A probiotic combination and a product thereof for intervening rosacea

Abstract

The invention discloses a probiotic combination and a product for intervening rosacea, and relates to the technical field of microorganisms. The invention provides a Lacticaseibacillus paracasei, and the preservation number of the Lacticaseibacillus paracasei is CGMCC No.27056. The invention also provides a Ligilactobacillus salivarius, and the preservation number of the Lactobacillus salivarius is CGMCC No.27886. The invention further provides a probiotic composition comprising Lactobacillus paracasei and Lactobacillus salivarius, and the probiotic composition has a good therapeutic effect on rosacea and has a wide range of application prospects in preparing products for improving rosacea.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a probiotic combination and a product thereof for intervening in rosacea. Background Art

[0002] Rosacea is a chronic inflammatory skin disease that mainly affects the blood vessels and hair follicles of the facial skin. It has a high incidence rate, and the prevalence rate is relatively high in the Chinese population, affecting tens of millions of patients. The clinical manifestations of rosacea are diverse, mainly paroxysmal flushing or persistent erythema in the central area of ​​the face (mainly including the cheeks, nose, and mouth), and papules, pustules, capillary dilation and other manifestations may occur. A small number of patients may show hypertrophic skin lesions; according to its clinical manifestations, it can be divided into type I erythematous capillary rosacea, type II papulopustular rosacea, type III hypertrophic rosacea and type IV ocular rosacea. The disease causes damage to the patient's facial appearance, increases the psychological burden, and even leads to psychological disorders such as anxiety and depression, causing great distress to the patient's physical and mental health.

[0003] There are relatively few systemic treatments for rosacea. Currently, modern medicine commonly uses doxycycline and retinoic acid for oral use, ivermectin and metronidazole for topical use, and modern methods such as photoelectric technology for systemic treatment. However, the disease is prone to recurrence, and for special populations such as adolescents, the drug selection is limited, adverse reactions are prone to occur, and the efficacy and prognosis are not satisfactory.

[0004] Probiotics are a class of active microorganisms that are beneficial to the host. They are a general term for active beneficial microorganisms that colonize in the human intestines and reproductive system, can produce definite health effects, thereby improving the host's microecological balance and exerting beneficial effects on the intestines.

[0005] Probiotics mainly play a probiotic role by reducing inflammation and enhancing the body's immunity. Probiotics have the good effect of regulating the structure of intestinal flora, improving the homeostasis of intestinal microecology, and thus promoting health. Rosacea is closely related to inflammatory bowel disease. Based on the good physiological characteristics and probiotic functions of salivary Lactobacillus and Lactobacillus paracasei, they can increase the phosphorylation level of tight junction proteins and protect the intestinal barrier function; they can also play an immune role by colonizing the intestine and enhancing the activity of immune cells. Therefore, probiotic intervention can be used to reduce inflammation and improve rosacea.

[0006] Lactobacillus salivae belongs to the family Lactobacillaceae and the genus Lactobacillus. It is a Gram-positive, non-spore-forming rod-shaped bacterium that can convert its own metabolized oligofructose into lactic acid and acetic acid. Lactobacillus paracasei is a facultative anaerobic, Gram-positive heterofermentative lactic acid bacterium with multiple probiotic functions. However, there are few studies on the therapeutic effects of probiotics on rosacea in the prior art. At present, there is an urgent need to provide a probiotic or probiotic composition with better effects in preventing and treating rosacea. Summary of the invention

[0007] The purpose of the present invention is to provide a probiotic combination and a product thereof for intervening rosacea.

[0008] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0009] In one aspect, the present invention provides a Lacticaseibacillus paracasei, wherein the Lacticaseibacillus paracasei has a deposit number of CGMCC No.27056.

[0010] Meanwhile, the present invention provides a preparation containing the above-mentioned Lactobacillus paracasei, wherein the preparation is selected from one or more of the following: fermentation broth of Lactobacillus paracasei, precipitate of fermentation broth of Lactobacillus paracasei, supernatant of fermentation broth of Lactobacillus paracasei, live bacteria of Lactobacillus paracasei, and freeze-dried powder of Lactobacillus paracasei.

[0011] On the other hand, the present invention provides a salivarius-associated Lactobacillus (Ligilactobacillus salivarius), and the preservation number of the salivarius-associated Lactobacillus is CGMCC No.27886.

[0012] At the same time, the present invention provides a preparation containing the above-mentioned salivarius-associated Lactobacillus, and the preparation is selected from one or more of the fermentation broth of salivarius-associated Lactobacillus, the fermentation broth precipitate of salivarius-associated Lactobacillus, the fermentation broth supernatant of salivarius-associated Lactobacillus, the live bacteria of salivarius-associated Lactobacillus, and the freeze-dried powder of salivarius-associated Lactobacillus.

[0013] Specifically, the fermentation liquid refers to the liquid obtained by inoculating the bacteria into the culture medium and culturing the culture medium.

[0014] More specifically, the fermentation broth supernatant refers to the clarified liquid on the upper layer of the fermentation broth after centrifugation; it contains abundant metabolites and some bacterial fragments during the bacterial growth and reproduction process.

[0015] More specifically, the fermentation liquid precipitation refers to the liquid precipitation after centrifugation, including free protein, residual bacteria, broken cells, and residues of the culture matrix.

[0016] The form of the preparation in the present invention is only for example, and in fact all products containing the Lactobacillus paracasei of the present invention are within the protection scope of the present invention.

[0017] In another aspect, the present invention provides a probiotic composition, comprising the above-mentioned Lactobacillus paracasei, the above-mentioned Lactobacillus salivarius and / or the above-mentioned preparation.

[0018] Specifically, the probiotic composition contains 1×10 6 -1×10 14 CFU / mL or 1×10 6 -1×10 14 CFU / g of Lactobacillus paracasei.

[0019] Furthermore, the probiotic composition comprises (1-10)×10 10 CFU / mL or (1-10)×10 10 CFU / g of Lactobacillus paracasei.

[0020] Furthermore, the probiotic composition comprises 10 10 CFU / mL or 10 10 CFU / g of Lactobacillus paracasei.

[0021] Specifically, the probiotic composition contains 1×10 6 -1×10 14 CFU / mL or 1×10 6 -1×10 14 CFU / g of salivary combined Lactobacillus.

[0022] Furthermore, the probiotic composition comprises (1-10)×10 9 CFU / mL or (1-10)×10 9 CFU / g of salivary combined Lactobacillus.

[0023] Furthermore, the probiotic composition comprises 10 9 CFU / mL or 10 9 CFU / g of salivary combined Lactobacillus.

[0024] In yet another aspect, the present invention provides a product for treating rosacea, wherein the product comprises the aforementioned Lactobacillus paracasei, the aforementioned Lactobacillus salivarius, the aforementioned preparation and / or the aforementioned probiotic composition.

[0025] Specifically, the products include medicines and cosmetics.

[0026] Furthermore, when the product is a medicine, the medicine also includes a pharmaceutically acceptable carrier.

[0027] Furthermore, the pharmaceutically acceptable carrier is selected from one or more of an excipient, a stabilizer, a diluent, a binder, a preservative, and a lubricant.

[0028] Specifically, the excipient is selected from at least one of microcrystalline cellulose, lactose, pregelatinized starch, cyclodextrin, carboxymethyl cellulose and mannitol.

[0029] Specifically, the stabilizer is selected from at least one of agar, alginic acid, cellulose ether, and carboxymethyl chitosan.

[0030] Specifically, the diluent is selected from at least one of erythritol, mannitol, sorbitol, xylitol, lactose, sucrose, corn starch, potato starch, calcium phosphate, calcium citrate, and crystalline cellulose.

[0031] Specifically, the binder is selected from at least one of ethanol, starch slurry, syrup, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, sodium alginate, and polyvinyl pyrrolidone.

[0032] Specifically, the preservative is selected from at least one of methylparaben, propylparaben, methylparaben, ethylparaben, propylparaben, chlorobutanol, thimerosal, mercuric cyanide, phenoxyethanol, chlorhexidine, benzoic acid, sodium benzoate, chlorocresol, benzalkonium bromide, benzalkonium chloride, and ethylparaben.

[0033] Specifically, the lubricant is selected from at least one of magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, and poloxamer.

[0034] Specifically, the dosage form of the drug is drops, mixtures, tinctures, injections, tablets, powders, oral liquids, capsules, granules, ointments, suspensions, powders, emulsions, solutions, pellets, pills, buccal tablets, lyophilized powder injections, gels, suppositories or aerosols.

[0035] Furthermore, the cosmetics can be conventional preparations such as lotion, toner, toner, essence water, face cream, face mask, cleansing cream, facial cleanser, shampoo and hair conditioner, shaving cream, hair milk, hair gel, lipstick, sunscreen, hand cream, etc.

[0036] Specifically, the cosmetics may further include base materials and conventional auxiliary materials.

[0037] Furthermore, the matrix raw materials include but are not limited to colloid raw materials, powdery raw materials, oil raw materials, wax raw materials, coagulants, and surfactants.

[0038] Furthermore, the conventional auxiliary materials include but are not limited to antioxidants, adhesives, lubricants, moisturizers, preservatives, whitening agents, film agents, emulsifiers, and cosmetic nutritional additives.

[0039] Specifically, the viable count of salivary combined lactobacilli in the product is 1×10 6 -1×10 14 CFU / mL or 1×10 6 -1×10 14 CFU / g, the viable count of Lactobacillus paracasei in the product is 1×10 6 -1×10 14 CFU / mL or 1×10 6 -1×10 14 CFU / g.

[0040] Furthermore, the viable count of salivary combined lactobacilli in the product is 1×10 9 CFU / mL or 1×10 9 CFU / g, the viable count of Lactobacillus paracasei in the product is 1×10 10 CFU / mL or 1×10 10 CFU / g.

[0041] In particular, the product is used to treat rosacea.

[0042] Furthermore, the rosacea includes type I erythematous capillary rosacea, type II papulopustular rosacea, type III hypertrophic rosacea and type IV ocular rosacea.

[0043] The beneficial effects of the present invention are:

[0044] The invention provides a strain of lactobacillus paracasei. The preservation number of the lactobacillus paracasei is CGMCC No. 27056. The lactobacillus paracasei has good activity, good acid resistance, good antibacterial performance on 8 pathogenic bacteria, good antioxidant performance, no hemolytic property, and is highly sensitive to antibiotics such as ampicillin, erythromycin, chloramphenicol, penicillin and tetracycline, but is insensitive to co-trimoxazole and gentamicin.

[0045] The invention also provides a salivary lactobacillus strain, the salivary lactobacillus strain has a preservation number of CGMCC No. 27886, has good activity and acid resistance, good antibacterial performance, strong antioxidant activity, no hemolytic property, and is highly sensitive to antibiotics such as ampicillin, erythromycin, chloramphenicol, penicillin and tetracycline.

[0046] The present invention further provides a probiotic composition, which contains Lactobacillus paracasei and Lactobacillus salivarius. The combined action of Lactobacillus paracasei and Lactobacillus salivarius on rosacea can alleviate the related symptoms of rosacea, inhibit the inflammatory response of rosacea through the TLR2 / MyD88 / NFκB pathway, improve intestinal microecological imbalance, and alleviate colon inflammation and intestinal permeability.

[0047] Collection Instructions

[0048] Deposit number: CGMCC No.27056;

[0049] Classification and nomenclature: Lacticaseibacillus paracasei;

[0050] Deposit date: April 11, 2023;

[0051] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0052] Abbreviation of depository unit: CGMCC;

[0053] Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0054] Collection Instructions

[0055] Deposit number: CGMCC No.27886;

[0056] Classification and nomenclature: Ligilactobacillus salivarius;

[0057] Preservation time: July 13, 2023;

[0058] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0059] Abbreviation of depository unit: CGMCC;

[0060] Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 is the optical density of bacterial suspension of L.salivarius-NCU-03.

[0062] Figure 2 It is the plate count method for L. salivarius-NCU-03.

[0063] Figure 3It is the acid resistance test of L.salivarius-NCU-03.

[0064] Figure 4 The antibacterial activity of L. salivarius-NCU-03 was determined by the Oxford cup method. a is the negative control and b is the cultured isolate of L. salivarius-NCU-03.

[0065] Figure 5 This is a statistical graph of the antibacterial activity of L.salivarius-NCU-03 determined by the Oxford cup method.

[0066] Figure 6 Determination of the antioxidant capacity of L.salivarius-NCU-03.

[0067] Figure 7 This is the hemolytic experiment of L. salivarius-NCU-03, a is the positive control Staphylococcus aureus, and b is the cultured isolate of L. salivarius-NCU-03.

[0068] Figure 8-Figure 9 To determine the drug resistance of L.salivarius-NCU-03.

[0069] Fig.10 Evaluation of the in vitro probiotic properties of L.paracasei-NCU-04; A. Optical density of bacterial suspension of A.paracasei-NCU-04; B. Plate count method of L.paracasei-NCU-04; C. Acid resistance determination of L.paracasei-NCU-04; D. Antibacterial activity of L.paracasei-NCU-04 determined by Oxford cup method, a is the negative control, b is the cultured isolate of L.paracasei-NCU-04; E. Antioxidant capacity determination of L.paracasei-NCU-04; F. Hemolysis experiment of L.paracasei-NCU-04, a is the positive control Staphylococcus aureus, b is the cultured isolate of L.paracasei-NCU-04; G. Determination of drug resistance of L.paracasei-NCU-04.

[0070] Fig.11 L. salivarius-NCU-03 combined with L. paracasei-NCU-04 alleviates LL37-induced rosacea-like phenotype in mice; A Animal experiment design; B Images of the mouse back taken 12 hours after the last injection of LL37; C H&E staining of mouse lesion skin (scale bar: 100μm); D CD4 +Immunofluorescence of T cells (Scale bar: 50 μm); E ELISA was used to detect the levels of inflammatory factors IL-6, IL-1β, and TNF-α in mouse serum; F ELISA was used to detect the level of inflammatory factor IL-1β in mouse serum; G ELISA was used to detect the level of inflammatory factor TNF-α in mouse serum; *p<0.05, **p<0.01, ***p<0.001.

[0071] Fig.12 The combination of L. salivarius-NCU-03 and L. paracasei-NCU-04 can reshape the intestinal microbiota; A species composition heat map; B principal coordinate analysis (PCoAanalysis); C non-metric multidimensional scaling analysis (NMDS analysis); D Venn diagram; E analysis of intestinal microbial flora abundance at the phylum level; F relative abundance of Firmicutes; G analysis of intestinal microbial flora abundance at the genus level; H. lactobacillus relative abundance; I. Coprococcus relative abundance; J. O. scillospira relative abundance *p<0.05, **p<0.01.

[0072] Fig.13 L. salivarius-NCU-03 and L. paracasei-NCU-04 postbiotics can alleviate LL37-induced rosacea-like phenotype in mice; A Animal experiment design; B Images of the back of mice taken 12 hours after the last injection of LL37; C H&E staining of mouse lesion skin (scale bar: 100μm); D CD4 + Immunofluorescence of T cells (scale bar: 50 μm); EELISA detected the inflammatory factor IL-6 in mouse serum; F ELISA detected the inflammatory factor IL-1β in mouse serum; G ELISA detected the inflammatory factor TNF-α in mouse serum; *p<0.05, **p<0.01, ***p<0.001.

[0073] Fig.14Postbiotics can reshape the gut microbiota for L. salivarius-NCU-03 and L. paracasei-NCU-04. A Species composition heat map; B Principal coordinate analysis (PCoAanalysis); C Non-metric multidimensional scaling analysis (NMDSanalysis); D Venn diagram; E Analysis of gut microbiota abundance at the phylum level; F Relative abundance of Firmicutes; G Relative abundance of Bacteroidetes; H Relative abundance of Proteobacteria; I Analysis of gut microbiota abundance at the genus level; J Relative abundance of Lactobacillus; *p<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0074] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0075] In the present invention, L. salivarius-NCU-03, Lactobacillus salivarius-NCU-03, and Ligilactobacillus salivarius-NCU-03 all refer to Lactobacillus salivarius with a deposit number of CGMCC No.27886.

[0076] In the present invention, L. paracasei-NCU-04 refers to Lactobacillus paracasei with a deposit number of CGMCC No.27056.

[0077] Basic Example

[0078] 1. Sample processing

[0079] Take two stool samples, add 5 mL of ordinary PBS to one and add 5 mL of PBS with pH=3 and 0.2% ox bile salt concentration to the other, and place them in a 37°C incubator for 2-3 hours to settle naturally.

[0080] 2. Dilute and coat the plate

[0081] The supernatant sample was diluted 100-107 times, and the appropriate concentration gradient was selected for coating. The gradients were 1, 3, 4, and 5, and 20-50 μL was coated.

[0082] Prepare solid culture medium based on 15 mL per plate. The required medium is as follows:

[0083] MRS-Lactobacillus (anaerobic + aerobic);

[0084] BSM (MRS + 0.1% cysteine) + mupirocin (50 mg / L) - Bifidobacterium (anaerobic);

[0085] LB (anaerobic + aerobic);

[0086] BHI-general screening (anaerobic + aerobic);

[0087] BHI + skim milk (5%) (anaerobic + aerobic);

[0088] BHI + sheep serum (5%) - Haemophilus (anaerobic + aerobic);

[0089] The above culture media were cultured aerobically and anaerobically for 48-72h;

[0090] TSB-Clostridium (anaerobic + aerobic) The diluted sample to be coated was placed in an 80°C water bath for 10 minutes (to kill non-spore-forming bacteria), inoculated into 5 mL of liquid TSB medium and cultured aerobically and anaerobically for 36 hours. After another 10 minutes of water bath at 80°C, it was coated into solid TSA medium and cultured aerobically and anaerobically for 24-48 hours.

[0091] 3. Bacterial activation

[0092] Select a plate with a colony count of about 200-400, and inoculate 20 single colonies into the corresponding 5 mL liquid culture medium in a sterile clean bench according to the shape, size, color, whether the edges are neat, whether they are raised, and the faster growing colonies, and culture them aerobically and anaerobically for 48-72 hours.

[0093] 4. Bacterial Identification

[0094] The activated bacterial solution was preserved (one tube was kept for each), and the remaining bacterial solution was centrifuged to extract nucleic acid DNA for 16S rDNA sequencing. The obtained sequence was compared with the BLAST sequence in NCBI to determine its species level.

[0095] Example 1 Evaluation of probiotic properties

[0096] 1 Experimental methods

[0097] 1.1 Probiotic growth curve

[0098] The growth curve was determined by turbidimetry. Since the concentration of the bacterial suspension is proportional to the turbidity, the optical density of the bacterial suspension can be measured by a spectrophotometer to infer the concentration of the bacterial solution. The strain was activated overnight, 1 mL was inoculated into 100 mL of MRS medium, cultured at 37°C, and the optical density was measured at 0, 2, 4, 6, 8, 16, 18, 20, and 24 hours at a wavelength of 600 nm. A growth curve was drawn with time as the horizontal axis and optical density as the vertical axis.

[0099] 1.2 Genetic stability and functional evaluation of probiotics

[0100] 1.2.1 Acid resistance test

[0101] The strain was cultured in a 37°C incubator overnight; 100 μL was taken out and diluted with PBS buffer for 10 1 , 10 3 , 10 5 times (take 100 μL and add it to 900 μL PBS buffer, then take 10 μL and add it to 990 μL PBS, then take 10 μL and add it to 990 μL PBS), centrifuge at 6000×g for 3 min and discard the supernatant; add PBS buffer with pH=2, 3, 4, 5, 7, let it stand for 4 hours, mix well, take 10 μL to spread on the plate, culture in a constant temperature incubator at 37°C for 12-48 hours, count the live bacteria, and keep experimental records.

[0102] 1.2.2 Antioxidant evaluation

[0103] Solution preparation: TSB culture medium, deionized water, 0.2mmol / L DPPH methanol solution, 2mmol / L ferrous sulfate solution, 6mmol / L hydrogen peroxide solution, 6mmol / L salicylic acid solution, 150mmol / L Tris-HCl solution at pH 8.0, 1.2mmol / L pyrogallol solution, 0.4% ferrous sulfate solution, 1% VC, 0.2mol / L sodium hydroxide solution, 10% trichloroacetic acid solution, 0.1% o-phenanthroline solution, pH 6.6 phosphate buffer, 1% potassium ferrocyanide solution, and 0.1% ferric chloride solution.

[0104] 1.2.2.1 Determination of DPPH free radical scavenging ability

[0105] Add 2 mL (0.2 mmol / L) DPPH methanol solution to 2 mL of the strain culture supernatant, react at room temperature in the dark for 30 min, take the supernatant to measure OD at 517 nm, and use 2 mL of deionized water plus DPPH methanol solution as a control.

[0106] DPPH free radical scavenging rate = [1-A517 (sample) / A517 (blank)] × 100%.

[0107] 1.2.2.2 Determination of hydroxyl radical scavenging ability

[0108] Take 1 mL of 2 mmol / L ferrous sulfate solution, 1 mL of 6 mmol / L hydrogen peroxide, and 1 mL of 6 mmol / L salicylic acid, add 1 mL of the strain culture supernatant, let it stand for 30 min, measure the absorbance at 510 nm using distilled water as a reference, and calculate the scavenging rate of hydroxyl free radicals.

[0109] Hydroxyl radical scavenging rate = [1-A510 (sample) / A510 (blank)] × 100%.

[0110] 1.2.2.3 Determination of superoxide free radical scavenging ability

[0111] Take 0.5mL of bacterial supernatant and add it to glass test tube No. 1, add 2mL of Tris-HCl solution and 1mL of pyrogallol solution, and react at room temperature for 30min. Add 0.5mL of deionized water, 1mL of pyrogallol and 2mL of 2mL Tris-HCl solution to test tube No. 2, add 1.5mL of deionized water and 2mL of Tris-HCl solution to test tube No. 3, and add 1mL of deionized water, 0.5mL of bacterial supernatant and 2mL of Tris-HCl solution to test tube No. 4.

[0112] Superoxide radical scavenging rate = [1-(A11-A10) / (A01-A00)] × 100%

[0113] A00: without sample and pyrogallol (3); A01: without sample and containing pyrogallol (2);

[0114] A10: contains the sample but not pyrogallol (4); A11: contains the sample and pyrogallol (1).

[0115] 1.2.2.4 Chelating ability of ferrous ions

[0116] Take 0.5mL of bacterial supernatant and add it to a glass tube. Add 0.1mL of ferrous sulfate solution and mix well. Then add 0.1mL of VC and 1mL of sodium hydroxide solution. React in a 37℃ oven for 20 minutes. Then add trichloroacetic acid and centrifuge to remove protein. The centrifugation conditions are 6000rpm, 10min, and 4℃. Take 0.4mL of supernatant and add 4mL of o-phenanthroline. Add 0.5mL of water to another test tube, and do the same as above. React at room temperature for 10 minutes and measure the absorbance at 536nm. Record the data of the control group and the experimental group:

[0117] Fe 2+ The chelating capacity = (A blank - A sample / A blank) × 100%.

[0118] 1.2.2.5 Determination of total reducing power

[0119] Take 1mL of bacterial supernatant, add 1mL of phosphate buffer and 1mL of potassium ferricyanide, mix well, keep warm at 50℃ for 2min, add 1mL of trichloroacetic acid, shake and mix well, take 1mL of the mixture, add 4mL of deionized water and 0.4mL of ferric chloride solution, and let stand for 10min. Add 1mL of deionized water to another test tube, and the other reagents and treatments are the same. After standing, measure the absorbance, and use deionized water as a blank. Measure the absorbance at 700nm, and record the data of the control group and the experimental group.

[0120] The absorbance A is measured at a wavelength of 700nm. The larger A is, the greater the reducing power is.

[0121] 1.3 Antibacterial experiment

[0122] The inoculated strain was cultured in the culture medium for 16 hours, and the supernatant was removed after centrifugation at 8000rpm for 10 minutes. Another 20 μL of 7 pathogenic bacteria (Escherichia coli O157, Escherichia coli K12, Staphylococcus aureus, Shigella flexneri, Staphylococcus epidermidis, Clostridium perfringens, and Enterococcus faecalis) cultured overnight were spread on LB solid culture medium plates, placed on Oxford cups, and 250 μL of bacterial supernatant was drawn into the Oxford cups. After culture at 37°C for 8 hours, the diameter of the inhibition zone was measured.

[0123] 1.4 Hemolysis test

[0124] The strain and the positive control Staphylococcus aureus were streaked onto a plate containing sheep blood to observe the growth and hemolysis of the strain.

[0125] 1.5 Drug resistance test

[0126] Spread the bacterial solution on a BHI solid plate, place a cotton piece containing antibiotics on the plate, and observe the diameter of the inhibition zone.

[0127] 2 Experimental results

[0128] 2.1 Properties of Saliva-Lactobacillus-NCU-03 Probiotics

[0129] Under normoxic culture conditions, saliva combined with Lactobacillus salivarius-NCU-03 reached the logarithmic growth phase in 6 hours ( Figure 1 ), which can reach 10 in the stable period 8 CFU / mL( Figure 2 ), indicating that L. salivarius-NCU-03 has good activity and can be used for subsequent experiments.

[0130] In order to explore the probiotic properties of L. salivarius-NCU-03, the present invention adopted acid resistance test, antioxidant, antibacterial, hemolytic and drug resistance test.

[0131] The results of acid resistance test showed that L.salivarius-NCU-03 could still reach 10 at pH=1.0. 3 CFU / mL( Figure 3 ).

[0132] In the antibacterial experiment, the inhibition zones of L.salivarius-NCU-03 against Shigella flexneri, Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, Shigella dysenteriae, Salmonella typhimurium, beta-hemolytic Streptococcus, and Salmonella enteritidis were 18.3mm, 19.3mm, 18.3mm, 20.7mm, 20.7mm, 19.3mm, 19.7mm, and 19mm, respectively, which were larger than those of the negative control group ( Figure 4-Figure 5 ).

[0133] The results of antioxidant experiments showed that the DPPH free radical scavenging rate of L. salivarius-NCU-03 was 80.76%, the -OH scavenging rate was 86.01%, and the -O2 - The clearance rate was 65.42%, Fe 2+ The chelation rate was 68.62% and the total reducing power OD was 0.71 ( Figure 6 ).

[0134] Hemolysis experiments showed that L.salivarius-NCU-03 did not have hemolytic properties compared to the positive control Staphylococcus aureus ( Figure 7 ).

[0135] In the drug resistance experiment, it was found that L. salivarius-NCU-03 was highly sensitive to antibiotics such as ampicillin (AMP), erythromycin (E), chloramphenicol (C), penicillin (PEN) and tetracycline (TET), but was not sensitive to trimethoprim-sulfamethoxazole (SXT) and gentamicin (GEN). Figure 8-Figure 9 ).

[0136] The above results show that L.salivarius-NCU-03 is a probiotic with good properties.

[0137] 2.2 Evaluation of the probiotic properties of Lactobacillus paracasei-NCU-04

[0138] Under normoxic culture conditions, L. paracasei-NCU-04 reached the logarithmic growth phase in 6 h ( Fig.10 A) in the stable period can reach 10 9 CFU / mL( Fig.10 B), indicating that L. paracasei-NCU-04 has good activity and can be used in subsequent experiments.

[0139] In the acid resistance experiment, it was found that L. paracasei-NCU-04 could still reach 10 at pH = 1.0. 4 CFU / mL( Fig.10 C).

[0140] The antibacterial experiments showed that the inhibition zones of L. paracasei-NCU-04 against Shigella flexneri, Listeria monocytogenes, Escherichia coli O157, Staphylococcus aureus, Shigella dysenteria, Salmonella Typhimurium, β-hemolytic sreptococcs and Salmonella enteritidis were 18.3 mm, 18.7 mm, 20.7 mm, 17 mm, 21.7 mm, 23.7 mm, 23.7 mm and 14 mm, respectively, and the diameters of the inhibition zones were larger than those of the negative control group ( Fig.10 D).

[0141] The results of the antioxidant experiment showed that the DPPH free radical scavenging rate of L. paracasei-NCU-04 was 84.23%, the -OH scavenging rate was 86.58%, and the -O2 - The clearance rate was 69.04%, Fe 2+ The chelation rate was 73.79% and the total reducing power OD was 0.69 ( Fig.10 E).

[0142] Hemolysis experiments showed that L. paracasei-NCU-04 did not have hemolytic properties compared to the positive control Staphylococcus aureus ( Fig.10 F).

[0143] The results of drug resistance experiments showed that L. paracasei-NCU-04 was highly sensitive to antibiotics such as ampicillin (AMP), erythromycin (E), chloramphenicol (C), penicillin (PEN) and tetracycline (TET). Fig.10 G in.

[0144] The above experimental results show that L.paracasei-NCU-04 has good probiotic properties.

[0145] Example 2 Effects of L. salivarius-NCU-03 and L. paracasei-NCU-04 on rosacea

[0146] 1 Experimental methods

[0147] 1.1 Establishment of rosacea mouse model

[0148] (1) Preparation of LL37 polypeptide

[0149] Before opening the lid of the LL37 polypeptide lyophilized powder, centrifuge at low speed to allow the lyophilized powder to settle to the bottom of the tube. Add 25 mg of LL37 to 8.6935 mL of PBS in a clean bench to prepare a solution with a concentration of 320 μM. After fully dissolved, divide the solution into portions and store it at -20°C.

[0150] (2) Establishment of rosacea mouse model induced by LL37

[0151] Fifty 8-week-old female BALB / c mice, weighing about 20 g, were fed normally for one week to adapt to the environment and randomly divided into groups of 10 mice per group. The back hair was shaved before the experiment to avoid skin damage as much as possible. After 24 hours, LL37 peptide (40 μL, 320 μM) or an equal amount of PBS was injected intradermally into the back skin of the mice, once every 12 hours, for a total of 4 injections. After the treatment, the formation of erythematous rosacea on the back of the mice was observed and photographed with a camera and a stereoscope.

[0152] 1.2 Grouping and treatment of rosacea mice

[0153] The probiotics were resuspended in a coating solution (0.9% saline solution containing 0.01% gelatin) and administered intragastrically, wherein the concentration of saliva combined with Lactobacillus solution was 10 9 CFU / mL, each mouse was given 10 8 CFU / mL bacteria, that is, 100 μL was administered to each mouse; the concentration of Lactobacillus paracasei liquid was 10 10 CFU / mL, each mouse was given 10 9 CFU / mL bacteria, that is, 100 μL was administered to each mouse by gavage once a day for 16 consecutive days, and the mice were killed and samples were taken for subsequent experiments.

[0154] The experimental animals were grouped and treated as shown in Table 1.

[0155] Table 1.

[0156]

[0157]

[0158] 1.3 Study on the specific mechanism of probiotic intervention in improving symptoms of rosacea in mice

[0159] 1.3.1 Phenotypic detection of rosacea mice

[0160] (1) Take photos to observe and assess the degree of inflammation

[0161] After the last injection of LL37, the mice were photographed and scored for erythematous rosacea on the back skin. After 21 days of probiotic intervention, the mice were photographed again for rosacea on the back and scored. The mice were photographed with a camera, and the local erythema was photographed with a stereoscope to analyze the area and severity of the erythema.

[0162] Skin inflammation in the rosacea mouse model was assessed by the severity of erythema and edema, i.e., the color of erythema was evaluated from 0 to 4 points, with 0 representing no erythema, 1 representing faintly visible erythema, 2 representing light erythema, 3 representing obvious and clear erythema, and 4 representing dark color with clear boundaries.

[0163] (2) HE staining

[0164] To observe the morphology of skin tissue and intestine and inflammatory cell infiltration in the lesion area, skin tissue and colorectal samples were taken for HE staining. The structure was observed under a 200x microscope, and 5 fields of view were randomly selected under a 400x microscope to count the number of immune cells in each field of view and take the average value.

[0165] 1.3.2 Probiotic intervention reduces skin inflammation in rosacea mice

[0166] (1) Detection of inflammatory factors (blood ELISA)

[0167] ELISA was used to detect the transcription levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β. If the levels of TNF-α, IL-6, and IL-1β were reduced, it indicated that probiotics reduced inflammation.

[0168] The TNF-α detection kit was purchased from Solarbio with the catalog number SEKM-0034; the IL-6 detection kit was purchased from Solarbio with the catalog number SEKM-0007; and the IL-1β detection kit was purchased from Neobioscience with the catalog number EMC001b.96.

[0169] 1.3.3 Effects of probiotic intervention on the intestinal tract of rosacea mice

[0170] (1) High-throughput sequencing

[0171] Studies have found that rosacea patients have intestinal bacterial overgrowth (SIBO), disordered intestinal flora, and increased pathogenic bacteria. Saliva combined with Lactobacillus can maintain the balance of intestinal flora. 3-4 pieces of mouse feces were collected at the time of successful modeling and at the end of intervention for high-throughput sequencing to detect intestinal flora.

[0172] The method for detecting intestinal flora by high-throughput sequencing includes the following steps:

[0173] Total genomic DNA from mouse fecal samples was extracted using the TIANamp Bacteria DNA Kit (QIAGEN) as instructed. The concentration and quality of the extracted genomic DNA were assessed by spectrophotometer (NanoDrop, Thermo Fisher Scientific, Inc., USA). The extracted DNA was subjected to 16S sequencing at Shanghai Paisono Biotechnology Co., Ltd. The PCR amplification products were sequenced using the Illumina MiSeq platform. FLASH was used to merge overlapping reads, and the UPARSE software package was used for sequence analysis. Reads with a quality score lower than 20, ambiguous bases, and inappropriate primers were discarded before clustering, and chimeras were checked and eliminated during the clustering process. ASV / OTU signature sequences were obtained using the DADA2 method and then processed using Quantitative Insights into Microbial Ecology (QIIME). The relative abundance of the total bacteria in each sample was classified into different taxonomic levels (phylum, class, order, family, and genus) using the Greengenes database v13.8 (19).

[0174] 2 Experimental results

[0175] 2.1 Effects of L. salivarius-NCU-03 and L. paracasei-NCU-04 on rosacea

[0176] To investigate the intervention effect of L. salivarius-NCU-03 and L. paracasei-NCU-04 on rosacea, 6-8 week-old BALB / c female mice were randomly divided into 5 groups and given PBS, L. salivarius-NCU-03, L. paracasei-NCU-04, and a combination of L. salivarius-NCU-03 and L. paracasei-NCU-04 for 16 days, respectively. On the 14th day after oral administration, the back skin of the mice was cleaned and the antimicrobial peptide LL37 (40 μL, 320 μM) was intradermally injected every 12 h for a total of 4 injections ( Fig.11 The results showed that 12 hours after the last injection of LL37, the mice in the M group showed obvious rosacea-like phenotype, and the erythema area of ​​the mice in the Ls, Lp and Ls&p groups decreased. The symptoms of the mice in the combined intervention group (Ls&p group) improved most significantly ( Fig.11B in the figure). Immunofluorescence and H&E results showed that compared with the M group, probiotic intervention reduced the infiltration of inflammatory cells in the dermis of mice, and the combined intervention group (Ls&p group) had the most significant effect ( Fig.11 C in Fig.11 D).

[0177] The combined intervention of probiotics significantly reduced the expression of inflammatory factors IL-6 (M vs.Ls&p=41.51pg / mL vs.22.01pg / mL), IL-1β (M vs.Ls&p=364.20pg / mL vs.83.72pg / mL), and TNF-α (M vs.Ls&p=70.41pg / mL vs.18.38pg / mL) in the serum of mice. Fig.11 EG in).

[0178] The above results indicate that L. salivarius-NCU-03 combined with L. paracasei-NCU-04 can alleviate the symptoms associated with LL37-induced rosacea in mice.

[0179] 2.2 L. salivarius-NCU-03 combined with L. paracasei-NCU-04 improves LL37-induced intestinal microecological imbalance

[0180] The study showed that intestinal flora disturbance may be closely related to the occurrence and development of rosacea. The present invention used 16SrRNA sequencing to detect changes in the intestinal microbiota of mice and found that there were differences in species composition between the groups ( Fig.12 A).

[0181] In the NMDS and PCoA score plots, the beta diversity of the intestinal microbiome community was visualized by different population clustering patterns. The study found that L. salivarius-NCU-03 and L. paracasei-NCU-04 changed the beta diversity of the intestinal microbiota in mice, but there was no significant difference ( Fig.12 B in Fig.12 In addition, the results of the Venn diagram showed that 256 common OTUs were found in the five groups, while there were 1225, 2474, 884, 1067 and 915 unique OUTs in C, M, Ls, Lp and Ls&p, respectively. Fig.12 D).

[0182] At the phylum level, Firmicutes, Proteobacteria, and Bacteroidetes were common microbiota in the mouse intestine, accounting for 94.85%, 88.41%, 85.10%, 92.71%, and 93.95% of the sequencing results in each group, respectively. Fig.12 E). It is worth noting that after oral administration of L. salivarius-NCU-03 and L. paracasei-NCU-04, the abundance of Firmicutes in the Ls&p group was higher than that in the M group (Ls&p vs. M = 87.49% vs. 80.59%) ( Fig.12 F), indicating that L. salivarius-NCU-03 and L. paracasei-NCU-04 could reverse the changes in Firmicutes induced by LL37.

[0183] At the genus level, compared with the M group, the abundance of Lactobacillus in the probiotic intervention groups, including the Ls group, the Lp group, and the Ls&p group, was increased (29.00% vs. 42.16%, 48.13%, and 63.12%) (p<0.01)( Fig.12 The G in Fig.12 In contrast, the abundance of Coprococcus (1.44% vs. 7.37%, 3.61%, 2.75%) (p<0.05) and Oscillospira (3.73% vs. 2.28%, 0.90%, 0.92%) (p<0.05) in the Ls, Lp, and Ls&p groups were lower than those in the M group ( Fig.12 These results suggest that L. salivarius-NCU-03 and L. paracasei-NCU-04 have the potential to counteract certain changes within the gut microbiota and restore intestinal homeostasis.

[0184] In summary, L. salivarius-NCU-03 and L. paracasei-NCU-04 help regulate and improve LL37-induced intestinal dysbiosis, thereby reshaping the diversity of intestinal microbiota, especially in reducing Coprococcus and Oscillospira and increasing Lactobacillus.

[0185] Example 3 Effects of L. salivarius-NCU-03 and L. paracasei-NCU-04 postbiotics on rosacea

[0186] 1 Experimental methods

[0187] 1.1 The groups and treatments of experimental animals are shown in Table 2.

[0188] Table 5.

[0189]

[0190] 1.2 Preparation of postbiotics

[0191] The preparation method of the postbiotics is as follows: inactivating the bacterial solution at 90° C. for 20 minutes to prepare the postbiotics.

[0192] 1.3 Study on the specific mechanism of postbiotic intervention in improving symptoms of rosacea in mice

[0193] 1.3.1 Phenotypic detection of rosacea mice

[0194] After the last injection of LL37, the mice were photographed and scored for erythematous rosacea on the back skin. After 21 days of probiotic intervention, the mice were photographed again for rosacea on the back and scored: the color of the erythema was evaluated from 0 to 4 points, with 0 representing no erythema, 1 representing faintly visible erythema, 2 representing light erythema, 3 representing obvious and clear erythema, and 4 representing dark color with clear boundaries.

[0195] The gross images of the mice were taken with a camera, and the local erythema was photographed with a stereoscope to analyze the area and severity of the erythema.

[0196] 1.3.2 HE staining

[0197] In order to observe the morphology of skin tissue and intestine and inflammatory cell infiltration in the lesion area, skin tissue and colorectal samples were taken for HE staining.

[0198] 1.3.3 Detection of inflammatory factors (blood ELISA)

[0199] ELISA was used to detect the transcription levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β. If the levels of TNF-α, IL-6, and IL-1β were reduced, it indicated that probiotics reduced inflammation.

[0200] 1.3.5 Effects of postbiotic intervention on the intestinal tract of rosacea mice

[0201] ① High-throughput sequencing

[0202] Studies have found that patients with rosacea have intestinal bacterial overgrowth (SIBO), disordered intestinal flora, and an increase in pathogenic bacteria. Saliva combined with Lactobacillus can maintain the balance of intestinal flora.

[0203] Three to four pellets of mouse feces were collected after successful modeling and at the end of intervention for high-throughput sequencing to detect whether postbiotics can regulate intestinal flora.

[0204] The detection method in Example 3 is the same as that in Example 2.

[0205] 2 Experimental results

[0206] 2.1 L. salivarius-NCU-03 and L. paracasei-NCU-04 postbiotics can inhibit the inflammatory response of rosacea

[0207] Postbiotics refer to non-living microorganisms or their components that are beneficial to the health of the host and have become a safer and more stable alternative to live bacteria. In this study, L. salivarius-NCU-03 and L. paracasei-NCU-04 were inactivated by high temperature to prepare their postbiotics, and their probiotic properties were determined.

[0208] Mice were orally treated with L. salivarius-NCU-03 and L. paracasei-NCU-04 postbiotics, and the phenotypes of mice were observed 12 h after the last injection of LL37 ( Fig.13 The results showed that both L. salivarius-NCU-03 and L. paracasei-NCU-04 postbiotics could improve the LL37-induced erythematous phenotype in mice. The combined use of postbiotics was better than that of either alone, but the combination of L. salivarius-NCU-03 and L. paracasei-NCU-04 had the most significant effect ( Fig.13 B).

[0209] In addition, H&E staining and immunofluorescence results showed that L. salivarius-NCU-03 and L. paracasei-NCU-04 postbiotics could alleviate the infiltration of inflammatory cells in the dermis induced by LL37 and the infiltration of CD4 + The expression of T Fig.13 C in Fig.13 D).

[0210] The study also found that L. salivarius-NCU-03 and L. paracasei-NCU-04 postbiotics can reduce the levels of inflammatory factors IL-6 (M vs. p-Ls, p-Lp, p-Ls&p=43.11 vs. 30.98, 33.20, 27.36 pg / mL), IL-1β (M vs. p-Ls, p-Lp, p-Ls&p=345.46 vs. 238.20, 240.80, 183.22 pg / mL) and TNF-α (M vs. p-Ls, p-Lp, p-Ls&p=70.28 vs. 42.10, 43.56, 35.87 pg / mL) in mouse serum. Fig.13 EG in).

[0211] 2.2 L. salivarius-NCU-03 and L. paracasei-NCU-04 postbiotics reverse LL37-induced intestinal flora imbalance and restore intestinal barrier

[0212] To further explore whether L. salivarius-NCU-03 and L. paracasei-NCU-04 postbiotics can regulate intestinal flora, this study performed 16S rRNA sequencing on mouse feces and found that species composition was different between different groups ( Fig.14 In the NMDS and PCoA score plots, the results showed that L. salivarius-NCU-03 and L. paracasei-NCU-04 postbiotics changed the β diversity of mouse intestinal microorganisms, but there was no significant difference ( Fig.14 B in Fig.14 In addition, the results of the Venn diagram showed that 237 common OTUs were found in the six groups, while there were 486, 1474, 1033, 508, 321 and 1651 unique OUTs in C, M, Ls&p, p-Ls, p-Lp and p-Ls&p, respectively. Fig.14 D).

[0213] At the phylum level, the common microbial communities Firmicutes, Bacteroidetes, and Proteobacteria in the mouse intestine accounted for 96.19%, 90.06%, 96.59%, 88.08%, 92.87%, and 92.33% of the sequencing results in each group, respectively. Fig.14 In addition, the study found that compared with the M group, the postbiotic intervention of L. salivarius-NCU-03 and L. paracasei-NCU-04 could upregulate the abundance of Firmicutes (M vs. p-Lp, p-Ls & p = 75.30% vs. 77.37%, 84.45%), while the p-Ls group was slightly decreased compared with the M group (M vs. p-Ls = 75.30% vs. 73.99%) (p < 0.01) ( Fig.14In contrast, postbiotic intervention with L. salivarius-NCU-03 and L. paracasei-NCU-04 could downregulate the abundance of Bacteroidetes (M vs. p-Ls, p-Lp, p-Ls & p = 11.51% vs. 7.24%, 7.84%, 3.97%) and Tenericutes (M vs. p-Ls, p-Lp, p-Ls & p = 4.43% vs. 0.17%, 0.93%, 1.54%). Fig.14 F in Fig.14 At the genus level, the abundance of Lactobacillus in the postbiotic intervention group was higher than that in the M group (M vs. p-Ls, p-Lp, p-Ls&p=24.40% vs. 33.31%, 31.54%, 53.94%) (p<0.001)( Fig.14 I in Fig.14 J in.

[0214] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Lactobacillus paracasei ( Lacticaseibacillus paracasei ), characterized in that, The deposit number of the Lactobacillus paracasei is CGMCC No. 27056.

2. A preparation comprising the Lactobacillus paracasei of claim 1, characterized in that The preparation is selected from one or more of the following: fermentation liquid of Lactobacillus paracasei, fermentation liquid precipitation of Lactobacillus paracasei, live bacteria of Lactobacillus paracasei, and freeze-dried powder of Lactobacillus paracasei.

3. A salivary lactobacillus ( Ligilactobacillus salivarius ), characterized in that, The deposit number of the salivary lactobacillus is CGMCC No. 27886.

4. A preparation comprising the salivary lactobacillus according to claim 3, characterized in that: The preparation is selected from one or more of the following: fermentation liquid of saliva-associated Lactobacillus, precipitate of fermentation liquid of saliva-associated Lactobacillus, live bacteria of saliva-associated Lactobacillus, and freeze-dried powder of saliva-associated Lactobacillus.

5. A probiotic composition, characterized in that: The probiotic composition comprises the Lactobacillus paracasei according to claim 1, the preparation according to claim 2, the Lactobacillus salivarius according to claim 3 and / or the preparation according to claim 4.

6. The probiotic composition according to claim 5, characterized in that The probiotic composition contains 1×10 6 -1×10 14 CFU / mL or 1×10 6 -1×10 14 CFU / g of Lactobacillus paracasei.

7. The probiotic composition according to claim 5, characterized in that The probiotic composition contains 1×10 6 -1×10 14 CFU / mL or 1×10 6 -1×10 14 CFU / g of salivary Lactobacillus.

8. A product for treating rosacea, characterized in that: The product comprises the Lactobacillus paracasei according to claim 1, the preparation according to claim 2, the Lactobacillus salivarius according to claim 3, the preparation according to claim 4 and / or the probiotic composition according to any one of claims 5-7.

9. The product according to claim 8, characterized in that The product is a medicine or a cosmetic.

10. The product according to claim 9, characterized in that When the product is a medicine, the medicine also includes a pharmaceutically acceptable carrier.

11. The product according to claim 10, characterized in that The pharmaceutically acceptable carrier is selected from one or more of an excipient, a stabilizer, a diluent, a binder, a preservative, and a lubricant.

12. The product according to claim 8, characterized in that The viable count of salivary combined lactobacilli in the product is 1×10 6 -1×10 14 CFU / mL or 1×10 6 -1×10 14 CFU / g, the viable count of Lactobacillus paracasei in the product is 1×10 6 -1×10 14 CFU / mL or 1×10 6 -1×10 14 CFU / g.

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