Microecological preparation for targeted promotion of proliferation of intestinal bifidobacteria and application thereof

Abstract

The invention discloses a microecological preparation capable of regulating and controlling abundance of intestinal bifidobacteria in a targeted mode and application of the microecological preparation, the microecological preparation is composed of intestinal symbiotic bacteria and prebiotics, the intestinal symbiotic bacteria are bacteroides, and the prebiotics are polysaccharide containing N-acetylglucosamine. The prebiotics and the bacteroides multiforme CCFM1441 are combined for use, the prebiotics N-acetylglucosamine can be effectively released, nutrition is provided for bifidobacterium longum, and proliferation of the bifidobacterium longum is promoted. Further, in vitro and in vivo experiments prove that there is a strong cross-feeding activity between Bacteroides multiforme CCFM1441 and Bifidobacterium longum based on prebiotics. The beneficial microecological preparation can promote proliferation of bifidobacterium longum in intestinal tracts in a targeted manner, improves intestinal flora imbalance caused by antibiotics, and further has a huge application prospect in preparation of products (such as food, drugs or health care products) for improving intestinal health.

Description

Technical Field

[0001] The present invention relates to a microecological preparation for promoting the proliferation of intestinal bifidobacteria in a targeted manner and application thereof, belonging to the technical field of microorganisms. Background Art

[0002] The human intestine is the host's largest microbial ecosystem. Bifidobacterium is one of the core bacterial genera in the intestine and is essential to the health of humans and other mammals. It breaks down complex carbohydrates that are difficult for the host to digest on its own and promotes the production of short-chain fatty acids, which are key substances for maintaining intestinal health. Bifidobacterium can also inhibit the growth of pathogens, protect the intestine from infection, and regulate immune cell activity through interactions with the intestinal immune system, reducing inflammatory responses and preventing autoimmune diseases and allergic reactions. With the advancement of science and technology, research on Bifidobacterium has deepened our understanding of the relationship between intestinal microorganisms and host health, showing its great potential in the prevention and treatment of various diseases.

[0003] In modern health management, methods for regulating intestinal bifidobacteria primarily include direct bifidobacterial supplementation or promoting their proliferation through prebiotics. Direct bifidobacterial supplementation is typically achieved through the ingestion of probiotic products containing active bifidobacteria, such as yogurt and probiotic supplements. However, this approach faces challenges related to the low survival rate of bifidobacteria in the intestine and their difficulty in long-term colonization. Another approach is through the ingestion of prebiotics—dietary ingredients that are not digested and absorbed by the body but promote the growth of beneficial bacteria such as bifidobacteria, such as oligosaccharides and inulin. Prebiotics act as "food" for bifidobacteria, helping to increase their numbers in the intestine. Although this approach can indirectly increase the abundance of bifidobacteria, it is relatively less targeted and cannot ensure the growth of only bifidobacteria without affecting other species of intestinal bacteria.

[0004] While these traditional approaches are effective to a certain extent, they all have limitations, such as the difficulty in precisely controlling the proliferation and activity of bifidobacteria. Therefore, developing more targeted and efficient regulatory strategies, such as preparing probiotics by analyzing the genomic information of bifidobacteria in the intestine and cross-feeding with bifidobacteria to enhance the function and stability of bifidobacteria, has become a new research direction. These highly targeted approaches may provide us with more effective solutions in the future to harness the health benefits of bifidobacteria.

[0005] Therefore, there is a need in the art to develop a microecological preparation that targets and regulates intestinal bifidobacteria based on an intestinal cross-feeding strategy. Summary of the Invention

[0006] In response to the above-mentioned deficiencies in the prior art, the present invention provides a microecological preparation that targets and promotes the proliferation of intestinal bifidobacteria and its application, aiming to solve the technical problem in the prior art that it is difficult to accurately control the proliferation and activity of bifidobacteria in products that regulate intestinal bifidobacteria.

[0007] The first technical solution provided by the present invention is Bacteroides thetaiotaomicron CCFM1441. The Bacteroides thetaiotaomicron CCFM1441 was deposited in the Guangdong Provincial Microbial Culture Collection Center on October 31, 2024, with the deposit number GDMCC No.65379.

[0008] The second technical solution provided by the present invention is a microbial preparation containing the Bacteroides thetaiotaomicron CCFM1441 described in the first technical solution.

[0009] In certain embodiments, the concentration of Bacteroides thetaiotaomicron CCFM1441 in the microbial preparation is not less than 1×10 6 CFU / g or 1×10 6 CFU / mL.

[0010] Furthermore, the concentration of the Bacteroides thetaiotaomicron CCFM1441 in the microbial preparation is not less than 1×10 8 CFU / g or 1×10 8 CFU / mL.

[0011] The third technical solution provided by the present invention is a probiotic preparation comprising intestinal symbiotic bacteria and prebiotics. The symbiotic bacteria include Bacteroides thetaiotaomicron CCFM1441 (Bt) described in the first technical solution or the microbial preparation described in the second technical solution. The prebiotic includes a polysaccharide containing N-acetylglucosamine, such as hyaluronic acid. It should be noted that a polysaccharide containing N-acetylglucosamine refers to a monosaccharide composition containing N-acetylglucosamine monomers.

[0012] In certain embodiments, in the microecological preparation, the amount of the Bacteroides thetaiotaomicron CCFM1441 added is not less than 1×10 6 CFU / g or 1×10 6 CFU / mL, and the added amount of the prebiotics is not less than 1%.

[0013] Optionally, the amount of Bacteroides thetaiotaomicron CCFM1441 added is 1×10 8 -5×10 12 CFU / g or 1×10 8 -5×10 12 CFU / mL, and the added amount of the prebiotics is 1-70wt%.

[0014] In certain embodiments, the symbiotic bacteria further include a functional strain capable of degrading dietary polysaccharides and promoting the proliferation of bifidobacteria, wherein the functional strain is at least one of Bacteroides faecalis, Bacteroides ovatus, and Bacteroides faecalis, and the added dosage of the functional strain does not exceed 50% of the added amount of Bacteroides thetaiotaomicron CCFM1441.

[0015] In certain embodiments, the prebiotics include different molecular weights and different salt forms of sodium salt, potassium salt, magnesium salt, calcium salt, zinc salt, bismuth salt, or a combination of two or more thereof.

[0016] The prebiotics are prebiotics with the same structure extracted by microbial fermentation, enzymatic method or chemical method.

[0017] The fourth technical solution provided by the present invention is a product containing the microecological preparation described in the third technical solution.

[0018] In certain embodiments, the product is a microbial agent, a medicine, a food or a health product.

[0019] Optionally, the food is yogurt, liquid beverage, solid beverage, compressed candy or meal replacement powder containing the probiotic preparation described in the third technical solution.

[0020] In certain embodiments, the total effective viable count in the product is 1.0×10 6 ~1.0×10 14 CFU / g or 1.0×10 6 ~1.0×10 14 CFU / mL, preferably 1.0×10 8 ~1.0×10 12 CFU / g or 1.0×10 8 ~1.0×10 12 CFU / mL.

[0021] The fifth technical solution provided by the present invention is the use of the probiotic preparation described in the third technical solution in the preparation of a product for targetedly promoting the proliferation of intestinal bifidobacteria.

[0022] In certain embodiments, the product is a medicine, a nutritional supplement, a functional food and / or a health food.

[0023] In certain embodiments, the drug further contains a drug carrier and / or a pharmaceutical excipient.

[0024] In certain embodiments, the drug carrier comprises a microcapsule, a microsphere, a nanoparticle and / or a liposome.

[0025] In certain embodiments, the pharmaceutical excipient comprises an excipient and / or an additive.

[0026] In certain embodiments, the excipient comprises a binder, a filler, a disintegrant, and / or a lubricant.

[0027] In certain embodiments, the additional agent comprises a solubilizer, a co-solvent, a co-solvent and / or a preservative.

[0028] In certain embodiments, the dosage form of the drug is powder, granules, capsules, tablets, pills or oral liquid.

[0029] The sixth technical solution provided by the present invention is the use of the proecological preparation described in the third technical solution in the preparation of a product for alleviating and / or treating intestinal Bifidobacterium longum damage and / or diseases related to Bifidobacterium longum damage.

[0030] In certain embodiments, the diseases associated with damage to Bifidobacterium longum include Parkinson's syndrome, dysbacteriosis, immunosuppression, diarrhea, constipation, enteritis, aging, chronic inflammation, metabolic syndrome, and the like.

[0031] In certain embodiments, the product is a medicine, a nutritional supplement, a functional food and / or a health food.

[0032] In certain embodiments, the drug further contains a drug carrier and / or a pharmaceutical excipient.

[0033] In certain embodiments, the drug carrier comprises a microcapsule, a microsphere, a nanoparticle and / or a liposome.

[0034] In certain embodiments, the pharmaceutical excipient comprises an excipient and / or an additive.

[0035] In certain embodiments, the excipient comprises a binder, a filler, a disintegrant, and / or a lubricant.

[0036] In certain embodiments, the additional agent comprises a solubilizer, a co-solvent, a co-solvent and / or a preservative.

[0037] In certain embodiments, the dosage form of the drug is powder, granules, capsules, tablets, pills or oral liquid.

[0038] The technical effects of the present invention are as follows:

[0039] 1. The present invention provides a probiotic preparation composed of intestinal symbiotic bacteria and prebiotics, wherein the symbiotic bacteria is Bacteroides thetaiotaomicron CCFM1441, and the prebiotic is a polysaccharide containing N-acetylglucosamine, such as hyaluronic acid; the present invention combines prebiotics with Bacteroides thetaiotaomicron, which can not only promote the proliferation of intestinal symbiotic bacteria Bacteroides thetaiotaomicron, but also through the synergistic effect of prebiotics, based on the cross-feeding strategy, Bacteroides thetaiotaomicron degrades prebiotics to produce N-acetylglucosamine to target and promote the proliferation of intestinal Bifidobacterium longum, significantly target and promote the abundance of intestinal Bifidobacterium, and further reduce the proliferation of harmful intestinal bacteria, maintain the steady-state balance of the intestinal environment, and thus promote the prevention and treatment of diseases related to intestinal flora.

[0040] 2. In the probiotic preparation product provided by the present invention, the prebiotics containing N-acetylglucosamine and Bacteroides thetaiotaomicron can be used together with other functional strains that have the ability to degrade dietary polysaccharides and promote the proliferation of bifidobacteria. The functional strains that have the ability to degrade dietary polysaccharides and promote the proliferation of bifidobacteria are at least one of Bacteroides faecalis, Bacteroides ovatus and Bacteroides faecalis. The amount of the functional strain that has the ability to degrade dietary polysaccharides and promote the proliferation of bifidobacteria does not exceed 50% of that of Bacteroides thetaiotaomicron. The synergistic effect of multiple strains can achieve a synergistic effect.

[0041] Biomaterial Deposit

[0042] Bacteroides thetaiotaomicron CCFM1441, taxonomically named Bacteroides thetaiotaomicron, was deposited in the Guangdong Provincial Microbial Culture Collection on October 31, 2024, with the deposit number GDMCC No. 65379, and the deposit address is Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0043] Bifidobacterium longum CCFM1442, taxonomically named Bifidobacterium longum, was deposited in the Guangdong Provincial Microbial Culture Collection on December 11, 2024. The culture collection number is GDMCC No. 65380, and the collection address is Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The utilization characteristics of hyaluronic acid by Bacteroides thetaiotaomicron CCFM1441; A: growth curves of 9 Bacteroides thetaiotaomicron strains on HA as the only carbon source; B: maximum growth of 9 Bacteroides thetaiotaomicron strains on different dietary polysaccharides.

[0045] Figure 2This is the cross-feeding network of Bacteroides thetaiotaomicron and Bifidobacterium longum based on HA; A: The supernatant and bacterial substances of Bacteroides thetaiotaomicron fermentation HA promote the growth of Bifidobacterium longum; B: Transwell experiment verifies the cross-feeding of Bacteroides thetaiotaomicron and Bifidobacterium longum; C: LC-MS / MS determination of the changes in metabolites of Bacteroides thetaiotaomicron fermentation HA; D: The sole carbon source verifies the promoting effect of differential metabolites on Bifidobacterium longum.

[0046] Figure 3 Probiotics containing HA and Bt can target and promote the proliferation of intestinal Bifidobacterium longum; A: Experimental design for verifying in vivo cross-feeding in pseudo-sterile mice; B: Absolute copy number of Bifidobacterium longum at different time points.

[0047] Figure 4 HA and Bacteroides thetaiotaomicron targeted the regulation of Bifidobacterium longum to alleviate antibiotic-induced dysbiosis; A: Determination of the absolute copy number of HA and Bacteroides thetaiotaomicron; B: β diversity (PCoA) analysis of the recovery of dysbiosis in different groups at different time points.

[0048] In all figures, asterisks indicate statistically significant differences between the data (ns p>0.05, **p<0.01, ***p<0.001). DETAILED DESCRIPTION

[0049] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0050] The culture medium involved in the following examples is:

[0051] 1. BHI medium: 38.5 g brain heart infusion medium, 1 g cysteine ​​hydrochloride, 10 mL hemin solution, 1 mL vitamin K1, add distilled water to 1 L, add 0.001% hemin chloride and 0.001% vitamin K1.

[0052] 2. The recipe for Brucella blood culture medium (LKV) has been modified. LKV is prepared as follows: 43.1g Brucella agar (or Brucella broth + 2% agar), 10mL hemin solution, 1mL vitamin K1, 1g cysteine ​​hydrochloride, and 1L distilled water. After all ingredients are uniformly dissolved, add 0.001% hemin chloride and 0.001% vitamin K1. Sterilize at 121°C for 15 minutes. When the temperature drops to 50°C, add 0.01% kanamycin, 0.00075% vancomycin, and 5% sheep blood. Mix thoroughly and pour into a sterile Petri dish.

[0053] 3. Sole carbon source medium: polysaccharide 5g (0.5%), potassium dihydrogen phosphate 13.61g, sodium chloride 0.877g, ammonium sulfate 1.123g, L-cysteine ​​0.7g, hematin 1.23mg, histidine 31mg, magnesium chloride 9.5mg, ferrous sulfate heptahydrate 0.4mg, calcium chloride 5.6mg, vitamin K 0.001g, vitamin B12 5μg, ATCC mixed vitamin solution 1mL, ATCC trace element mixed solution 10mL, distilled water 1L, pH to 7.0 (0.2 floating).

[0054] The strains and animals involved in the following examples are:

[0055] 1. The strain deposit number of Bifidobacterium longum CCFM1442 is GDMCC No. 65380.

[0056] 2. SPF-grade 8-week-old male C57BL / 6J mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0057] The following embodiments relate to the method:

[0058] Metabolome analysis: 0.1 mL of the supernatant from the HA fermentation with B. thetaiotaomicron was added to 0.4 mL of organic solvent (methanol:acetonitrile = 1:1, pre-cooled to 20°C), shaken for 40 seconds, and incubated at 20°C for 1 hour to precipitate proteins. The sample was resuspended in 0.2 mL of solvent (acetonitrile:water = 4:1) and shaken for 35 seconds. The sample was centrifuged at 15,000 × g for 15 minutes at 4°C, and the supernatant was filtered through a 0.22 μm filter into an injection vial.

[0059] LC-MS analysis was modified from previous studies. MS parameters were as follows: ionization mode, ESI (electrospray); scan range, 70–1050; resolution, 70,000. The separation column used was an Atlantis Hilic Silica (3.0 μm, 100 × 2.1 mm). The mobile phases in positive mode were: A, 10 mM ammonium acetate, 0.1% formic acid, ACN:H₂O = 95:5; B, 10 mM ammonium acetate, 0.1% formic acid, ACN:H₂O = 50:50. The mobile phases in negative mode were: A, 10 mM ammonium acetate, ACN:H₂O = 95:5 (pH 9.0, adjusted with aqueous ammonia); B, 10 mM ammonium acetate, ACN:H₂O = 50:50 (pH 9.0, adjusted with aqueous ammonia). CompoundDiscoverer software was used to convert the raw LC-MS data into visual results and then into a data format to export the metabolite data of the samples.

[0060] 2. Absolute quantification analysis by RT-qPCR: DNA from all samples was diluted 1:1000 in nuclease-free sterile water to dilute the concentration of any PCR inhibitors. Standards were prepared using custom synthetic plasmids containing portions of the 16S rRNA gene of specific bacteria. Standards were diluted from 1:10 to 1:10M to prepare a 10-log standard curve. Appendix 1 shows the specific qPCR primers for bacteria. All qPCR samples were run using QuantStudio 12K Flex (Applied Biosystems) and SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, 1725270). qPCR analysis was performed using QuantStudio Design & Analysis 2.6.0 (Thermo Fisher Scientific). To calculate the total microbial load, the Cq value of each sample was converted to the number of copies of the specific gene per milliliter or per gram using a standard curve.

[0061] Example 1 Isolation and identification of Bacteroides thetaiotaomicron CCFM1441

[0062] The specific steps are as follows:

[0063] The common isolation method of Bacteroides is as follows: fecal samples are diluted with physiological saline (NaCl, pH = 7.0) in a gradient dilution (from 10 -1 -10 -9 ), 150 μL of stool sample suspension was spread on LKV agar culture plates and incubated anaerobically at 37°C for 48 hours. Based on the colony morphology, 10 single colonies were randomly selected (ensuring that colonies of each colony morphology were picked) and streaked onto new plates for culture; then a single colony was picked from the new plate for further streak purification; finally, a single colony was picked and transferred to the corresponding 5 mL liquid culture medium and cultured for 48 hours. 0.5 mL of the bacterial solution (48 hours) was transferred to a 2 mL culture storage tube, 0.5 mL of 40% glycerol (containing 0.1% cysteine) was added, mixed thoroughly, and cultured in 3-5 replicates at -80°C. Further, the strain information was identified by 16s sequencing.

[0064] Identification of bacterial 16s rDNA: After isolation and purification, the culture medium was centrifuged at 10,000 g for 1 minute, the supernatant discarded, and the culture resuspended in an equal volume of sterile water. 0.5 μL of the culture medium was used as a template for PCR amplification of the 16s rDNA (approximately 1500 bp) using universal primers 27F (AGA GTT TGA TCCTGG CCT CA) and 1492R (GGT TAC CTT GTT ACG ACT T). The reaction system (25 μL) was as follows: 12.5 μL 2× Premix Taq, 1 μL 27F, 1 μL 1492R, 0.5 μL genomic DNA template, and 10 μL ddH2O. PCR reaction conditions were as follows: 95°C for 5 minutes, 95°C for 30 seconds, 55°C for 30 seconds, and 30 cycles of 72°C for 2 minutes and 72°C for 10 minutes. After the PCR reaction, the product was confirmed by 1.0% agarose gel electrophoresis and then sequenced by Suzhou Jinweizhi Biotechnology Co., Ltd. The nucleic acid sequence of the strain was identified to be 100% similar to that of Bacteroides thetaiotaomicron and was named Bacteroides thetaiotaomicron CCFM1441.

[0065] Example 2 Analysis of the Utilization Characteristics of Hyaluronic Acid by Bacteroides thetaiotaomicron CCFM1441

[0066] The specific steps are as follows:

[0067] Bacteroides thetaiotaomicron CCFM1441 was cultured anaerobically in BHI medium until the exponential growth phase (OD 600 The culture was transferred to pre-reduced MM + 0.5% (w / v) HA in a total volume of 300 μL, with an initial OD of 0.05. The culture was incubated anaerobically at 37°C in a multi-well plate. Bacterial growth was monitored using an Infinite F50 microplate reader, with data collected every 0.5 h for a total of 30 h. The original OD of each well was calculated by 600 Subtract the average blank OD 600 To correct the OD under each polysaccharide condition and draw the growth curve. All cultures were repeated three times ( Figure 1 -A).

[0068] In order to further demonstrate the targeted regulatory effect of HA on Bacteroides thetaiotaomicron CCFM1441, the effects of common dietary polysaccharides HA, chondroitin sulfate, inulin, fructan, xyloglucan, rhamnogalacturonic acid, arabinogalactan, glucomannan and β-glucan on the growth of Bacteroides thetaiotaomicron were compared horizontally ( Figure 1-B). The results showed that after 24 hours of culture, HA showed a consistent and strong effect in promoting the growth of Bacteroides thetaiotaomicron, which was significantly better than other tested polysaccharides. Specifically, Bacteroides thetaiotaomicron showed the strongest growth in HA medium, with OD 600 Close to or exceeding 0.8. This result is not only significantly higher than the control group, but also significantly better than other polysaccharide substrates. It is worth noting that although some other polysaccharides, such as galactomannan and xyloglucan, also show a certain degree of promoting effect, their effects are not as significant as HA. The above results indicate that HA may play a key role as a high-quality prebiotic in regulating the balance of intestinal flora, especially in promoting the growth of beneficial Bacteroides thetaiotaomicron CCFM1441.

[0069] Example 3: Cross-feeding network based on HA Bacteroides thetaiotaomicron and Bifidobacterium longum

[0070] The specific steps are as follows: First, culture Bacteroides thetaiotaomicron CCFM1441 anaerobically at 37°C in BHI medium for at least 12 hours, collect the supernatant and bacterial lysate by centrifugation at 12,000 r / min, and sterile filter them separately. The cultured and activated Bifidobacterium longum CCFM1442 was inoculated into the supernatant of Bacteroides thetaiotaomicron CCFM1441 and the bacterial lysate of Bacteroides thetaiotaomicron CCFM1441, and its growth condition was measured, and it was found that the proliferation of Bifidobacterium longum could not be promoted. Similarly, after the initial BHI medium was replaced with MM medium containing 0.5% HA, the above culture process was repeated and it was found that Bifidobacterium longum could proliferate significantly. The results show that the metabolites of HA degraded by Bacteroides thetaiotaomicron CCFM1441 can effectively promote the proliferation of Bifidobacterium longum. Specific results show that when HA is present, the absolute amount of Bifidobacterium longum in 24 hours is increased to 10 8 , is 10 of the initial inoculum 4 times( Figure 2 -A and B).

[0071] In order to analyze the material basis for promoting the proliferation of Bifidobacterium longum, the metabolome analysis of single bacteria revealed that N-acetylglucosamine was a significantly different substance among the metabolites produced by the degradation of HA by Bacteroides thetaiotaomicron CCFM1441. The results of LC-MS showed that compared with 0h before fermentation, the concentration of the key different substance N-acetylglucosamine increased by more than 100 times after 24h of fermentation; and D-glucuronic acid increased by more than 10 times. The sole carbon source experiment proved that N-acetylglucosamine is the material basis for promoting the proliferation of Bifidobacterium longum. The results showed that N-acetylglucosamine can significantly promote the proliferation of Bifidobacterium longum, and the maximum value of the growth curve reached 0.5. Compared with D-glucuronic acid, the promotion effect was poor, and the maximum value only reached 0.21( Figure 2-C and D). In summary, N-acetylglucosamine produced by the degradation of HA by Bacteroides thetaiotaomicron is the key material basis for the targeted promotion of the proliferation of Bifidobacterium longum.

[0072] Example 4: In vivo experiments in mice demonstrated that the probiotic preparation composed of HA and Bt can target and promote the proliferation of Bifidobacterium longum in the intestine

[0073] To further validate the role of HA and B. thetaiotaomicron in targeting and promoting the growth of B. longum in mice, SPF-grade C57BL / 6J mice were inoculated with a mixed antibiotic Abx for 10 consecutive days to create pseudo-germ-free mice. The mice were then randomly divided into three groups (B. longum, B. longum + HA, and B. longum + HA + B. thetaiotaomicron), with five mice in each group. All mice were then inoculated with B. longum once on day 11. On day 14, the B. longum group was gavaged with PBS, the B. longum + HA group was gavaged with HA, and the B. longum + HA + B. thetaiotaomicron group was first inoculated with B. thetaiotaomicron and then gavaged with HA for three consecutive days.

[0074] The results of absolute quantitative analysis of RT-qPCR using species-specific primers of Bifidobacterium longum showed that it was difficult for Bifidobacterium longum to proliferate effectively in the intestine when inoculated alone. HA was supplemented at the same time, because the proliferation of Bacteroides thetaiotaomicron in the intestine would further degrade HA and promote the proliferation of Bifidobacterium longum. If HA and Bacteroides thetaiotaomicron were supplemented at the same time, the proliferation of Bifidobacterium longum could be significantly targeted and promoted. Specifically, compared with the supplementation of Bifidobacterium longum alone, the synergistic intervention of HA and Bacteroides thetaiotaomicron can increase the absolute abundance of Bifidobacterium longum in the intestine by more than 100 times ( Figure 3 ).

[0075] Example 5: HA and Bt microecological preparations targeted regulation of Bifidobacterium longum to restore bacterial flora caused by antibiotics

[0076] To further demonstrate the physiological effects of a probiotic formulation composed of HA and B. thetaiotaomicron targeting Bi. longum, we established an antibiotic-induced gut dysbiosis mouse model. Conventionally healthy mice (C57BL / 6J, with normal intestinal development and mucin production) were treated with antibiotics for five days and then randomly assigned to four different groups: oral gavage with: PBS; HA; B. thetaiotaomicron + B. longum; and HA + B. thetaiotaomicron + B. longum. As expected, all treatment groups exhibited a >2-log reduction in microbial biomass following antibiotic treatment.

[0077] Feces of mice were collected on days 0, 6, 16, and 22, and 16S rRNA absolute quantitative sequencing was performed, combined with RT-qPCR to determine the absolute abundance of specific strains of Bacteroides thetaiotaomicron and Bifidobacterium. Figure 4As shown in the figure, the absolute abundance of Bacteroides thetaiotaomicron and Bifidobacterium longum was analyzed using strain-specific primers. The results showed that when Bt+Bl was supplemented alone, the abundance of Bt could be well maintained, while the abundance of Bl was difficult to maintain. However, the simultaneous supplementation of HA+Bt+Bl could well maintain the absolute abundance of Bl at a high level. Compared with the Bt+Bl group, the absolute abundance of Bl in the HA+Bt+Bl group on day 22 was 7.8 times higher ( Figure 4 In summary, the maintenance of Bl abundance in the intestine requires the presence of HA and Bt to provide a suitable growth environment for it.

[0078] β diversity analysis of the changes in intestinal flora structure showed that antibiotic intervention significantly changed the structure of the intestinal flora. The intestinal flora structure of the PBS group on days 16 and 22 was still significantly different from that before antibiotics, while the intestinal flora structure of the HA+Bacteroides thetaiotaomicron+Bifidobacterium longum group returned to the same level as before antibiotics on day 22 ( Figure 4 -B), indicating that HA+ Bacteroides thetaiotaomicron targeted regulation of Bifidobacterium longum can quickly help reshape the intestinal flora affected by antibiotics.

[0079] Example 6: Preparation of capsule products containing the probiotic preparation of the present invention

[0080] The Bacteroides thetaiotaomicron CCFM1441 of the present invention was cultured anaerobically in BHI medium at 37°C for 24 hours, centrifuged at 5000 rpm at 4°C for 15 minutes, washed 1-2 times with sterile phosphate buffer (pH 7.2), and resuspended with a protective agent to a final concentration of 10 10 CFU / mL. Add the bacterial suspension to a 3% sodium alginate solution to make the bacterial concentration no less than 1×10 6 The mixture was stirred thoroughly to uniformly disperse the cells of Bacteroides thetaiotaomicron CCFM1441 in the sodium alginate solution to obtain a mixed solution, which was then extruded into a 2% calcium chloride solution to form micelles. The formed micelles were allowed to solidify for 30 minutes, and then filtered to collect the micelles. The collected micelles were freeze-dried for 48 hours to obtain a powder containing Bacteroides thetaiotaomicron. The powder was then loaded into a commercially available pharmaceutical capsule together with hyaluronic acid to obtain a capsule product.

[0081] The protective agent comprises: 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.

[0082] Example 7: Preparation of tablets using the probiotic preparation of the present invention

[0083] 25.7 parts by weight of a Bacteroides thetaiotaomicron CCFM1441 powder preparation prepared by a freeze-drying method, 55.0 parts by weight of hyaluronic acid, 4.5 parts by weight of a cellulose derivative, 12.0 parts by weight of sodium carboxymethyl starch, 0.8 parts by weight of talc, 1.0 parts by weight of sucrose, and 1.0 parts by weight of water were respectively weighed and mixed, and wet granules were prepared by a conventional method. The granules were then compressed using a tablet press produced by Zhongnan Pharmaceutical Machinery Factory, dried using a small drug dryer produced by Qingzhou Yikang Traditional Chinese Medicine Machinery Co., Ltd., and packaged to obtain the tablets of the present invention.

[0084] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Bacteroides thetaiotaomicron CCFM1441, characterized in that It was deposited in Guangdong Provincial Microbiological Culture Collection on October 31, 2024, with the deposit number GDMCC No.65379.

2. A microbial preparation containing the Bacteroides thetaiotaomicron CCFM1441 according to claim 1.

3. A microecological preparation, characterized in that: The invention is composed of intestinal symbiotic bacteria and prebiotics, wherein the symbiotic bacteria include the Bacteroides thetaiotaomicron CCFM1441 described in claim 1 or the microbial preparation described in claim 2, and the prebiotics include polysaccharides containing N-acetylglucosamine; optionally, the prebiotics include hyaluronic acid.

4. The microecological preparation according to claim 3, characterized in that In the microecological preparation, the amount of the Bacteroides thetaiotaomicron CCFM1441 added is not less than 1×10 6 CFU / g or 1×10 6 CFU / mL, and the added amount of the prebiotics is not less than 1%.

5. The microecological preparation according to claim 3, characterized in that The symbiotic bacteria further include a functional strain capable of degrading dietary polysaccharides and promoting the proliferation of bifidobacteria, wherein the functional strain is at least one of Bacteroides faecalis, Bacteroides ovatus and Bacteroides faecalis.

6. The probiotic preparation according to claim 5, characterized in that The added amount of the functional strain shall not exceed 50% of the added amount of Bacteroides thetaiotaomicron CCFM1441.

7. A medicine, characterized in that: The medicine contains the microecological preparation according to any one of claims 3 to 6.

8. Use of the probiotic preparation according to any one of claims 3 to 6 in the preparation of a medicine for targetedly promoting the proliferation of intestinal bifidobacteria.

9. Use of the proecological preparation according to any one of claims 3 to 6 in the preparation of a medicament for alleviating and / or treating intestinal Bifidobacterium longum damage and / or diseases associated with Bifidobacterium longum damage.

10. The use according to claim 9, characterized in that The diseases associated with damage to Bifidobacterium longum include Parkinson's syndrome, dysbacteriosis, immunosuppression, diarrhea, constipation, enteritis, aging, chronic inflammation and metabolic syndrome.

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