Lactobacillus paracasei SINEFREE with anti-inflammatory and anti-allergy effects and application of lactobacillus paracasei SINEFREE

Abstract

The invention relates to lactobacillus paracasei SINEFREE with anti-inflammatory and anti-allergy effects and application of the lactobacillus paracasei SINEFREE. The lactobacillus paracasei SINEFREE is classified and named as lactobacillus paracasei, and the preservation number of the lactobacillus paracasei SINEFREE is CGMCC (China General Microbiological Culture Collection Center) No.31279. The lactobacillus paracasei SINEFREE is lactobacillus paracasei. The strain plays a remarkable role in regulating and improving the aspects of reducing the level of inflammatory cytokines in a mouse body, regulating the level of serum-related inflammatory antibodies, reducing the nose wiping frequency of an allergic mouse and relieving lung tissue injury. Therefore, the lactobacillus paracasei strain can be used for preparing a product with anti-inflammatory and anti-allergy effects, and provides a coping strategy and a reference basis for prevention and treatment of related allergic diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial culture, and relates to a Lactobacillus paracasei SINEFREE with anti-inflammatory and allergy-relieving effects and application thereof. Background Art

[0002] As the largest micro-ecological system in the human body, the balance of intestinal flora is crucial to maintaining the health of the host. Intestinal flora not only participates in the digestion and absorption process of the intestine, but is also closely related to nutrient metabolism, regulation of the immune system, and prevention and treatment of various diseases. In addition, the richness of intestinal flora has a complex interaction with the occurrence and development of allergic diseases.

[0003] Hyaluronidase is an enzyme that can decompose hyaluronic acid. It plays a key role in various inflammatory and allergic reactions. In the pathological process of allergic diseases such as allergic rhinitis, the activity of hyaluronidase increases, resulting in increased permeability of the nasal mucosa, making it easier for inflammatory cells and mediators to invade, thereby exacerbating allergic symptoms. This state not only damages the metabolic balance, but may also have serious effects on organs such as lung tissue or liver.

[0004] Probiotics refer to living microorganisms that can provide health benefits to the host. They work through a variety of mechanisms. For example, probiotics can improve the intestinal microecological balance of the host, improve the health level of the body, and affect the host's health and metabolism by regulating the composition and abundance of intestinal microbiota, participating in immune regulation, or improving gastrointestinal physiological functions. However, in the prior art, there are relatively few reports on probiotic products for the effective prevention and treatment of allergic diseases or some inflammatory reactions. Therefore, how to develop a probiotic product that can effectively resist inflammation and relieve allergies, and has the ability to prevent, relieve or treat allergic diseases and inflammatory reactions has become a problem that needs to be solved urgently. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide a Lactobacillus paracasei SINEFREE with anti-inflammatory and allergy-relieving effects and an application thereof.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a Lactobacillus paracasei SINEFREE with anti-inflammatory and allergy-relieving effects. The classification name of the Lactobacillus paracasei SINEFREE is Lactobacillus paracasei, the preservation number is CGMCC No. 31279, and the preservation date is July 12, 2024.

[0008] The present invention isolates and preserves a new strain of Lactobacillus paracasei with anti-inflammatory and allergy-relieving effects from human breast milk samples, and names it as the Lactobacillus paracasei SINEFREE strain. The strain has excellent anti-inflammatory and allergy-relieving effects and can significantly improve allergic rhinitis reactions, specifically by (1) reducing the level of inflammatory cytokines in mice; (2) regulating serum OVA-IgE and MCP-1 levels; (3) reducing the number of nose rubbings in allergic mice; and (4) alleviating lung tissue damage. In addition, the strain is a probiotic, so when it is used to prepare related efficacy products, it has high safety and is not easy to generate dependence.

[0009] Among them, in accordance with the provisions of the "List of Bacteria that can be used in Food" issued by the National Health Commission of the People's Republic of China in 2022, the above-mentioned Lactobacillus paracasei is now named Lactobacillus paracasei.

[0010] In a second aspect, the present invention provides a culture of Lactobacillus paracasei SINEFREE as described in the first aspect, wherein the culture is prepared by the following method: inoculating Lactobacillus paracasei SINEFREE into a culture medium and culturing at 30-38° C. for 18-26 hours;

[0011] The above-mentioned "30-38°C" can be, for example, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 35.5°C, 36°C, 36.5°C, 37°C, 37.5°C, 38°C, etc. Other specific point values ​​within the numerical range can be selected, and they will not be described one by one here;

[0012] The above-mentioned "18-26h" can be, for example, 18h, 19h, 20h, 21h, 21.5h, 22h, 22.5h, 23h, 23.5h, 24h, 24.5h, 25h, 25.5h, 26h, etc. Other specific point values ​​within the numerical range can be selected, and they will not be elaborated here one by one.

[0013] In a third aspect, the present invention provides a probiotic with anti-inflammatory and allergy-relieving effects, wherein the strain in the probiotic with anti-inflammatory and allergy-relieving effects comprises the Lactobacillus paracasei SINEFREE strain with anti-inflammatory and allergy-relieving effects as described in the first aspect.

[0014] Preferably, in the probiotic, the viable count of the Lactobacillus paracasei SINEFREE is not less than 1×10 10 CFU / mL or 1×10 10 CFU / g, for example 1×10 10 CFU / mL (CFU / g), 2×10 10CFU / mL (CFU / g), 5×10 10 CFU / mL (CFU / g), 8×10 10 CFU / mL (CFU / g), 1×10 11 CFU / mL (CFU / g), 5×10 11 CFU / mL (CFU / g), 1×10 12 CFU / mL (CFU / g), etc. Other specific point values ​​within this numerical range can be selected and will not be described in detail here.

[0015] Preferably, the strains in the probiotic with anti-inflammatory and allergy-relieving effects further include any one or a combination of at least two of Bifidobacterium longum subsp. longum, Lactobacillus plantarum, Bifidobacterium longum subsp. infantis, Pediococcus pentosaceus or Lactobacillus rhamnosus.

[0016] In the present invention, when Lactobacillus paracasei SINEFREE is compounded with the above-mentioned probiotics, synergistic effects can be achieved, and better effects are achieved in related anti-inflammatory and allergy-relieving effects.

[0017] Preferably, the dosage form of the probiotic includes solution, lyophilized powder, capsule, tablet or granule.

[0018] Preferably, the probiotic further comprises a protective agent and / or a prebiotic.

[0019] Preferably, the protective agent comprises skim milk.

[0020] Preferably, the prebiotics include any one of xylooligosaccharides, fructooligosaccharides, galactooligosaccharides, mannose oligosaccharides, trehalose, soybean oligosaccharides, resistant dextrin, polydextrose, α-lactalbumin, and lactoferrin, or a combination of at least two thereof.

[0021] In a fourth aspect, the present invention provides a use of the Lactobacillus paracasei SINEFREE as described in the first aspect, the culture as described in the second aspect, or the probiotic agent as described in the third aspect in the preparation of a product for preventing, alleviating or treating allergic rhinitis.

[0022] In a fifth aspect, the present invention provides a drug having the efficacy of preventing, alleviating or treating allergic rhinitis, wherein the drug comprises the Lactobacillus paracasei SINEFREE strain described in the first aspect.

[0023] Preferably, the drug further comprises pharmaceutically acceptable excipients;

[0024] Preferably, the auxiliary materials include any one or a combination of at least two of fillers, pH regulators, antioxidants, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, colorants, antibacterial agents or buffers.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention isolates and preserves a new strain of Lactobacillus paracasei with anti-inflammatory and allergy-relieving effects from human breast milk samples, and names it as the Lactobacillus paracasei SINEFREE strain. The strain has excellent anti-inflammatory and allergy-relieving effects, and can significantly improve allergic rhinitis reactions, specifically by reducing the level of inflammatory cytokines in mice; regulating serum OVA-IgE and MCP-1 levels; reducing the number of nose rubbings in allergic mice and relieving lung tissue damage. In addition, the strain is a probiotic, so when it is used to prepare related efficacy products, it has high safety and is not easy to generate dependence.

[0027] The SINEFREE strain involved in the present invention is classified and named as Lactobacillus paracasei, the preservation unit is the General Microbiology Center of China Microorganism Culture Collection Administration, the preservation number is CGMCC No.31279, the preservation date is July 12, 2024, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a graph showing the gastric acid resistance test results of each group of strains;

[0029] Figure 2 It is the test result diagram of hyaluronidase inhibition ability of each group of strains;

[0030] Figure 3 This is a graph showing the test results of each group of strains' ability to promote the secretion of interferon-γ and IL-10;

[0031] Figure 4 This is a graph showing the test results of each group of strains' ability to promote macrophage proliferation;

[0032] Figure 5 This is the test result of serum inflammatory factor levels in each group of mice;

[0033] Figure 6 This is the test result of serum OVA-IgE and MCP-1 levels in each group of mice;

[0034] Figure 7 This is the HE staining microscopic examination result of lung tissue sections of mice in each group;

[0035] Figure 8 This is the PAS staining microscopic examination result of lung tissue sections of mice in each group;

[0036] Fig. 9 These are the results of Tunel staining microscopic examination of lung tissue sections of mice in each group. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0038] The strain information involved is as follows:

[0039] ①The classification name of SINEFREE strain is Lactobacillus paracasei, and the deposit number is CGMCC No.31279;

[0040] ②The 2409 strain is the Lactobacillus paracasei 2409 strain;

[0041] ③The 2410 strain is the Lactobacillus paracasei 2410 strain;

[0042] ④GG strain is Lactobacillus rhamnosus GG strain;

[0043] ⑤ATCC 25302 strain is Lactobacillus paracasei ATCC 25302 strain.

[0044] The following MRS solid culture medium: weigh 10 g of peptone, 10 g of beef extract, 20 g of glucose, 2 g of sodium acetate, 5 g of yeast powder, 2 g of diammonium hydrogen citrate, 2.6 g of K2PO4·3H2O, 0.1 g of MgSO4·7H2O, 0.05 g of MnSO4, 20 g of agar and 0.5 g of cysteine ​​hydrochloride, dissolve them in deionized water, add 1 mL of Tween 80, make up to 1 L, sterilize and cool, and pour into a sterilized culture dish for later use;

[0045] The following MRS liquid culture medium involved: weigh 10 g of peptone, 10 g of beef extract, 20 g of glucose, 2 g of sodium acetate, 5 g of yeast powder, 2 g of diammonium hydrogen citrate, 2.6 g of K2PO4·3H2O, 0.1 g of MgSO4·7H2O, 0.05 g of MnSO4 and 0.5 g of cysteine ​​hydrochloride, dissolve them in deionized water, add 1 mL of Tween 80, make up to 1 L, sterilize and cool for use.

[0046] The preparation method of bacterial suspension and bacterial powder involved below is: after activating the strain, inoculate it into culture medium for cultivation to obtain culture solution; centrifuge the culture solution, resuspend the bacteria to obtain bacterial suspension, or further add protective agent for freeze drying to obtain freeze-dried bacterial powder product.

[0047] The experimental results were statistically analyzed using ggplot2 in R language. Compared with the model group / control group, *** represents p<0.001, ** represents p<0.01, * represents p<0.05, and ns represents no significant difference. Lowercase letters in the result graph indicate significant differences (p<0.05). When the letters in each group are the same, the difference is not significant, and when the letters are completely different, the difference is significant.

[0048] Example 1

[0049] In this embodiment, a strain of Lactobacillus paracasei with anti-inflammatory and allergy-relieving effects was isolated and screened, and the steps were as follows:

[0050] (1) Selected from human breast milk samples, diluted 10 times with 0.9% saline, diluted 3 times, spread on MRS solid medium, cultured at 37°C for 48 hours, picked out colonies of different morphologies and streaked on the surface of MRS solid medium for purification, picked out single colonies and expanded them at 37°C with MRS liquid medium, and then preserved with 30% glycerol.

[0051] (2) Further in vitro physiological property tests were performed on the three bacterial strains (strain 2409, strain 2410, and strain 2411) preserved in step (1) to select a single strain with the best gastric acid resistance. The specific steps are as follows:

[0052] Preparation of artificial gastric juice: dissolve pepsin into 0.5% physiological saline (containing NaCl), adjust the pH to 2.5 with dilute hydrochloric acid solution, the final concentration of pepsin in the prepared artificial gastric juice is 0.3wt%, and the final concentration of NaCl is 0.2wt%. After fully dissolved, filter and sterilize with a 0.22μm microporous filter membrane and set aside.

[0053] The bacterial suspensions of three strains (strain 2409, strain 2410, and strain 2411) were mixed with 9.0 mL of artificial gastric juice with pH values ​​of 2.0, 2.5, and 3.0, respectively, and incubated anaerobically at 37°C. Samples were taken at the beginning of incubation (0 h) and 3 h after treatment, and the number of viable bacteria was determined by the pour culture method and the survival rate was calculated by the following formula. The test results are shown in the figure. Figure 1 shown.

[0054] Survival rate (%) = N1 / N0×100%

[0055] Among them, N1 is the number of viable bacteria after 3 hours of artificial gastric juice treatment; N0 is the number of viable bacteria after 0 hours of artificial gastric juice treatment.

[0056] From the statistical results in the figure, we can see that in the simulated gastric juice environment, the survival rates of the three strains range from 95% to 104%, indicating that all three strains have the ability to tolerate human gastric juice, among which strain 2411 has the best tolerance effect.

[0057] Example 2

[0058] In this example, the strains screened in Example 1 were subjected to morphological identification and 16S rRNA molecular biological identification, and the steps were as follows:

[0059] (1) Morphological identification:

[0060] The 2411 strain was inoculated into MRS medium, cultured at 37°C for 48 hours, and then observed under a microscope. After smear and Gram staining, it was observed that the Gram staining was positive, the strain was short rod-shaped, and it appeared as milky white, smooth, moist, semicircular protrusions, and neatly edged colonies on the MRS medium.

[0061] (2) 16S rRNA molecular biological identification:

[0062] The 2409 strains, 2410 strains and 2411 strains preserved at -80°C were taken out, inoculated in a centrifuge tube equipped with 20mL MRS liquid medium at a ratio of 2% (v / v), centrifuged at 37°C for 18h, centrifuged at 8000rpm for 10min, and the supernatant was removed to collect the thalline. The genome of the strain was extracted, and a bacterial universal primer was added to carry out PCR amplification, and the amplified product was sent to a sequencing company for sequencing identification. The strain was sequenced and analyzed, and the 16S rRNA sequence of the 2411 strain was as shown in SEQ ID No: 1, and the 16S rRNA sequences of the 2409 strains and the 2410 strains were as shown in SEQ ID No: 2 and SEQ ID No: 3, respectively. The sequence obtained by sequencing was compared with the nucleic acid sequence in GeneBank, and the result showed that the three strains were all Lactobacillus paracasei.

[0063] SEQ ID No: 1:

[0064]

[0065] SEQ ID No:2:

[0066]

[0067] SEQ ID No:3:

[0068]

[0069] Based on the results of 16S rRNA molecular biological identification and morphological identification in Example 2, it was confirmed that strains 2409, 2410 and 2411 all belonged to Lactobacillus paracasei, and strain 2411 was named Lactobacillus paracasei SINEFREE strain.

[0070] Test Example 1

[0071] Physiological and biochemical index tests

[0072] (1) Resistance to intestinal fluid

[0073] Trypsin was dissolved in PBS buffer at pH 8.0 to a final concentration of 0.8 g / L, and the solution was sterilized by filtration through a 0.22 μm filter membrane to prepare simulated intestinal fluid.

[0074] Take 1.0mL of Lactobacillus paracasei SINEFREE suspension (concentration of 1×10 9 CFU / mL, the concentration of bacterial solution was measured by the method in the national standard "GB4789.35-2016 National Food Safety Standard Food Microbiology Detection Lactic Acid Bacteria Detection") added to 9mL simulated intestinal fluid (pH 8.0), mixed and cultured at 37℃ for 8h, and then the number of viable bacteria was detected. MRS solid medium was used for culture at 37℃ for 48h, and the survival rate of probiotics was calculated using the following formula:

[0075] Survival rate (%) = [log CFU N1 / log CFU N0] × 100%;

[0076] Among them, N1 is the number of viable bacteria after 3 hours of artificial intestinal fluid treatment, and N0 is the number of viable bacteria after 0 hours of artificial intestinal fluid treatment. The test results are shown in Table 1:

[0077] Table 1

[0078] Viable bacteria count N0(0h) Viable count N1(3h) Survival rate (%) SINEFREE strains <![CDATA[(5.68±0.22)×10 8 ]]> <![CDATA[(4.22±0.31)×10 8 ]]> 82.82

[0079] It can be seen from the statistical data in the table that the Lactobacillus paracasei SINEFREE strain provided by the present invention has good intestinal fluid tolerance. After incubation in artificial simulated intestinal fluid for 3 hours, the survival rate can still reach 82.82%. Good intestinal fluid tolerance is one of the prerequisites for it to exert its anti-inflammatory and allergy-relieving effects in the body.

[0080] (2) Choline tolerance

[0081] 1 mL of a concentration of 1×10 9The SINEFREE strain and GG strain with CFU / mL were inoculated into 100 mL of MRS-THIO medium (MRS containing 0.2% sodium thioglycolate) containing 0.3% bile salts, cultured at 37°C for 4 h, and the absorbance was detected at 600 nm using an ELISA reader. OD 600 The larger the value, the stronger the strain's ability to tolerate bile salts. The results are shown in Table 2:

[0082] Table 2

[0083] SINEFREE strains GG strain <![CDATA[OD 600 Value(4h)]]> 0.38 0.22

[0084] From the data in the table, we can see that after 4 hours of choline environment stimulation culture, the OD of SINEFREE strain 600 The value is 0.38, which is higher than the effect of the rhamnosus Lactobacillus GG strain in the positive control group. It can be seen that the Lactobacillus paracasei SINEFREE strain screened by the present invention has excellent bile salt tolerance.

[0085] Test Example 2

[0086] Hyaluronidase inhibition

[0087] (1) Add 0.1 mL of 2.5 mmol / L CaCl2 solution and 0.5 mL of 600 U / mL hyaluronidase to the test tube, incubate at 37°C for 20 min, and then add 0.5 mL of SINEFREE bacterial suspension (OD 600 =1), incubate at 37°C for 20 min after mixing, then add 0.5 mL of 0.5 mg / mL sodium hyaluronate solution, incubate at 37°C for 30 min, and then stand at 25°C for 5 min;

[0088] (2) Add 0.5 mL of acetylacetone solution (1.4 mL of acetylacetone dissolved in 20 mL of 1.0 mol / L Na2CO3 solution, prepared on the spot) and 0.1 mL of 0.4 mol / L NaOH solution to the static treatment solution, boil in a 100°C water bath for 15 min, then immediately transfer to an ice bath for treatment for 5 min;

[0089] (3) 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, mix well, prepare before use), color at 25°C for 20 min, and use an ELISA reader to measure the sample absorbance at a wavelength of 530 nm. At the same time, strains GG, 2409, and 2410 were used as control strains for synchronous testing. The results are as follows: Figure 2 shown.

[0090] Hyaluronidase is an enzyme that can hydrolyze hyaluronic acid and can participate in most type I allergic reactions mediated by immunoglobulin E (IgE) and type IV allergic reactions mediated by T cells. Therefore, detecting the in vitro inhibition rate of hyaluronidase is an effective method for rapid in vitro screening of anti-allergic substances. From the test statistical results in the figure, it can be seen that the three strains of Lactobacillus paracasei have different degrees of inhibition on hyaluronic acid, among which the SINEFREE strain has the best inhibition effect.

[0091] Test Example 3

[0092] In vitro anti-inflammatory level test

[0093] (1) Stimulate the secretion of interferon-γ

[0094] Extraction of primary cells from mouse spleen: 6-week-old SPF-grade BALB / c mice were killed by cervical dislocation, and the spleen was removed and placed in pre-cooled RPMI-1640 medium containing 5% FBS. The spleen was ground using a tissue grinder until there were no obvious tissue blocks, and the tissue homogenate was filtered through a 70 μm mesh into a centrifuge tube, centrifuged at 4°C, 500g for 5 min, and the supernatant was discarded; 4 mL of red blood cell lysis buffer was added to react for 5 min, PBS was added to terminate the reaction, centrifuged at 4°C, 500g for 5 min, the supernatant was discarded, and 10 mL of complete cell culture medium (RPMI-1640 medium containing 10% FBS + 1% penicillin-streptomycin + 50 μM β-mercaptoethanol) was added, and a single-cell suspension was obtained after mixing.

[0095] Spleen cells were cultured at 4 × 10 4 Cells at a concentration of 100 cells / mL were added to a 96-well plate and cultured in a 5% CO2, 37°C incubator for 24 h until the cells were stable.

[0096] Experimental groups: blank control group-normal saline (CK), positive control group a-4μg / mL concanavalin A (conA), positive control group b-2μg / mL lipopolysaccharide (LPS), Lactobacillus rhamnosus suspension (GG), Lactobacillus paracasei 2409 suspension, Lactobacillus paracasei 2410 suspension and Lactobacillus paracasei SINEFREE suspension. The amount of treatment solution added to each group was 500μL. In the four probiotic treatment groups, the live bacteria concentration of the bacterial suspension was 4×10 7 CFU / mL, the treatment solution of each group was mixed with the cell suspension, incubated in a 5% CO2, 37°C incubator for 48h, the cell suspension was taken out, centrifuged at 4°C, 500g for 5min, the supernatant was taken out, and the content of γ-interferon in the cell supernatant of each group was detected according to the steps in the ELISA kit manual. The results are as follows Figure 3 shown.

[0097] (2) Stimulate the secretion of interleukin-10 (IL-10)

[0098] Experimental groups: blank control group (DMEM medium), Lactobacillus rhamnosus suspension (GG), Lactobacillus paracasei 2409 suspension, Lactobacillus paracasei 2410 suspension and Lactobacillus paracasei SINEFREE suspension. The amount of treatment solution added to each group was 500 μL. In the four probiotic treatment groups, the live bacteria concentration of the bacterial suspension was 1×10 7 CFU / mL.

[0099] Macrophage RAW264.7 cells were inoculated into 24-well plates, and 500 μL of cell suspension (cell concentration was 1×10 6 The cells were placed in an incubator and cultured for 24 h. The treatment solutions of each group were added and cultured for 24 h at 37°C and 5% CO2. After the culture, the cells were centrifuged at 1000 rpm for 5 min at 4°C. The supernatant was taken out and the IL-10 content in the supernatant of each group of cells was detected according to the instructions of the ELISA kit. The results are as follows. Figure 3 shown.

[0100] Interferon-γ is a lymphokine with a wide range of immunomodulatory effects; IL-10 secretion mainly comes from immune cells in the body and is an important anti-inflammatory cytokine that plays an important role in regulating and terminating inflammatory responses. From the test statistical results in the figure, it can be seen that the three types of Lactobacillus paracasei have different degrees of promoting effects on stimulating the secretion of interferon-γ and IL-10, among which the SINEFREE strain has the best inhibitory effect.

[0101] Test Example 4

[0102] In vitro immunomodulation level test

[0103] The experimental groups were as follows: blank control group (DMEM medium), positive control group (4 μg / mL concanavalin A), Lactobacillus rhamnosus suspension (GG), Lactobacillus paracasei 2409 suspension, Lactobacillus paracasei 2410 suspension and Lactobacillus paracasei SINEFREE suspension. The amount of treatment solution added to each group was 100 μL. In the four probiotic treatment groups, the live bacterial concentration of the bacterial suspension was 4×10 7 CFU / mL.

[0104] The concentration of macrophage RAW264.7 cells was adjusted to 4 × 10 6 / mL, 100μL / well was inoculated in a 96-well plate, and cultured at 5% CO2 and 37℃ for 24h. The supernatant in the well plate was aspirated, and the well plate was washed and centrifuged twice with PBS buffer. The treatment solutions of each group were added respectively, and cultured at 37℃ and 5% CO2 for 24h. After the culture, it was washed with PBS buffer for 3 times, and 100μL PBS solution and 10μL CCK-8 reagent were added to each well, and incubated at 37℃ in the dark for 1h. The OD value of each group of samples at a wavelength of 450nm was measured with an enzyme marker, and the cell proliferation rate of macrophage RAW264.7 was counted. The results are as follows Figure 4 shown.

[0105] As part of the innate immune system, macrophages participate in early defense responses and also regulate long-term immune responses through interaction with the adaptive immune system. As shown in the test statistical results in the figure, the three types of Lactobacillus paracasei can induce the in vitro proliferation of macrophages RAW264.7 to a certain extent, among which the SINEFREE strain has the best effect. It can be seen that the Lactobacillus paracasei SINEFREE strain provided by the present invention can promote macrophage proliferation and has potential immunoregulatory ability.

[0106] Test Example 5

[0107] Intervention effect on mice with allergic rhinitis

[0108] (1) Animal modeling

[0109] ①Solution preparation:

[0110] Ovalbumin (OVA) storage solution: Mix 1 g of OVA with 100 mL of PBS, place at 25°C for 30 min until OVA is fully dissolved, filter (0.2 μm) to sterilize, and set aside;

[0111] Sensitization solution: 2 mg of aluminum hydroxide adjuvant adsorbed with 50 μg of OVA was suspended in 200 μL of phosphate buffered saline (PBS: pH 7.4);

[0112] Stimulation solution: Dilute OVA stock solution and PBS buffer at a volume ratio of 1:4, filter (0.2 μm) for sterilization, and set aside;

[0113] ② Sensitization by intraperitoneal injection: After 7 days of adaptive feeding, 200 μL of sensitization solution was injected intraperitoneally on days 7, 14 and 21. From day 29 to day 38, mice were challenged by nasogastric administration of the provocation solution once a day, 50 μL each time, to obtain mice with allergic rhinitis model.

[0114] (2) Experimental groups and intervention methods:

[0115] Allergic rhinitis model mice were divided into 6 groups, 10 mice in each group, including model group (MG): distilled water, positive control group (PG): 300 μg / mL cetirizine solution, ATCC 25302 group, 2409 group, 2410 group and SINEFREE group. The live bacterial concentration of the bacterial suspension in the four probiotic treatment groups was 2×10 10 CFU / mL, the treatment solution intervention method of each group was intragastric administration, the dose was 0.2mL / animal / day, intragastric administration for 10 days;

[0116] At the same time, a sham operation group (CG) was set up. After 7 days of adaptive feeding, the mice in the sham operation group were injected with sensitization solution three times in the abdomen on the 7th, 14th and 21st days respectively. From the 29th to the 38th day, the mice were stimulated by nasogastric administration of PBS buffer once a day, 50 μL each time. The mice obtained were intervened by intragastric administration of distilled water 0.2 mL / mouse / day for 10 days.

[0117] (3) Index detection

[0118] ① Serum inflammatory index levels

[0119] After the gavage, the mice were anesthetized by gas, blood was collected from the eyeballs, and the blood was kept in a 37°C water bath for 1 h, then refrigerated at 4°C for 3.5 h until serum separation. The blood was centrifuged at 4000 rpm for 10 min, the supernatant was collected, and kept at 4°C for later use. ELISA kits were used to detect the levels of tumor necrosis factor-α (TNF-α), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-1β (IL-1β), ovalbumin-specific IgE antibody (OVA-IgE) and monocyte activating protein-1 (MCP-1). The results were as follows: Figure 5 and Figure 6 shown.

[0120] As can be seen from the statistical results in the figure, the levels of various indicators of the model group mice showed significant differences compared with the sham operation group mice, proving that the allergic rhinitis mouse modeling was successful; the SINEFREE strain inhibited the secretion levels of TNF-α and IL-5 most significantly, and the inhibitory secretion levels of IL-1β and IL-6 factors were significantly better than the model strain ATCC 25302 (p<0.001). The results showed that the three strains of Lactobacillus paracasei screened out all had the ability to reduce inflammatory response, among which the effect of SINEFREE strain was the most significant; in the mouse model of OVA allergy, the immune system would produce IgE antibodies against OVA, and detecting the IgE antibody level in mice was also one of the main means to directly judge the allergy level of biological organisms. After testing, the three strains of Lactobacillus paracasei screened out could reduce the IgE antibody level in mice with allergic rhinitis to varying degrees, among which the SINEFREE strain inhibited the IgE antibody level most significantly; the MCP-1 level of the SINEFREE group was close to that of the sham operation group (p<0.01), indicating that the strain SINEFREE could significantly reduce the expression of MCP-1. It can be seen that the SINEFREE strain provided by the present invention can alleviate the relevant symptoms of allergic rhinitis by regulating the secretion of pro-inflammatory cytokines, reducing the IgE antibody level, and inhibiting the recruitment and activation of inflammatory cells.

[0121] ②Pathological examination of lung tissue

[0122] Mouse lung tissue slice processing: (1) Sampling and fixation: The mouse after blood collection was dissected, and the lung tissue was taken and cut into tissue blocks with a side length of about 0.5 cm. Rinse with 0.85% saline. Fix the tissue with FAA (formalin-acetic acid-ethanol) fixative, with the ratio of the amount of fixative to the volume of the tissue block being 20:1. Fix for 18 hours, and then wash off the fixative with 70% ethanol;

[0123] (2) Dehydration and hardening: The tissue is dehydrated and hardened by sequentially treating with 70% ethanol, 95% ethanol, anhydrous ethanol, and xylene for clearing;

[0124] (3) Wax immersion and embedding: The transparent lung tissue samples were immersed in molten paraffin for 10 h until a tissue block was formed.

[0125] (4) Sectioning: Use a microtome to cut the tissue block into thin slices;

[0126] (5) HE staining;

[0127] (6) PAS staining;

[0128] (7) Tunel staining;

[0129] The results of the above three staining conditions of HE staining, PAS staining and Tunel staining on the pathological reaction of mouse lung tissue are shown in the figure below. Figure 7 , Figure 8 and Fig. 9 shown.

[0130] Depend on Figure 7 Analysis of the HE staining results showed that the sham group had intact alveolar structure, normal distribution of inflammatory cells, unobstructed capillaries in the alveolar wall, and no obvious inflammation, edema, or other pathological changes related to lung injury or disease; in the model group, there were a large number of inflammatory cells in the lung tissue, thickening of the bronchial wall, and obvious pathological characteristics; both the ATCC 25302 group and the positive group showed mild inflammatory reactions; the 2410 group had large structural cavities, showing obvious airway inflammation and mild airway remodeling; the 2409 group had relatively intact alveolar structure, and obvious inflammatory cell infiltration could be observed in the alveolar septum and around the alveoli; in the SINEFREE group, dense red blood cells with clear boundaries were seen, and a small number of inflammatory cells infiltrated around the alveoli and in the interstitium, indicating that mild inflammation may occur in this area. It can be seen that the SINEFREE strain has the best effect in alleviating inflammatory infiltration and pathological lesions in the lung tissue of mice with allergic rhinitis.

[0131] Depend on Figure 8 Analysis of the PAS staining results showed that in the sham operation group, the airway epithelial cells had normal morphology, intact lung tissue, and normal alveolar structure and mucus distribution; in the model group, a large amount of mucus infiltration could be seen around the airways and in the inner cavity, the epithelial cells around the airways might show hyperplasia, a large number of inflammatory cells infiltrated, and typical allergic disease characteristics and inflammatory reactions were present, indicating a successful modeling; in the 2409 group, 2410 group, and SINEFREE group, the alveolar structure was normal and uniform, the mucus distribution was normal, only mild inflammatory cells were present, and the SINEFREE strain treatment had the best effect; the experimental results showed that Lactobacillus paracasei had a more significant effect in alleviating mucus infiltration and improving and repairing the state of lung tissue.

[0132] Depend on Fig. 9 Analysis of the Tunel staining results showed that there was no significant apoptosis tendency in the sham operation group, and the alveolar cavity structure was obvious, showing normal size and shape; the green fluorescence signal in the model group increased significantly, especially in some areas, where a large number of cells were apoptotic; the ATCC 25302 group had obvious green fluorescence aggregation; the green fluorescence signal of the 2409 group was close to that of the model group, indicating that there were more apoptotic cells and the overall structure of the tissue was slightly collapsed or abnormal; the number of Tunel-positive cells in the SINEFREE group and the 2410 group was not large, indicating a low level of apoptosis, and the blue nuclei were evenly distributed, indicating that the cells were in a healthy state, without obvious aggregation or abnormality, and the lung tissue of the mice in the SINEFREE group was in the best state.

[0133] Through the above three different staining conditions, multi-angle pathological reaction analysis of the lung tissue of the experimental mice was carried out. It can be seen that the SINEFREE strain group is the most effective in alleviating the allergic pathological reaction of the lung tissue, and has an excellent effect on improving the related symptoms of allergic mice.

[0134] ③Statistical analysis of the number of nose rubbing in mice

[0135] The number of times the mice rubbed their noses was counted on the 0th, 5th and 10th days after oral gavage. Each mouse was observed individually for 10 minutes each time, and the sum of the number of times the mice rubbed their noses during this period was counted. On the 10th day, the model group (MG) had a significant increase in the mean number of nose rubbings compared with the first two statistical means, while the other groups had a decrease. The results of the univariate mean (ANOVA) analysis showed a significant difference (p=0.001584), and a subsequent multiple comparison Tukey HSD test analysis was performed. The results are shown in Table 3. It can be seen that the nose rubbing symptoms of the mice in the SINEFREE strain intervention treatment group were significantly improved compared with other groups, and were better than the positive drug cetirizine.

[0136] Table 3

[0137] Comparison Group Mean Difference 95% confidence interval lower limit 95% confidence interval upper limit p-value MG vs CG 14 4.07437197 23.925628 0.000911613 MG vs 2409 11.2 1.27437197 21.125628 0.0162114 MG vs 2410 11.5 1.57437197 21.425628 0.012204963 MG vs SINEFREE 13.5 3.57437197 23.425628 0.001576999 MG vs ATCC25302 11 1.07437197 20.925628 0.019515313 MG vs PG 9.7 0.22562803 19.625628 0.060149008

[0138] In summary, the present invention separates and obtains and preserves a new strain of Lactobacillus paracasei with anti-inflammatory and allergy-relieving effects, which is named as Lactobacillus paracasei. The strain has excellent anti-inflammatory and allergy-relieving effects, can significantly improve allergic rhinitis reactions, and has played a significant regulating and improving role in reducing the level of inflammatory cytokines in mice, regulating serum OVA-IgE and MCP-1 levels, reducing the number of nose rubbing times of allergic mice, and alleviating lung tissue damage. Therefore, this Lactobacillus paracasei strain can be used to prepare products with anti-inflammatory and allergy-relieving effects.

[0139] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0140] 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, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0141] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A Lactobacillus paracasei SINEFREE with anti-inflammatory and allergy-relieving effects, characterized in that: The classification name of the Lactobacillus paracasei SINEFREE is Lactobacillus paracasei, the preservation number is CGMCC No.31279, and the preservation date is July 12, 2024.

2. A culture of Lactobacillus paracasei SINEFREE as claimed in claim 1, characterized in that The culture is prepared by the following method: Lactobacillus paracasei SINEFREE is inoculated into a culture medium and cultured at 30-38° C. for 18-26 hours.

3. A probiotic with anti-inflammatory and allergy-relieving effects, characterized in that: The strain in the probiotic with anti-inflammatory and allergy-relieving effects includes the Lactobacillus paracasei SINEFREE strain with anti-inflammatory and allergy-relieving effects as claimed in claim 1.

4. The probiotic agent with anti-inflammatory and allergy-relieving effects as claimed in claim 3, characterized in that: In the probiotic, the viable count of the Lactobacillus paracasei SINEFREE is not less than 1×10 10 CFU / mL or 1×10 10 CFU / g.

5. The probiotic agent with anti-inflammatory and allergy-relieving effects as claimed in claim 3, characterized in that: The strains in the probiotic with anti-inflammatory and allergy-relieving effects also include any one or a combination of at least two of Bifidobacterium longum subsp. longum, Lactobacillus plantarum, Bifidobacterium longum subsp. infantis, Pediococcus pentosaceus or Lactobacillus rhamnosus.

6. The probiotic agent with anti-inflammatory and allergy-relieving effects as claimed in claim 3, characterized in that: The dosage form of the probiotics includes solution, lyophilized powder, capsule, tablet or granule.

7. The probiotic agent with anti-inflammatory and allergy-relieving effects as claimed in claim 3, characterized in that: The probiotics also include protective agents and / or prebiotics; Preferably, the protective agent comprises skim milk; Preferably, the prebiotics include any one of xylooligosaccharides, fructooligosaccharides, galactooligosaccharides, mannose oligosaccharides, trehalose, soybean oligosaccharides, resistant dextrin, polydextrose, α-lactalbumin, and lactoferrin, or a combination of at least two thereof.

8. Use of the Lactobacillus paracasei SINEFREE according to claim 1, the culture according to claim 2, or the probiotic according to any one of claims 3 to 7 in the preparation of a product for preventing, alleviating or treating allergic rhinitis.

9. A drug for preventing, alleviating or treating allergic rhinitis, characterized in that: The medicine comprises the Lactobacillus paracasei SINEFREE strain according to claim 1.

10. The drug according to claim 9, characterized in that The drug also includes pharmaceutically acceptable excipients; Preferably, the auxiliary materials include any one or a combination of at least two of fillers, pH regulators, antioxidants, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, colorants, antibacterial agents or buffers.

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