A type of Lactobacillus plantarum YYS-B1 with hyaluronidase inhibitory activity and its application

Abstract

This invention relates to the field of microbial technology, specifically to a *Lactobacillus plantarum* YYS-B1 strain with hyaluronidase inhibitory activity and its applications. *Lactobacillus plantarum* (… Lactiplantibacillus plants YYS-B1 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 12, 2023, with accession number CGMCC No. 27599. This *Lactobacillus plantarum* YYS-B1 exhibits excellent acid and bile salt resistance, enabling it to successfully reach the intestines and exert its probiotic effects. It also demonstrates outstanding anti-allergic, anti-inflammatory, and antioxidant capabilities, specifically exhibiting high hyaluronidase inhibition, strong free radical scavenging ability, and a good inhibitory effect on inflammatory responses caused by allergies. Therefore, it has significant application value and can be used in the development of functional products.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a plant lactobacillus YYS-B1 with hyaluronidase inhibitory activity and its applications. Background Technology

[0002] Allergies are complex immune diseases with symptoms including asthma, urticaria, food allergies, and inflammatory reactions such as rhinitis and dermatitis, severely impacting patients' quality of life. Current allergy treatments have side effects and risks; therefore, there is a need to find safe and effective anti-allergy strategies.

[0003] In recent years, research on the health benefits of probiotics has garnered significant attention. Studies have shown that probiotics exert their anti-allergic effects by inhibiting hyaluronidase activity. Hyaluronidase is an enzyme that hydrolyzes hyaluronic acid; its overexpression leads to excessive breakdown of hyaluronic acid, triggering allergic reactions. Inhibiting hyaluronidase can suppress allergic reactions, playing a crucial role in type I and type IV hypersensitivity reactions, and also exhibiting a certain degree of anti-inflammatory effect. While some natural products and compounds have been found to inhibit hyaluronidase activity, their application is limited. Current research confirms that probiotics such as *Lactobacillus plantarum* can effectively inhibit hyaluronidase activity, reduce IgE levels, and alleviate allergy symptoms.

[0004] Lei Wenping et al. have confirmed that probiotics have the function of inhibiting hyaluronidase, and Lactobacillus plantarum ( Lactiplantibacillus plantarum ) and Lactobacillus rhamnosus ( Lactobacillus rhamnosus These drugs have good inhibitory effects and can also reduce serum IgE levels.

[0005] Furthermore, allergic diseases are often accompanied by inflammatory responses; probiotics with anti-inflammatory activity can reduce allergic inflammatory responses, regulate the balance of the immune system, promote tissue repair, and protect the mucosal barrier. Through these mechanisms, probiotics can reduce the release of inflammatory mediators, inhibit the activation and migration of inflammatory cells, thereby reducing the inflammatory response caused by allergies, helping to alleviate symptoms and improve patients' quality of life.

[0006] Chinese invention patent application number CN201510613663, published on August 5, 2017, discloses that: when the amount of agarwood extract with soothing and anti-allergic effects reaches 8.0 mg / mL, the hyaluronidase inhibition rate can reach up to 92.0%; and the DPPH free radical scavenging rate reaches 78.0%.

[0007] Currently, there are very few reports on probiotics that inhibit hyaluronidase, and even fewer reports on probiotic strains that simultaneously possess anti-allergic properties, soothe allergic reactions and inflammation, and reduce oxidative damage caused by allergies and inflammation. Summary of the Invention

[0008] To address the shortcomings of the prior art mentioned in the background section, this invention provides a *Lactobacillus plantarum* YYS-B1, the technical solution of which is as follows:

[0009] The *Lactobacillus plantarum* YYS-B1 provided by this invention is derived from Wujiang pickled mustard tuber in Xiamen City, Fujian Province, and is classified and named *Lactobacillus plantarum* YYS-B1. Its Latin scientific name is: Lactiplantibacillus plantarum It was deposited on June 12, 2023 at the China General Microbiological Culture Collection Center, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, accession number: CGMCC No. 27599.

[0010] This strain exhibits the following characteristics: ability to inhibit hyaluronidase; reduction of IgE levels; acid and bile salt resistance; scavenging of DPPH and ABTS free radicals; and reduction of the release of inflammatory mediators.

[0011] Therefore, it has the following functions: it can inhibit hyaluronidase and reduce IgE levels, thus having anti-allergic ability; it can inhibit inflammatory responses caused by allergies, thus having anti-inflammatory ability; it can prevent oxidative damage caused by allergic reactions, thus having antioxidant ability; it has good acid and bile salt resistance, and can be used to prepare probiotic products, anti-allergy or anti-inflammatory products, providing new directions and more possibilities for the development of anti-allergy or anti-inflammatory products.

[0012] This invention provides the application of *Lactobacillus plantarum* YYS-B1 as described above in the preparation of probiotic products.

[0013] This invention provides the application of *Lactobacillus plantarum* YYS-B1 as described above in the preparation of anti-allergy products.

[0014] This invention provides the application of *Lactobacillus plantarum* YYS-B1 as described above in the preparation of anti-inflammatory products.

[0015] This invention provides the application of *Lactobacillus plantarum* YYS-B1 as described above in the preparation of antioxidant products.

[0016] The present invention also provides a freeze-dried powder, the components of which include *Lactobacillus plantarum* YYS-B1 as described above.

[0017] In one embodiment, the viable count of the *Lactobacillus plantarum* YYS-B1 is not less than 1 × 10⁻⁶. 11 CFU / g.

[0018] The present invention also provides a microbial agent, wherein the components of the microbial agent comprise the freeze-dried powder as described above, and / or, the components of the microbial agent comprise *Lactobacillus plantarum* YYS-B1 as described above.

[0019] In one embodiment, the microbial agent further includes other ingredients, which are existing ingredients suitable for microbial agents, such as at least one of prebiotics, fillers, acidulants, solvents, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, lubricants, wetting agents, stabilizers, flow aids, flavoring agents, preservatives, coating materials, fragrances, anti-adhesion agents, binding agents, thickeners, and inclusion agents.

[0020] This invention provides the application of *Lactobacillus plantarum* YYS-B1 as described above in the preparation of functional products.

[0021] In one embodiment, the functional product includes at least one of the following functions:

[0022] (1) It has the ability to inhibit hyaluronidase;

[0023] (2) Reduce IgE levels;

[0024] (3) Acid and bile salt resistance;

[0025] (4) Scavenging free radicals DPPH;

[0026] (5) Scavenging free radicals ABTS;

[0027] (6) Reduce the release of inflammatory mediators.

[0028] Based on the above, compared with the prior art, the *Lactobacillus plantarum* YYS-B1 provided by the present invention has the following beneficial effects:

[0029] The *Lactobacillus plantarum* YYS-B1 strain exhibits outstanding anti-allergic, anti-inflammatory, and antioxidant capabilities. Specifically, it demonstrates high hyaluronidase inhibition, strong free radical scavenging ability, and anti-inflammatory activity. This gives it both anti-allergic properties and a strong inhibitory effect on allergic-induced inflammatory responses, effectively reducing oxidative damage caused by allergic and inflammatory reactions. Furthermore, its good acid and bile salt resistance allows it to easily reach the intestines and exert its probiotic effects. This makes it suitable for use in the preparation of probiotic products, anti-allergy products, and anti-inflammatory products. As a natural probiotic, it has no side effects, making it safer and healthier. It provides an excellent microbial resource for the development of anti-allergy or anti-inflammatory products, offering new directions and more possibilities for product development.

[0030] Other features and beneficial effects of the invention will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other beneficial effects of the invention can be realized and obtained by means of the structures particularly pointed out in the description, claims and drawings. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Unless otherwise specified, the positional relationships shown in the drawings in the following description are based on the direction in which the components are drawn in the figure.

[0032] Figure 1 This is a photograph of the calcium dissolution zone during the isolation process of Lactobacillus plantarum YYS-B1.

[0033] Figure 2 This is a photograph of the colony morphology of Lactobacillus plantarum YYS-B1.

[0034] Figure 3 The image shows the Gram staining results of Lactobacillus plantarum YYS-B1.

[0035] Figure 4 Agarose gel electrophoresis image of the 16S rRNA amplification product of Lactobacillus plantarum YYS-B1;

[0036] Figure 5 The graph shows the effect of Lactobacillus plantarum YYS-B1 on the activity of superoxide dismutase (SOD) in mouse serum and liver.

[0037] Figure 6 The graph shows the effect of Lactobacillus plantarum YYS-B1 on the activity of glutathione peroxidase (GSH-PX) in mouse serum and liver.

[0038] Figure 7 A schematic diagram of a phylogenetic tree of *Lactobacillus plantarum* YYS-B1 based on its 16S rRNA sequence. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. The technical features designed in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0040] In the description of this invention, it should be noted that all terms used in this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and should not be construed as limiting the invention; it should be further understood that the terms used in this invention should be understood to have the same meaning as those in the context of this specification and in the relevant field, and should not be understood in an idealized or overly formal sense, except as expressly defined in this invention.

[0041] This invention provides a *Lactobacillus plantarum* YYS-B1:

[0042] The Latin scientific name of Lactobacillus plantarum YYS-B1 is: Lactiplantibacillus plantarum It was deposited on June 12, 2023 at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 27599.

[0043] Source: Separated from Wujiang pickled mustard tuber from Xiamen City, Fujian Province;

[0044] Colony morphology: The colony surface is raised, milky white, round, and smooth;

[0045] This strain has the following characteristics: it has the ability to inhibit hyaluronidase; it reduces IgE levels; it is acid and bile salt resistant; it scavenge free radical DPPH; it scavenge free radical ABTS; and it reduces the release of inflammatory mediators.

[0046] Therefore, it has the following functions: it can inhibit hyaluronidase and reduce IgE levels, thus having anti-allergic ability; it can inhibit inflammatory responses caused by allergies, thus having anti-inflammatory ability; it can prevent oxidative damage caused by allergic reactions, thus having antioxidant ability; it has good acid and bile salt resistance, and can be used to prepare probiotic products, anti-allergy or anti-inflammatory products, providing new directions and more possibilities for the development of anti-allergy or anti-inflammatory products.

[0047] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0048] Example 1: Isolation of Lactobacillus plantarum YYS-B1

[0049] Aseptic sampling was performed on Wujiang pickled mustard tuber using the spread plate method. 5 g of Wujiang pickled mustard tuber sample was placed in a sterile homogenizing bag, labeled, and 45 mL of 0.85% physiological saline was added. The sample was then thoroughly mixed by tapping.

[0050] Then, 100 μL of sample was taken and serially diluted 10-fold. -4 10-5 10 -6 100 μL of the sample was spread onto an MRS plate containing 2.5% CaCO3 and incubated upside down at 37°C for 24 h. Colonies with good growth and large calcium-dissolving zones were selected (see [reference]). Figure 1 The colonies were repeatedly isolated and purified using the streak plate method until single colonies were obtained. The colony morphology was characterized by a raised, milky-white, round surface with a smooth surface (see [link]). Figure 2 The isolated strain was named YYS-B1, numbered, and stored in a bacterial bank at -80℃ with glycerol.

[0051] Example 2: Identification of Lactobacillus plantarum YYS-B1

[0052] 2.1 Physiological and Biochemical Tests

[0053] The purified strain YYS-B1 was subjected to Gram staining (see [link to Gram staining]). Figure 3 The bacterial strain YYS-B1 underwent a catalase test and its physiological and biochemical indicators were measured. The results were compared with those in Bergey's Manual of Systematic Bacteriology, 8th Edition, for preliminary identification of the bacterial species. The test showed that the selected strain YYS-B1 stained purple with a Gram stain, indicating a positive result. Its cells were rod-shaped, catalase-negative, and did not form spores.

[0054] 5.2 Identification of 16S rRNA

[0055] YYS-B1 DNA was extracted according to the instructions of the bacterial gene DNA extraction kit, and PCR amplification of the 16S rRNA gene was performed using the DNA as a template.

[0056] The amplification primers used are universal primers:

[0057] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1).

[0058] 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO. 2).

[0059] The PCR reaction system consisted of: 2 μL DNA, 2 μL 27F, 2 μL 1492R, 25 μL Premix Ex Taq, and 19 μL ddH2O.

[0060] PCR reaction conditions: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles; final extension at 72℃ for 5 min.

[0061] YYS-B1 DNA genome extraction followed by 16S rRNA amplification fragment (see...) Figure 4 Then, the PCR amplification products were sent for DNA sequencing (Guangzhou Qingke Biotechnology Co., Ltd.). The sequencing results were used to search for similar sequences in the NCBI database using Blast software. The obtained sequences were compared with the 16S rRNA gene sequences of related species obtained from gene banks. A phylogenetic tree was constructed using Mega 11.0 software (see [link to documentation]). Figure 7 ).

[0062] The results of 16S rRNA gene sequencing are as follows:

[0063]

[0064] Based on a comprehensive analysis of the strain's colony and cell morphology, physiological and biochemical characteristics, 16S rRNA gene sequence, and with reference to Bergey's Manual of Systematic Bacteriology, 8th Edition, the strain was identified as *Lactobacillus plantarum*. Lactiplantibacillus plantarum ).

[0065] Example 3: Hyaluronidase Inhibition Test by Lactobacillus plantarum YYS-B1

[0066] 3.1 In vitro inhibition analysis of hyaluronidase

[0067] The *Lactobacillus plantarum* YYS-B1 strain was activated to the second generation, following the Elson-Morgan modified method. 0.1 mL of 2.5 mmol / L CaCl₂ was added to the test tube. 2， Add 0.5 mL of 600 U / mL hyaluronidase and treat at 37℃ for 20 min; add 0.5 mL of bacterial suspension and treat at 37℃ for 20 min; add 0.5 mL of 0.5 mg / mL sodium hyaluronate solution and treat at 37℃ for 30 min; let stand at room temperature for 5 min; add 0.5 mL of acetylacetone solution (1.4 mL of acetylacetone dissolved in 20 mL of 1.0 mol / L Na2CO3 solution, freshly prepared) and 0.1 mL of 0.4 mol / L NaOH solution; boil in a water bath for 15 min, then immediately place on ice for 5 min; add 1 mL of Ehrlich reagent (0.8 g of p-dimethylaminobenzaldehyde dissolved in 15 mL of concentrated hydrochloric acid and 15 mL of anhydrous ethanol, freshly prepared); develop color at room temperature for 20 min; and measure its absorbance at 530 nm.

[0068] The inhibition rate is calculated as follows:

[0069] Inhibition rate = [(AB) - (CD)] / (AB) × 100%;

[0070] Where A is the absorbance value of the control group (acetic acid buffer replaced bacterial suspension), B is the absorbance value of the control blank group (acetic acid buffer replaced enzyme solution and bacterial suspension), C is the absorbance value of the sample group, and D is the absorbance value of the sample blank group (acetic acid buffer replaced enzyme solution).

[0071] 3.2 Solution Preparation

[0072] Prepare an acetate buffer solution (pH 5.6) by mixing and diluting 4.8 mL of solution A (11.55 mL of 0.2 mol / L acetic acid dissolved in 1 L of distilled water) and 45.2 mL of solution B (16.4 g of anhydrous sodium acetate or 27.2 g of sodium acetate trihydrate dissolved in 1 L of distilled water). For the acetylacetone solution, mix 50 mL of 1.0 mol / L sodium carbonate solution and 3.5 mL of acetylacetone solution thoroughly (prepare fresh before use). For the P-DAB colorimetric reagent, dissolve 0.8 g of p-dimethylaminobenzaldehyde in 15 mL of concentrated hydrochloric acid and 15 mL of anhydrous ethanol thoroughly.

[0073] MRS liquid medium: glucose 20.0 g, tryptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, Tween 80 1.0 mL, dipotassium hydrogen phosphate 2.0 g, ammonium citrate 2.0 g, anhydrous sodium acetate 5.0 g, magnesium sulfate 0.5 g, manganese sulfate monohydrate 0.25 g, deionized water 1 L, pH=6.5 (add 1.5% agar to make MRS solid medium).

[0074] In this experiment, hyaluronic acid was used as a substrate to carry out an enzyme reaction under specific conditions, and changes in its activity were measured. Based on the experimental results, the activity was analyzed using absorbance OD. 530nm Tests showed that YYS-B1 fermentation significantly reduced hyaluronidase activity, with an inhibition rate of up to 76.2%.

[0075] The *Lactobacillus plantarum* YYS-B1 provided by this invention has excellent ability to inhibit hyaluronidase and can be used to prepare anti-allergy products.

[0076] Example 4: Determination of DPPH free radical scavenging rate of Lactobacillus plantarum YYS-B1

[0077] 4.1 Solution Preparation

[0078] DPPH solution: Dissolve 0.002 g of DPPH in 50 mL of ethanol each time, and store in the dark. Dilute with ethanol to an absorbance of about 0.7 before use, and prepare fresh each time.

[0079] 4.2 Sample Detection and Analysis

[0080] Lactobacillus plantarum YYS-B1 was activated to the second generation, centrifuged at 8000 r / min for 10 min, the supernatant was discarded, and the bacterial pellet was resuspended in sterile PBS to a bacterial concentration of 1x10⁻⁶. 8 cfu / mL. The specific experiment was divided into three groups: A, B, and C. The dosage of each reactant is shown in Table 1. Each reactant was added sequentially to a standard 96-well plate and incubated for 30 min at room temperature in the dark. The absorbance at 517 nm was then measured.

[0081] The formula for calculating the DPPH free radical scavenging rate of Lactobacillus plantarum YYS-B1 is as follows:

[0082] Inhibition rate = [1 - (AC) / B] × 100%;

[0083] In the formula, A is the absorbance value of the sample group, B is the absorbance value of the blank group, and C is the absorbance value of the control group.

[0084] Table 1

[0085]

[0086] By wavelength OD 517nm The test results showed that YYS-B1 achieved a DPPH free radical scavenging rate of 86.2% ± 1.3%. The *Lactobacillus plantarum* YYS-B1 provided by this invention exhibits excellent DPPH free radical scavenging effects and can be applied to the development of antioxidant products.

[0087] Example 5: Determination of ABTS Free Radical Scavenging Rate by Lactobacillus plantarum YYS-B1

[0088] 5.1 Solution Preparation

[0089] ABTS stock solution (7 mmol / L): Weigh 0.0384 g of ABTS and dilute to 10 mL with distilled water. K₂S₂O₈ stock solution (2.45 mmol / L): Weigh 0.0066 g of K₂S₂O₈ and dilute to 10 mL with distilled water. ABTS working solution: Mix the ABTS stock solution and K₂S₂O₈ stock solution 1:1, let stand for 12–16 h, and prepare the ABTS working solution.

[0090] 5.2 Sample Detection and Analysis

[0091] Lactobacillus plantarum YYS-B1 was activated to the second generation, centrifuged at 8000 r / min for 10 min, the supernatant was discarded, and the bacterial pellet was resuspended in sterile PBS to a bacterial concentration of 1x10⁻⁶. 8 cfu / mL. Take an appropriate amount of ABTS working solution and dilute it with PBS solution to OD. 734 nm The value was 0.7. The experiment was divided into three groups: A, B, and C. The dosage of each reactant is shown in Table 2. Each reactant was added sequentially to a standard 96-well plate, and the plates were incubated at room temperature in the dark for 6 minutes. The absorbance at 734 nm was then measured. The formula for calculating the ABTS free radical scavenging rate of *Lactobacillus plantarum* YYS-B1 is as follows:

[0092] Inhibition rate = [1 - (AC) / B] × 100%;

[0093] Where A is the absorbance of the sample group, B is the absorbance of the blank group, and C is the absorbance of the control group.

[0094] Table 2

[0095]

[0096] By wavelength OD 734nm The test results showed that YYS-B1 achieved an ABTS free radical scavenging rate of 77.3% ± 1.4%. The *Lactobacillus plantarum* YYS-B1 provided by this invention further demonstrates its excellent ABTS free radical scavenging effect and can be applied to the development of antioxidant products.

[0097] Example 6: Preparation method of Lactobacillus plantarum YYS-B1 bacterial powder

[0098] 6.1 Materials used in preparation

[0099] The culture medium and lyophilization protectant used in this embodiment are as follows:

[0100] The seed culture medium, by weight percentage, consists of: 3% glucose, 2% peptone, 1.5% yeast extract, 1% beef extract, 0.5% anhydrous sodium acetate, 0.05% magnesium sulfate, 0.06% manganese sulfate, 0.1% Tween 80, and the balance being water, with a pH of 6.8.

[0101] The fermentation medium, by weight percentage, consists of: 2.3% glucose, 2.5% soybean peptone, 2% yeast extract, 0.6% anhydrous sodium acetate, 0.05% magnesium sulfate, 0.03% manganese sulfate, 0.1% Tween 80, and the balance being water;

[0102] The freeze-drying protectant comprises, by weight percentage: 10% trehalose, 8% skim milk powder, 0.5% glycerol, and the balance being water.

[0103] 6.2 Preparation process:

[0104] The freeze-dried powder preparation process includes the following steps:

[0105] S1. Inoculate Lactobacillus plantarum YYS-B1 into a test tube containing seed culture medium for activation, and culture in a constant temperature incubator for 20 h to obtain the first-grade seed liquid.

[0106] S2. Inoculate the primary seed culture into an Erlenmeyer flask containing seed culture medium at a rate of 3% (by mass). Incubate in a constant temperature incubator for 18 hours to obtain the secondary seed culture.

[0107] S3. Inoculate the secondary seed solution into an Erlenmeyer flask containing seed culture medium at a rate of 5% (by mass). Incubate in a constant temperature incubator for 16 hours to obtain the tertiary seed solution.

[0108] S4. Inoculate the tertiary seed culture into a fermenter containing fermentation medium at an inoculation rate of 5% (by mass). The preferred stirring speed is 50 r / min. The initial pH of fermentation is 6.0. Incubate at 37°C for 8 h.

[0109] S5, viable bacterial count in the fermentation broth ≥ 2.9 × 10⁻⁶ 9 When the cfu / mL level is reached, stop fermentation and collect the cells by centrifugation (8000 r / min, 15 min).

[0110] S6. The bacterial cells and the freeze-drying protectant were emulsified and encapsulated at a mass ratio of 1:0.6. The emulsification temperature was 20℃ and the emulsification time was 20 min.

[0111] S7. Pre-freeze the emulsion for 6 hours, then freeze-dry it under vacuum at -30℃ for 30 hours at a vacuum degree of 0.2 mbar. The resulting powder is then pulverized to obtain *Lactobacillus plantarum* YYS-B1 bacterial powder with a viable count of 2.9 × 10⁻⁶. 11 cfu / g.

[0112] Example 7: Effects of Lactobacillus plantarum YYS-B1 on allergic and inflammatory responses in mice

[0113] 7.1 Experimental Preparation and Procedure

[0114] Forty male BALB / c mice were used as experimental subjects and divided into four groups: normal control group, SEB+OVA model group, model control group, and Lactobacillus plantarum YYS-B1 group.

[0115] Except for the normal control group, other groups were administered 0.2 mL of sterile saline containing 10 μg of Staphylococcus aureus enterotoxin B (SEB) and 20 μg of ovalbumin (OVA) via intraperitoneal injection on days 0, 3, 9, 11, and 14. After day 14, the model group was administered 0.2 mL of sterile saline containing 10 μg of Staphylococcus aureus enterotoxin B (SEB) and 20 μg of ovalbumin (OVA); the model control group was injected with 0.2 mL of sterile saline; the *Lactobacillus plantarum* YYS-B1 group was prepared by adding 0.9% physiological saline to the lyophilized *Lactobacillus plantarum* YYS-B1 powder obtained in Example 6 to prepare a 2% bacterial powder suspension with a concentration of 5.8 × 10⁻⁶. 9 Administer 0.2 mL once daily for 7 consecutive days at a concentration of cfu / mL.

[0116] 7.1 Testing Process

[0117] The total IgE level in mouse serum was determined using the ELISA method. This indicator is often used to assess allergic reactions (test results are shown in Table 3).

[0118] The levels of inflammatory factors such as IL-4 and IFN-γ in mouse serum were detected by ELISA (the test results are shown in Table 4).

[0119] In addition, the level of diamine oxidase (DAO) in mouse serum was measured, and this enzyme is closely related to allergic reactions (test results are shown in Table 3).

[0120] Table 3 Comparison of serum IgE and DAO levels in mice treated with and controlled by *Lactobacillus plantarum* YYS-B1 (n=10, mean±SD)

[0121]

[0122] Table 4 Comparison of mice treated with and controlled by *Lactobacillus plantarum* YYS-B1 (n=10, mean±SD)

[0123]

[0124] 7.3 Test Results:

[0125] Compared with the normal control group, the IgE level in the SEB+OVA experimental group was significantly increased, and the difference between the groups was statistically significant. P <0.05). This indicates that the method successfully induced food allergy in mice.

[0126] After gavage administration of *Lactobacillus plantarum* YYS-B1, IgE levels decreased significantly compared to the experimental group. P <0.05). IL-4 is a cellular inflammatory cytokine. In allergic organisms, IL-4 levels are significantly elevated, while IFN-γ levels are decreased. In this study, after gavage administration of *Lactobacillus plantarum* YYS-B1, serum IFN-γ levels in sensitized mice significantly increased, while IL-4 levels significantly decreased.

[0127] Experimental results show that:

[0128] Lactobacillus plantarum YYS-B1 can downregulate elevated Th2 cytokines (i.e., reduce the release of inflammatory mediators), correct Th1 / Th2 imbalance, and alleviate allergy symptoms. Our experiments show that Lactobacillus plantarum YYS-B1 can reduce IgE allergic reactions and allergic-induced inflammatory responses in allergic mice.

[0129] Example 8: Determination of Superoxide Dismutase (SOD) and Glutathione Peroxidase (GSH-PX) Activities in Mouse Serum and Liver by Lactobacillus plantarum YYS-B1

[0130] 8.1 Experimental Materials and Procedures

[0131] Healthy male BALB / c mice were randomly divided into an experimental group and a control group, with 20 mice in each group. The *Lactobacillus plantarum* YYS-B1 bacterial powder obtained in Example 6 was added to 0.9% physiological saline to prepare a 2% bacterial powder suspension with a concentration of 5.8 × 10⁻⁶. 9 Mice in the experimental group were given 0.2 mL of 0.9% saline solution at a concentration of cfu / mL, while the control group received the same volume. Treatment continued for 4 weeks, once daily. At the end of the experiment, serum and liver tissue samples were collected from the mice. Relevant indicators were measured using a SOD and GSH-PX activity assay kit.

[0132] 8.1 Test Results:

[0133] The results of the mouse serum SOD activity assay are as follows: Figure 5 , Figure 6 As shown, compared with the control group, the serum and liver SOD and GSH-PX activities of mice in the experimental group were significantly increased, indicating that consuming *Lactobacillus plantarum* YYS-B1 powder can significantly improve the body's SOD and GSH-PX activities and enhance the body's antioxidant capacity.

[0134] Example 9: Acid and bile salt resistance test of Lactobacillus plantarum YYS-B1

[0135] 9.1 Simulated gastric juice acid resistance test

[0136] Weigh 0.2 g NaCl and 0.35 g pepsin and dissolve them in an appropriate amount of distilled water to prepare three portions. Adjust the pH to 1.5, 2.5, and 3.5 respectively with 1.0 mol / L hydrochloric acid, and then dilute to the mark with 100 mL volumetric flasks. Inoculate the *Lactobacillus plantarum* YYS-B1 bacterial powder obtained in Example 6 into simulated gastric acid solutions with different pH values ​​at an inoculation rate of 1% (w / v). After incubation at 37°C for 0, 2, and 4 h, dilute 10-fold serially, shake well, and then pour 100 μL of the bacterial solution onto a plate. Incubate at 37°C for 24 h and calculate the viable count of YYS-B1. The results are shown in Table 5.

[0137] Table 5

[0138]

[0139] As can be seen from Table 5:

[0140] The viable count of *Lactobacillus plantarum* YYS-B1 varied in simulated gastric fluid with different pH values. As the pH increased, the viable count and survival rate continuously improved. However, in simulated gastric fluid at the same pH, the viable count decreased with increasing treatment time.

[0141] However, in simulated gastric juices with different pH values, the number of viable bacteria remained at a high level after 2 hours of treatment, indicating that YYS-B1 can still have a high number of viable bacteria after being digested by human gastric juice and has excellent tolerance to gastric acid.

[0142] 9.2 Simulated intestinal fluid bile salt tolerance test

[0143] The lyophilized powder of *Lactobacillus plantarum* YYS-B1 obtained in Example 6 was inoculated at a rate of 1% (w / v) into porcine bile salt solutions with concentrations of 0.03 g / mL, 0.3 g / mL, and 0.5 g / mL, respectively. After incubation at 37°C for 0, 2, and 4 h, the solutions were serially diluted 10-fold, shaken thoroughly, and 100 μL of the bacterial solution was poured onto a plate and cultured at 37°C for 24 h. The viable count of YYS-B1 was then calculated, and the results are shown in Table 6.

[0144] Table 6

[0145]

[0146] As can be seen from Table 6:

[0147] The viable count and survival rate of *Lactobacillus plantarum* YYS-B1 varied under different initial concentrations of bile salts in simulated intestinal fluid. As the concentration of bile salts in the simulated intestinal fluid increased, the initial viable count gradually decreased, and the survival rate also gradually declined with longer treatment times.

[0148] However, even after treatment with bile salt concentrations closest to those in the human body (typically 0.03–0.3 g / mL in the human small intestine) for 2 hours, a high number of viable bacteria remained, indicating that YYS-B1 retains a significant number of live bacteria after digestion in the human gastrointestinal tract. The *Lactobacillus plantarum* YYS-B1 of this invention exhibits excellent tolerance to gastric acid and bile salts, enabling it to successfully reach the intestines, regulate intestinal flora, and exert probiotic effects.

[0149] Example 10: Preparation of probiotic products using *Lactobacillus plantarum* YYS-B1 of the present invention

[0150] 10.1 Probiotic Products

[0151] The probiotic product is a composition of Lactobacillus plantarum YYS-B1 and other ingredients; in the product, the viable count of Lactobacillus plantarum YYS-B1 is not less than 1×10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g.

[0152] The ingredients include, but are not limited to, one or more of the following: prebiotics, fillers, acidulants, solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, coating materials, fragrances, anti-adhesion agents, binding agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, and filter aids.

[0153] It should be noted that probiotic products can be in the form of solid beverages, liquid beverages, compressed candies, granules, capsules, tablets, pills, or oral liquids, which are food types suitable for and designed for human consumption.

[0154] 10.2 Example of Probiotic Products in Solid Beverages:

[0155] In this embodiment, a probiotic product in the form of a solid beverage (Lactobacillus plantarum YYS-B1 solid beverage) is provided, which, by weight, includes 10 parts of active bacteria powder, 24.8 parts of xylitol, 24 parts of whole milk powder, 12 parts of fermented mulberry powder, 23 parts of maltodextrin, 5.6 parts of galactooligosaccharides, 0.1 parts of fructooligosaccharides, and 0.5 parts of anhydrous citric acid.

[0156] The preparation method of this solid beverage is as follows:

[0157] S1. The *Lactobacillus plantarum* YYS-B1 strain is cultured in liquid culture medium, the bacterial cells are collected and washed, excipients (protectant in Example 8) are added, and the strain is dried to prepare active bacterial powder.

[0158] S2. After thoroughly mixing the active bacteria powder, xylitol, whole milk powder, fermented mulberry powder, maltodextrin, galactooligosaccharides, fructooligosaccharides, and anhydrous citric acid, a viable count of *Lactobacillus plantarum* YYS-B1 is obtained, which is not less than 1 × 10⁻⁶. 8 Solid beverage with CFU / g.

[0159] It should be noted that when adding *Lactobacillus plantarum* YYS-B1 freeze-dried powder or other forms of *Lactobacillus plantarum* YYS-B1 to food, the ingredients can be existing food additives and excipients, including but not limited to the substances described in the examples.

[0160] In summary, the *Lactobacillus plantarum* YYS-B1 provided by this invention has the following properties, effects, and applications:

[0161] 1. From the perspective of mechanism of action, *Lactobacillus plantarum* YYS-B1 has the following functions:

[0162] (1) It has the ability to inhibit hyaluronidase; its main efficacy is anti-allergy.

[0163] (2) Reduce IgE levels;

[0164] (3) Acid and bile salt resistance;

[0165] (4) Scavenging free radicals DPPH; its main function is antioxidant.

[0166] (5) Scavenging free radicals ABTS; its main manifestation is its antioxidant effect;

[0167] (6) Reduce the release of inflammatory mediators; for example, the use of this bacterium can reduce the content of pro-inflammatory factor IL-4 and increase the content of anti-inflammatory factor IFN-γ, thereby inhibiting the response and proliferation of Th2 cells, thereby reducing the release of cytokines (i.e. inflammatory mediators) secreted by Th2 cells and reducing the production of IgE antibodies, thus playing an anti-inflammatory role.

[0168] 2. From a functional perspective, it possesses the following functional characteristics:

[0169] It has outstanding anti-allergy, anti-inflammatory and antioxidant capabilities. It not only has anti-allergy ability, but also has a good inhibitory effect on the inflammatory response caused by allergies. It can also effectively reduce the oxidative damage to the body caused by allergic and inflammatory reactions.

[0170] Lactobacillus plantarum YYS-B1 exhibits excellent tolerance to gastric acid and bile salts, enabling it to successfully reach the intestines, regulate gut microbiota, and exert probiotic effects.

[0171] 3. Compared with the prior art, the advantages of this invention are:

[0172] Currently, no oral probiotic strains have been reported to simultaneously possess hyaluronidase inhibition, anti-allergy, and anti-inflammatory effects. Unlike existing commercially available probiotic anti-allergy preparations, the *Lactobacillus plantarum* YYS-B1 strain in this invention has the following advantages:

[0173] (1) High hyaluronidase inhibition rate;

[0174] (2) It can prevent and inhibit inflammatory reactions caused by allergies;

[0175] (3) It has excellent antioxidant capacity and can effectively reduce the oxidative damage to the body caused by allergic and inflammatory reactions.

[0176] In summary, the strains of the present invention, when used alone, can effectively achieve anti-allergic, anti-inflammatory, and antioxidant effects.

[0177] Anti-allergy: Lactobacillus plantarum YYS-B1 has outstanding performance in anti-allergy, surpassing most other strains on the market. Its hyaluronidase inhibition rate reaches 76.2%, which can reduce the mediators released by mast cells, thereby reducing allergic reactions.

[0178] Antioxidant and anti-inflammatory:

[0179] Antioxidant experiments showed that the strain achieved free radical scavenging rates of 86.2% for DPPH and 77.3% for ABTS. Mouse experiments demonstrated that the strain possesses antioxidant and anti-inflammatory capabilities, which are of great significance for preventing and reducing the occurrence of allergies and inflammation.

[0180] In summary, the *Lactobacillus plantarum* YYS-B1 exhibits outstanding anti-allergic, anti-inflammatory, and antioxidant capabilities. Specifically, it demonstrates high hyaluronidase inhibition, strong free radical scavenging ability, and anti-inflammatory activity, thus possessing both anti-allergic properties and a strong inhibitory effect on allergic-induced inflammatory responses. It can also effectively reduce oxidative damage to the body caused by allergic and inflammatory reactions. Furthermore, its good acid and bile salt resistance allows it to successfully reach the intestines and exert its probiotic effects, making it suitable for use in the preparation of probiotic products, anti-allergy products, or anti-inflammatory products. As a natural probiotic, it has no side effects, making it safer and healthier. It provides an excellent microbial resource for the development of anti-allergy or anti-inflammatory products, offering new directions and more possibilities for product development.

[0181] 4. Based on its function and properties, *Lactobacillus plantarum* YYS-B1 can be applied to functional products that include at least one of the following functions:

[0182] (1) It has the ability to inhibit hyaluronidase (the main effect of which is anti-allergy).

[0183] (2) Reduce IgE levels;

[0184] (3) Acid and bile salt resistance;

[0185] (4) Scavenging free radicals DPPH (antioxidant);

[0186] (5) Scavenging free radicals ABTS (antioxidant);

[0187] (6) Reduce the release of inflammatory mediators.

[0188] It should be noted that the bacteria YYS-B1 with the above-mentioned (1)-(6) effects can be used in antioxidant products, anti-allergy, anti-inflammatory and probiotic functional products to play a significant role in antioxidant, anti-inflammatory and anti-allergy effects; it can also have other significant effects based on the mechanism of action of (1)-(6).

[0189] It should be noted that:

[0190] (1) Definition:

[0191] The term "food" as used herein is used in a broad sense, including human food and drink. In some embodiments, the food is in a dosage form suitable for and designed for human consumption, including but not limited to solid beverages, liquid beverages, compressed candies, granules, capsules, tablets, pills, or oral liquids.

[0192] “DPPH” stands for free radical 2,2-biphenyl-1-picrylhydrazyl, and “ABTS” stands for free radical 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid.

[0193] "IgE" refers to immunoglobulin, which is an antibody in the human body. It exists in the blood and is the immunoglobulin with the lowest content in the serum of normal people.

[0194] "OD" is an abbreviation for optical density, also known as absorbance. The energy difference before and after light passes through a analyte is the energy absorbed by the analyte. At a specific wavelength, there is a quantitative relationship between the concentration of the same analyte and the absorbed energy, which can be used to determine the concentration of the analyte. "ODx" is the optical density value measured when the wavelength is set to x nm. It is a standard indicator for tracking the density of microorganisms in liquid cultures, usually used to indicate bacterial cell density. The method for measuring "OD" values ​​is existing technology, and its principles and methods will not be elaborated here.

[0195] Unless otherwise specified, the experimental procedures involved in the embodiments of the present invention are conventional experimental procedures in the art, and the reagents or instruments involved can be obtained from legitimate channels.

[0196] Furthermore, those skilled in the art should understand that although many problems exist in the prior art, each embodiment or technical solution of the present invention can be improved in only one or a few aspects, without necessarily solving all the technical problems listed in the prior art or the background art simultaneously. Those skilled in the art should understand that any content not mentioned in a claim should not be construed as a limitation on that claim.

[0197] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A plant lactobacillus with hyaluronidase inhibitory activity ( Lactiplantibacillus plantarum YYS-B1, characterized in that: Its accession number is CGMCC No. 27599.

2. The application of Lactobacillus plantarum YYS-B1 as described in claim 1 in the preparation of probiotic products.

3. The application of Lactobacillus plantarum YYS-B1 as described in claim 1 in the preparation of anti-allergy products.

4. The application of Lactobacillus plantarum YYS-B1 as described in claim 1 in the preparation of antioxidant products.

5. A freeze-dried powder, characterized in that: Its components include *Lactobacillus plantarum* YYS-B1 as described in claim 1.

6. A microbial agent, characterized in that: The microbial agent comprises the lyophilized powder as described in claim 5, and / or the microbial agent comprises *Lactobacillus plantarum* YYS-B1 as described in claim 1.

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