Lactobacillus rhamnosus LRa19 for inhibiting hyaluronidase activity and tryptase release and application of lactobacillus rhamnosus LRa19

Abstract

The invention relates to a lactobacillus rhamnosus LRa19 capable of inhibiting hyaluronidase activity and tryptase release and application of the lactobacillus rhamnosus LRa19. The lactobacillus rhamnosus LRa19 is classified and named as lactobacillus rhamnosus, the preservation number of the lactobacillus rhamnosus LRa19 is CGMCC (China General Microbiological Culture Collection Center) No.33767, and the preservation date of the lactobacillus rhamnosus LRa19 is March 10, 2025. The strain has good tolerance in gastric juice and cholate, and has the potential of smoothly entering the gastrointestinal tract of a human body. The strain can inhibit hyaluronidase activity and tryptase release, adjust Th1 / Th2 imbalance, improve the abundance of effective microbial communities in intestinal tracts, and effectively relieve and improve atopic dermatitis symptoms.

Description

Technical Field

[0001] The present invention belongs to the technical field of new strain development and application, and relates to a Lactobacillus rhamnosus LRa19 capable of inhibiting hyaluronidase activity and trypsin release and an application thereof. Background Art

[0002] At present, the main measure for treating allergic diseases is the use of anti-allergic drugs, but adverse reactions may occur, such as drowsiness, metabolic disorders, excessive gastric acid secretion, etc., especially in infants and pregnant women. There is a correlation between intestinal flora and the occurrence of allergic diseases. The use of probiotics may change the composition of intestinal flora, thereby regulating the responsiveness of the immune system. Allergic reactions can be divided into four types according to their occurrence mechanism and clinical characteristics. The allergic reactions referred to in the study of probiotics improving allergic diseases belong to type I hypersensitivity reactions, which are mainly IgE-mediated diseases such as atopic dermatitis, allergic rhinitis, allergic asthma, etc. Probiotics can interact with specific microorganisms or epithelial cells in the intestines of organisms to stimulate the body to mount a correct immune response.

[0003] Hyaluronidase is a player in type I hypersensitivity reactions and tumor cell proliferation, and it is strongly correlated with inflammation and allergies. The breakdown of hyaluronic acid can cause numerous diseases, such as allergies, inflammation, and degenerative arthritis. Furthermore, tryptase is a specific active substance of mast cells and basophils. When activated, it is released along with other mediators in the form of mast cell degranulation. It promotes airway repair, regulates the tone and reactivity of airway smooth muscle cells, and stimulates mast cell activation. It is associated with conditions such as bronchial asthma, chronic urticaria, anaphylactic shock, and systemic allergic reactions. Therefore, the development of probiotic strains that inhibit hyaluronidase activity and tryptase release is crucial. This would not only provide anti-inflammatory, anti-allergic, and degenerative arthritis-relieving benefits, but also improve the course of allergic inflammation. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the object of the present invention is to provide a Lactobacillus rhamnosus LRa19 that inhibits hyaluronidase activity and tryptase release and its application.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a rhamnosus Lactobacillus LRa19 that inhibits hyaluronidase activity and trypsin release. The rhamnosus Lactobacillus LRa19 is classified as Lacticaseibacillus rhamnosus, with a preservation number of CGMCC No. 33767 and a preservation date of March 10, 2025.

[0007] The present invention obtains and preserves a new Lactobacillus rhamnosus with the effect of suppressing hyaluronidase activity and tryptase release from infant feces, and is named as Lactobacillus rhamnosus Lacticaseibacillusrhamnosus LRa19 bacterial strain, and this bacterial strain has good tolerance in gastric juice and bile salts, and has the potentiality of smoothly entering the human gastrointestinal tract. This bacterial strain has excellent hyaluronidase inhibitory activity, and therefore has the potentiality of intervening and improving diseases related to hyaluronic acid or hyaluronidase, and can also be used as a hyaluronidase inhibitor for scientific research. Meanwhile, this bacterial strain can also suppress tryptase release, has the potentiality of intervening and improving diseases related to mast cell activation, and can also be used as a tryptase release inhibitor for scientific research. Because hyaluronidase can participate in most type I and T cell-mediated type IV allergic reactions, tryptase is a specific marker of mast cell activation and an inflammatory marker in allergic reactions. Based on the fact that the LRa19 strain has the effect of inhibiting hyaluronidase activity and tryptase release, it can effectively relieve and improve allergic symptoms, especially atopic dermatitis symptoms.

[0008] In addition, Lactobacillus rhamnosus is a probiotic bacterium, so the Lactobacillus rhamnosus LRa19 strain screened and obtained in the present invention is highly safe when used in preparing relevant efficacy products.

[0009] In a second aspect, the present invention provides a bacterial agent for inhibiting hyaluronidase activity and / or inhibiting trypsin release, wherein the strain in the bacterial agent includes the Lactobacillus rhamnosus LRa19 described in the first aspect.

[0010] Preferably, the number of viable bacteria of Lactobacillus rhamnosus LRa19 in the bacterial agent is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g, for example 1×10 8 CFU / mL, 2×10 8 CFU / mL, 5×10 8 CFU / mL, 8×10 8 CFU / mL, 1×10 9 CFU / mL, 5×10 9 CFU / mL, 1×10 10 CFU / mL, etc. Other specific point values within this numerical range can be selected and will not be described here one by one.

[0011] Preferably, the dosage form of the bacterial agent includes a solution, powder, tablet, granule or capsule. The dosage form of the bacterial agent involved in the present invention is not limited, including the most commonly used solution, powder, or further prepared capsule, tablet or granule.

[0012] Preferably, the bacterial agent may also contain excipients; the excipients include any one or a combination of at least two of a protective agent, a filler, a binder, a disintegrant, an emulsifier, a solubilizer, a solubilizing agent, an osmotic pressure regulator, a colorant, a pH regulator, an antioxidant, an antibacterial agent or a buffer.

[0013] In a third aspect, the present invention provides use of the Lactobacillus rhamnosus LRa19 according to the first aspect or the bacterial agent according to the second aspect in the preparation of a hyaluronidase activity inhibitor.

[0014] According to the research results of the present invention, Lactobacillus rhamnosus LRa19 can inhibit the activity of hyaluronidase at the in vitro level, that is, Lactobacillus rhamnosus LRa19 or its bacterial agent can be made into a preparation for exploring the physiological metabolic processes or disease mechanisms related to hyaluronic acid / hyaluronidase.

[0015] In a fourth aspect, the present invention provides use of the Lactobacillus rhamnosus LRa19 according to the first aspect or the bacterial agent according to the second aspect in the preparation of a trypsin release inhibitor.

[0016] According to the research results of the present invention, Lactobacillus rhamnosus LRa19 can inhibit the release of trypsin at the in vitro level (cellular level), that is, Lactobacillus rhamnosus LRa19 or its bacterial agent can be made into a preparation for exploring the physiological metabolic processes or disease mechanisms related to mast cell activation / trypsin release.

[0017] In a fifth aspect, the present invention provides use of the Lactobacillus rhamnosus LRa19 according to the first aspect or the bacterial agent according to the second aspect in the preparation of a product for preventing, improving or assisting in improving atopic dermatitis.

[0018] According to the research results of the present invention, Lactobacillus rhamnosus LRa19 can alleviate the symptoms of atopic dermatitis in mice, including alleviating the infiltration of mast cells in the mouse skin; alleviating the symptoms of dermatitis on the back of mice; reducing the swelling of the mouse ears caused by local inflammatory infiltration; restoring the balance between Th1 and Th2 immune responses; improving the imbalance of the mouse intestinal flora, increasing the abundance of beneficial bacteria in the intestine, and restoring the uniformity of the mouse intestinal flora.

[0019] In a sixth aspect, the present invention provides use of the Lactobacillus rhamnosus LRa19 described in the first aspect or the bacterial agent described in the second aspect in the preparation of a product for preventing, improving or assisting in improving allergic reactions.

[0020] In a seventh aspect, the present invention provides a method for inhibiting hyaluronidase activity, comprising adding the Lactobacillus rhamnosus LRa19 described in the first aspect or the bacterial agent described in the second aspect to a system containing hyaluronidase.

[0021] In an eighth aspect, the present invention provides a method for inhibiting degranulation of mast tumor cells, comprising administering the Lactobacillus rhamnosus LRa19 described in the first aspect or the bacterial agent described in the second aspect to mast tumor cells.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention isolates and preserves a new strain of Lactobacillus rhamnosus LRa19 from infant feces. The strain has good tolerance in gastric juice and bile salts and has the potential to smoothly enter the human gastrointestinal tract. The strain has excellent hyaluronidase inhibitory activity and therefore has the potential to intervene in and improve diseases related to hyaluronic acid or hyaluronidase, and can also be used as a hyaluronidase inhibitor for scientific research. At the same time, the strain can also inhibit the release of tryptase, has the potential to intervene in and improve diseases related to mast cell activation, and can also be used as a tryptase release inhibitor for scientific research. Based on the fact that the LRa19 strain has the effect of inhibiting hyaluronidase activity and tryptase release, it can effectively relieve and improve allergic symptoms, especially the symptoms of atopic dermatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a colony morphology of Lactobacillus rhamnosus LRa19 on a plate;

[0025] Figure 2 This is a bacterial image of Lactobacillus rhamnosus LRa19 under a microscope (10×100);

[0026] Figure 3 This is a bar graph showing the inhibition rate of hyaluronidase by Lactobacillus rhamnosus LRa19 and Lactobacillus rhamnosus LGG;

[0027] Figure 4 is a bar graph showing the effects of Lactobacillus rhamnosus LRa19 and Lactobacillus rhamnosus LGG on tryptase release;

[0028] Figure 5 is a picture of the skin lesions on the back of mice in different treatment groups;

[0029] Figure 6 is a picture of ear swelling in mice in different treatment groups;

[0030] Figure 7 is the right ear thickness analysis result of mice in different treatment groups;

[0031] Figure 8 These are the pathological sections of the back skin tissues of mice in different treatment groups;

[0032] Figure 9This is the toluidine blue staining of mast cells in the lesional area on the back of mice in different treatment groups;

[0033] Figure 10 This is the result of analyzing the number of mast cells in the skin lesions on the back of mice in different treatment groups.

[0034] Figure 11 This is a graph showing the analysis results of total IgE in the serum of mice in different treatment groups;

[0035] Figure 12 This is a graph showing the results of serum interferon-γ analysis in mice in different treatment groups;

[0036] Figure 13 This is the Chao1 index analysis result of the intestinal flora of mice in different treatment groups;

[0037] Figure 14 This is the Simpson index analysis result of the intestinal flora of mice in different treatment groups;

[0038] Figure 15 This is the species composition analysis result at the phylum level of the intestinal flora of mice in different treatment groups;

[0039] Figure 16 This is the analysis result of the ratio of Firmicutes to Bacteroidetes in the intestinal flora of mice in different treatment groups;

[0040] Figure 17 This is the result of relative abundance analysis of Lactobacillus in the intestines of mice in different treatment groups;

[0041] The LRa19 strain involved in the present invention is classified and named Lacticaseibacillus rhamnosus, the preservation time is March 10, 2025, the preservation number is CGMCC No. 33767, the preservation unit is the General Microbiology Center of the China Culture Collection Administration, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0042] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0043] The LRa19 strain involved in the following experiments is classified as Lacticaseibacillus rhamnosus and its deposit number is CGMCC No. 33767.

[0044] The P815 cells involved in the following experiments were purchased from the China Center for Type Culture Collection; BALB / c female mice were purchased from Beijing Sibeifu Biotechnology Co., Ltd.

[0045] LBS medium (g / L) used in the following experiments: 5.0 g yeast extract, 10.0 g tryptone, 6.0 g potassium dihydrogen phosphate, 0.034 g ferrous sulfate, 0.575 g magnesium sulfate, 20.0 g glucose, 25.0 g sodium acetate, 2.0 g ammonium citrate, 0.12 g manganese sulfate, pH adjusted to 5.5. Add 1.0 mL Tween-80 and 1.3 mL glacial acetic acid, dissolve in 1000 mL distilled water, and autoclave at 121°C for 15 min. For solid medium, add 15.0 g agar.

[0046] MRS medium (g / L) used in the following experiments: 20.0 g glucose, 10.0 g yeast peptone, 10.0 g beef extract powder, 5.0 g yeast extract powder, 5.0 g anhydrous sodium acetate, 1.0 g Tween-80, 2.0 g diammonium hydrogen citrate, 2.0 g dipotassium hydrogen phosphate, 0.58 g magnesium sulfate heptahydrate, and 0.19 g manganese sulfate monohydrate. Dissolved in 1000 mL of distilled water, adjusted to pH 6.8, and autoclaved at 115°C for 20 min. Solid medium was supplemented with 1.5% agar.

[0047] Preparation of LRa19 bacterial suspension used in the following experiments:

[0048] The rhamnosus Lactobacillus LRa19 strain was inoculated into MRS liquid culture medium and cultured at 37°C for 24 hours for activation. The activation was repeated twice to obtain rhamnosus Lactobacillus LRa19 bacterial liquid. The liquid was centrifuged at 5000 rpm for 5 minutes at 4°C. The bacterial slurry was collected, resuspended with physiological saline and adjusted to the required concentration.

[0049] Unless otherwise specified, all reagents and consumables used in the following experiments are commercially available, and the relevant experimental steps are common operations in the field and have technical meanings that can be generally understood by those skilled in the art.

[0050] Example 1

[0051] Isolation and screening of Lactobacillus rhamnosus LRa19:

[0052] Fecal samples collected from infants were aseptically diluted with 0.85% saline, plated on LBS agar plates, and incubated at 37°C for 72 hours. Colony morphology was visually observed, and suspected single colonies were selected for microscopic examination before initial screening and purification. Purified strains were incubated in MRS medium at 37°C for 12 hours, centrifuged, and resuspended in sterile 30% glycerol solution and stored at -80°C.

[0053] Example 2

[0054] Morphological characteristics and 16S rDNA identification of Lactobacillus rhamnosus LRa19:

[0055] The colony morphology of Lactobacillus rhamnosus on MRS medium and the bacteria under microscope (10×100) are shown as follows: Figure 1 and Figure 2 Lactobacillus rhamnosus grows well on MRS solid plates. Individual colonies are milky white, round, raised, medium-sized, with neat edges and a smooth surface. Microscopic examination reveals rod-shaped cells arranged singly, in pairs, or in chains, consistent with the staining characteristics of Gram-positive bacteria.

[0056] The screened target strain was cultured in liquid, the cells were collected, the genomic DNA was extracted, and the PCR amplification reaction was performed using the universal primer 27F / 1492R. The PCR amplification program was as follows: 94°C pre-denaturation for 5 minutes, 94°C denaturation for 30 seconds, 55°C annealing for 30 seconds, 72°C extension for 90 seconds, for a total of 35 cycles, and finally extension at 72°C for 10 minutes. The content and purity of the PCR amplification product were then tested, and after passing the test, it was sent to Wuhan Jinkairui Bioengineering Co., Ltd. for sequencing. The sequence was: SEQ ID NO.1. Based on the sequencing results, the BLAST tool in the NCBI database was used for homology comparison, and the obtained strain was identified as rhamnosus lactobacillus. The strain was named rhamnosus lactobacillus LRa19 and sent for preservation.

[0057] Example 3

[0058] Gastrointestinal tolerance experiment of Lactobacillus rhamnosus LRa19:

[0059] (1) Preparation of artificial gastric juice and artificial bile: Prepare 0.5% normal saline, adjust the pH to 3 with dilute hydrochloric acid (HCl), then add pepsin to a final concentration of 0.3%, fully dissolve, filter and sterilize with a 0.22 μm microporous filter, and set aside; add 0.2% (w / v) sodium thioglycolate to MRS medium, then add ox bile powder to a final concentration of 0.3%, filter and sterilize, and set aside;

[0060] (2) Take 1 mL of LRa19 bacterial suspension (1×10 8 CFU / mL), centrifuge, discard the supernatant, add 1mL artificial gastric juice, mix well, place in 37℃ incubator, and perform tolerance test of gastric juice 0h and gastric juice 3h respectively. Take another 1mL LRa19 bacterial suspension (1×10 8 CFU / mL), centrifuge, discard the supernatant, add 1 mL of artificial bile, mix well, place in a 37°C incubator, and perform tolerance tests for 0 h and 3 h, respectively.

[0061] Strain tolerance rate (%) = Nt / N0×100%, where N0 represents the viable bacterial count of the strain at 0 h (log CFU / mL), and Nt represents the viable bacterial count of the strain at 3 h (log CFU / mL).

[0062] The experimental results showed that the tolerance rates of Lactobacillus rhamnosus LRa19 after treatment in gastric juice and bile for 3 hours were 99.22±0.31% and 99.93±0.26%, respectively, indicating that the strain has the potential to successfully enter the human gastrointestinal tract.

[0063] Example 4

[0064] In vitro inhibition experiment of hyaluronidase by Lactobacillus rhamnosus LRa19:

[0065] Add 0.1 mL of 2.5 mmol / L CaCl2 and 0.5 mL of 600 U / mL hyaluronidase to a clean centrifuge tube and treat at 37°C for 20 min; add 0.5 mL of LRa19 bacterial suspension (1×10 8 CFU / mL) and incubate at 37°C for 20 minutes. Add 0.5 mL of 0.5 mg / mL sodium hyaluronate solution and incubate at 37°C for 30 minutes. Allow to stand at room temperature for 5 minutes. Add 0.5 mL of acetylacetone solution and 0.1 mL of 0.4 mol / L NaOH solution and boil in water for 15 minutes, then immediately place on ice for 5 minutes. Add 1 mL of Ehrlich's reagent and color develop at room temperature for 20 minutes. The absorbance is measured at 530 nm. The inhibition rate of commercially available Lactobacillus rhamnosus LGG strains against hyaluronidase was determined using the same method.

[0066] Inhibition rate / %=[(AB)-(CD)] / (AB).

[0067] Where: A is the absorbance of the control group (acetate buffer replaces the bacterial suspension); B is the absorbance of the blank group (acetate buffer replaces the enzyme solution and bacterial suspension); C is the absorbance of the experimental group; D is the absorbance of the sample blank group (acetate buffer replaces the enzyme solution).

[0068] The results are as follows Figure 3 As shown, the inhibition rate of LRa19 on hyaluronidase in vitro was 65.71±3.42%, and its inhibitory effect on hyaluronidase activity was better than that of the LGG group (62.84±1.36%), indicating that the LRa19 strain has a better inhibitory effect on hyaluronidase activity.

[0069] Example 5

[0070] Inhibition experiment of tryptase secretion by Lactobacillus rhamnosus LRa19:

[0071] (1) Mastoma cell culture: The mastoma cell P815 suspension in the culture flask was collected into a centrifuge tube and centrifuged (800 rpm, 3 min). The supernatant was discarded and the suspension was resuspended in culture medium (90% DMEM + 10% FBS). The suspension was placed in a 24-well plate and cultured in an incubator.

[0072] (2) After 24 h of culture, the P815 cells were taken out of the constant temperature incubator. The experiment set up four treatments, namely, the LRa19 group (0.5 mL of bacterial solution was inoculated into each well to make the bacterial solution concentration reach 2×10 8 CFU / mL), LGG group (0.5 mL of bacterial solution was inoculated into each well to make the bacterial solution concentration reach 2×10 8 CFU / mL), control group-1 (positive control, added 0.5mL of DMEM medium) and control group-2 (negative control, added 0.5mL of DMEM medium). The 24-well plate was placed in a 37°C constant temperature incubator and incubated for 3 hours. After the incubation, 100μg / mL of C48 / 80 was added to the probiotic treatment group, LGG group and control group-1, and sterile water was added to the control group-2, and the cells were incubated at 37°C for 1 hour. After the incubation, the cells were centrifuged at 1000rpm for 20 minutes, and the concentration of tryptase in the supernatant was determined using the corresponding ELISA kit.

[0073] The concentration of tryptase in the cell supernatant of different treatment groups was as follows Figure 4 As shown in the results, the tryptase content in the positive control group (C48 / 80) was significantly higher than that in the negative control group (CK) (p<0.05), indicating that compound C48 / 80 induced mast cell degranulation, released tryptase, and caused allergic symptoms. The tryptase content in the LRa19-treated group was significantly lower than that in the C48 / 80 group (p<0.05), and was not different from the CK group, indicating that the LRa19 strain can inhibit the release of tryptase and mast cell degranulation, and has the potential to alleviate allergies. In addition, its effect of inhibiting mast cell degranulation and tryptase release is better than that of the LGG-treated group.

[0074] Example 6

[0075] Lactobacillus rhamnosus LRa19 alleviates atopic dermatitis in mice:

[0076] (1) Specific pathogen-free BALB / c female mice (18-20 g) were housed in the animal room of the Hubei Provincial Food and Drug Safety Evaluation Center. The temperature was maintained at 22 ± 2°C, the humidity was 50 ± 5%, and the light-dark cycle was 12 h. They were allowed to drink water and eat freely. The methods used in this animal experiment were reviewed and approved by the Ethics Committee of the Hubei Center for Disease Control and Prevention (Ethics Committee No. 202410199).

[0077] (2) Before the experiment, the mice were adaptively fed for one week and then randomly divided into four groups: normal control group (NC), dermatitis model group (MC), drug group (Keto, ketotifen) and Lactobacillus rhamnosus LRa19 group (LRa19).

[0078] (3) During the entire experimental period, mice in the NC and MC groups were gavaged with 0.2 mL of sterile saline daily, mice in the Keto group were gavaged with an equal volume of drug solution (1 mg / kg), and mice in the LRa19 group were gavaged with an equal volume of Lactobacillus rhamnosus suspension (viable count of 1 × 10 9 CFU / mL) until the end of the experiment, a total of 3 weeks.

[0079] Before modeling, the mice's backs were shaved with a shaver. A base solution was prepared using acetone and olive oil in a 4:1 ratio. 2,4-dinitrofluorobenzene (DNFB) sensitizers were then prepared using the base solution and 0.5% and 0.2% DNFB. Modeling began in the second week. On the first day of modeling, a 0.5% DNFB solution was applied to the back skin of the mice to induce atopic dermatitis. On days 5, 8, 11, and 14, a 0.2% DNFB solution was applied to the back skin and right ears of the mice to induce atopic dermatitis. Mice in the control group were treated with an equal amount of the base solution. After the experiment, the mice were observed for skin lesions, and orbital blood was collected. The mice were sacrificed and samples collected for subsequent analysis.

[0080] (4) Evaluation of experimental results:

[0081] (4.1) Skin lesions on the back of mice

[0082] DNFB destroyed the skin barrier of mice, causing significant skin lesions on the back of the mice. Figure 5 Observation revealed that the back skin of mice in the MC group and the model group showed typical dermatitis symptoms, accompanied by varying degrees of scratches and crusting scales, while the skin of mice in the control group was smooth, rosy, and scratch-free. The positive drug (Keto group) significantly alleviated the dermatitis symptoms on the back of the mice, and the skin returned to normal. Compared with the model group, the pathological symptoms of the back epidermis of mice in the LRa19 group were alleviated, and no significant differences were observed with the Keto group.

[0083] (4.2) Effect of ear swelling on mice

[0084] Repeated topical application of DNFB can induce clinical symptoms of atopic dermatitis in mice. Changes in ear thickness reflect the degree of ear swelling caused by local inflammatory infiltration. At the end of the experiment, the ear thickness of mice in different treatment groups was as follows: Figure 6 As shown. The thickness of the ears of mice in the MC group was significantly higher than that in the NC group (p<0.01), and the positive drug Keto significantly reduced the swelling of the mice's ears (p<0.05) ( Figure 7 ). Compared with the MC group, LRa19 intervention significantly reduced the ear thickness of mice (p<0.01), and was closest to the healthy group ( Figure 7These results suggest that Lactobacillus rhamnosus LRa19 can alleviate the symptoms of atopic dermatitis after ingestion.

[0085] (4.3) Evaluation of pathological symptoms on the back of mice

[0086] The back skin of different treatment groups was stained with hematoxylin-eosin, and the results were as follows: Figure 8 As shown in the figure, compared to the NC group, DNFB-induced skin inflammation in the MC group resulted in a significant increase in dorsal skin thickness in mice, accompanied by extensive inflammatory cell infiltration, such as spongioma and acanthosis. Similar to the mitigating effect of the positive drug (Keto), LRa19 intervention significantly reduced dorsal skin thickness in mice, indicating that the strain significantly suppressed immune inflammation in the lesions on the mice's backs, alleviating skin inflammation.

[0087] (4.4) Mast cell inflammatory infiltration in the back skin

[0088] Mast cells, primarily distributed in the skin and mucous membranes, are important effector cells in various allergic diseases, particularly atopic dermatitis. Activated mast cells release multiple inflammatory mediators, which further promote the development of skin lesions and the itch associated with atopic dermatitis. Therefore, we evaluated the effect of LRa19 on mast cell inflammatory infiltration in the dorsal skin of mice with atopic dermatitis.

[0089] The inflammatory infiltration of mast cells in the back skin of mice in different treatment groups is shown in Figure 2. Figure 9 As shown in the figure, toluidine blue staining showed that compared with the NC group, the MC group mice showed significant pathological symptoms of mast cell inflammatory infiltration in the back lesions, and the number of mast cells increased significantly (p < 0.05). LRa19 intake alleviated the infiltration of mast cells in the mouse skin ( Figure 9 ), significantly reduced the number of mast cells in the back skin of mice ( Figure 10 ), thereby helping to improve and alleviate the clinical skin lesions of atopic dermatitis on the back of mice.

[0090] (4.5) Cellular inflammatory factors

[0091] After the experiment, orbital blood was collected from the mice, and the blood was allowed to stand at room temperature for 30 min, then centrifuged (3000 r / min, 10 min), and the upper serum was collected. The content of inflammatory factors in the serum of each group of mice was determined according to the detection method in the ELISA kit manufacturer's instructions.

[0092] The core immune mechanism of atopic dermatitis is an excessive Th2 immune response. Th2 immune cells release cytokines such as IL-4, IL-5, and IL-13 to induce the synthesis of IgE and the infiltration of inflammatory cells, thereby destroying the normal skin barrier and leading to pathological symptoms of severe skin lesions. Therefore, the large amount of IgE produced in the serum has been recognized as a typical sign of atopic dermatitis. Figure 11 It can be seen that after local DNFB sensitization of mice, the total serum IgE level of the model group mice increased significantly, while the total serum IgE level of the mice was significantly reduced after LRa19 intervention (p < 0.05), and there was no significant difference in the total serum IgE level compared with the control group, indicating that LRa19 can inhibit Th2 immune response after intake.

[0093] In inflammatory responses, regulatory T lymphocytes can play an important role in immune regulation by producing cytokines, especially IFN-γ, which can inhibit Th2 cells and effector cells of allergic inflammation. Figure 12 Compared with the NC group, the IFN-γ level in the MC group was significantly lower (p < 0.05). LRa19 intervention significantly increased the IFN-γ level in the mouse serum (p < 0.05). Increased IFN-γ levels indicate an upregulation of the Th1 immune response, thus helping to restore the balance between the Th1 and Th2 immune responses and alleviate the pathological symptoms of atopic dermatitis.

[0094] (4.6) Changes in intestinal flora

[0095] After the experiment, the mice were killed and dissected, and their cecal contents were collected. The 16S rRNA was sequenced and analyzed.

[0096] In order to investigate the effect of Lactobacillus rhamnosus LRa19 on the α diversity of intestinal flora in mice with atopic dermatitis, the Chao1 index was used to characterize the changes in the α diversity of intestinal flora. Figure 13 As shown in the figure, compared with the MC group, there was no significant difference in the Chao1 index of intestinal microorganisms in the NC group, Keto group and LRa19 group, indicating that LRa19 intake had no significant effect on the α-diversity of the intestinal flora in mice.

[0097] The Simpson index indicates the uniformity of intestinal flora species. The Simpson index of mice in different treatment groups is as follows: Figure 14As shown in the results, the Simpson index of the intestinal flora of mice in the MC group was significantly lower than that in the NC group, indicating that the symptoms of atopic dermatitis caused by DNFB reduced the uniformity of the intestinal flora (p<0.05). After the intake of LRa19, the Simpson index of the flora was significantly increased (p<0.05), with no difference from the NC group and the Keto group, indicating that LRa19 helps to restore the uniformity of the intestinal flora and thus alleviate the symptoms of atopic dermatitis.

[0098] The relative abundance of different bacterial groups in the intestines of mice in different treatment groups is as follows Figure 15 As shown in the figure, at the phylum level, the mouse intestinal flora is mainly composed of Firmicutes, Bacteroidota, Campylobacterota, and also contains other low-abundance intestinal microorganisms such as Proteobacteria, Actinobacteriota, etc. The intake of LRa19 changes the composition of the intestinal flora of mice with atopic dermatitis.

[0099] The intestinal flora Firmicutes / Bacteroidetes (F / B ratio) ratio has the potential to serve as a biomarker of intestinal flora in pathological conditions. The intestinal flora F / B ratio of mice in different treatment groups is as follows Figure 16 As shown in the data, compared with the NC group, the relative abundance of Bacteroidetes in the intestinal flora of mice in the MC group increased, while the relative abundance of Firmicutes decreased, resulting in a decrease in the F / B ratio; compared with the decreased F / B ratio in the MC group, LRa19 intake could significantly increase the F / B ratio.

[0100] The intake of Lactobacillus and Bifidobacterium not only improves the abundance of beneficial bacteria in the intestine, but also regulates the balance of intestinal flora, and has been used by researchers to alleviate the clinical symptoms of atopic dermatitis. To further explore the effect of LRa19 intake on the beneficial flora in the intestine of mice, the changes in the relative abundance of Lactobacillus were analyzed at the genus level. Figure 17 The results showed that LRa19 consumption significantly increased the relative abundance of Lactobacillus in the intestinal flora of mice (p<0.05). This increase in the relative abundance of beneficial intestinal flora can regulate the intestinal microecology of mice to maintain a balanced state and maintain a healthy state of the body. These results indicate that the consumption of Lactobacillus rhamnosus LRa19 can help improve the imbalance of intestinal flora in mice caused by DNFB and alleviate the symptoms of atopic dermatitis.

[0101] The applicant declares that the present invention is illustrated by the above-described embodiments, but the present invention is not limited to the above-described embodiments. This does not mean that the present invention must rely on the above-described embodiments in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements for raw materials in the present invention, additions of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0102] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0103] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A Lactobacillus rhamnosus LRa19 that inhibits hyaluronidase activity and trypsin release, characterized in that: The classification name of the Lactobacillus rhamnosus LRa19 is Lacticaseibacillus rhamnosus, the preservation number is CGMCC No.33767, and the preservation date is March 10, 2025.

2. A bacterial agent for inhibiting hyaluronidase activity and / or inhibiting trypsin release, characterized in that: The strain in the bacterial agent includes the Lactobacillus rhamnosus LRa19 described in claim 1.

3. The microbial agent according to claim 2, characterized in that The number of viable bacteria of Lactobacillus rhamnosus LRa19 in the inoculum is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g.

4. The microbial agent according to claim 2, characterized in that The dosage form of the bacterial agent includes solution, powder, tablet, granule or capsule.

5. Use of the Lactobacillus rhamnosus LRa19 according to claim 1 or the bacterial agent according to any one of claims 2 to 4 in the preparation of a hyaluronidase activity inhibitor.

6. Use of the Lactobacillus rhamnosus LRa19 according to claim 1 or the bacterial agent according to any one of claims 2 to 4 in the preparation of a trypsin release inhibitor.

7. Use of the Lactobacillus rhamnosus LRa19 according to claim 1 or the bacterial agent according to any one of claims 2 to 4 in preparing a product for preventing, improving or assisting in improving atopic dermatitis.

8. Use of the Lactobacillus rhamnosus LRa19 according to claim 1 or the bacterial agent according to any one of claims 2 to 4 in preparing a product for preventing, improving or assisting in improving allergic reactions.

9. A method for inhibiting hyaluronidase activity, characterized in that: The method comprises: adding the Lactobacillus rhamnosus LRa19 according to claim 1 or the bacterial agent according to any one of claims 2 to 4 to a system containing hyaluronidase.

10. A method for inhibiting mast cell degranulation, characterized in that: The method comprises: administering the Lactobacillus rhamnosus LRa19 according to claim 1 or the bacterial agent according to any one of claims 2 to 4 to mastoma cells.

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