A compound probiotic preparation capable of alleviating antibiotic-associated diarrhea

Abstract

The present invention discloses a compound probiotic preparation capable of alleviating antibiotic-associated diarrhea, belonging to the fields of microbial technology and pharmaceutical technology. The present invention provides a strain of Bifidobacterium adolescentis CCFM1285, which is deposited in the Guangdong Provincial Culture Collection of Microorganisms. The combination of Bifidobacterium adolescentis CCFM1285 and yeast β-glucan of the present invention can significantly reduce the fecal water content of mice with antibiotic-associated diarrhea, alleviate the colonic tissue damage caused by antibiotics and restore the mucus distribution in the colon, reduce the content of pro-inflammatory cytokines in the colonic tissue, restore the short-chain fatty acid content in the cecal contents, and effectively regulate the α and β diversities of the intestinal flora. The compound probiotic preparation provided by the present invention has great application prospects in the preparation of products for preventing and / or treating antibiotic-associated diarrhea.

Description

Technical Field

[0001] The present invention relates to a compound probiotic preparation capable of alleviating antibiotic-associated diarrhea, belonging to the technical fields of microbiology and medicine. Background Art

[0002] Antibiotic-associated diarrhea (AAD) refers to diarrhea of unknown cause that occurs during the use of antibiotics. The main mechanisms of AAD include the disruption of the resident gastrointestinal microbiota and mucosal integrity, overgrowth of pathogens, and metabolic imbalance. It is estimated that approximately 5 - 35% of patients will develop AAD during or at the end of antibiotic treatment, and the clinical symptoms range from mild diarrhea without complications to severe colitis, fulminant pseudomembranous colitis, and even death. Any antibiotic can cause AAD, but broad-spectrum antibiotics that mainly target anaerobes and have poor absorption (such as clindamycin, cephalosporins, and amoxicillin-clavulanate) have a higher incidence of AAD.

[0003] Currently, the main method for treating AAD is to discontinue or replace the antibiotic. In cases where the cause of AAD is known, specific antibiotics against these pathogens can be used. For example, patients with AAD related to Clostridioides difficile infection can be treated with oral vancomycin and metronidazole. Antibiotic treatment strategies for recurrent Clostridioides difficile-related AAD include repeating the antibiotic course using an extended, gradually decreasing dose regimen or a pulsed (every other day) regimen. However, due to the frequent use of antibiotics, it is easy to lead to the emergence of drug-resistant strains and is accompanied by certain adverse reactions. Given the various problems of traditional treatment methods, it is particularly important to explore a new and effective method for treating AAD.

[0004] Although it is disclosed in the prior art that Bacteroides uniformis can synergistically alleviate AAD, it is a strain that cannot be used in food and drugs and has potential safety hazards. Therefore, providing a probiotic preparation that can be added to food and drugs and has the function of treating AAD has important application value for alleviating or treating AAD. Summary of the Invention

[0005] The present invention provides a strain of Bifidobacterium adolescentis CCFM1285, which is preserved in the Guangdong Provincial Culture Collection of Microorganisms, with the preservation number GDMCC No: 62967 and the preservation date of November 13, 2022.

[0006] The Bifidobacterium adolescentis CCFM1285 was isolated from the fecal sample of a healthy adult male in Guancheng Hui District, Zhengzhou City, Henan Province. The genome of the screened Bifidobacterium adolescentis was extracted, and its 16S rDNA was amplified and sequenced (completed by Shanghai Sangon Biotech Co., Ltd.). Through sequencing analysis, the 16S rDNA sequence of this strain is shown in SEQ ID NO.1. When this sequence was compared in GenBank, the homology with the genus Bifidobacterium was 98.17%. The results showed that the strain was Bifidobacterium adolescentis, named Bifidobacterium adolescentis CCFM1285.

[0007] The cell characteristics of the Bifidobacterium adolescentis CCFM1285: Gram-positive rod-shaped bacteria, rarely showing a bifurcated or curved rod shape, and no spore formation.

[0008] The colony characteristics of the Bifidobacterium adolescentis CCFM1285: The colonies on the modified MRS solid medium are porcelain white, round and raised, moist, with neat and smooth edges.

[0009] The growth characteristics of the Bifidobacterium adolescentis CCFM1285: This strain is a strict anaerobe, sensitive to oxygen, and grows best at 37°C.

[0010] The present invention provides a microbial preparation, which contains the Bifidobacterium adolescentis CCFM1285 or its fermentation broth or its freeze-dried powder.

[0011] In one embodiment of the present invention, in the microbial preparation, the viable count of Bifidobacterium adolescentis CCFM1285 in the microbial preparation is not less than 5×10 8 CFU / mL or 5×10 8 CFU / g.

[0012] In one embodiment of the present invention, the microbial agent is a powder prepared by a conventional freeze-drying process or other processes from a bacterial liquid containing the Bifidobacterium adolescentis CCFM1285.

[0013] The present invention provides a compound probiotic preparation, which contains Bifidobacterium adolescentis CCFM1285 and yeast β-glucan (commercially available); by mass percentage, the addition amount of Bifidobacterium adolescentis CCFM1285 is 1% - 10%, and the addition amount of yeast β-glucan is 90% - 99%.

[0014] In one embodiment of the present invention, the yeast β-glucan is purchased from Angel Yeast Co., Ltd.

[0015] The present invention also provides a product, which contains Bifidobacterium adolescentis CCFM1285 or the microbial preparation or the compound probiotic preparation.

[0016] In one embodiment of the present invention, the product is a food, a drug or a health product.

[0017] In one embodiment of the present invention, the viable count of Bifidobacterium adolescentis CCFM1285 in the product is not less than 5×10 8 CFU / mL or 5×10 8 CFU / g.

[0018] In one embodiment of the present invention, the drug is composed of the above compound probiotic preparation and a pharmaceutically acceptable carrier.

[0019] In one embodiment of the present invention, in the drug, the addition amount of the compound probiotic preparation is 15 - 35% or 20 - 30% of the weight of the drug.

[0020] In one embodiment of the present invention, the pharmaceutically acceptable carrier is one or more carriers selected from fillers, binders, wetting agents, disintegrants, lubricants and flavoring agents commonly used in pharmacy.

[0021] In one embodiment of the present invention, the filler should be understood as an auxiliary diluent for increasing the weight and volume of tablets to facilitate tabletting, or an auxiliary absorbent for absorbing excess liquid components in the raw materials.

[0022] In one embodiment of the present invention, the filler is selected from starch, sucrose, lactose, calcium sulfate or microcrystalline cellulose.

[0023] In one embodiment of the present invention, the binder should be understood as a raw material drug that has no stickiness or insufficient stickiness itself, and a sticky substance needs to be added to facilitate granulation. The binder is selected from cellulose derivatives, alginates, gelatin, or polyvinylpyrrolidone.

[0024] In one embodiment of the present invention, the wetting agent should be understood as a liquid that has no stickiness in the raw material drug itself, but can moisten its drug raw materials and excipients and induce its stickiness to form granules. The wetting agent is selected from water, ethanol, starch, or syrup.

[0025] In one embodiment of the present invention, the disintegrant should be understood as an excipient that can be added to tablets to promote the rapid disintegration of the tablets into fine particles in gastrointestinal fluids.

[0026] In one embodiment of the present invention, the disintegrant is selected from sodium carboxymethyl starch, carboxypropyl cellulose, cross-linked carboxymethyl cellulose, agar, calcium carbonate, or sodium bicarbonate.

[0027] In one embodiment of the present invention, the lubricant should be understood as a chemical substance that is beneficial to improving the fluidity of tablets during the granulation process and facilitating the demolding of tablets.

[0028] In one embodiment of the present invention, the lubricant is selected from talc powder, calcium stearate, magnesium stearate, colloidal silica, or polyethylene glycol.

[0029] In one embodiment of the present invention, the flavoring agent should be understood as a pharmaceutical excipient used to improve or mask the unpleasant odor and taste of drugs in pharmaceuticals.

[0030] In one embodiment of the present invention, the flavoring agent is selected from: sweetening agents such as simple syrup, sucrose, lecithin, orange peel syrup, or cherry syrup; aromatic agents such as lemon, fennel, or peppermint oil; mucilage agents such as sodium alginate, gum arabic, gelatin, methylcellulose, or sodium carboxymethylcellulose; and effervescent agents such as a mixture of citric acid, tartaric acid, or sodium bicarbonate.

[0031] The present invention also provides a Bifidobacterium adolescentis cryopreservative. In the Bifidobacterium adolescentis cryopreservative, it contains Bifidobacterium adolescentis CCFM1285 with a viable count of not less than 10 10 CFU / mL.

[0032] In one embodiment of the present invention, the cryopreservative is prepared by inoculating the above-mentioned Bifidobacterium adolescentis CCFM1285 into a culture medium, culturing it for 24 - 30 h, washing it twice with a phosphate buffer solution at pH 7.0 - 7.2, adding a cryoprotectant, and storing it at 80 °C for standby.

[0033] In one embodiment of the present invention, the protective agent contains 1 g / L of cysteine hydrochloride and 200 g / L of glycerol.

[0034] The present invention also provides a method for culturing the above-mentioned Bifidobacterium adolescentis CCFM1285, which is to inoculate the Bifidobacterium adolescentis CCFM1285 into a culture medium and anaerobically culture it at 37°C.

[0035] In one embodiment of the present invention, the strain reaches the stationary phase after culturing for 24 - 30 h.

[0036] In one embodiment of the present invention, the modified MRS medium is used for culturing.

[0037] In one embodiment of the present invention, 1 g / L of cysteine hydrochloride, 0.01 g / L of hemin, and 0.002 g / L of vitamin K1 are further added to the modified MRS medium.

[0038] The present invention also provides the use of the above-mentioned Bifidobacterium adolescentis CCFM1285, or the above-mentioned microbial preparation, or the above-mentioned compound microecological preparation in the preparation of products for relieving and / or treating antibiotic-associated diarrhea, or in the preparation of probiotic health products, or in the preparation of probiotic foods.

[0039] In one embodiment of the present invention, the product is a food, a drug, or a health product.

[0040] In one embodiment of the present invention, the viable count of the Bifidobacterium adolescentis CCFM1285 in the product is not less than 5×10 8 CFU / mL or 5×10 8 CFU / g.

[0041] The present invention also provides the use of the above-mentioned Bifidobacterium adolescentis CCFM1285, or the above-mentioned microbial preparation, or the above-mentioned compound microecological preparation in the preparation of probiotic health products and probiotic foods for relieving and / or treating antibiotic-associated diarrhea.

[0042] The present invention also provides the use of the above-mentioned compound microecological preparation in the fields of foods and health products.

[0043] Beneficial effects

[0044] (1) The compound probiotic preparation described in the present invention contains the Bifidobacterium adolescentis CCFM1285 and yeast β-glucan, and has the following properties:

[0045] 1) It can significantly reduce the water content of feces in AAD mice.

[0046] 2) It can protect the colon tissue damage caused by antibiotics and restore the mucus distribution in the colon.

[0047] 3) It can reduce the content of pro-inflammatory cytokines in the colon tissue caused by antibiotics.

[0048] 4) It can restore the content of short-chain fatty acids in the cecal contents.

[0049] 5) It can effectively regulate the α and β diversities of the intestinal flora.

[0050] (2) The present invention exerts a role in alleviating AAD through the synergistic effect of yeast β-glucan and Bifidobacterium adolescentis CCFM1285. Since both yeast β-glucan and Bifidobacterium adolescentis are additives that can be used in food, the compound probiotic preparation of the present invention has a wide application range and high safety.

[0051] Biological material preservation

[0052] A strain of Bifidobacterium adolescentis CCFM1285, taxonomically named Bifidobacterium adolescentis, was deposited at the Guangdong Provincial Culture Collection of Microorganisms on November 13, 2022, with the deposit number GDMCC No: 62967, and the deposit address is: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology. Description of the drawings

[0053] Figure 1 : Effect of the combination of Bifidobacterium adolescentis CCFM1285 and yeast β-glucan on the water content of feces in AAD mice.

[0054] Figure 2 : Effect of the combination of Bifidobacterium adolescentis CCFM1285 and yeast β-glucan on the pathological structure of the colon tissue in AAD mice.

[0055] Figure 3 : Effect of the combination of Bifidobacterium adolescentis CCFM1285 and yeast β-glucan on the content of pro-inflammatory cytokines in the colon tissue of AAD mice.

[0056] Figure 4 : Effects of the combination of Bifidobacterium adolescentis CCFM1285 and yeast β-glucan on the production of short-chain fatty acids in AAD mice.

[0057] Figure 5 : Effects of the combination of Bifidobacterium adolescentis CCFM1285 and yeast β-glucan on the composition and diversity of the intestinal flora in AAD mice. Specific embodiments

[0058] The yeast β-glucan involved in the following examples was purchased from: Angel Yeast Co., Ltd. The Bacteroides uniformis FGDLZ48B1 involved in the following examples was preserved in the culture collection of the Food Biotechnology Center of Jiangnan University.

[0059] The culture media involved in the following examples are as follows:

[0060] Bifidobacterium-specific screening medium (1L): 10 g peptone, 10 g beef extract, 5 g yeast extract, 20 g glucose, 5 g sodium acetate, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 2 g diammonium citrate, 0.1 g magnesium sulfate heptahydrate, 0.05 g manganese sulfate monohydrate, distilled water: 1000 mL; pH: 6.2 - 6.4; sterilized at 115 °C for 20 min.

[0061] When preparing the solid medium, add 15 g agar, and before pouring the plates, add sterile mupirocin and sterile nystatin to the medium at 1‰ and 0.5‰ of the volume of the medium respectively, that is, the final concentrations are 100 μg / mL mupirocin and 25 U / mL nystatin.

[0062] Modified MRS solid medium (1L): 10 g peptone, 20 g glucose, 0.5 g L-cysteine hydrochloride, 10 g beef extract, 2 g potassium dihydrogen phosphate, 2 g anhydrous sodium acetate, 5 g yeast powder, 2 g diammonium hydrogen citrate, 0.58 g magnesium sulfate, 0.25 g manganese sulfate, 1 mL Tween 80, 20 g agar.

[0063] Modified MRS liquid medium (1L): 10 g peptone, 20 g glucose, 0.5 g L-cysteine hydrochloride, 10 g beef extract, 2 g potassium dihydrogen phosphate, 2 g anhydrous sodium acetate, 5 g yeast powder, 2 g diammonium hydrogen citrate, 0.58 g magnesium sulfate, 0.25 g manganese sulfate, 1 mL Tween 80.

[0064] Modified brain heart infusion solid medium (1L): 38.5 g brain heart infusion, 1 g L-cysteine hydrochloride, 10 mL hemin solution (1 mg / mL), 1 mL vitamin K1 (2 mg / mL), 20 g agar.

[0065] Improved Brain Heart Infusion Liquid Medium (1L): 38.5 g of Brain Heart Infusion, 1 g of L-cysteine hydrochloride, 10 mL of hematin solution (1 mg / mL), 1 mL of vitamin K1 (2 mg / mL).

[0066] Example 1: Screening and Identification of Bifidobacterium adolescentis CCFM1285

[0067] 1. Sample Collection

[0068] Collect a fecal sample from a healthy adult male in Guancheng Hui District, Zhengzhou City, Henan Province, place it in a sample collection tube containing 30% glycerol, store it in a thermo box with an ice pack, and quickly place it in a -80 °C refrigerator for isolation and screening after bringing it back to the laboratory.

[0069] 2. Isolation and Purification of Strains

[0070] (1) Dilution Coating: Take about 0.5 g of the fecal sample stored in a 30% glycerol tube, mix it with 4.5 mL of normal saline in a 10 mL centrifuge tube under sterile conditions to obtain a 10 -1 dilution. Repeat the above operation to obtain 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 dilutions in sequence;

[0071] (2) Coating Culture: Respectively pipette 100 μL of the 10 -4 , 10 -5 , 10 -6 three gradient dilutions in step (1) onto a Bifidobacterium-specific screening medium, spread evenly with a spreading rod, and culture under anaerobic conditions at 37 °C for 48 h to obtain dilution coating plates;

[0072] (3) Purification Culture: Take the dilution coating plates with the number of colonies in the range of 30 - 300. Randomly select 10 porcelain-white, smooth and moist surfaces, and neatly edged colonies from each sample and streak them on the improved MRS solid medium, then culture under anaerobic conditions at 37 °C for 48 h to obtain single colonies. Then inoculate the single colonies into the improved MRS liquid medium and culture under anaerobic conditions at 37 °C for 24 h. The obtained is the purified culture solution.

[0073] 3. Strain Preservation and Identification

[0074] The genome of the screened Bifidobacterium adolescentis was extracted, and its 16S rDNA was amplified and sequenced (completed by Shanghai Sangon Biotech Co., Ltd.). Through sequencing analysis, the 16S rDNA sequence of this strain is shown in SEQ ID NO.1. When this sequence was compared in GenBank, the homology with the genus Bifidobacterium was 98.17%. The results showed that the strain was Bifidobacterium adolescentis, named Bifidobacterium adolescentis CCFM1285.

[0075] Example 2: Effect of Bifidobacterium adolescentis CCFM1285 combined with yeast β-glucan on fecal water content in AAD mice.

[0076] The specific steps are as follows:

[0077] (1) Preparation of gastric lavage fluid:

[0078] 1) Streak inoculate the preserved Bifidobacterium adolescentis CCFM1285 strain on the improved MRS solid medium. After anaerobic culture at 37°C for 48 h, pick single colonies and inoculate them in MRS liquid medium, continue anaerobic culture for 24 h, and activate for three consecutive generations.

[0079] Perform colony counting and subculture on the activated strain. Centrifuge at 6000g for 5 - 10 min to collect the bacterial sludge, and according to the colony counting results, resuspend the strain in sterile phosphate buffer saline (PBS) to the specified concentration: 2.5×10 9 CFU / mL.

[0080] 2) Configure yeast β-glucan into an aqueous solution with a concentration of 80 mg / mL using sterile PBS, let it stand for 12 h for overnight hydration.

[0081] 3) Streak inoculate the Bacteroides uniformis FGDLZ48B1 strain preserved in the laboratory on the improved BHI solid medium. After anaerobic culture at 37°C for 48 h, pick single colonies and inoculate them in the improved BHI liquid medium, continue anaerobic culture for 18 h, and activate for three consecutive generations.

[0082] Perform colony counting and subculture on the activated strain. Centrifuge at 6000g for 5 - 10 min to collect the bacterial sludge, and according to the colony counting results, resuspend the strain in sterile PBS to the specified concentration: 2.5×10 9 CFU / mL.

[0083] (2) Experimental animals:

[0084] SPF-grade 7-week-old male BALB / C mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and raised in an SPF-grade barrier environment with an environmental temperature of 23 ± 2°C and a relative humidity of 50 ± 10%.

[0085] (3) Experimental method:

[0086] The newly introduced SPF-grade BALB / c mice were first fed adaptively for 7 days, and then 60 mice were randomly and evenly divided into 6 groups, namely: normal control group, model group, positive reference (Bacteroides uniformis + Bifidobacterium adolescentis; Bu + Ba) group, CCFM1285 (Bifidobacterium adolescentis; Ba) group, β-glucan (Glu) group, β-glucan + CCFM1285 (β-glucan + Bifidobacterium adolescentis; Glu + Ba) group.

[0087] After the adaptation period ended, except for the normal control group, the mice in other groups were intragastrically administered with lincomycin hydrochloride (LH) solution twice a day at 8:00 am and 20:00 pm for 3 consecutive days, and during the recovery period, the mice were intragastrically administered with 0.2 mL of PBS or a bacterial suspension resuspended with PBS; the specific operations are shown in Table 1.

[0088] Table 1: Experimental protocol for the alleviating effect of β-glucan and Bifidobacterium adolescentis on AAD mice

[0089]

[0090] The mice were sacrificed 7 days after intragastric administration.

[0091] One day before sacrificing the mice, the feces of the mice were collected, and a vacuum freeze dryer was used to measure the water content of the feces. The results are as Figure 1 shown.

[0092] The results showed that after different treatments on AAD mice, the water content of the feces of the mice in the normal control group was: 57.29%;

[0093] The water content of the feces of the mice in the positive reference group, CCFM1285 group (Ba group), β-glucan group (Glu group), and the combination group with β-glucan (Glu + Ba group) was significantly decreased compared with that of the model group (which was: 68.20%), and they were 60.91%, 68.16%, 62.24%, and 61.95% respectively, indicating that each group improved the diarrhea state of the mice. Among them, the decrease level of the combination group of CCFM1285 and β-glucan (Glu + Ba group) was better than that of the β-glucan group and was comparable to that of the positive reference group.

[0094] Since Bacteroides uniformis in the positive reference group is not an edible strain and cannot be directly used in food, medicine, and health products, while the CCFM1285 strain and β-glucan in the present invention are both edible products, therefore, the composite microecological preparation composed of CCFM1285 and β-glucan in the present invention has a wide range of applications.

[0095] Example 3: Effects of the combination of Bifidobacterium adolescentis CCFM1285 and yeast β-glucan on the histopathological structure of the colon tissue of AAD mice.

[0096] The specific steps are as follows:

[0097] The specific implementation method is the same as that in Example 2. The mice were sacrificed and a part of their colon tissue was placed in 4% Carnoy's fixative for colon section observation. The AB-PAS staining method was used to evaluate the damage of the colon tissue of mice caused by antibiotic stimulation and the protective effect of microecological agents intervention on the colon tissue. The results are as Figure 2 shown.

[0098] The results showed that the structure of the mice in the normal control group was intact, the intestinal villi were neat, the goblet cells were abundant, and the mucus was evenly and richly distributed;

[0099] However, in the model group treated with antibiotics, the intestinal epithelial villi of the mice became shorter and the mucus distribution area decreased.

[0100] The combination of β-glucan and CCFM1285 (Glu+Ba group) could significantly restore the length of intestinal epithelial villi, increase the proportion of mucus, and restore the mucus barrier of the intestine.

[0101] Example 4: Effects of the combination of Bifidobacterium adolescentis CCFM1285 and yeast β-glucan on the content of pro-inflammatory cytokines in the colon tissue of AAD mice.

[0102] The specific steps are as follows:

[0103] The specific implementation method is the same as that in Example 2. The mice were sacrificed, their colon tissue was taken and quickly stored in liquid nitrogen, and then added with normal saline and ground into tissue homogenate. The content of pro-inflammatory cytokines in the colon tissue was measured by the Elisa method. The results are as Figure 3 shown.

[0104] The results showed that the use of β-glucan alone (Glu group) and in combination with CCFM1285 (Glu+Ba group) significantly reduced the increase in the level of IL-6 in the colon tissue caused by antibiotics compared with the model group (77.11 pg / mg), which were 40.90 pg / mg and 24.90 pg / mg respectively. Among them, the combination group of CCFM1285 and β-glucan was lower than the positive reference group and close to the normal control group; while the levels of IL-6 in the colon tissue of the mice after intervention in the normal control group and CCFM1285 group were 23.89 pg / mg and 61.24 pg / mg respectively.

[0105] In addition, the combination of β-glucan and CCFM1285 (Glu+Ba group) also significantly reduced the level of IL-17 (to 21.77 pg / mg), approaching the level of the normal control group (21.15 pg / mg). The levels of IL-17 in the colon tissues of mice after intervention in the model group, β-glucan alone (Glu group), and CCFM1285 group (Ba group) were 71.94 pg / mg, 35.28 pg / mg, and 55.39 pg / mg, respectively.

[0106] In summary, the combination of β-glucan and CCFM1285 has a certain regulatory effect on antibiotic-induced colon tissue inflammation.

[0107] Example 5: Effects of the combination of Bifidobacterium adolescentis CCFM and yeast β-glucan on the production of short-chain fatty acids in AAD mice.

[0108] The specific steps are as follows:

[0109] The specific implementation method was the same as that in Example 2. The mice were sacrificed, and the cecal contents were collected and quickly stored in liquid nitrogen. The short-chain fatty acids in the contents were extracted and measured by GC-MS. The results are as Figure 4 shown in Table 2.

[0110] Table 2: Contents of short-chain fatty acids in mice treated with different groups (μmol / g)

[0111]

[0112]

[0113] The results showed that after antibiotic treatment (model group), the levels of acetic acid, propionic acid, isovaleric acid, and isobutyric acid in the intestines of AAD mice decreased significantly.

[0114] As Figure 4 shown in A-D, the levels of acetic acid and propionic acid in the positive reference group, β-glucan group, and the combination group with CCFM1285 increased significantly. In Figure 4 D, the levels of isobutyric acid in all experimental groups increased significantly compared with the model group.

[0115] In summary, β-glucan alone and in combination with CCFM1285 can also restore to a certain extent the decrease in the levels of SCFAs in the cecal contents caused by antibiotics and regulate the microecological environment in the intestine, approaching the effect of the positive control group.

[0116] Example 6: Effects of the combination of Bifidobacterium adolescentis CCFM1285 and yeast β-glucan on the intestinal flora composition and diversity of AAD mice.

[0117] The specific steps are as follows:

[0118] The specific implementation method was the same as that in Example 2. The mice were sacrificed, and the colonic contents were collected and quickly stored in liquid nitrogen. The DNA in the contents was extracted and subjected to machine testing.

[0119] The results were as Figure 5 shown in A and B. Gavage with antibiotics (model group) led to a significant decrease in the Shannon index and Fisher index of mice. However, after co-gavage with β-glucan and CCFM1285 (Glu+Ba group), the α-diversity of the intestinal flora of AAD mice was significantly improved, indicating that the co-gavage treatment had a significant improving effect on the diversity of the flora composition.

[0120] As Figure 5 shown in C, distance clustering based on the Jaccard index showed that the β-glucan group and the group used in combination with CCFM1285 improved the flora disorder in mice, making the flora structure closer to that of healthy mice.

[0121] Example 7: Preparation of a capsule product containing the probiotic preparation of the present invention

[0122] Bifidobacterium adolescentis CCFM1285 of the present invention was anaerobically cultured in a modified MRS medium at 37 °C for 24 hours, centrifuged at 5000 rpm for 15 min at 4 °C, rinsed 1-2 times with sterile phosphate buffer (pH 7.2), and the cells were resuspended with a cryoprotectant to make the final cell concentration reach 10 10 CFU / mL. The bacterial suspension was added to a 3% sodium alginate solution so that the cell concentration was not less than 1×10 6 CFU / mL, and stirred well to evenly disperse Bacteroides vulgatus CCFM1285 cells in the sodium alginate solution to obtain a mixed solution. Then, this mixed solution was extruded into a 2% calcium chloride solution to form gel particles. After the formed gel particles were allowed to stand and solidify for 30 min, the gel particles were collected by filtration. The collected gel particles were freeze-dried for 48 hours to obtain a powder containing Bifidobacterium adolescentis CCFM1285. The powder was filled into a commercially available medicinal capsule together with yeast β-glucan to obtain the capsule product.

[0123] The components of the cryoprotectant include: 100 g / L skim milk powder, 30 mL / L glycerol, 100 g / L maltodextrin, 150 g / L trehalose, and 10 g / L sodium L-glutamate.

[0124] Example 8: Preparation of tablets using the probiotic preparation of the present invention

[0125] Weigh 25.7 parts by weight of the Bifidobacterium adolescentis CCFM1285 powder preparation prepared by the freeze-drying method, 55.0 parts by weight of yeast β-glucan, 4.5 parts by weight of cellulose derivative, 12.0 parts by weight of sodium carboxymethyl starch, 0.8 parts by weight of talc powder, 1.0 part by weight of sucrose and 1.0 part by weight of water respectively, mix them, make wet granules by the conventional method, then use a tablet press produced by Zhongnan Pharmaceutical Machinery Factory for tableting, use a small drug dryer produced by Qingzhou Yikang Traditional Chinese Medicine Machinery Co., Ltd. for drying, and then package to obtain the tablets of the present invention.

[0126] Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A compound microecological preparation, characterized in that, The composite probiotic preparation contains Bifidobacterium adolescentis CCFM1285 and yeast β-glucan; by mass percentage, the addition amount of Bifidobacterium adolescentis CCFM1285 is 1% - 10%, and the addition amount of yeast β-glucan is 90% - 99%; Bifidobacterium adolescentis CCFM1285 is preserved in the Guangdong Provincial Culture Collection Center of Microorganisms, with the preservation number GDMCC No: 62967, and the preservation date is November 13, 2022.

2. A drug, characterized in that, The drug contains the composite probiotic preparation described in claim 1; Bifidobacterium adolescentis CCFM1285 is preserved in the Guangdong Provincial Culture Collection Center of Microorganisms, with the preservation number GDMCC No: 62967, and the preservation date is November 13, 2022.

3. The drug according to claim 2, characterized in that, The viable count of Bifidobacterium adolescentis CCFM1285 in the drug is not less than 5×10 8 CFU / mL or 5×10 8 CFU / g.

4. Use of the compound microecological preparation according to claim 1 in the preparation of a medicament for relieving and / or treating antibiotic-associated diarrhea, characterized in that, Bifidobacterium adolescentis CCFM1285 is preserved in the Guangdong Provincial Culture Collection Center of Microorganisms, with the preservation number GDMCC No: 62967, and the preservation date is November 13, 2022.

5. The application according to claim 4, characterized in that, The viable count of the Bifidobacterium adolescentis CCFM1285 in the drug is not less than 5×10 8 CFU / mL or 5×10 8 CFU / g.

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