Composition containing probiotics and application thereof in regulating intestinal flora

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a composition containing probiotics and application of the composition in regulating intestinal flora. The active polypeptide disclosed by the invention can be directly absorbed and utilized by small intestines without being digested by a human body, is high in absorption speed and high in bioavailability, and can effectively promote growth and reproduction of bifidobacterium and also promote generation of acetic acid. When the active polypeptide is independently used or combined with probiotics, the number recovery of bifidobacteria in intestinal tracts can be promoted, the growth and reproduction of enterobacter can be inhibited, and the flora balance of the intestinal tracts can be maintained.

Description

Technical Field

[0001] The present invention belongs to the field of biological medicine technology, and particularly relates to a composition containing probiotics and its application in regulating intestinal flora. Background Art

[0002] The characteristics of intestinal flora mainly include high flora density, wide diversity and complex interactions, etc. It plays an important role in the growth, development, health and physiological metabolism of the host, and is regarded as an important part of the host, known as the "forgotten organ". Research has found that in the intestines of higher mammals, Firmicutes and Bacteroidetes are the dominant flora, and Actinobacteria and Proteobacteria are the sub-dominant flora. Normal intestinal flora has various physiological regulatory functions on the host, such as antagonism, immunity, detoxification and anti-aging. The antagonistic effect is mainly manifested in that normal flora can adhere, colonize and reproduce at a specific position of the host to form a bacterial film barrier to inhibit and exclude the invasion and enrichment of passing flora, thus maintaining the balance between the host and microorganisms; the immune effect is that the body can produce immune and clearance functions under the stimulation of normal flora; the detoxification effect is reflected in that Bifidobacterium can reduce the excessive Gram-negative bacilli in the intestine to the normal level, thereby reducing the absorption of endotoxin; the anti-aging effect is mainly manifested in that some normal flora of the body can synthesize various vitamins necessary for human growth and development, such as B vitamins, vitamin K, nicotinic acid and pantothenic acid, and can also synthesize non-essential amino acids such as aspartic acid, alanine, valine and threonine from protein residues, and at the same time can participate in the metabolism of carbohydrates and proteins, and promote the absorption of mineral elements such as iron, magnesium and zinc. Under normal circumstances, there is a dynamic balance relationship among intestinal flora, maintaining the normal physiological functions of the host such as nutrition, immunity and digestion. Once this balance relationship is broken and intestinal flora dysregulation occurs, the host will develop corresponding diseases. For example, the abuse of antibiotics causes intestinal microbial dysregulation, thus increasing the probability of intestinal diseases.

[0003] Small molecule peptides, polypeptides and other substances produced by the enzymatic hydrolysis of proteins can promote the growth of intestinal probiotics and maintain the balance of the intestinal flora. As early as the beginning of the 21st century, Comelia Liepke et al. used pepsin to hydrolyze breast milk, and after obtaining the hydrolysis products, three bioactive peptide fragments that can promote the proliferation of Bifidobacterium were isolated by chromatography (Comelia Liepke et al 2002). In recent years, Xiao et al. used alkaline protease to hydrolyze faba bean protein, and a tripeptide that can promote the proliferation of Lactobacillus bulgaricus was isolated from its hydrolysis products, which can significantly increase the viable count of Lactobacillus bulgaricus (Xiao et al 2015). Usually, probiotics cannot grow and reproduce rapidly due to the lack of nutrients required for their growth (Ghyselinck et al 2021). At the same time, probiotics can play a probiotic role in the body only when the viable count reaches a certain order of magnitude. Therefore, choosing the correct and efficient way to increase the viable count of probiotics is one of the focuses of current probiotic research. Summary of the Invention

[0004] In view of the situation of the prior art, the purpose of the present invention is to provide an active polypeptide LG57, its composition with probiotics, and its application in regulating the intestinal flora. The active polypeptide of the present invention can be directly absorbed and utilized by the small intestine without being digested by the human body, has a fast absorption rate and high bioavailability, can effectively promote the growth and reproduction of Bifidobacterium, and at the same time promote the production of acetic acid. The active polypeptide of the present invention can be used alone or in combination with probiotics to promote the recovery of the number of Bifidobacterium in the intestine, inhibit the growth and reproduction of Enterobacter, and maintain the balance of the intestinal flora.

[0005] The present invention first provides an active polypeptide LG57, characterized in that the amino acid sequence of the polypeptide is DEKINPTFGESTASMNA, as shown in SEQ ID NO:1.

[0006] Another aspect of the present invention also provides the use of the polypeptide for preparing a drug for regulating the intestinal flora.

[0007] Another aspect of the present invention provides a pharmaceutical composition, which comprises a probiotic and an active polypeptide LG57 as active ingredients, and the amino acid sequence of the polypeptide is as shown in SEQ ID NO:1.

[0008] In some embodiments, the probiotic is Bifidobacterium or Lactobacillus.

[0009] In some embodiments, the probiotic is Bifidobacterium.

[0010] In some embodiments, the Bifidobacterium is ATCC 29521.

[0011] The polypeptide of the present invention or a pharmaceutical composition comprising the polypeptide is administered orally.

[0012] In some embodiments, the oral dosage form includes solutions, emulsions, powders, capsules, tablets, pills, granules, and lyophilized powders.

[0013] Another aspect of the present invention also provides the use of the polypeptide or the pharmaceutical composition in the preparation of a drug for regulating the intestinal flora.

[0014] In some embodiments, the regulation of the intestinal flora can be manifested as promoting the growth and reproduction of Bifidobacterium in the intestine and / or inhibiting the growth and reproduction of Enterobacter. Description of the Drawings

[0015] Figure 1 Shown is the effect of the bioactive polypeptide LG57 on the OD600 value of the Bifidobacterium culture solution.

[0016] Figure 2 Shown is the effect of the bioactive polypeptide LG57 on the pH value of the Bifidobacterium culture solution.

[0017] Figure 3 Shown is the effect of the bioactive polypeptide LG57 on the viable count of the Bifidobacterium culture solution.

[0018] Figure 4 Shown is the effect of the bioactive polypeptide LG57 on the acetic acid production in the Bifidobacterium culture solution. Detailed Description of the Invention

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0020] Except in the operating examples or as otherwise indicated, all numbers expressing amounts of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about". When used in conjunction with percentages, the term "about" can mean ±1%.

[0021] The term "probiotic" refers to live microorganisms that confer health benefits on the host when ingested in appropriate amounts. Generally, probiotics are mainly part of the gut microbiota such as lactic acid bacteria and bifidobacteria. There is a wide variety of probiotics. In the early days, the term "probiotic" was limited to lactic acid bacteria. With the identification of new bacteria and the increasing research on probiotic species, probiotics also include some yeasts. Among them, lactic acid bacteria include 17 genera and 273 species: Enterococcus, Lactobacillus, Bacillus, Clostridium, Lactococcus lactis, Leuconostoc, Pediococcus, Carnobacterium, Vagococcus, Tetragenococcus, Bifidobacterium, Atopobium, Weissella, Abiotrophia, Granulicatella, Oenococcus, Paralactobacillus. In addition, it also includes some Saccharomyces such as Saccharomyces boulardii.

[0022] The pharmaceutical composition for regulating gut microbiota of the present invention may contain probiotics. In some embodiments, the probiotic is bifidobacterium or lactic acid bacteria. In some embodiments, the probiotic is bifidobacterium. The bifidobacterium ATCC 29521 strain of the present invention can be recovered through processes such as centrifugation after cultivation and can be used in the form of an oral preparation. In some embodiments, the recovered bifidobacterium ATCC 29521 strain can be prepared into the form of a viable bacteria agent by drying, for example, by freeze-drying. Relative to the total weight of the composition, the pharmaceutical composition of the present invention may contain 10 8 CFU to 10 12 CFU of bifidobacterium ATCC 29521 or a culture with an equivalent number of viable bacteria as an active ingredient.

[0023] The term "regulating the intestinal flora" refers to restoring or maintaining the diversity and functional stability of the microbial community in the intestine through intervention means (such as drugs, diet, etc.), so that beneficial bacteria, neutral bacteria and harmful bacteria are in a dynamic balance. This balance is crucial for digestion and absorption, immune regulation, metabolic function and the health of the nervous system. When the flora is unbalanced, it may lead to various health problems such as diarrhea, constipation, inflammatory bowel disease, obesity, etc. The promotion of digestion and absorption by the balance of intestinal flora means that beneficial bacteria (such as Bifidobacterium, Lactobacillus) can decompose dietary fiber to produce short-chain fatty acids, which can supply energy for intestinal cells and help digest substances such as lactose. The enhancement of immune function by the balance of intestinal flora means that the intestinal flora can help resist pathogens and reduce the risk of autoimmune diseases by stimulating the development of the immune system and regulating immune responses. The influence of the balance of intestinal flora on metabolic regulation refers to affecting the metabolism and storage of nutrients, participating in hormone synthesis, reducing cholesterol and blood sugar levels, and reducing the risk of obesity and diabetes. The influence of the balance of intestinal flora on nerve and mental health means that the intestinal flora affects neurotransmitters (such as serotonin) through the gut-brain axis, improves mood and cognitive function, and may relieve mental diseases such as anxiety and depression. The anti-pathogenic and anti-inflammatory effects of the balance of intestinal flora refer to inhibiting the proliferation of pathogenic bacteria (such as Staphylococcus aureus, Salmonella), and relieving chronic inflammation such as inflammatory bowel disease.

[0024] The term "oral preparation" refers to a drug dosage form administered through the oral route (ingested through the digestive tract), and its active ingredient needs to be absorbed through the gastrointestinal tract and enter the bloodstream, or directly act locally (such as in the intestine). Such dosage forms need to meet requirements such as safety, stability, palatability, etc., and ensure the effective release and absorption of the drug in the body. "Oral preparations" can be divided into solid preparations, liquid preparations, and special function dosage forms. Among them, solid preparations include tablets, capsules, granules / powders; liquid preparations include oral solutions / syrups, suspensions, emulsions; special function dosage forms include orally disintegrating tablets, microecological preparations. Microecological preparations refer to tablets, capsules or granules containing probiotics, which need to be stored at low temperature to maintain their activity.

[0025] In some examples of the present invention, the pharmaceutical composition containing the above-mentioned Bifidobacterium ATCC 29521 strain and the active polypeptide LG57 can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc., external preparations, suppositories or sterilized injection solutions according to the usual methods, but not limited thereto.

[0026] The pharmaceutical composition of the present invention can be formulated into an enteral or oral pharmaceutical product. Moreover, the pharmaceutical composition of the present invention can be formulated into enteric packaging by a known method to rapidly release the microorganism as an active substance in the intestine after passing through the gastrointestinal tract and reaching the small intestine.

[0027] In some examples of the present invention, the liquid preparations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used as simple diluents, excipients can also be included, such as wetting agents, sweeteners, fragrances, preservatives, etc., but are not limited thereto.

[0028] In the present invention, the forms of the medicament include but are not limited to tablets, powders, capsules, granules or coated agents. The forms of food or health products include but are not limited to tablets, powders, capsules, granules, coated agents, beverages, pastries, biscuits, candies, chocolates or jellies. Preferably, the food containing the probiotic preparation containing bioactive peptides is made into various forms of snacks.

[0029] The present invention has no special limitation on how to make the probiotic preparation containing bioactive peptides into corresponding foods, medicaments, health products or fermenting agents, and the preparation methods known in the art can be adopted. The present invention has no special limitation on the excipients in the food, medicament, health product or fermenting agent, and the food excipients, drug excipients and health product excipients well-known in the art can be adopted.

[0030] In other embodiments, the pharmaceutical composition for regulating intestinal flora of the present invention may further include other probiotic drugs, among which the bifidobacterium group includes Golden Bifid (Bifidobacterium + Lactobacillus), Bifidobacterium triple / quadruple viable bacteria tablets; the bacillus subtilis group includes Chang'an (Bacillus subtilis + Enterococcus faecalis); the bacillus licheniformis group includes Zhengchangsheng; the saccharomyces boulardii group; the clostridium butyricum viable bacteria; it can also be used in combination with other drugs for relieving diarrhea caused by dysbacteriosis, such as montmorillonite powder.

[0031] When the pharmaceutical composition of the present invention is administered in combination with other therapeutic agents, it can be administered sequentially or simultaneously, and can be administered once or multiple times.

[0032] In addition to the above active ingredients, the pharmaceutical composition of the present invention may further contain pharmaceutically acceptable carriers and / or excipients. In addition, it can also be formulated together with various additives commonly used in pharmacy, such as binders, disintegrants, coating agents, lubricants, etc.

[0033] The acceptable excipients in the present invention include sugars such as sucrose, lactose, mannitol, glucose, etc. and starches such as corn starch, potato starch, rice starch, partially pregelatinized starch, etc. The binders include polysaccharides such as dextran, sodium alginate, carrageenan, guar gum, gum arabic, agar, etc., natural macromolecular substances such as tragacanth gum, gelatin, gluten, etc., cellulose derivatives such as hydroxypropyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, hydroxypropyl ethyl cellulose, sodium carboxymethyl cellulose, etc. and polymers such as polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl acetate, polyethylene glycol, polyacrylic acid, polymethacrylic acid and vinyl acetate resin.

[0034] Acceptable disintegrants in the present invention may include cellulose derivatives such as carboxymethyl cellulose, calcium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose, and starches such as sodium carboxymethyl starch, hydroxypropyl starch, corn starch, potato starch, rice starch, and partially pre-gelatinized starch.

[0035] Examples of acceptable lubricants in the present invention include talc, stearic acid, calcium stearate, magnesium stearate, colloidal silica, hydrated silica, various waxes, and hydrogenated oils.

[0036] Coating agents include non-water-soluble polymers such as dimethylaminoethyl methacrylate-methacrylic acid copolymer, polyvinyl acetal diethylaminoacetate, ethyl acrylate-methacrylic acid copolymer, ethyl acrylate-methyl methacrylate-ethyl methacrylate trimethylammonium chloride copolymer, ethyl cellulose, enteric polymers such as methacrylic acid-ethyl acrylate copolymer, hydroxypropyl cellulose phthalate, hypromellose acetate succinate, and water-soluble polymers such as methyl cellulose, hydroxypropyl methyl cellulose, polyvinyl pyrrolidone, and polyethylene glycol, but are not limited thereto.

[0037] In the pharmaceutical composition for regulating intestinal flora of the present invention, the dosage of the above-mentioned strain as an active ingredient can be determined according to factors including the type of various diseases, the age, weight, gender of the patient, the medical condition of the patient, the severity of the condition, the sensitivity to the drug, the administration time, the administration route and the metabolic ratio, the treatment period, the drugs used simultaneously, and other factors well-known in the medical field. Therefore, although the dosage and treatment method can vary within a wide range, it is important to administer the drug in an amount that can obtain the maximum effect with the minimum amount without side effects after considering all the above factors, which can be easily determined by relevant practitioners using standard methods.

[0038] As used herein, "promote" or "increase" or "promoting" or "increasing" may be used interchangeably herein. These terms refer to an increase in a measured parameter of a treated cell, tissue, or individual compared to an untreated cell, tissue, or individual. The same cell or tissue or individual can also be compared before and after treatment. In some embodiments, the increase in the treated cell, or tissue, or individual compared to the untreated cell, or tissue, or individual is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold or more.

[0039] As used herein, "treatment" or "treating" may be used interchangeably herein. These terms refer to a course of action for obtaining a beneficial or desired result, including but not limited to a therapeutic benefit and / or a prophylactic benefit.

[0040] In this application, unless specifically stated otherwise, the use of the singular also includes the plural. In this application, unless otherwise stated, the use of "or" means "and / or". Additionally, the use of the term "including" and other forms such as "includes" and "included" is not restrictive. Further, unless specifically stated otherwise, terms such as "element" or "component" encompass both elements and components that include one unit and elements and components that include more than one subunit. Additionally, the use of the term "portion" may include a part of a portion or the whole portion. Throughout this specification, unless the context requires otherwise, the word "comprise" or variants such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0041] The pharmaceutical composition of the present invention is formulated to have a pH of 5.5 to 7.5. In one embodiment, the pH of the aqueous medium can be adjusted by a low concentration of a suitable biocompatible buffering component, non-limiting examples of which are tromethamine, sodium carbonate and sodium bicarbonate, and sodium dihydrogen phosphate and disodium hydrogen phosphate.

[0042] The compositions of the present invention can be administered daily or intermittently, and the frequency of administration can be once a day or 2 to 3 times a day. If each of the two active ingredients is a single formulation, their frequencies of administration can be the same as or different from each other. In addition, the compositions of the present invention can be used alone or in combination with other drugs that regulate the intestinal flora. Considering all of the above factors, it is important to administer at the lowest dose without side effects to achieve the best therapeutic effect, which can be easily determined by those skilled in the art. In some embodiments, the dosing regimen is repeated, for example, once, twice, three times or more; for example, it is repeated during the remaining life cycle of an individual in need.

[0043] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited in any way. Any transformation or improvement made based on the teachings of the present invention falls within the protection scope of the present invention.

[0044] Example 1 Preparation of Active Polypeptide

[0045] The yam was homogenized and centrifuged to obtain the supernatant. 2 mol / L HCl was added to adjust the pH to 3.5, and then centrifuged at 4000 rpm for 10 min. After discarding the supernatant, it was redissolved with distilled water. 2 mol / L NaOH was added to adjust the pH to 7.0, and the supernatant was washed 3 times with distilled water to remove salts. The crude extract of yam protein was obtained after freeze-drying. After ultrasonically treating the crude extract of yam protein for 15 min, trypsin and papain were added for enzymatic hydrolysis for 2 h. After terminating the enzymatic hydrolysis, it was centrifuged at 8000 rpm for 10 min. The supernatant was the enzymatic hydrolysate of yam protein, and the refined extract of yam protein was obtained after freeze-drying. After LC-MS / MS analysis of the refined extract of yam protein, a series of polypeptide sequences were obtained. After chemical synthesis, the antibacterial activity was measured, and finally the active polypeptide LG57 was screened out, and its amino acid sequence is shown in SEQ ID NO: 1.

[0046] The active polypeptide LG57 was synthesized by Novoprotein Scientific Inc. After mass spectrometry and high performance liquid analysis, the correctness of the polypeptide compound was ensured. The polypeptide compound was a white powder with a purity of ≥95%. The lyophilized powder was dissolved in physiological saline and filtered through a sterile filter for later use.

[0047] Example 2. Effect of LG57 on probiotic flora

[0048] Under sterile and anaerobic operating conditions, the bacterial liquid of Bifidobacterium (ATCC 29521) stored in the liquid nitrogen tank was taken and inoculated into MRS liquid medium at an inoculation amount of 1‰ for continuous cultivation. After 24 h, it was inoculated into MRS liquid medium at an inoculation amount of 1‰ for continuous cultivation again, and the third-generation bacterial liquid was taken for later use. The activated strain was inoculated into 10 mL of MRS liquid medium at an inoculation amount of 10 μL, and different concentrations of active polypeptide LG57 (0 mg / mL, 2.5 mg / mL, 5 mg / mL) were added to the medium. After static cultivation at 37°C for 24 h, the OD value and pH value of the culture solution at 600 nm were measured, and viable cell counting was carried out to determine the proliferation effect of different concentrations of active polypeptide LG57 on Bifidobacterium. The formula of the MRS liquid medium used in the experiment was as follows: yeast powder 10.0 g / L, glucose 25.0 g / L, beef extract powder 10.0 g / L, ammonium citrate 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, sodium acetate 5.0 g / L, Tween-80 1.08 g / L, magnesium sulfate (MgSO4·7H2O) 0.2 g / L, manganese sulfate (MnSO4·4H2O) 0.05 g / L, made up to 1000 mL with distilled water, pH 5.7 - 5.9.

[0049] The results of the change in OD600 value are as Figure 1 shown. After culturing for 24 h, the OD600 values in the Bifidobacterium culture solutions added with active polypeptide LG57 were all significantly higher than those in the control group, indicating that Bifidobacterium could grow well in the culture solution added with active polypeptide LG57.

[0050] The results of pH value changes are as Figure 2 shown. As the addition amount of bioactive polypeptide LG57 increases, the pH value of the Bifidobacterium culture solution shows a downward trend.

[0051] The results of the viable count changes are as Figure 3 shown. In the Bifidobacterium culture solution, as the addition amount of bioactive polypeptide LG57 increases, the viable count of Bifidobacterium shows an upward trend.

[0052] Example 3. LG57 promotes Bifidobacterium to produce acetic acid

[0053] Under sterile and anaerobic operating conditions, take the Bifidobacterium (ATCC 29521) bacterial liquid stored in a liquid nitrogen tank, inoculate it into MRS liquid medium at an inoculation amount of 1%, and continue to culture. After 24 h, inoculate it into MRS liquid medium at an inoculation amount of 1% again and continue to culture. Take the third-generation bacterial liquid for standby. After the strain activation is completed, under sterile conditions, inoculate it into the fermentation culture solution at an inoculation amount of 2%, and add 5 mg / mL bioactive polypeptide LG57 to the culture solution. After anaerobic culture for 0 h, 6 h, 12 h, 24 h, and 48 h, take the culture solution and place it in a sterilized polyethylene tube for storage and analysis. Take 1 mL of the culture solution, centrifuge it at 10000 r / min at 4 °C for 15 min. Take the supernatant, filter it with a 0.22 μm aqueous phase filter membrane, and then perform gas chromatography analysis. Detect the standard product and the sample under the same chromatographic conditions, and determine the corresponding acetic acid content in the sample according to the retention time and peak area. The gas chromatography analysis conditions are as follows: The chromatographic column is a DB-FFAP chromatographic column; the detector is FID, and the carrier gas is N2; the flow rate of N2 is 30.0 mL / min, and the split ratio is 1:5. The detector temperature is 240 °C, and the injection port temperature is 240 °C; the temperature programming is 70 °C (1 min)-240 °C (5 °C / min); the flow rate of air is 400 mL / min, and the flow rate of H2 is 30 mL / min; the sample injection volume is 1 μL, and the measurement time for each determination is 43 min.

[0054] The results are as Figure 4 shown. The acetic acid content in the Bifidobacterium culture solution shows an upward trend with the increase of the culture time, and tends to be stable after reaching the maximum value. The acetic acid concentration reaches the peak value of 5.035 mg / mL at 24 h.

[0055] Example 4. The effect of LG57 on the microorganisms in the cecum of mice

[0056] Fifteen Kunming mice aged 4 - 6 weeks were randomly divided into 3 groups, with 5 mice in each group, namely the control group gavaged with normal saline, the low - dose group of active polypeptide LG57 gavaged, and the high - dose group of active polypeptide LG57 gavaged. Among them, the dose of normal saline was 10 mg / kg, the low - dose of active polypeptide LG57 was 0.5 g / kg, and the high - dose of active polypeptide LG57 was 1 g / kg. All experimental mice were administered once a day at a fixed time, 0.3 mL each time, and gavaged continuously for 4 weeks. The mice were sacrificed by cervical dislocation, the cecal part was aseptically removed, and the surface blood was aspirated with a sterilized filter paper. The cecal contents were collected and placed in a sterile centrifuge tube. Total DNA was extracted using a fecal genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd.). PCR amplification was performed using Bifidobacterium primers SEQ ID Nos: 2 - 3 to analyze the number of Bifidobacterium in the cecal contents of mice.

[0057] The results are shown in Table 1. After treatment with active polypeptide LG57, the number of Bifidobacterium in the cecum of mice increased, and showed a dose - dependence, indicating that active polypeptide LG57 can promote the proliferation of Bifidobacterium and improve the intestinal flora of mice.

[0058] Table 1

[0059]

[0060] Example 5. Effects of LG57 and probiotic composition on mice with intestinal flora disorder

[0061] Preparation of LG57 and probiotic composition: Under sterile and anaerobic operating conditions, take the Bifidobacterium (ATCC 29521) bacterial liquid stored in a liquid nitrogen tank, inoculate it into MRS liquid medium at an inoculation amount of 1‰ and continue culturing. After 24 h, inoculate it into MRS liquid medium again at an inoculation amount of 1‰ and continue culturing. Inoculate the activated strain into 100 mL of MRS liquid medium at an inoculation amount of 200 μL. Take the Bifidobacterium in the logarithmic growth phase, centrifuge at 4000 rpm for 10 min, collect the bacterial cells, resuspend and wash the bacterial cells with normal saline, centrifuge at 4000 rpm for 10 min, collect the bacterial cells, and resuspend with normal saline. Add the dry powder of active polypeptide LG57 to the above - mentioned bacterial cells to obtain the LG57 and probiotic composition.

[0062] Twenty-five Kunming mice aged 4 - 6 weeks were randomly divided into 5 groups with 5 mice in each group. Among them, 4 groups were experimental groups. The mice in the experimental groups were gavaged with ampicillin at a dose of 8 g / kg every day for 7 consecutive days to establish a mouse intestinal flora dysbiosis model. The mice in 1 group were the blank control group. The mice in the blank control group (G1) were gavaged with an equal dose of normal saline in the same way. After modeling, the mice in the experimental groups were divided into a model group (G2) gavaged with normal saline, a group (G3) gavaged with LG57 and probiotic composition, a group (G4) gavaged with LG57 polypeptide, and a group (G5) gavaged with probiotics. Among them, the dose of normal saline in the G2 group was 10 mg / kg, the dose of probiotics in the G3 group was 125 mg / kg and the dose of LG57 polypeptide was 0.5 g / kg, the dose of LG57 polypeptide in the G4 group was 1 g / kg, and the dose of probiotics in the G5 group was 250 mg / kg. They were continuously gavaged for 21 days. The G1 group was gavaged with 10 mg / kg normal saline in the same way. After the gavage was completed, 0.5 g of fresh feces of the mice was aseptically collected, dissolved in normal saline, and diluted 10 9 times. 1 mL of the diluted solution was respectively inoculated into the Bifidobacterium medium and Enterococcus agar medium, and after culturing for 48 h, the count was carried out to calculate CFU / g, that is, the total number of bacterial colonies contained in each gram of feces. The results were presented after taking the logarithm, that is, in the unit of lgCFU / g.

[0063] As shown in Table 2, compared with the blank control group (G1), the number of Bifidobacterium in the mice of the intestinal flora dysbiosis model group (G2) decreased significantly, and the number of Enterobacter increased. Compared with the mice of the intestinal flora dysbiosis model group (G2), after treatment with probiotics (G5), LG57 polypeptide (G4) or the combination of both (G3), the number of Bifidobacterium in the mice increased significantly, and the number of Enterobacter decreased. Moreover, the effect of the group of mice treated with the combination of probiotics and LG57 polypeptide was the best, and it basically recovered to the level comparable to that of the blank control group (G1). The experimental results showed that the combination of probiotics and LG57 polypeptide effectively promoted the growth of Bifidobacterium, while inhibited the proliferation of Enterobacter and promoted the balance of intestinal flora.

[0064] Table 2

[0065] Group Bifidobacterium Enterobacter G1 8.537±0.126 6.781±0.053 G2 6.324±0.523 8.765±0.737 G3 8.727±0.378 6.865±0.676 G4 8.323±0.423 7.076±0.531 G5 8.276±0.578 7.891±0.546

Claims

1. An active polypeptide LG57, which consists of the amino acid sequence shown in SEQ ID NO:

1.

2. A pharmaceutical composition comprising an effective amount of the polypeptide according to claim 1 and probiotics.

3. The pharmaceutical composition according to claim 2, wherein the probiotics are bifidobacteria or lactic acid bacteria. The pharmaceutical composition according to claim 3 , wherein the probiotic is Bifidobacterium. The pharmaceutical composition according to claim 4 , wherein the Bifidobacterium is ATCC 29521.

6. The pharmaceutical composition according to any one of claims 2 to 5, which is administered orally.

7. The pharmaceutical composition according to claim 6, wherein the oral dosage form comprises solution, emulsion, powder, capsule, tablet, pill, granule and lyophilized powder.

8. Use of the polypeptide according to claim 1 or the pharmaceutical composition according to claims 2-7 for preparing a medicament for regulating intestinal flora.

9. The use according to claim 8, wherein the regulating intestinal flora can be manifested as promoting the growth and reproduction of bifidobacteria in the intestine and / or inhibiting the growth and reproduction of enterobacteriaceae.

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