Lactobacillus paracasei LC97 for relieving allergy and application, product and method thereof

Abstract

The invention relates to a Lactobacillus paracasei LC97, and an application, a product and a method thereof, and belongs to the technical field of microorganisms. The preservation number of the lactobacillus paracasei LC97 for relieving allergy is CGMCC (China General Microbiological Culture Collection Center) NO. 32421. Based on the strain, the invention also provides application and related products of the strain. The LC97 strain has broad-spectrum antibacterial performance, has the functions of enhancing immune function and relieving atopic dermatitis, and can relieve atopic dermatitis by relieving skin lesion, inhibiting inflammation, reducing ear swelling degree and regulating cell factor level. And by reducing the total levels of serum IgE and IL-4, the levels of IFN-gamma and IL-10 are improved, and the levels of cytokines are regulated, so that the immunity is enhanced.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a Lactobacillus paracasei LC97 for allergy relief and an application, product and method thereof. Background Art

[0002] The prevalence of allergic diseases has gradually increased in recent years. Common allergic diseases include allergic rhinitis, allergic asthma, allergic eczema, food allergy, atopic dermatitis and contact dermatitis. Among them, atopic dermatitis is a common inflammatory skin disease. Atopic dermatitis is characterized by severe itching and repeated attacks, dry skin, erythema, and papules. In addition, the scratching behavior causes the patient's skin barrier to be damaged and the condition is aggravated, causing the patient to fall into a vicious cycle of "itching-scratching-severe itching". In addition, the condition is prone to recurrence, which increases the cost of treatment. Therefore, atopic dermatitis not only seriously affects the patient's sleep quality, but also greatly increases the economic burden on the patient's family.

[0003] At present, the internationally recognized treatment management for atopic dermatitis is "step-by-step treatment", and the ultimate goal is to control the clinical symptoms of atopic dermatitis, relieve skin itching, and improve the quality of life of patients. Atopic dermatitis is characterized by repeated attacks, so the treatment of atopic dermatitis is a long-term care and control process. In this process, different treatment methods are used for different degrees of symptoms. At present, the drugs for the treatment of atopic dermatitis include glucocorticoids, topical immune drugs, and immunosuppressants that antagonize sensitizing effectors. However, long-term use of these drugs will cause great side effects to patients, such as skin burning, damage to the central nervous system, etc., and the efficacy of specific drugs for atopic dermatitis has not yet been confirmed. Therefore, exploring dietary intervention methods for relieving atopic dermatitis may be a safer and more widely used strategy.

[0004] Atopic dermatitis belongs to type I allergic reaction. This type of allergic disease is usually characterized by elevated IgE levels, which is mainly mediated by type II helper T (Th2) cells. These cells synthesize high levels of interleukin (IL)-4, IL-5 and IL-13, leading to the production of allergen-specific IgE and the release of histamine and leukotrienes by mast cells. Interferon-γ (IFN-γ) can inhibit the overexpression of cytokines in Th2 lymphocytes, especially inhibiting the secretion of IL-4, thereby reducing the proliferation of B cells. IFN-γ can also stimulate the immune response of type I helper T (Th1) cells and inhibit the synthesis of IgE.

[0005] Mast cells are considered to be the key mediators of allergic inflammation and hypersensitivity reactions in the human respiratory tract and are the main effector cells of allergic reactions. Activation of the IgE high-affinity receptor FcεRI on mast cells triggers type I allergic reactions. Tryptase is a specific marker of mast cell activation and an inflammatory marker in allergic reactions. Tryptase is a serine protease unique to mast cell secretory granules, and is usually only secreted in small amounts into the blood. When allergies cause mast cell degranulation, a large amount enters the blood, causing a sharp increase in serum concentrations. Serum tryptase has been widely used in clinical practice to diagnose mast cell-related diseases, including anaphylaxis, mastocytosis, and many other secondary or idiopathic mast cell activation syndromes. Therefore, the level of this enzyme in serum can be used as one of the key indicators to determine the degree of allergic reactions in the body.

[0006] Probiotics are defined as "live microorganisms that, when ingested in sufficient quantities, are beneficial to the health of the host". They are tolerant to gastric acid and bile in the gastrointestinal tract, can colonize in the intestines, and regulate intestinal flora. Lactobacillus, as a representative of probiotics, is an important component of the intestinal microorganisms of humans and animals. Studies have found that lactobacillus can stimulate the secretion of IFN-γ and participate in regulating the body's immune response.

[0007] Lactobacillus paracasei is an important functional probiotic, and its role in relieving hapten-induced atopic dermatitis and its mechanism of action are still unclear. Therefore, providing a strain that promotes cell secretion of IFN-γ, inhibits mast cell degranulation and releases tryptase, and relieves atopic dermatitis has broad application prospects. Summary of the invention

[0008] In order to meet the above-mentioned needs of the prior art, the present invention provides a Lactobacillus paracasei LC97 for allergy relief and its application, product and method.

[0009] The technical solution of the present invention is as follows: A Lactobacillus paracasei strain that relieves allergies Lacticaseibacillus paracasei )LC97, its deposit number is CGMCC No. 32421.

[0010] The Lactobacillus paracasei with a deposit number of CGMCC No. 32421 ( Lacticaseibacillus paracasei ) Application of LC97 in the preparation of foods for enhancing immune function, products for alleviating atopic dermatitis, antibacterial agents or antibacterial agents for non-therapeutic purposes.

[0011] The immunity enhancement includes: regulating cytokine levels; Preferably, the relief of atopic dermatitis includes: relieving skin lesions, inhibiting inflammation, reducing ear swelling, and regulating cytokine levels; Preferably, the regulating cytokine levels comprises: reducing serum total IgE and IL-4 levels, and increasing IFN-γ and IL-10 levels; Preferably, the antibacterial spectrum includes: Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, Bacteroides fragilis, Streptococcus pasteurianus, Streptococcus pharyngitis, and Streptococcus Parisi.

[0012] A method for preparing an immune function enhancing food, comprising a functional active ingredient; the functional active ingredient comprises Lactobacillus paracasei with a preservation number of CGMCC No. 32421 ( Lacticaseibacillus paracasei )LC97.

[0013] The method for preparing food for enhancing immune function also includes auxiliary materials.

[0014] A product for relieving atopic dermatitis, comprising an active ingredient; the active ingredient comprises Lactobacillus paracasei with a preservation number of CGMCC No. 32421 ( Lacticaseibacillus paracasei )LC97.

[0015] The product for alleviating atopic dermatitis further comprises: auxiliary materials.

[0016] An antibacterial agent, comprising an antibacterial active ingredient; the antibacterial active ingredient comprises Lactobacillus paracasei ( Lacticaseibacillus paracasei )LC97.

[0017] The antibacterial agent further comprises: auxiliary materials.

[0018] A non-therapeutic antibacterial method, using Lactobacillus paracasei with a preservation number of CGMCC No. 32421 ( Lacticaseibacillus paracasei )LC97 for antibacterial effect.

[0019] The beneficial effects of the present invention are as follows: The present invention screened a strain from a breast milk sample and identified that it belongs to Lactobacillus paracasei ( Lacticaseibacillus paracasei), and named it LC97. A large number of experiments have proved that the LC97 strain of the present invention has a broad-spectrum antibacterial property, and can well inhibit pathogens including Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, Bacteroides fragilis, Streptococcus pasteurianus, Streptococcus pharyngitis, and Streptococcus Parisi. The LC97 strain also has the function of relieving atopic dermatitis, specifically by relieving skin lesions, inhibiting inflammation, reducing the degree of ear swelling, and regulating cytokine levels to relieve atopic dermatitis. At the same time, the LC97 strain can also regulate cytokine levels by reducing serum total IgE and IL-4 levels, and increasing IFN-γ and IL-10 levels to enhance immunity. The preservation information of the Lactobacillus paracasei LC97 of the present invention is as follows: Deposit number: CGMCC No. 32421 Classification name: Lactobacillus paracasei Lacticaseibacillus paracasei Depository: China National Microbiological Culture Collection Administration General Microbiology Center Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing Date of preservation: October 30, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a colony morphology photograph of Lactobacillus paracasei LC97 of Experimental Example 1 of the present invention on MRS culture medium.

[0021] Figure 2 This is a photo of the Lactobacillus paracasei LC97 of Experimental Example 1 of the present invention stained under a microscope.

[0022] Figure 3 It is a bar graph of tryptase concentrations in the cell supernatants of different treatment groups in Experimental Example 2 of the present invention; the labels in the figure are as follows: C48 / 80: positive control group, CK: negative control group, LC97: LC97 group.

[0023] Figure 4 It is a bar graph of the concentration of IFN-γ in different treatment groups of Experimental Example 3 of the present invention; the labels in the figure are as follows: NC: blank control group, CK: negative control group, LC97: LC97 group, LPS: positive control group-1, ConA: positive control group-2.

[0024] Figure 5 It is a bar graph of the adhesion ability of Lactobacillus paracasei LC97 and Lactobacillus rhamnosus LGG in Experimental Example 6 of the present invention; the labels in the figure are listed as follows: LGG: Lactobacillus rhamnosus LGG, LC97: Lactobacillus paracasei LC97.

[0025] Figure 6These are photos of skin lesions on the backs of mice in different treatment groups in Experimental Example 7 of the present invention.

[0026] Figure 7 These are photos of pathological sections of the back skin of mice in different treatment groups in Experimental Example 7 of the present invention.

[0027] Figure 8 It is a bar graph of ear thickness of mice in different treatment groups in Experimental Example 7 of the present invention.

[0028] Fig. 9 It is a bar graph of the serum total IgE levels of mice in different treatment groups in Experimental Example 7 of the present invention.

[0029] Fig.10 It is a bar graph of serum IL-4 levels of mice in different treatment groups in Experimental Example 7 of the present invention.

[0030] Fig.11 It is a bar graph of serum IFN-γ levels of mice in different treatment groups in Experimental Example 7 of the present invention.

[0031] Fig.12 It is a bar graph of serum IL-10 levels of mice in different treatment groups in Experimental Example 7 of the present invention.

[0032] Figure 6-Figure 12 The markers are listed as follows: NC: normal control group, MC: dermatitis model group, Keto: drug group, LC97: Lactobacillus paracasei group. DETAILED DESCRIPTION

[0033] The specific contents and technical effects of the present invention are further described in detail below in conjunction with specific embodiments, experimental examples and drawings, but the protection scope of the present invention is not limited thereto.

[0034] 1. Sources of biological materials (I) The mastoma cells P815 used in Experimental Example 2 of the present invention were purchased from China Center for Type Culture Collection.

[0035] (ii) The mice used in Experiment 3 of the present invention were purchased from Beijing Sibeifu Biotechnology Co., Ltd.

[0036] (III) The Streptococcus pasteurianus used in Experimental Example 5 of the present invention is Streptococcus pasteurianus 12758, which has been registered in NCBI with an accession number of MW445225; the Streptococcus Parisi is Streptococcus Parisi 21178, which has been registered in NCBI with an accession number of OM403351; the Streptococcus anginosus is Streptococcus pharyngitis strain 4997, which has been registered in NCBI with an accession number of MT459365; it is currently preserved in the applicant's laboratory, and the applicant promises to release it to the public for verification of the technical effects of the present invention within 20 years from the date of application of the present invention; Escherichia coli, Salmonella enteritidis and Staphylococcus aureus were all purchased from Guangdong Provincial Microbiological Culture Collection Center; Bacteroides fragilis ATCC25285 was purchased from China General Microbiological Culture Collection Management Center.

[0037] (IV) The Caco-2 cells used in Experimental Example 6 of the present invention were purchased from China Center for Type Culture Collection, and Lactobacillus rhamnosus LGG was purchased from Guangdong Microbial Culture Collection Center.

[0038] (V) The BALB / c female mice used in Experimental Example 7 of the present invention were purchased from Beijing Sibeifu Biotechnology Co., Ltd.

[0039] The first group of examples, Lactobacillus paracasei LC97 of the present invention This group of embodiments provides a method for allergy relief of Lactobacillus paracasei ( Lacticaseibacillus paracasei )LC97, its deposit number is CGMCC No. 32421.

[0040] Any act of culturing, propagating, fermenting, enriching, producing, preparing, using, inoculating, amplifying, transforming, modifying, transforming, selling, promising to sell a strain of Lactobacillus paracasei LC97 with a preservation number of CGMCC NO. 32421, and / or using a strain of Lactobacillus paracasei LC97 with a preservation number of CGMCC NO. 32421 in combination with other probiotics, and / or using a strain of Lactobacillus paracasei LC97 with a preservation number of CGMCC NO. 32421 to antagonize various pathogens, and / or prepare an antibacterial agent, and / or prepare an immune function enhancing food, and / or a product for alleviating atopic dermatitis all fall within the scope of protection of the present invention.

[0041] The other probiotics include but are not limited to: Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii lactic acid subsp., Lactobacillus helveticus, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus salivarius, Saccharomyces cerevisiae, Delb's spore-forming torula, Candida, Wickham's yeast, Pichia, Saccharomyces boulardii, Torulopsis spp., Saccharomyces cerevisiae, Rhodotorula, Schizosaccharomyces pombe , Saccharomyces spp., Bacillus thuringiensis, Bacillus laterosporus, Bacillus megaterium, Bacillus mucilaginosus, Bacillus azotobacterium, Bacillus sphaericus, Clostridium butyricum, Bifidobacterium adolescentis, Bifidobacterium angularis, Bifidobacterium animalis, Bifidobacterium asteroides, Bifidobacterium bifidum, Bifidobacterium bovis, Bifidobacterium breve, Bifidobacterium dentate, Bifidobacterium infantis (i.e., Bifidobacterium longum infantis subsp. infantis), Bifidobacterium lactis (i.e., Bifidobacterium animalis subsp. cremoris), Bifidobacterium longum, Bifidobacterium pseudocatenulatum, as well as Bifidobacterium thermophilum and Bifidobacterium acidothermophilum.

[0042] Those skilled in the art can make conventional selections or adjustments to pharmaceutical excipients according to actual production needs, in combination with conventional technical means or basic common sense of production processes in the pharmaceutical field (for example, "Encyclopedia of Preparation Technology", "Encyclopedia of Food and Food Production", "Lactic Acid Bacteria and Fermented Foods", "Research and Application of Microbial Agent Technology", etc.), and then make a strain of Lactobacillus paracasei LC97 with a preservation number of CGMCC NO. 32421 into products with different dosage forms, different storage conditions, and different shelf lives. This is possible and easy to do without technical barriers for those skilled in the art.

[0043] The second group of examples, Lactobacillus paracasei LC97 of the present invention This group of embodiments provides a Lactobacillus paracasei with a deposit number of CGMCC No. 32421 ( Lacticaseibacillus paracasei ) Application of LC97 in the preparation of foods for enhancing immune function, products for alleviating atopic dermatitis, antibacterial agents or antibacterial agents for non-therapeutic purposes.

[0044] In a specific embodiment, the enhancing immunity comprises: regulating cytokine levels; Preferably, the relief of atopic dermatitis includes: relieving skin lesions, inhibiting inflammation, reducing ear swelling, and regulating cytokine levels; Preferably, the regulating cytokine levels comprises: reducing serum total IgE and IL-4 levels, and increasing IFN-γ and IL-10 levels; Preferably, the antibacterial spectrum includes: Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, Bacteroides fragilis, Streptococcus pasteurianus, Streptococcus pharyngitis, and Streptococcus Parisi.

[0045] The third group of embodiments, the preparation of the food for enhancing immune function of the present invention This group of embodiments provides a method for preparing a food for enhancing immune function. All embodiments of this group have the following common features: the method for preparing a food for enhancing immune function includes a functional active ingredient; the functional active ingredient includes Lactobacillus paracasei (CGMCC No. 32421) Lacticaseibacillus paracasei )LC97.

[0046] In some embodiments, the method for preparing a food for enhancing immune function further includes: auxiliary materials.

[0047] In a more specific embodiment, the excipient is selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculating agents, filter aids, release retardants, etc.

[0048] According to the content of the present invention, for different needs in actual production and application, combined with conventional technical means in the field of preparation preparation (for example, "Encyclopedia of Preparation Technology", "Encyclopedia of Food and Food Production", "Lactic Acid Bacteria and Fermented Foods", "Research and Application of Microbial Agent Technology", etc.), those skilled in the art can select and formulate the above-mentioned auxiliary materials, and prepare Lactobacillus paracasei LC97 with a deposit number of CGMCC NO. 32421 into different dosage forms, such as powders, tablets, oral liquids, gels, patches, sprays, lotions, granules, etc.

[0049] In a specific embodiment, the dosage form of the food for enhancing immune function is selected from: one or more of powder, tablet, liquid, and capsule.

[0050] The fourth group of embodiments, the product for relieving atopic dermatitis of the present invention This group of embodiments provides a product for relieving atopic dermatitis. All embodiments in this group have the following common features: the product for relieving atopic dermatitis includes an active ingredient; the active ingredient includes Lactobacillus paracasei ( Lacticaseibacillus paracasei )LC97.

[0051] In a further embodiment, the product for alleviating atopic dermatitis further comprises: auxiliary materials.

[0052] In a more specific embodiment, the excipient is selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculating agents, filter aids, release retardants, etc.

[0053] According to the content of the present invention, for different needs in actual production and application, combined with conventional technical means in the field of food preparation (for example, "Encyclopedia of Preparation Technology", "Encyclopedia of Food and Food Production", "Lactic Acid Bacteria and Fermented Foods", "Research and Application of Microbial Agent Technology", etc.), those skilled in the art can select and formulate the above-mentioned auxiliary materials, and prepare different functional food dosage forms such as solid beverages, milk beverages, powders, tablets, oral liquids, gels, patches, sprays, granules, etc. with the Lactobacillus paracasei LC97 with a deposit number of CGMCC NO. 32421.

[0054] In a specific embodiment, the dosage form of the product for alleviating atopic dermatitis is selected from: one or more of: solid beverages, milk beverages, tablets, liquids, and capsules.

[0055] In a specific embodiment, the product for alleviating atopic dermatitis is selected from: food or bacterial agent.

[0056] The fifth group of embodiments, the antibacterial agent of the present invention This group of embodiments provides an antibacterial agent. All embodiments of this group have the following common features: the antibacterial agent includes an antibacterial active ingredient; the antibacterial active ingredient includes Lactobacillus paracasei ( Lacticaseibacillus paracasei )LC97.

[0057] In a further embodiment, the antibacterial agent further comprises: auxiliary materials.

[0058] In a more specific embodiment, the excipient is selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculating agents, filter aids, release retardants, etc.

[0059] According to the content of the present invention, for different needs in actual production and application, combined with conventional technical means in the field of food preparation (for example, "Encyclopedia of Preparation Technology", "Encyclopedia of Food and Food Production", "Lactic Acid Bacteria and Fermented Foods", "Research and Application of Microbial Agent Technology", etc.), those skilled in the art can select and formulate the above-mentioned auxiliary materials, and prepare Lactobacillus paracasei LC97 with a preservation number of CGMCC NO. 32421 into different functional food dosage forms, such as solid beverages, milk beverages, powders, tablets, oral liquids, gels, patches, sprays, lotions, granules, etc.

[0060] In a specific embodiment, the dosage form of the antibacterial agent is selected from: one or more of: solid beverages, milk beverages, tablets, liquids, and capsules.

[0061] The sixth group of embodiments, the antibacterial method of the present invention This group of embodiments provides a non-treatment destination antibacterial method. All embodiments in this group have the following common features: using Lactobacillus paracasei (CGMCC No. 32421) Lacticaseibacillus paracasei )LC97 for antibacterial effect.

[0062] In a specific embodiment, the antibacterial spectrum includes: Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, Bacteroides fragilis, Streptococcus pasteurianus, Streptococcus pharyngitis, and Streptococcus Parisi.

[0063] The use of a strain of Lactobacillus paracasei LC97 with a preservation number of CGMCC NO. 32421 in combination with other probiotics for in vitro bacteriostasis falls within the protection scope of the present invention.

[0064] The other probiotics include but are not limited to: Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii lactic acid subsp., Lactobacillus helveticus, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus salivarius, Saccharomyces cerevisiae, Delb's spore-forming torula, Candida, Wickham's yeast, Pichia, Saccharomyces boulardii, Torulopsis spp., Saccharomyces cerevisiae, Rhodotorula, Schizosaccharomyces pombe , Saccharomyces spp., Bacillus thuringiensis, Bacillus laterosporus, Bacillus megaterium, Bacillus mucilaginosus, Bacillus azotobacterium, Bacillus sphaericus, Clostridium butyricum, Bifidobacterium adolescentis, Bifidobacterium angularis, Bifidobacterium animalis, Bifidobacterium asteroides, Bifidobacterium bifidum, Bifidobacterium bovis, Bifidobacterium breve, Bifidobacterium dentate, Bifidobacterium infantis (i.e., Bifidobacterium longum infantis subsp. infantis), Bifidobacterium lactis (i.e., Bifidobacterium animalis subsp. cremoris), Bifidobacterium longum, Bifidobacterium pseudocatenulatum, as well as Bifidobacterium thermophilum and Bifidobacterium acidothermophilum.

[0065] Experimental Example 1: Isolation, screening and identification of Lactobacillus paracasei (1) Isolation and screening of strains Lactobacillus paracasei was isolated from breast milk samples. The sample was diluted with 0.85% saline gradient under sterile conditions, and the appropriate gradient was selected and spread on LBS agar plates, and cultured at 37°C for 48-72 hours. The colony morphology was observed with the naked eye, and suspected single colonies were picked. After microscopic observation, preliminary screening and purification culture were performed. The purified strain was cultured in MRS liquid at 37°C for 8-12 hours, centrifuged to remove the supernatant, resuspended in sterile 30% glycerol aqueous solution, and stored at -80°C.

[0066] LBS medium formula (g / L): 5.0g yeast extract, 10.0g tryptone, 6.0g potassium dihydrogen phosphate, 0.034g ferrous sulfate, 0.575g magnesium sulfate, 20.0g glucose, 25.0g sodium acetate, 2.0g ammonium citrate, 0.12g manganese sulfate, pH = 5.5. Then add 1.0mL Tween-80 and 1.3mL glacial acetic acid, heat and stir to dissolve in 1000mL distilled water, and sterilize at 121℃ for 15min. Add 15.0g agar to the solid medium.

[0067] MRS medium formula (g / L): 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, 0.19 g manganese sulfate monohydrate, dissolved in 1000 mL distilled water, adjusted to pH = 6.8, and sterilized at 115 ° C for 20 min. Add 1.5% agar to the solid medium.

[0068] (2) Morphological characteristics and 16S rDNA identification The colony morphology of Lactobacillus paracasei on MRS medium and the bacteria under a microscope (10×100) are shown in Figure 2. Figure 1 , Figure 2 Lactobacillus paracasei grows well on MRS solid plates. Single colonies are milky white, round and convex, medium in size, with neat edges and smooth surfaces. Microscopic examination shows that the bacteria are rod-shaped, arranged singly, in pairs or in chains, which conforms to the staining characteristics of Gram-positive bacteria.

[0069] The screened target strains were cultured in liquid, the cells were collected, the genomic DNA was extracted, and the universal primers 27F and 1492R recorded in paragraph 58 of Chinese invention patent ZL202210478937.4 were used for PCR amplification reaction. The PCR amplification program was: 94°C pre-denaturation for 5 min, 94°C denaturation for 30s, 55°C annealing for 30s, 72°C extension for 90s, a total of 35 cycles, and finally extended at 72°C for 10min. 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. According to the sequencing results, the BLAST tool in the NCBI database was used for homology comparison, and the obtained strain was identified as Lactobacillus paracasei. The strain was named Lactobacillus paracasei LC97 and sent for preservation. The preservation information is as follows: Deposit number: CGMCC No. 32421 Classification name: Lactobacillus paracasei Lacticaseibacillus paracasei Depository: China General Microbiological Culture Collection Management Center Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing Date of preservation: October 30, 2024.

[0070] Experimental Example 2: Inhibitory ability of Lactobacillus paracasei LC97 on trypsin secretion (1) Mastoma cell culture: Collect the mastoma cell P815 suspension in the culture bottle into a centrifuge tube and centrifuge (800r / min, 3min). Discard the supernatant, add culture medium (90% DMEM + 10% FBS) to resuspend, and place in an incubator for culture.

[0071] (2) Preparation of LC97 bacterial suspension: activate the strain for three generations, collect the cells by centrifugation in 1 mL of bacterial suspension, resuspend in DMEM medium, and adjust the bacterial suspension concentration to 10 8 CFU / mL.

[0072] (3) After 24 h of culture, the P815 cells were removed from the incubator. The experiment was set up with three treatments, namely, the LC97 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 above 24-well plate was placed in a 37℃ constant temperature incubator for 3 h. After the incubation, 100μg / mL of C48 / 80 was added to the probiotic treatment group and control group 1, and sterile water was added to control group 2, and they were placed in a constant temperature incubation of 37℃ for 1h. After the incubation, centrifuged at 1000rpm / min for 20min, and the concentration of tryptase in the supernatant was determined by the corresponding ELISA kit.

[0073] Tryptase (TPS) is a specific marker of mast cell activation and an inflammatory marker in allergic reactions. The level of this enzyme in serum can be used as one of the key indicators for judging the degree of allergic reactions in the body. C48 / 80 compound is a polyamine synthetic complex, often used as a mast cell mediator releaser. The concentration of tryptase in the cell supernatant of different treatment groups is as follows: Figure 3 As shown in the figure, the content of tryptase in the positive control group (C48 / 80) was significantly higher than that in the negative control group (CK), indicating that compound C48 / 80 induced the release of tryptase in mastoma cells and caused allergic symptoms. The content of tryptase in the group treated with Lactobacillus paracasei LC97 was significantly lower than that in the group treated with C48 / 80 ( p <0.05), indicating that the LC97 strain could inhibit the degranulation of mastoma cells and reduce the release of tryptase.

[0074] Experimental Example 3: Lactobacillus paracasei LC97 stimulates the secretion of interferon-γ The mice were killed by cervical dislocation, and the spleen was removed. The spleen tissue was mechanically crushed to make free spleen cells and stored in isotonic saline to form a spleen cell suspension. The spleen cells were concentrated at 4×10 6 cells / mL was added to a 96-well plate, and then the bacterial solution was diluted to 4×10 6 cfu / well concentration was added to a 96-well plate. The same volume of culture medium was used as the blank control group (NC), 2 μg / mL lipopolysaccharide (LPS) was used as the positive control group-1, and 4 μg / mL concanavalin A (ConA) was used as the positive control group-2. The treated spleen cells were cultured for 48 hours and the supernatant was collected by centrifugation. The concentration of IFN-γ in the supernatant was determined according to the detection method of the ELISA kit manufacturer's instructions.

[0075] The concentration of IFN-γ in different treatment groups was as follows Figure 4As shown, the dotted line represents the concentration of IFN-γ in the negative control group (CK). Compared with the negative control CK, strain LC97 can promote the secretion of IFN-γ, and the concentration of IFN-γ in the LC97 group is close to that in the positive control LPS group, indicating that Lactobacillus paracasei LC97 can stimulate the secretion of IFN-γ to a certain extent, promote Th1 immune response, regulate Th1 / Th2 balance, and thus improve allergic symptoms.

[0076] Experimental Example 4: Gastrointestinal tolerance test of LC97 1. Preparation of artificial gastric juice and ox bile: prepare 0.5% physiological saline, adjust the pH to 2.5 with dilute hydrochloric acid (HCl), then add pepsin with a final concentration of 0.3%, fully dissolve it, filter it with a 0.22μm microporous filter membrane for sterilization, and set aside; add 0.2% (w / v) sodium thioglycolate to MRS medium, then add ox bile with a final concentration of 0.3%, and sterilize it at 115℃ for 20 minutes; 2. Experimental operation: (1) Take 1 mL of the cultured third-generation bacterial solution, centrifuge it, discard the supernatant, add 1 mL of artificial gastric juice, mix it evenly, place it in a 37°C incubator, and perform tolerance tests on gastric juice at 0 h and gastric juice at 3 h.

[0077] (2) Take 1 mL of the cultured 3rd generation bacterial solution, centrifuge, discard the supernatant, add 1 mL of artificial bile, mix well, place in a 37°C incubator, and perform tolerance tests at 0 h and 3 h, respectively. Strain survival rate (%) = Nt / N0 × 100%, where N0 represents the number of viable bacteria of the strain at 0 h (log CFU / mL), and Nt represents the number of viable bacteria of the strain at 3 h (log CFU / mL).

[0078] The tolerance results of Lactobacillus paracasei LC97 in gastric juice and bile salts are shown in Table 1. As shown in Table 1, the survival rates of the strain after 3 h of treatment in gastric juice and bile salts were 101.42% and 96.60%, respectively, indicating that the strain has the potential to successfully enter the human gastrointestinal tract.

[0079] Table 1. Tolerance of Lactobacillus paracasei LC97 to gastric juice and bile salts

[0080] Experimental Example 5: Inhibitory effect of LC97 on pathogenic bacteria (1) Preparation of indicator bacteria suspension: Preparation of bacterial suspension of Escherichia coli 21652, Salmonella enteritidis 21651 and Staphylococcus aureus 21654: Escherichia coli 21652, Salmonella enteritidis 21651 and Staphylococcus aureus 21654 were activated on LB solid plate medium, cultured at 37°C for 24 h, and the bacterial lawn was picked up and placed in physiological saline to prepare bacterial suspension for 108 CFU / mL.

[0081] Preparation of Bacteroides fragilis 21645 suspension: Bacteroides fragilis 21645 was inoculated on a BHI plate medium containing 5% defibrinated sheep blood for activation, cultured at 37°C for 24 h, and the bacterial moss was picked up and placed in physiological saline to prepare a bacterial suspension. The bacterial suspension concentration was adjusted to 10 8 CFU / mL.

[0082] Preparation of Streptococcus pasteurianus 12758 suspension: Streptococcus pasteurianus 12758 was inoculated on a BHI plate medium containing 5% defibrinated sheep blood for activation, cultured at 37°C for 24 h, and the bacterial moss was picked up and placed in saline to prepare a bacterial suspension. The concentration of the bacterial suspension was adjusted to 10 8 CFU / mL.

[0083] Preparation of Streptococcus anginosus 4997 bacterial suspension: Streptococcus anginosus was inoculated into TSA blood plates containing 5% defibrinated sheep blood and cultured at 37°C for 16-20 hours. The bacterial suspension concentration was adjusted to 10 8 CFU / mL.

[0084] Preparation of Streptococcus Parisi 21178 bacterial suspension: Streptococcus Parisi 21178 was inoculated into TSA blood plates containing 5% defibrinated sheep blood and cultured at 37°C for 16-20 hours. The bacterial suspension concentration was adjusted to 10 8 CFU / mL.

[0085] (2) Preparation of fermentation broth: Lactobacillus paracasei LC97 was inoculated into MRS liquid culture medium at 1% (v / v) and cultured at 37°C for 12 h.

[0086] Antibacterial experiment: (1) Cool the MRS broth containing 1.5% agar to about 55°C and mix it with the suspension of Escherichia coli, Salmonella enteritidis and Staphylococcus aureus in a certain proportion so that the number of viable indicator bacteria is 10 6 CFU / mL, and then quickly poured into the plate with the Oxford cup placed in advance. After the culture medium cooled and solidified, take out the Oxford cup and inject 200μL of the strain fermentation liquid (the number of viable bacteria is 10 8 CFU / mL level), and the diameter of the inhibition zone was measured after overnight culture at 37°C.

[0087] (2) Cool the TSA medium containing 5% defibrinated sheep blood and 1.5% agar to about 55°C and mix it with the suspension of Bacteroides fragilis, Streptococcus pasteurianus, Streptococcus Parisi and Streptococcus anginosus in a certain proportion so that the number of viable indicator bacteria is 10 6CFU / mL, and then quickly poured into the plate with the Oxford cup placed in advance. After the culture medium cooled and solidified, take out the Oxford cup and inject 200μL of the strain fermentation liquid (the number of viable bacteria is 10 8 CFU / mL level), and the diameter of the inhibition zone was measured after overnight culture at 37°C.

[0088] As shown in Table 2, the antagonistic diameter range of Lactobacillus paracasei LC97 to pathogenic bacteria is 15-32 mm, indicating that Lactobacillus paracasei LC97 has a good inhibitory effect on the growth of these seven pathogenic bacteria.

[0089] Table 2. Inhibitory effect of Lactobacillus paracasei LC97 on pathogenic bacteria

[0090] Experimental Example 6: Adhesion ability of LC97 to intestinal epithelial cells In this experimental example, Caco-2 cells were used as epithelial cells, which have functional microvilli and hydrolytic enzymes that attach to epithelial cells. The ability of probiotics to adhere to human epithelial cells is considered to be a prerequisite for strains to colonize in the human gastrointestinal tract and produce beneficial effects, and probiotics can prevent the colonization of harmful microorganisms by competing for binding sites on relevant epithelial cells.

[0091] (1) Preparation of bacterial suspension: Inoculate the first generation bacterial suspension into fresh culture medium at a 2% inoculum volume and incubate at 37°C for 15 h. Take the cultured target bacterial suspension, collect the cells by centrifugation at room temperature, wash twice with sterile PBS, and finally resuspend in DMEM culture medium; (2) Preparation of monolayer: Caco-2 cells were inoculated into DMEM medium supplemented with 20% (v / v) fetal bovine serum and transferred to a 12-well cell culture plate. The amount of cells added was 2.4×10 cells per well in 1 mL. 5 cell / mL of cells, 5% CO 2 , culture at 37°C to obtain a single cell layer for later use; (3) Co-culture: After washing the 12-well plate with PBS, add 1 mL of bacterial suspension to each well and incubate at 37°C and 5% CO 2 After incubation for 2 hours under the same conditions, add sterile PBS to wash the 12-well plate thoroughly, add 0.2 mL / well of trypsin cell digestion solution, digest for 10 minutes, and collect the sample solution; (4) Perform gradient dilution and live bacterial count on the collected samples. Adhesion rate and adhesion capacity are calculated according to the following formula: Adhesion capacity (CFU / cell) = number of bacteria adhering to cells (CFU) / number of cells in the well Adhesion rate (%) = number of bacteria adhering to cells (CFU) / total number of bacteria added to the well × 100% Lactobacillus rhamnosus LGG (LGG) is an internationally recognized probiotic strain with strong adhesion ability. It was used as a positive control strain to evaluate the adhesion ability of Lactobacillus paracasei LC97. Figure 5 As shown, the adhesion ability of strain LC97 to Caco-2 cells is close to that of the control strain LGG, indicating that the bacteria can colonize well in the human intestine.

[0092] Experimental Example 7: Application of LC97 in alleviating atopic dermatitis in mice Specific pathogen-free BALB / c female mice (18–20 g) were purchased from Beijing Sibeifu Biotechnology Co., Ltd. and housed in the animal room of Hubei Food and Drug Safety Evaluation Center. The temperature was maintained at 22 ± 2 °C, the humidity was 50 ± 5%, the light-dark cycle was 12 h, and free access to water and food was allowed.

[0093] Before the experiment, the mice were first adaptively fed for one week, and then randomly divided into three groups: normal control group (NC), dermatitis model group (MC), drug group (Keto), and Lactobacillus paracasei group (LC97). During the entire experiment, mice in the NC and MC groups were gavaged with 0.2 mL of sterile saline every day, the Keto group was gavaged with an equal volume of drug solution, and the LC97 group was gavaged with an equal amount of Lactobacillus paracasei suspension (the number of viable bacteria was 1×10 9 CFU / mL) until the end of the experiment, a total of 3 weeks. Before modeling, the hair on the back of the mouse was shaved with a shaver. Acetone and olive oil were prepared into a base solution in a ratio of 4:1, and then 2,4-dinitrofluorobenzene (DNFB) and the base solution were used to prepare 0.5% and 0.2% DNFB sensitizers. Modeling began in the week. On the first day of modeling, 0.5% DNFB was used to smear the back skin of the mouse for sensitization. Then, on the 5th, 8th, 11th, and 14th days, 0.2% DNFB solution was used to smear the back skin and right ear of the mouse to induce atopic dermatitis. The blank group mice were smeared with the same amount of base solution. During the experiment, the thickness of the right ear of the mouse was measured with a digital spiral micrometer. After the experiment, the mice were killed and samples were collected for subsequent analysis. The detailed animal experiments are shown in Table 3.

[0094] Table 3. Animal experiment design

[0095] (1) Skin lesions on the back of mice DNFB destroyed the mouse skin barrier, causing significant skin lesions on the back of the mice. Figure 6As shown. Through observation, it was found that the back skin of the mice in the MC group and the model group had typical dermatitis symptoms, accompanied by scratches and scabs of varying degrees, while the skin of the mice in the control group was smooth, ruddy and without scratches. The positive drug Keto group significantly alleviated the dermatitis symptoms on the back of the mice and basically returned to normal. Compared with the model group, the LC97 group had no obvious scab symptoms, and there was no difference in naked eye observation with the drug group.

[0096] (2) Evaluation of pathological symptoms on the back of mice Back skin sections of different treatment groups Figure 7 As shown. Compared with the NC group, the skin inflammation induced by DNFB (MC group) led to a significant increase in the thickness of the mouse back skin, accompanied by a large number of inflammatory cell infiltrations, such as spongioma, acanthosis, etc. Similar to the relief effect of the positive drug (Keto), Lactobacillus paracasei LC97 significantly reduced the thickness of the mouse back skin, indicating that the strain significantly inhibited the immune inflammation of the mouse back skin lesions after intake, and had a relieving effect on skin inflammation.

[0097] (3) Effect of the degree of ear swelling on mice Repeated topical application of DNFB induced clinical symptoms of atopic dermatitis in mice, and changes in ear thickness reflected the degree of ear swelling caused by local inflammatory infiltration. Figure 8 The thickness of the ears of mice in the MC group was significantly higher than that in the NC group ( p <0.01), the positive drug Keto significantly reduced the swelling of the mouse ears ( p <0.05). Compared with the MC group, LC97 intervention significantly reduced the ear thickness of mice ( p <0.01), and was closest to the healthy group. These results suggest that Lactobacillus paracasei LC97 can alleviate the symptoms of atopic dermatitis after ingestion.

[0098] (4) Cellular inflammatory factors After the experiment, orbital blood was collected from 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.

[0099] The core immune mechanism of atopic dermatitis is the 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 causing severe pathological symptoms of skin lesions. Therefore, the large amount of IgE produced in serum has been recognized as a typical sign of atopic dermatitis. Fig. 9 and Fig.10 It can be seen that after sensitizing mice with DNFB, the serum total IgE and IL-4 levels of the model group mice increased significantly, while the intervention of Lactobacillus paracasei LC97 significantly reduced the serum total IgE and IL-4 levels of the mice ( p < 0.05), while there was no significant difference in IgE and IL-10 levels compared with the control group, indicating that LC97 intake can inhibit Th2 immune response.

[0100] In inflammatory responses, regulatory T lymphocytes can play an important role in immunomodulation by producing cytokines, especially IFN-γ and IL-10, which can inhibit Th2 cells and effector cells of allergic inflammation. Fig.11 and Fig.12 Compared with the NC group, the levels of IFN-γ and IL-10 in the MC group were significantly decreased ( p <0.05). After intervention with Lactobacillus paracasei LC97, the levels of IFN-γ and IL-10 in the serum of mice were significantly increased ( p <0.05), and the increased levels of IFN-γ and IL-10 mean that the Th1 immune response is upregulated, thus helping to restore the balance between Th1 and Th2 immune responses and alleviate the pathological symptoms of atopic dermatitis.

Claims

1. A Lactobacillus paracasei strain for allergy relief ( Lacticaseibacillus paracasei ) LC97, characterized in that Its deposit number is CGMCC No. 32421.

2. Lactobacillus paracasei with a deposit number of CGMCC No. 32421 ( Lacticaseibacillus paracasei ) Application of LC97 in the preparation of foods for enhancing immune function, products for alleviating atopic dermatitis, antibacterial agents or antibacterial agents for non-therapeutic purposes.

3. according to claim 2, the deposit number is CGMCC No. 32421 Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Application of LC97 in the preparation of foods for enhancing immune function, products for relieving atopic dermatitis, antibacterial agents or non-therapeutic antibacterial agents, characterized in that: The immunity enhancement includes: regulating cytokine levels; And / or, the relief of atopic dermatitis includes: relieving skin lesions, inhibiting inflammation, reducing ear swelling, and regulating cytokine levels; And / or, the regulating cytokine levels includes: reducing serum total IgE and IL-4 levels, and increasing IFN-γ and IL-10 levels; And / or, the antibacterial spectrum includes: Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, Bacteroides fragilis, Streptococcus pasteurianus, Streptococcus pharyngitis, and Streptococcus Parisi.

4. A method for preparing a food for enhancing immune function, characterized in that: Including functional active ingredients; the functional active ingredients include Lactobacillus paracasei with a preservation number of CGMCC No. 32421 ( Lacticaseibacillus paracasei )LC97.

5. The method for preparing a food for enhancing immune function according to claim 4, characterized in that: Also includes: Auxiliary materials.

6. A product for relieving atopic dermatitis, characterized in that: The invention comprises an active ingredient; the active ingredient comprises Lactobacillus paracasei having a preservation number of CGMCC No. 32421 ( Lacticaseibacillus paracasei )LC97.

7. A product for relieving atopic dermatitis according to claim 6, characterized in that: Also includes: Auxiliary materials.

8. An antibacterial agent, characterized in that Including antibacterial active ingredients; the antibacterial active ingredients include Lactobacillus paracasei with a preservation number of CGMCC No. 32421 ( Lacticaseibacillus paracasei )LC97.

9. An antibacterial agent according to claim 8, characterized in that: Also includes: Auxiliary materials.

10. A non-therapeutic antibacterial method, characterized in that: The Lactobacillus paracasei with a preservation number of CGMCC No. 32421 ( Lacticaseibacillus paracasei )LC97 for antibacterial effect.

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