comprises the following steps: washing the Lactobacillus crispatus JYLU-888 bacterial bodies with sterile physiological saline, resuspending the bacterial bodies in reconstituted skim milk to obtain a bacterial suspension, and freeze-drying and pulverizing the bacterial suspension.

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

5. A use of Lactobacillus crispatus JYLU-888 as claimed in claim 1 in the preparation of 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 inoculant also contains maltodextrin.

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

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