Anti-inflammatory compound probiotics and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to an anti-inflammatory compound probiotic and application thereof.Lactobacillus rhamnosus LGD-0817, lactobacillus rhamnosus LDL-1708 and lactobacillus plantarum LEP-1208 serve as basic dominant bacteria and then are compounded with multiple bacteria such as lactobacillus casei, lactobacillus delbrueckii and lactobacillus paracasei, the multiple bacteria can be matched and promoted mutually, and the anti-inflammatory compound probiotic has the anti-inflammatory effect. The traditional Chinese medicines jointly play a significant role in improving ulcerative colitis; the anti-inflammatory compound probiotics are used for traditional Chinese medicine fermentation, and the obtained traditional Chinese medicine fermentation composition can regulate intestinal flora, enhance the barrier function of the intestinal tract, inhibit release of proinflammatory factors, improve the shortening of the colon length of a mouse with colitis, inhibit the levels of proinflammatory cytokines IL-1beta, IL-6 and TNF-alpha, competitively inhibit colonization of pathogenic bacteria and improve the colonization of colitis. The method shows a good comprehensive intervention effect in a DSS-induced mouse ulcerative colitis model, and has clinical practical value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganisms, in particular to an anti-inflammatory composite probiotic and application thereof. BACKGROUND

[0002] Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory disease, and patients often have digestive symptoms such as diarrhea and hematochezia, as well as systemic symptoms such as weight loss and fever. In recent years, the incidence of IBD has increased significantly. Ulcerative colitis (UC) is a typical inflammatory bowel disease, and its lesion range is wide, and its characteristics are persistent inflammation of the colonic mucosa and ulcer formation. Common anti-inflammatory drugs include aminosalicylic acid derivatives, glucocorticoids, immunosuppressants and biological agents, etc. However, long-term use of non-steroidal anti-inflammatory drugs can easily cause gastrointestinal and kidney damage, liver function abnormalities, cardiovascular system risks, blood system effects, allergic and immune reactions, nervous system symptoms and other side effects; long-term use of steroids can cause metabolic disorders, bone and muscle damage, immune system suppression, eye complications, skin and endocrine problems and other side effects.

[0003] Intestinal flora disorder is an important manifestation of UC. Compared with healthy people, the diversity of intestinal flora of UC patients is reduced, and pro-inflammatory bacteria such as Enterobacteriaceae and Fusobacteriaceae and their metabolites can further exacerbate intestinal inflammation. Therefore, by taking live probiotics to regulate intestinal flora, enhance intestinal barrier function, regulate immunity and other mechanisms to relieve intestinal inflammation.

[0004] Therefore, the development of more anti-inflammatory composite probiotics can provide new ideas and methods for the treatment of colitis. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides an anti-inflammatory composite probiotic, and studies its biological activity and medical use.

[0006] In a first aspect, the present application provides an anti-inflammatory composite probiotic, which is composed of active bacteria group A and active bacteria group B: The active bacteria group A is composed of Lactobacillus rhamnosus LGD-0817, Lactobacillus rhamnosus LDL-1708 and Lactobacillus plantarum LEP-1208; The active bacteria group B is selected from one or more of Lactobacillus casei LPM-0188, Lactobacillus delbrueckii subsp. bulgaricus RHM-1068, Paracaseicola lactis LPA-2088, Lactobacillus fermentum LAF-1387, Lactobacillus plantarum MCA-3066 and Paracaseicola lactis BMC-2020.

[0007] In the present application, the Lactobacillus rhamnosus LGD-0817 is classified as Lactobacillus rhamnosus Lactobacillus rhamnosus , and is preserved in the China General Microbiological Culture Collection Center, located at No. 3, Beichen West Road, Haidian District, Beijing, with a preservation number of CGMCC No. 23632 and a preservation date of October 20, 2021; The Lactobacillus rhamnosus LDL-1708 is classified as Lactobacillus rhamnosus Lactobacillus rhamnosus , and is preserved in the China General Microbiological Culture Collection Center, located at No. 3, Beichen West Road, Haidian District, Beijing, with a preservation number of CGMCC No. 23960 and a preservation date of November 24, 2021; The Lactobacillus plantarum LEP-1208 is classified as Lactobacillus plantarum Lactobacillus plantarum , and is preserved in the China General Microbiological Culture Collection Center, located at No. 3, Beichen West Road, Haidian District, Beijing, with a preservation number of CGMCC No. 24012 and a preservation date of December 1, 2021; The Lactobacillus casei LPM-0188 is classified as Lactobacillus casei Lactobacillus casei , and is preserved in the China General Microbiological Culture Collection Center, located at No. 3, Beichen West Road, Haidian District, Beijing, with a preservation number of CGMCC No. 23959 and a preservation date of November 24, 2021; The Lactobacillus delbrueckii subsp. bulgaricus RHM-1068 is classified as Lactobacillus delbrueckii subsp. bulgaricus Lactobacillus delbrueckii subsp. bulgaricus , and is preserved in the China General Microbiological Culture Collection Center, located at No. 3, Beichen West Road, Haidian District, Beijing, with a preservation number of CGMCC No. 24013 and a preservation date of December 1, 2021; The Lactobacillus paracasei LPA-2088 is classified as Lactobacillus paracasei Lactobacillus paracasei , and is preserved in the China General Microbiological Culture Collection Center, located at No. 3, Beichen West Road, Haidian District, Beijing, with a preservation number of CGMCC No. 24057 and a preservation date of December 8, 2021; The Lactobacillus fermentum LAF-1387 is classified as Lactobacillus fermentum Lactobacillus fermentum , and is preserved in the China General Microbiological Culture Collection Center, located at No. 3, Beichen West Road, Haidian District, Beijing, with a preservation number of CGMCC No. 24056 and a preservation date of December 8, 2021; The Lactobacillus plantarum MCA-3066 is classified as Lactobacillus plantarum Lactobacillus plantarum , and is preserved in the China General Microbiological Culture Collection Center, located at No. 3, Beichen West Road, Haidian District, Beijing, with a preservation number of CGMCC No. 24489 and a preservation date of March 9, 2022; The Lactobacillus paracasei BMC-2020 is classified as Lactobacillus paracaseiLactobacillus paracasei , the preservation number is CGMCC No. 24488, the address is No. 3, Beichen West Road, Chaoyang District, Beijing, and the preservation date is March 9, 2022.

[0008] Further, the total viable bacteria number of the anti-inflammatory complex probiotic is 10e 3 -10e 12 CFU / mL, or 10e 6 -10e 12 CFU / mL, or 10e 6 -10e 9 CFU / mL.

[0009] Further, the mass ratio of the active bacteria group A to the active bacteria group B is (1-10):(1-10), or (1-8):(1-10), or (1-6):(1-8), or (1-4):(1-6), or (1-3):(1-6).

[0010] Further, the active bacteria group A consists of the following components in terms of weight parts: 1-10 parts of Lactobacillus rhamnosus LGD-0817, 1-10 parts of Lactobacillus rhamnosus LDL-1708, and 1-10 parts of Lactobacillus plantarum LEP-1208.

[0011] In a second aspect, the present application provides an application of the anti-inflammatory complex probiotic in the preparation of a drug for preventing, improving or treating colitis.

[0012] Further, the drug is prepared into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable excipient.

[0013] Further, the excipient includes one or more of a solvent, a disintegrant, a flavoring agent, a preservative, a coloring agent, a binder, a lubricant, a diluent, and a drug carrier.

[0014] Further, the dosage form is a solution, a lyophilized powder, a capsule, a tablet or a granule.

[0015] The following drug preparation method is also within the protection scope of the present application: Further, the anti-inflammatory complex probiotic is fermented from a traditional Chinese medicine extract, and the traditional Chinese medicine extract includes an Astragalus extract and a Glycyrrhiza extract.

[0016] Further, the traditional Chinese medicine extract consists of the following raw materials in terms of mass parts: 1-10 parts of an Astragalus extract and 1-5 parts of a Glycyrrhiza extract.

[0017] In a third aspect, the present application provides a traditional Chinese medicine fermentation composition, which is fermented from the anti-inflammatory probiotic compound and a traditional Chinese medicine extract. The traditional Chinese medicine extract is composed of Astragalus membranaceus extract 1-10 parts and Glycyrrhiza uralensis extract 1-5 parts.

[0018] In a fourth aspect, the present application provides use of the traditional Chinese medicine fermentation composition in the preparation of a medicament for preventing, improving or treating colitis.

[0019] Further, the medicament is prepared by using the traditional Chinese medicine fermentation composition as an active ingredient and pharmaceutically acceptable excipients to form a pharmaceutically acceptable dosage form.

[0020] Further, the dosage form is a solution.

[0021] In the present specification, the "weight parts" of each pharmaceutical component represent the ratio of the amount of each component, rather than the actual unit of mass. According to the actual situation, the unit weight parts can be any mass, such as 1 weight part can be 1 g, 500 g or 1 kg, or even 15 g, 30 g, etc.

[0022] As fully appreciated by those skilled in the art, excipients (e.g., pharmaceutical excipients) include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, antioxidants, flavoring agents, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavorings, preservatives, suspending agents, coating materials, fragrances, antiadherents, integrating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickeners, complexing agents, humectants, absorbents, diluents, flocculating and deflocculating agents, filtration aids, and release retarders.

[0023] The diluent can be one or more of mannitol, sucrose, lactose, sorbitol, xylitol, polyethylene glycol, propylene glycol, vegetable oil, mineral oil; the disintegrant can be one or more of crosscarmellose sodium, colloidal silicon dioxide, citric acid; the binder can be one or more of starch paste, ethanol, water, povidone solution; the preservative can be one or more of ethylparaben, propylparaben, sorbic acid, potassium sorbate, calcium propionate, sodium dehydroacetate, sodium diacetate, sodium lactate; the antioxidant can be one or more of ethylenediaminetetraacetic acid, disodium edetate, butylated hydroxytoluene, glycine, inositol, ascorbic acid, sodium ascorbate, lecithin, malic acid, hydroquinone, citric acid, succinic acid, sodium metabisulfite; the flavoring agent can be one or more of aspartame, sucrose, xylitol, steviol glycosides, cyclamate, sorbitol, cocoa, pure vanilla, vanillin, ethyl vanillin, chocolate, malt, peppermint; the suspending agent can be one or more of xanthan gum, polyvinylpyrrolidone, sodium alginate, aluminum stearate, hydrogenated vegetable oil; the emulsifying agent can be one or more of alkyl sulfate, soap, dodecylbenzenesulfonate, lactylate, sulfosuccinate, monoglyceride sulfonate, phosphate ester, silicone, taurinate.

[0024] The dosage administered will depend on a number of factors, such as the gender, age, body weight, and individual response of the patient or animal, the route of administration, and the number of doses given. The above dosages are exemplary of the average case. Some patients, and modern medical practice, will require smaller doses, and others, less severe, require larger doses. The dosages, per se, will depend on the severity of the condition being treated, the stage of the condition, and the patient's general health status and prior medical history. However, it is to be understood that the specific therapeutically effective dose level for any particular patient will depend upon the severity of the condition being treated, the stage of the condition, and the patient's general health status and prior medical history.

[0025] It will be recognized by one of ordinary skill in the art that the total daily usage of the compositions of the application will be decided by the attending physician within the scope of sound medical judgment. For any particular patient, the specific therapeutically effective dose level will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion; the duration of the treatment; drugs used in combination or coincidental with the treatment; and like factors well known in the medical arts. For example, it is to be understood that the use of doses that are less than the maximum human therapeutic dose can be resorted to in some cases, and that the dosage can be increased until the desired effect is achieved.

[0026] Generally, the effective component of the present application calculates the dosage of mammals, especially humans, which can be between 1-1000 mg / kg / day, for example, between 1-500 mg / kg / day, for example, between 50-500 mg / kg / day, or 100-500 mg / kg, or 150-500 mg / kg, or 200-500 mg / kg, or 250-500 mg / kg.

[0027] Compared with the prior art, the present application has the following beneficial effects: (1) The anti-inflammatory probiotic complex of the present application takes Lactobacillus rhamnosus LGD-0817, Lactobacillus rhamnosus LDL-1708 and Lactobacillus plantarum LEP-1208 as the basic dominant bacterial species, and is compounded with Lactobacillus casei, Lactobacillus delbrueckii, Paralactobacillus casei and other bacteria, which can cooperate and promote each other, and jointly play a significant role in improving ulcerative colitis; (2) The traditional technology is single dependent on live bacteria, and the natural anti-inflammatory substances such as prebiotics, licorice extract and astragalus extract in the traditional Chinese medicine fermentation composition obtained by the present application can synergistically reduce intestinal inflammation with probiotics; (3) Through animal experiments and clinical verification, the traditional Chinese medicine fermentation composition obtained by the present application can regulate intestinal flora, enhance intestinal barrier function, inhibit the release of pro-inflammatory factors, improve the shortening of colon length in colitis mice, inhibit the levels of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, and competitively inhibit pathogenic bacteria colonization, which shows good comprehensive intervention effect in DSS-induced mouse ulcerative colitis model and has clinical practical value. DETAILED DESCRIPTION

[0028] In order for those skilled in the art to better understand the technical solutions of the present application, the present application will be described in detail below in combination with specific embodiments. The experimental methods not specified in the following examples are generally carried out according to conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, the test materials used in the following examples are purchased from conventional biochemical reagent stores. Unless otherwise specified, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as understood by those skilled in the art. In addition, any method and material similar or equivalent to those described can be used in the present application. The preferred methods and materials described herein are only used as an example.

[0029] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0030] Gut microbiota imbalance is a core mechanism of inflammatory bowel diseases (such as ulcerative colitis and Crohn's disease). This invention selects functionally complementary strains—Lactobacillus rhamnosus LGD-0817, Lactobacillus rhamnosus LDL-1708, and Lactobacillus plantarum LEP-1208—as a base to enhance immunomodulation and anti-inflammatory functions. Further, it selects multiple genera of bacteria, including Lactobacillus casei LPM-0188, Lactobacillus delbrueckii subsp. bulgaricus RHM-1068, Lactobacillus paracasei LPA-2088, Lactobacillus fermentum LAF-1387, Lactobacillus plantarum MCA-3066, and Lactobacillus paracasei BMC-2020, to create a complex formula covering intestinal barrier enhancement, immune regulation, and metabolic support. Through synergistic effects, this formula effectively improves ulcerative colitis.

[0031] Based on this invention, *Lactobacillus rhamnosus* LGD-0817 functions to regulate the balance of intestinal flora, relieve diarrhea and constipation, and enhance the intestinal barrier; *Lactobacillus paracasei* BMC-2020 functions to regulate immunity, inhibit pathogen colonization, and improve allergic reactions; and *Lactobacillus plantarum* MCA-3066 functions to promote the fermentation of plant-based raw materials, produce short-chain fatty acids (such as acetic acid and propionic acid), and support metabolic health.

[0032] Based on this invention, the total live bacteria count of the anti-inflammatory compound probiotic is 10e. 3 -10e 12 CFU / mL, or 10e 6 -10e 12 CFU / mL, or 10e 6 -10e 9 CFU / mL, for example, 1×10e 3 CFU / mL (CFU / g), 2×10e 4 CFU / mL (CFU / g), 3×10e 5 CFU / mL (CFU / g), 4×10e 6 CFU / mL (CFU / g), 5×10e 7 CFU / mL (CFU / g), 6×10e 8 CFU / mL (CFU / g), 7×10e 9 CFU / mL (CFU / g), 8×10e 10CFU / mL (CFU / g), 9 x 10e 11 CFU / mL (CFU / g), 1 x 10e 12 CFU / mL (CFU / g), 1 x 10e

[0033] According to the present application, the mass ratio of the active bacteria group A to the active bacteria group B is (1-10):(1-10), or (1-8):(1-10), or (1-6):(1-8), or (1-4):(1-6), or (1-3):(1-6), for example, 1:1, 2:1, 4:1, 6:1, 8:1, 10:1, 1:2, 1:4, 1:8, 1:6, 1:10, and the like, and other specific point values within the numerical range can be selected, which will not be repeated here.

[0034] According to the present application, the active bacteria group A is composed of Lactobacillus rhamnosus LGD-0817, Lactobacillus rhamnosus LDL-1708 and Lactobacillus plantarum LEP-1208. In some specific embodiments, the Lactobacillus rhamnosus LGD-0817 in the active bacteria group A of the present application can be 1-10 parts by weight, for example, but not limited to, 1 part, 2 parts, 4 parts, 6 parts, 8 parts, 10 parts. In some specific embodiments, the Lactobacillus rhamnosus LDL-1708 in the active bacteria group A of the present application can be 1-10 parts by weight, for example, but not limited to, 1 part, 2 parts, 4 parts, 6 parts, 8 parts, 10 parts. In some specific embodiments, the Lactobacillus plantarum LEP-1208 in the active bacteria group A of the present application can be 1-10 parts by weight, for example, but not limited to, 1 part, 2 parts, 4 parts, 6 parts, 8 parts, 10 parts.

[0035] According to the present application, the Astragalus extract is obtained by extracting Astragalus with an extraction solvent; the extraction solvent is selected from one or more of water, methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, methyl formate, ethyl formate, methyl acetate, ethyl acetate, dichloromethane, chloroform, carbon tetrachloride, dichloroethane, acetone, butanone, pentanone; In some embodiments, the total amount of Astragalus saponins in the Astragalus extract can be 40%-90% by weight, for example, 45%-85%, or 50%-80%, or 60%-70%.

[0036] Based on the present application, the liquorice extract is extracted by an extraction solvent; the extraction solvent is selected from one or more of water, methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, methyl formate, ethyl formate, methyl acetate, ethyl acetate, dichloromethane, chloroform, carbon tetrachloride, dichloroethane, acetone, butanone, pentanone; In some embodiments, the total amount of glycyrrhizic acid in the liquorice extract can be 40%-90% by weight, for example 45%-85%, or 50%-80%, or 60%-70%.

[0037] Based on the present application, the traditional Chinese medicine extract is composed of the astragalus extract and the liquorice extract.

[0038] In some specific embodiments, the radix scrophulariae extract can also be added for compounding.

[0039] In some specific embodiments, the astragalus extract in the traditional Chinese medicine extract of the present application can be 1-10 parts by weight, for example, but not limited to, 1 part, 2 parts, 4 parts, 6 parts, 8 parts, 10 parts; In some specific embodiments, the liquorice extract in the traditional Chinese medicine extract of the present application can be 1-5 parts by weight, for example, but not limited to, 1 part, 2 parts, 3 parts, 4 parts, 5 parts; Based on the present application, the traditional Chinese medicine fermentation composition is fermented from the anti-inflammatory compound probiotic bacteria and the traditional Chinese medicine extract, wherein the traditional Chinese medicine extract is composed of 1-10 parts of the astragalus extract and 1-5 parts of the liquorice extract.

[0040] Based on the present application, a preparation method of the traditional Chinese medicine fermentation composition is provided, which comprises separately preparing a traditional Chinese medicine fermentation substrate and a compound probiotic bacteria, inoculating the prepared compound probiotic bacteria into the traditional Chinese medicine fermentation substrate, mixing, fermenting, and obtaining the traditional Chinese medicine fermentation composition.

[0041] In some specific embodiments, the germinated barley is crushed, water is added, and enzymatic hydrolysis is carried out at 55℃ for 2 hours, then the wort is obtained by boiling and filtering, and then the lonicera caerulea pulp and the traditional Chinese medicine extract are added, mixed, sterilized, and then the traditional Chinese medicine fermentation substrate is prepared; the compound probiotic bacteria is inoculated into the traditional Chinese medicine fermentation substrate, and anaerobic fermentation is carried out at 37℃ for 48 hours, and then the traditional Chinese medicine fermentation composition is prepared.

[0042] In some specific embodiments, the addition amount of the compound bacteria liquid in the above-mentioned substrate is 2-10% (v / w); for example, but not limited to, 2%, 4%, 6%, 8%, 10%.

[0043] The technical solutions of the present application are further described in detail below in combination with specific embodiments and the accompanying tables. It should be understood that the following embodiments are only used to explain the present application, and are not used to limit the present application.

[0044] Example 1 Compound probiotic bacteria liquid A composite probiotic, consisting of 1 part Lactobacillus rhamnosus LGD-0817, 1 part Lactobacillus rhamnosus LDL-1708, 1 part Lactobacillus plantarum LEP-1208, 1 part Lactobacillus casei LPM-0188, 1 part Lactobacillus delbrueckii subsp. bulgaricus RHM-1068, 1 part Lactobacillus paracasei LPA-2088, 1 part Lactobacillus fermentum LAF-1387, 1 part Lactobacillus plantarum MCA-3066, 1 part Lactobacillus paracasei BMC-2020, by mass parts.

[0045] Example 2 Composite probiotic A composite probiotic, consisting of 1 part Lactobacillus rhamnosus LGD-0817, 1 part Lactobacillus rhamnosus LDL-1708, 1 part Lactobacillus plantarum LEP-1208, 1 part Lactobacillus casei LPM-0188, 1 part Lactobacillus delbrueckii subsp. bulgaricus RHM-1068, 1 part Lactobacillus paracasei LPA-2088, 1 part Lactobacillus fermentum LAF-1387, 1 part Lactobacillus plantarum MCA-3066, 1 part Lactobacillus paracasei BMC-2020, by mass parts.

[0046] Example 3 Composite probiotic liquid A composite probiotic, consisting of 3 parts Lactobacillus rhamnosus LGD-0817, 2 parts Lactobacillus rhamnosus LDL-1708, and 4 parts Lactobacillus plantarum LEP-1208, by mass parts.

[0047] Example 4 A composite probiotic, consisting of 1 part Lactobacillus rhamnosus LGD-0817, 1 part Lactobacillus rhamnosus LDL-1708, 1 part Lactobacillus plantarum LEP-1208, 1 part Lactobacillus casei LPM-0188, by mass parts. Example 5 A composite probiotic, consisting of 1 part Lactobacillus rhamnosus LGD-0817, 1 part Lactobacillus rhamnosus LDL-1708, 1 part Lactobacillus plantarum LEP-1208, 1 part Lactobacillus delbrueckii subsp. bulgaricus RHM-1068, 1 part Lactobacillus paracasei LPA-2088, by mass parts. Example 6 A composite probiotic, consisting of 8 parts Lactobacillus rhamnosus LGD-0817, 8 parts Lactobacillus rhamnosus LDL-1708, 8 parts Lactobacillus plantarum LEP-1208, 1 part Lactobacillus fermentum LAF-1387, by mass parts.

[0048] Example 7 Chinese medicine fermentation composition Take 200 g of malted barley powder, add 1 L of water, and enzymatically hydrolyze (cellulase: pectinase = 1:1) at 55°C for 2 h. Boil and filter to obtain wort. Add 100 g of Lonicera caerulea pulp and 50 mL of traditional Chinese medicine extract (10% Radix Astragali + 5% liquorice water extract) to the wort, mix well, sterilize at 115°C for 15 min, and obtain a traditional Chinese medicine fermentation substrate. Inoculate the bacteria liquid of Example 1 into the traditional Chinese medicine fermentation substrate at an inoculation amount of 2-3%, and anaerobically ferment at 37°C for 48 h. After fermentation, a liquid probiotic preparation (final viable bacterial count ≥ 5 × 10 8 CFU / mL) is obtained.

[0049] Example 8 Traditional Chinese medicine fermentation composition The preparation method is the same as that of Example 7, except that the bacteria liquid of Example 2 is inoculated into the traditional Chinese medicine fermentation substrate.

[0050] Comparative Example 1 The preparation method is the same as that of Example 7, except that after fermentation, the bacteria are collected by centrifugation (8000 rpm, 10 min), resuspended with physiological saline to the original volume, and the viable bacterial count is adjusted to ≥ 5 × 10 8 CFU / mL.

[0051] Comparative Example 2 The preparation method is the same as that of Example 7, except that after fermentation, the bacteria are collected by centrifugation (8000 rpm, 10 min), and the supernatant is taken and the bacteria are discarded.

[0052] Comparative Example 3 The preparation method is the same as that of Example 7, except that after fermentation, the bacteria are freeze-dried to obtain a freeze-dried powder, and before use, the freeze-dried powder is resuspended with physiological saline to a viable bacterial count of ≥ 5 × 10 8 CFU / mL.

[0053] The examples and comparative examples of the present application are subjected to efficacy verification.

[0054] Test animals: SPF C57BL / 6J mice (23-26 g), male, 6 weeks old.

[0055] Animal grouping and administration: After adaptive feeding for one week (temperature (23±2℃, humidity 50±10%, day and night alternation 12 h), the mice were randomly divided into healthy blank group (CON group), model group (DSS group), Example 1, Example 2, Example 7, Example 8, Comparative Example 1, Comparative Example 2, Comparative Example 3, each group of 6. Except for the healthy blank group, the rest of the groups of mice freely drank 2% DSS-containing sterilized water for 7 days, to construct a mouse ulcerative colitis model. After the DSS modeling was completed, the Example and Comparative Example groups were given the test substance by gavage (dose: 0.3 mL / each / day), and the CON / DSS group: the same volume of normal saline. All data were expressed as mean ± standard deviation (Mean ± SD), and single factor analysis of variance (ANOVA) was performed using SPSS 27 software. When P<0.05, the difference was considered statistically significant.

[0056] (1) Investigation of the effect of administration on the colon length of ulcerative colitis mice After the experiment, the mice in each group were dissected, and the colon samples were taken, and the colon lengths of each group were counted. The colon length can reflect the severity of inflammation in mice, and the results are shown in Table 1.

[0057] Table 1

[0058] Note: * indicates P<0.05 compared with the CON group; ** indicates P<0.05 compared with the DSS group; *** indicates P<0.01 compared with the DSS group.

[0059] From the above table, compared with the CON group of mice, the colon of the DSS group of mice was significantly shortened. Compared with the DSS group, the colon length of the mice in the Example group increased, indicating that the combination of probiotics and traditional Chinese medicine fermentation products can effectively improve the colon shortening of the ulcerative colitis mouse model, and the colon length of the mice in Example 7-8 group is close to the blank group, showing that the traditional Chinese medicine fermentation product has the best repair effect on intestinal damage, indicating that the probiotics and traditional Chinese medicine active ingredients in the traditional Chinese medicine fermentation composition synergistically act, further improving the anti-inflammatory effect.

[0060] (2) Investigation of the effect of administration on inflammatory factors in ulcerative colitis mice The mouse colon samples were taken, and physiological saline was used at a ratio of 1:9 (w / v) to perform ultrasonic crushing. The ultrasonically crushed samples were centrifuged at 10000 r / min at 4℃ for 10 min, and the supernatant was collected. The total protein content of the supernatant was detected according to the BCA protein concentration kit instructions, as a background correction of the inflammatory factor content. In addition, according to the corresponding ELISA kit operation instructions, the contents of inflammatory cytokines TNF-α, IL-1β and IL-10 in the supernatant were determined, and the results are shown in Table 2.

[0061] Table 2

[0062] Note: * means P<0.05 compared with CON group; ** means P<0.05 compared with DSS group; *** means P<0.01 compared with DSS group.

[0063] From the above table, it can be seen that the levels of TNF-a, IL-6 and IL-1β in the colon tissues of the mice in the DSS group were significantly increased compared with the CON group (P<0.05), indicating that the mice in the DSS group had obvious inflammatory reactions; compared with the DSS group, the levels of TNF-a, IL-6 and IL-1β in the colon tissues of the mice in the example groups were significantly reduced, and the inhibitory effects of the traditional Chinese medicine ferment of examples 7-8 on the inflammatory cytokines of the mice were more obvious, and the effects thereof were better than those of examples 1-2.

[0064] (3) Investigation of the effect of administration on the intestinal colonization of the mice with ulcerative colitis After the experiment, the cecal contents of the mice in each group were taken under sterile conditions for colonization experiments, specifically: Sample processing: 100 mg of the contents were added to 900 μL of PBS buffer, vortexed and gradiently diluted; Selective culture: probiotic bacteria quantification: MRS + 0.3% oxgallate medium, 37°C anaerobic culture for 48 h; pathogenic bacteria quantification: MacConkey medium, 37°C aerobic culture for 24 h; Specific identification: randomly picked colonies were subjected to strain-specific PCR verification; Colonization rate = (target probiotic CFU / total bacterial CFU) x 100%; pathogenic bacteria inhibition rate = (control group CFU-experimental group CFU) / control group CFU x 100%. The results are shown in Table 3.

[0065] Table 3

[0066] Note: * means P<0.05 compared with example 5; ** means P<0.01 compared with the DSS group.

[0067] As can be seen in the above table, the liquid traditional Chinese medicine fermentation composition of Example 7 is more conducive to the colonization of exogenous probiotics, has the highest colonization rate and the shortest time to reach stable colonization, can effectively colonize and proliferate in the intestinal tract in vivo, and has long-term effectiveness; and the colon length recovery rate of the mice in the Example 7 group (up 37.5%) is significantly higher than the colon length recovery rate of the mice in the Comparative Example 3 group (up 23.4%), and the colonization of pathogenic bacteria is inhibited competitively, proving that the live bacteria metabolites can effectively enhance intestinal repair, and the anti-inflammatory active substances (such as bacteriocins and polysaccharides) released by the bacteria liquid fermentation and the traditional Chinese medicine components (astragalus saponins and glycyrrhizic acid) synergistically act to improve ulcerative colitis; compared with the DSS group, the colon tissue TNF-α of the mice in the Comparative Example 2 group is reduced by 51.1%, indicating that the fermentation supernatant still has an anti-inflammatory effect, and the live bacteria metabolites are the core synergistic components.

[0068] Finally, it should be noted that the above description is only a preferred embodiment of the present application, and those skilled in the art can make various similar representations under the inspiration of the present application without departing from the purpose and scope of the claims. Such changes fall within the scope of the present application.

Claims

1. An anti-inflammatory probiotic complex, characterized in that, consists of (1-10): (1-10) active bacteria group A and active bacteria group B by weight, the active bacteria group A consists of 1-10 parts of Lactobacillus rhamnosus LGD-0817, 1-10 parts of Lactobacillus rhamnosus LDL-1708 and 1-10 parts of Lactobacillus plantarum LEP-1208; The active bacteria group B is selected from one or more of Lactobacillus casei LPM-0188, Lactobacillus delbrueckii subsp. bulgaricus RHM-1068, Lactobacillus paracasei LPA-2088, Lactobacillus fermentum LAF-1387, Lactobacillus plantarum MCA-3066 and Lactobacillus paracasei BMC-2020; Lactobacillus rhamnosus Lactobacillus rhamnosus LGD-0817, classified as Lactobacillus rhamnosus Lactobacillus rhamnosus , and having a deposit accession number of CGMCC No. 23632; Lactobacillus rhamnosus LDL-1708, classified as Lactobacillus rhamnosus Lactobacillus plantarum , with the accession number CGMCC No. 23960; Lactobacillus plantarum LEP-1208, classified as Lactobacillus plantarum, with a preservation number of CGMCC No. 24012 Lactobacillus casei ; Lactobacillus casei LPM-0188, classified as Lactobacillus casei Lactobacillus delbrueckii subsp. bulgaricus , with the accession number CGMCC No. 23959; Lactobacillus delbrueckii subsp. bulgaricus RHM-1068, classified as Lactobacillus delbrueckii subsp. bulgaricus Lactobacillus paracasei , with the accession number CGMCC No. 24013; Lactobacillus paracasei LPA-2088, classified as Lactobacillus paracasei Lactobacillus fermentum , and having the accession number CGMCC No. 24057; Lactobacillus fermentum LAF-1387, classified as Lactobacillus fermentum, with a preservation number of CGMCC No. 24056 Lactobacillus plantarum ; Lactobacillus plantarum MCA-3066, taxonomically named Lactobacillus plantarum, with a deposit accession number of CGMCC No. 24489; Lactobacillus paracasei Lactobacillus plantarum MCA-3066, taxonomically named Lactobacillus plantarum, with a deposit accession number of Lactobacillus paracasei BMC-2020, classified as Lactobacillus paracasei 4. Use of the anti-inflammatory complex probiotic bacteria according to any one of claims 1-3 in the preparation of a medicament for preventing, ameliorating or treating colitis. , and has a preservation number of CGMCC No. 24488.

2. The anti-inflammatory probiotic complex of claim 1, wherein, The total viable count of the anti-inflammatory probiotic complex is 10e 3 -10e 12 CFU / mL.

3. The anti-inflammatory probiotic complex of claim 1, wherein, The total viable count of the anti-inflammatory probiotic complex is 10e 6 -10e 9 CFU / mL. The medicament is prepared by fermenting the anti-inflammatory complex probiotic bacteria with traditional Chinese medicine extracts, and the traditional Chinese medicine extracts include Astragalus extract and licorice extract.

5. Use according to claim 4, characterized in that, The traditional Chinese medicine extracts consist of 1-10 parts of Astragalus extract and 1-5 parts of licorice extract by mass fraction.

6. Use according to claim 5, characterized in that, The anti-inflammatory complex probiotic bacteria according to any one of claims 1-3 are fermented with traditional Chinese medicine extracts, and the traditional Chinese medicine extracts consist of 1-10 parts of Astragalus extract and 1-5 parts of licorice extract.

7. A traditional Chinese medicine fermentation composition, characterized in that, 8. Use of the traditional Chinese medicine fermentation composition according to claim 7 in the preparation of a medicament for preventing, ameliorating or treating colitis. The medicament takes the traditional Chinese medicine fermentation composition as the active ingredient and is prepared into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.

9. Use according to claim 8, characterized in that, The dosage form is a solution.

10. Use according to claim 9, characterized in that: ​

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