Isolation method and application of intestinal probiotic escherichia

Abstract

The present application relates to the technical field of microbial separation, and particularly relates to a separation method and application of intestinal probiotic escherichia; the separation method of intestinal probiotic escherichia is to take bovine large intestine content as raw material, to realize pre-screening of potential colonization strains through mucus anaerobic targeted enrichment, then to obtain resistant colonies through bile salt-short chain fatty acid stress primary screening, to scrape the colonies with metallic luster and fluorescence quenching ring through FITC-labeled mucus fluorescence indication secondary screening, and finally to obtain target strains through multiple PCR screening of negative virulence gene-positive functional gene, to realize directional separation of intestinal probiotic escherichia after purification of nutrient agar; the present application realizes targeted enrichment of intestinal colonization bacteria, environmental tolerance pressure screening and mucus binding visualization identification, solves the problems of strong randomness of traditional probiotic screening and inability to predict colonization potential, and provides a new strategy of efficient and safe probiotic strain screening for development of functional food and microecological preparation.

Description

Technical Field

[0001] This invention relates to the field of microbial isolation technology, specifically to a method for isolating and applying beneficial intestinal Escherichia coli. Background Technology

[0002] In the field of intestinal microecological preparation development, Escherichia coli, as a typical representative of the intestinal flora, plays a crucial role. The isolation and screening of specific probiotic strains directly determines the reliability of subsequent probiotic efficacy and the safety of application. Escherichia coli is widely distributed in the intestines of humans and animals. Although most strains are harmless, some serotypes, such as O157:H7, are pathogenic and can cause serious foodborne illnesses. Therefore, precise screening of probiotic Escherichia coli from the complex intestinal flora has become a core element of refined intestinal health intervention. Traditional methods for isolating probiotic Escherichia coli rely on selective culture media for initial screening and morphological observation. This screening model based on a single phenotypic characteristic has inherent defects. MacConkey medium can only distinguish lactose fermentation phenotypes, and EMB medium can only observe metallic luster characteristics. Although the strains obtained by such methods belong to the Escherichia genus, their intestinal colonization potential cannot be predicted, and there is a lack of effective identification of potential virulence genes. The screening process is highly random and lacks specific targeting.

[0003] In recent years, researchers have attempted to introduce molecular biology identification into the probiotic screening process in order to improve strain safety. Patent CN112961809A discloses a selective isolation and culture method for Shiga toxin-producing Escherichia coli, which uses acid-treated culture medium to improve the isolation rate of specific strains. However, this method depends on bacterial growth on the culture medium, and it takes several hours to several days to observe colony growth results, resulting in a slow output speed. Moreover, the use of specific culture medium cannot completely avoid competition or inhibition with other bacteria, and non-probiotic strains can still grow under traditional culture conditions. In complex samples, it is impossible to effectively distinguish morphologically similar bacteria, leading to misjudgment and cross-contamination during the isolation process. Existing technical solutions are still limited to using PCR detection only as a final verification method and tolerance testing only as an independent step, lacking the ability to make early predictions on the intestinal adaptability of strains from the functional mechanism level. Furthermore, safety screening and functional verification are separated in the screening process, resulting in a long screening cycle and low hit rate. Therefore, how to construct a multi-level synergistic screening system that combines targeted enrichment, environmental stress screening, and functional gene verification, and achieve integrated prediction of colonization potential, tolerance assessment, and safety screening in the early stages of isolation, has become a core technological bottleneck that urgently needs to be overcome in the development of functional microecological preparations.

[0004] To address the above problems, the present invention provides a solution. Summary of the Invention

[0005] The purpose of this invention is to provide a method and application for isolating beneficial Escherichia coli from the intestine, which can effectively isolate beneficial Escherichia coli from the intestine.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for isolating probiotic Escherichia coli from the intestines uses bovine large intestine contents as raw material. Potential colonizing strains are pre-screened through anaerobic targeted enrichment of mucin. Subsequently, tolerant colonies are obtained through bile salt-short-chain fatty acid stress screening. Then, colonies with metallic luster and fluorescence quenching zones are scraped off using FITC-labeled mucin fluorescence indicators for secondary screening. Finally, target strains are obtained through multiplex PCR screening with negative virulence genes and positive functional genes. After purification with nutrient agar, the targeted isolation of probiotic Escherichia coli from the intestines is achieved.

[0007] A method for isolating beneficial intestinal Escherichia coli includes the following steps: S1: Take a fresh bovine large intestine tissue sample and place it in a sterile container. Sterilize it with ultraviolet light in a biosafety cabinet for 30 minutes. Weigh 10g of bovine large intestine contents and add it to 90ml of modified M9 basal medium. Incubate in an anaerobic incubator at 37℃ for 48 hours to obtain an enriched bacterial culture medium. S2: Dilute the enriched bacterial culture medium sequentially to 10 using sterile physiological saline. -3 10 -4 10 -5 Acetic acid, propionic acid and butyric acid were mixed and homogenized for 10 min to obtain short-chain fatty acid mixtures with a mass ratio of 5:2:3. Then, 100 μL of the enriched bacterial culture medium of different gradients was spread on modified MacConkey agar plates containing 0.4 wt% porcine bile salts and 10 mM short-chain fatty acid mixtures. The plates were spread evenly with a spreader and then inverted in a 37°C incubator for aerobic incubation for 24 h. S3: Dissolve mucin in 0.1M carbonate buffer at pH 9, add FITC (fiber sulfide), with a mass ratio of FITC to mucin of 1:50, stir at 4℃ in the dark for 8 hours, dialyze to remove free FITC, and freeze dry to obtain FITC-labeled mucin. After culture, observe modified MacConkey agar plates, scrape the pink colonies growing on the plates with a sterile inoculation loop, transfer them to EP tubes containing 1ml of sterile physiological saline, vortex for 1min to prepare a bacterial suspension, weigh 38g of EMB agar powder and add it to 1L of pure water, heat and stir until completely dissolved, then add 1g of FITC-labeled mucin, mix well and put it into an autoclave for autoclaving at 121℃ for 15min. After sterilization, cool the culture medium to 50℃, pour EMB agar into sterile petri dishes in the operating table, and allow it to cool and solidify naturally to obtain fluorescently indicated EMB agar medium. S4: Use an inoculation loop to pick up the bacterial suspension, streak it onto fluorescent indicator EMB agar medium, and incubate it in a 37°C incubator for 24 hours. After the incubation is complete, observe it under a UV lamp. Use a sterile inoculation loop to scrape off colonies with a purplish-black metallic luster and a fluorescent quenching zone around them, and transfer them to sterile physiological saline. S5: Genomic DNA was extracted from the colonies obtained in S5 using a bacterial genomic DNA extraction kit and detected using a multiplex PCR system. The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min, followed by 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 35 cycles, and finally 72℃ final extension for 10 min. S6: The PCR products were detected by agarose gel electrophoresis. Strains that were negative for all virulence genes stx1, stx2, eaeA, elt, and est, and positive for functional genes colicin, mucV, and fimH were selected as candidate strains of enteroprobiotic Escherichia coli. 38g of nutrient agar powder was weighed and added to 1L of pure water. The mixture was heated and stirred until completely dissolved. The mixture was sterilized at 121℃ for 15min. After cooling to 45℃, the mixture was poured into plates. The candidate strains of enteroprobiotic Escherichia coli were streaked onto nutrient agar plates and incubated at 37℃ for 24h. The streaking was repeated 3 times until a single colony with consistent morphology was obtained. The single colony was scraped off and transferred to a cryovial containing 15% glycerol. The colonies were stored at -80℃ for long-term storage to obtain enteroprobiotic Escherichia coli.

[0008] Furthermore, the modified M9 basal culture medium described in step S1 includes 12.8g Na2HPO4·7H2O, 3g KH2PO4, 0.5g NaCl, 1g NH4Cl, 0.24g MgSO4, 0.011g CaCl4 and 5g mucin, diluted with distilled water to 1L, with a pH of 7.2; Furthermore, the PCR reaction system described in step S5 includes 12.5 μL 2×Taq PCR Master Mix, 2 μL virulence gene primer mixture, 2 μL functional gene primer mixture, 2 μL template DNA, and sterile double-distilled water to a final volume of 25 μL; the virulence gene primer mixture includes stx1 primer, stx2 primer, eaeA primer, elt primer, and est primer; the functional gene primer mixture includes colicin primer, mucV primer, and fimH primer. Furthermore, the present invention provides a compound freeze-dried powder of intestinal probiotic Escherichia coli, wherein the compound freeze-dried powder of intestinal probiotic Escherichia coli uses intestinal probiotic Escherichia coli as an active ingredient. Furthermore, a method for preparing a compound lyophilized powder of intestinal probiotic Escherichia coli includes the following steps: A1: Inoculate probiotic Escherichia coli into seed culture medium and culture at 37℃ with shaking for 12 h to obtain seed liquid. Then, inoculate the seed liquid into fermentation medium, where the mass ratio of seed liquid to fermentation medium is 1:20. High-density fermentation culture is carried out at 37℃ for 24 h. During the fermentation process, the pH is controlled at 7.2 and the dissolved oxygen concentration is 30%. A2: After fermentation, centrifuge the fermentation broth at 8000 rpm for 10 min at 4℃, and collect the cell precipitate and fermentation supernatant separately. Wash the cell precipitate three times with sterile physiological saline, resuspend it in a cryoprotectant, and adjust the bacterial concentration to 1×10⁻⁶. 10 -1×10 11 CFU / ml was used to obtain a bacterial suspension; A3: Add the bacterial suspension and fermentation supernatant to the reaction vessel, followed by the addition of fructooligosaccharides. The volume ratio of bacterial suspension, fermentation supernatant, and fructooligosaccharides is 10:10:1. Set the stirrer speed to 300 rpm and stir for 30 minutes. After stirring and mixing, dispense the mixture into freeze-drying trays and perform vacuum freeze-drying. The vacuum freeze-drying process includes: pre-freezing at -40℃ for 3 hours, primary drying at -20℃ for 24 hours, and desorption drying at 20℃ for 8 hours. After vacuum freeze-drying, the intestinal probiotic Escherichia coli compound freeze-dried powder is obtained.

[0009] Furthermore, the fermentation medium described in step A1 comprises 15 g / L tryptone, 10 g / L yeast extract, 5 g / L glucose, 2 g / L K2HPO4, 0.5 g / L MgSO4·7H2O, 0.05 g / L MnSO4·H2O and 1 mL / L Tween-80, with a pH of 7.2; Furthermore, the protective agent described in step A2 consists of the following components by weight percentage: 10% skim milk powder, 5% trehalose, 1% monosodium glutamate, 1% glycerol and 83% sterile distilled water; In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are as follows: 1. This invention creates a novel three-level targeted screening model: mucin-targeted enrichment, initial screening under stress, and secondary screening using fluorescence indicators. It achieves targeted enrichment of strains with intestinal colonization potential through anaerobic culture with mucin as the sole carbon source. It uses bile salts and short-chain fatty acids as dual stress to simulate the real intestinal environment for initial screening of tolerance. Finally, it uses FITC-labeled mucin-indicating medium to visualize the mucin-binding ability of strains for secondary screening. This invention solves the key problems of high randomness, unpredictable colonization potential, and low screening efficiency in traditional probiotic screening. 2: This invention uses bovine large intestine contents as raw material to construct an isolation system integrating phenotypic screening and genotypic verification. Each screening step works in precise synergy in terms of mechanism of action: mucin-targeted enrichment is responsible for the pre-screening of potential colonizing strains; bile salt-short chain fatty acid stress is responsible for the stress screening of intestinal environmental tolerance; FITC fluorescence indication is responsible for the visual identification of mucin binding ability; and multiplex PCR detection is responsible for the molecular verification of virulence gene negative to functional gene positive. This multi-target, cascaded design achieves precise control over the entire process of probiotic Escherichia coli from colonization potential to safety in the screening logic. The obtained strains are significantly better than traditional random isolation methods in terms of gastrointestinal tolerance, intestinal adhesion ability, and safety. 3. This invention, through the innovative integration of targeted enrichment culture, environmental stress screening, and molecular biological identification, successfully integrates intestinal microecological simulation technology, fluorescence indicator technology, and multiplex PCR detection technology into the same technical system. The isolated probiotic Escherichia coli were all negative in virulence gene screening and carried multiple colonization-related functional genes. The survival rate in gastrointestinal tolerance tests all exceeded 75%, which can effectively restore the intestinal microbiota balance of patients with dysbiosis. This provides a new strategy for screening probiotic strains that is efficient, safe, and clearly targeted for the development of functional foods and microecological preparations. Detailed Implementation

[0010] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific embodiments.

[0011] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0012] Example 1 The fresh bovine large intestine tissue sample in this embodiment was obtained from healthy yellow cattle in Fuyang, Anhui Province. 1: Take a fresh bovine large intestine tissue sample and place it in a sterile container. Sterilize it with ultraviolet light in a biosafety cabinet for 30 minutes. Weigh 10g of bovine large intestine contents and add it to 90ml of modified M9 basal medium. Incubate in an anaerobic incubator at 37℃ for 48 hours to obtain an enriched bacterial culture medium. 2: Use sterile physiological saline to sequentially dilute the enriched bacterial culture medium to 10. -3 10 -4 10 -5Three gradients were used to homogenize 50g acetic acid, 20g propionic acid and 30g butyric acid for 10 min to obtain short-chain fatty acid mixtures. Then, 100 μL of the enriched bacterial culture medium of different gradients was spread on modified MacConkey agar plates containing 0.4 wt% porcine bile salt and 10 mM short-chain fatty acid mixture. The plates were spread evenly with a spreader and then inverted in a 37°C incubator for aerobic incubation for 24 h. 3: Dissolve 50g of mucin in 0.1M carbonate buffer (pH 9), add 1g of FITC, stir at 4℃ in the dark for 8 hours, dialyze to remove free FITC, and freeze-dry to obtain FITC-labeled mucin. After culture, observe the modified MacConkey agar plate, scrape the pink colonies growing on the plate with a sterile inoculation loop, transfer them to an EP tube containing 1ml of sterile physiological saline, vortex for 1min to prepare a bacterial suspension, weigh 38g of EMB agar powder and add it to 1L of pure water, heat and stir until completely dissolved, then add 1g of FITC-labeled mucin, mix well and put it into an autoclave for autoclaving at 121℃ for 15min. After sterilization, cool the culture medium to 50℃, pour the EMB agar into a sterile petri dish in the operating table, and allow it to cool and solidify naturally to obtain fluorescently indicated EMB agar medium. 4: Use an inoculation loop to dip the bacterial suspension and streak it onto fluorescent indicator EMB agar medium. Incubate at 37°C for 24 hours. After incubation, observe under UV light. Use a sterile inoculation loop to scrape off colonies with a purplish-black metallic luster and a fluorescent quenching zone around them, and transfer them to sterile physiological saline. 5: Genomic DNA was extracted from the colonies obtained from S5 using a bacterial genomic DNA extraction kit and detected using a multiplex PCR system. The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min, followed by 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 35 cycles, and finally 72℃ final extension for 10 min. 6. The PCR products were detected by agarose gel electrophoresis. Strains that were negative for all virulence genes stx1, stx2, eaeA, elt, and est, and positive for functional genes colicin, mucV, and fimH were selected as candidate strains of enteroprobiotic Escherichia coli. 38g of nutrient agar powder was weighed and added to 1L of pure water. The mixture was heated and stirred until completely dissolved, sterilized at 121℃ for 15min, cooled to 45℃, and poured into plates. The candidate strains of enteroprobiotic Escherichia coli were streaked onto nutrient agar plates and incubated at 37℃ for 24h. The streaking was repeated 3 times until a single colony with consistent morphology was obtained. The single colony was scraped off and transferred to a cryovial containing 15% glycerol and stored at -80℃ for long-term storage to obtain the enteroprobiotic Escherichia coli isolated in Example 1.

[0013] Example 2 The fresh bovine large intestine tissue sample in this embodiment was obtained from healthy dairy cows in Dali, Yunnan Province. 1: Take a fresh bovine large intestine tissue sample and place it in a sterile container. Sterilize it with ultraviolet light in a biosafety cabinet for 30 minutes. Weigh 10g of bovine large intestine contents and add it to 90ml of modified M9 basal medium. Incubate in an anaerobic incubator at 37℃ for 48 hours to obtain an enriched bacterial culture medium. 2: Use sterile physiological saline to sequentially dilute the enriched bacterial culture medium to 10. -3 10 -4 10 -5 Three gradients were used to homogenize 50g acetic acid, 20g propionic acid and 30g butyric acid for 10 min to obtain short-chain fatty acid mixtures. Then, 100 μL of the enriched bacterial culture medium of different gradients was spread on modified MacConkey agar plates containing 0.4 wt% porcine bile salt and 10 mM short-chain fatty acid mixture. The plates were spread evenly with a spreader and then inverted in a 37°C incubator for aerobic incubation for 24 h. 3: Dissolve 50g of mucin in 0.1M carbonate buffer (pH 9), add 1g of FITC, stir at 4℃ in the dark for 8 hours, dialyze to remove free FITC, and freeze-dry to obtain FITC-labeled mucin. After culture, observe the modified MacConkey agar plate, scrape the pink colonies growing on the plate with a sterile inoculation loop, transfer them to an EP tube containing 1ml of sterile physiological saline, vortex for 1min to prepare a bacterial suspension, weigh 38g of EMB agar powder and add it to 1L of pure water, heat and stir until completely dissolved, then add 1g of FITC-labeled mucin, mix well and put it into an autoclave for autoclaving at 121℃ for 15min. After sterilization, cool the culture medium to 50℃, pour the EMB agar into a sterile petri dish in the operating table, and allow it to cool and solidify naturally to obtain fluorescently indicated EMB agar medium. 4: Use an inoculation loop to dip the bacterial suspension and streak it onto fluorescent indicator EMB agar medium. Incubate at 37°C for 24 hours. After incubation, observe under UV light. Use a sterile inoculation loop to scrape off colonies with a purplish-black metallic luster and a fluorescent quenching zone around them, and transfer them to sterile physiological saline. 5: Genomic DNA was extracted from the colonies obtained from S5 using a bacterial genomic DNA extraction kit and detected using a multiplex PCR system. The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min, followed by 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 35 cycles, and finally 72℃ final extension for 10 min. 6. The PCR products were detected by agarose gel electrophoresis. Strains that were negative for all virulence genes stx1, stx2, eaeA, elt, and est, and positive for functional genes colicin, mucV, and fimH were selected as candidate strains of enteroprobiotic Escherichia coli. 38g of nutrient agar powder was weighed and added to 1L of pure water. The mixture was heated and stirred until completely dissolved, sterilized at 121℃ for 15min, cooled to 45℃, and poured into plates. The candidate strains of enteroprobiotic Escherichia coli were streaked onto nutrient agar plates and incubated at 37℃ for 24h. The streaking was repeated 3 times until a single colony with consistent morphology was obtained. The single colony was scraped off and transferred to a cryovial containing 15% glycerol and stored at -80℃ for long-term storage to obtain the enteroprobiotic Escherichia coli isolated in Example 2.

[0014] Example 3 1: The intestinal probiotic Escherichiae isolated in Example 1 were inoculated into seed culture medium and cultured at 37°C with shaking for 12 hours to obtain seed liquid. Subsequently, 50g of seed liquid was inoculated into 1000g of fermentation medium and fermented at high density at 37°C for 24 hours. During the fermentation process, the pH was controlled at 7.2 and the dissolved oxygen concentration was controlled at 30%. 2. After fermentation, the fermentation broth was centrifuged at 8000 rpm for 10 min at 4℃. The bacterial precipitate and fermentation supernatant were collected separately. The bacterial precipitate was washed three times with sterile physiological saline, resuspended in a cryoprotectant, and the bacterial concentration was adjusted to 1×10⁻⁶. 10 CFU / ml was used to obtain a bacterial suspension; 3: Add 100ml of bacterial suspension and 100ml of fermentation supernatant to the reaction vessel, then add 10ml of oligofructose. Set the stirrer speed to 300rpm and stir for 30min. After stirring and mixing, dispense into freeze-drying trays and perform vacuum freeze-drying. The vacuum freeze-drying includes pre-freezing at -40℃ for 3h, primary drying at -20℃ for 24h, and desorption drying at 20℃ for 8h. After vacuum freeze-drying, the intestinal probiotic Escherichia coli compound freeze-dried powder prepared in Example 3 is obtained.

[0015] Example 4 1: The intestinal probiotic Escherichiae isolated in Example 2 were inoculated into seed culture medium and cultured at 37°C with shaking for 12 hours to obtain seed liquid. Subsequently, 50g of seed liquid was inoculated into 1000g of fermentation medium and fermented at high density at 37°C for 24 hours. During the fermentation process, the pH was controlled at 7.2 and the dissolved oxygen concentration was controlled at 30%. 2. After fermentation, the fermentation broth was centrifuged at 8000 rpm for 10 min at 4℃. The bacterial precipitate and fermentation supernatant were collected separately. The bacterial precipitate was washed three times with sterile physiological saline, resuspended in a cryoprotectant, and the bacterial concentration was adjusted to 1×10⁻⁶. 11 CFU / ml was used to obtain a bacterial suspension; 3: Add 100ml of bacterial suspension and 100ml of fermentation supernatant to the reaction vessel, then add 10ml of fructooligosaccharide. Set the stirrer speed to 300rpm and stir for 30min. After stirring and mixing, dispense into freeze-drying trays and perform vacuum freeze-drying. The vacuum freeze-drying includes pre-freezing at -40℃ for 3h, primary drying at -20℃ for 24h, and desorption drying at 20℃ for 8h. After vacuum freeze-drying, the intestinal probiotic Escherichia coli compound freeze-dried powder prepared in Example 4 is obtained.

[0016] Comparative Example 1 Example 1 of Chinese Patent Publication No. CN109497149A was selected as Comparative Example 1.

[0017] Comparative Example 2 Example 2 of Chinese Patent Publication No. CN102229898A was selected as Comparative Example 2.

[0018] Experiment on improvement of intestinal discomfort The intestinal probiotic Escherichia coli compound freeze-dried powder prepared in Examples 3 and 4 were respectively used to prepare yogurt. The yogurt was composed of the following percentage ingredients: 2% intestinal probiotic Escherichia coli compound freeze-dried powder, 2% lactic acid bacteria, 1.5% bifidobacteria, 2.5% strawberry chunks, 2% vitamin C, 0.5% potassium sorbate, 0.5% prebiotics and 89% pure milk. Eighty patients with intestinal discomfort were recruited and randomly divided into four groups of 20 each. Each group consumed yogurt prepared using Comparative Example 1, yogurt prepared using Example 3, yogurt prepared using Example 4, and blank yogurt (containing only lactic acid bacteria and bifidobacteria) without the addition of Escherichia coli freeze-dried powder. They drank the yogurt for 14 consecutive days, twice a day, 240 ml each time. The improvement of intestinal discomfort after 14 days was recorded. Table 1. Improvement of intestinal discomfort in volunteers

[0019] As shown in Table 1, the yogurt prepared using the intestinal probiotic Escherichia coli compound freeze-dried powder prepared in Examples 3 and 4 can significantly improve the intestinal discomfort symptoms of volunteers. The diarrhea improvement rate in the Example 3 group reached 90.9%, and the constipation improvement rate reached 88.9%; the diarrhea improvement rate in the Example 4 group reached 90.0%, and the constipation improvement rate reached 100%, both of which were significantly better than those in Comparative Example 1 and the blank control group.

[0020] Gut microbiota modulation experiment The intestinal probiotic Escherichia coli compound freeze-dried powders prepared in Examples 3 and 4 were transferred to MacConkey plates and blood agar plates, respectively, and incubated at 37°C for 18 hours. After the incubation was completed, intact colonies were selected and inoculated into 5 mL of broth medium and incubated at 37°C for 6 hours. Subsequently, different concentration gradients of bacterial suspensions were prepared using broth medium, as shown in Table 2. Table 2. Relationship between the amount of bacterial suspension used in the control and experimental phases.

[0021] Treatment efficacy for patients with grade II dysbiosis Seventy-five patients with grade II gut microbiota dysbiosis were recruited and randomly divided into three groups of 25 each. Experimental group 1 used bacterial solution from Example 3-1, experimental group 2 used bacterial solution from Example 4-1, and the control group used bacterial solution from Comparative Example 2-1. Enemas were administered once daily for three consecutive days, and then changes in gut microbiota were detected. The average value of the results was taken. Table 3. Changes in gut microbiota in patients with grade II dysbiosis (1g CFU / g stool)

[0022] Treatment efficacy for patients with grade III dysbiosis Seventy-five patients with grade III gut microbiota dysbiosis were recruited and randomly divided into three groups of 25 patients each. Experimental group 1 used the bacterial suspension from Example 3-2, experimental group 2 used the bacterial suspension from Example 4-2, and the control group used the bacterial suspension from Comparative Example 2-2. Enemas were administered once daily for three consecutive days, and changes in gut microbiota were subsequently assessed. Table 4. Changes in gut microbiota in patients with grade III dysbiosis (1g CFU / g stool)

[0023] Treatment efficacy for patients with grade II gut microbiota dysbiosis Seventy-five patients with grade II gut microbiota dysbiosis were recruited and randomly divided into three groups of 25 each. Experimental group 1 used the bacterial suspension from Example 3-3, experimental group 2 used the bacterial suspension from Example 4-3, and the control group used the bacterial suspension from Comparative Example 2-3. Enemas were administered once daily for three consecutive days, followed by monitoring of changes in gut microbiota. Table 5. Changes in gut microbiota in patients with grade II dysbiosis (1g CFU / g stool)

[0024] Treatment efficacy for patients with grade III gut microbiota dysbiosis Seventy-five patients with grade III gut microbiota dysbiosis were recruited and randomly divided into three groups of 25 patients each. Experimental group 1 used bacterial suspensions from Examples 3-4, experimental group 2 used bacterial suspensions from Examples 4-4, and the control group used bacterial suspensions from Comparative Examples 2-4. Enemas were administered once daily for three consecutive days, and changes in gut microbiota were subsequently assessed. Table 6. Changes in gut microbiota in patients with grade III dysbiosis (1g CFU / g stool)

[0025] Analysis of Table 3-6 shows that: The bacterial enema solutions prepared in Examples 3 and 4 significantly restored the intestinal flora. After treatment, the total bacterial count, Bifidobacteria, Lactobacillus, and Enterobacteriaceae all increased significantly, approaching normal levels. The treatment effect of the Example 3 group was significantly better than that of the Comparative Example 2 group. Taking a patient with grade III dysbiosis as an example, after treatment with Examples 3-4, the total bacterial count recovered from 7.52 lgCFU / g to 20.43 lgCFU / g, and the Bifidobacteria count recovered from 2.18 lgCFU / g to 9.73 lgCFU / g; while after treatment with Comparative Examples 2-4, the total bacterial count only recovered to 19.38 lgCFU / g, and the Bifidobacteria count only recovered to 9.24 lgCFU / g. Examples 3 and 4 showed significant therapeutic effects on different degrees of dysbiosis and gut microbiota disorder (grade II and grade III), indicating that the gut probiotic Escherichia coli screened by the present invention has good broad-spectrum applicability. The therapeutic effect of Example 4 was slightly better than that of Example 3. Enema treatment with the bacterial solution prepared by the gut probiotic Escherichia coli isolated by the method of the present invention can effectively restore the balance of gut microbiota and has a good therapeutic effect on dysbiosis and gut microbiota disorder.

[0026] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for isolating beneficial intestinal Escherichia coli, characterized in that, Using bovine large intestine contents as raw material, potential colonizing strains were pre-screened through anaerobic targeted enrichment of mucin. Subsequently, tolerant colonies were obtained through bile salt-short chain fatty acid stress screening. Then, colonies with metallic luster and fluorescence quenching zone were scraped out using FITC-labeled mucin fluorescence indicator for secondary screening. Finally, target strains were obtained through multiplex PCR screening with negative virulence genes and positive functional genes. After purification with nutrient agar, the targeted isolation of beneficial intestinal Escherichia coli was achieved.

2. The method for isolating beneficial intestinal Escherichia coli according to claim 1, characterized in that, Includes the following steps: S1: Take a fresh bovine large intestine tissue sample and place it in a sterile container. Sterilize it with ultraviolet light in a biosafety cabinet for 30 minutes. Weigh 10g of bovine large intestine contents and add it to 90ml of modified M9 basal medium. Incubate in an anaerobic incubator at 37℃ for 48 hours to obtain an enriched bacterial culture medium. S2: Dilute the enriched bacterial culture medium sequentially to 10 using sterile physiological saline. -3 10 -4 10 -5 Acetic acid, propionic acid and butyric acid were mixed and homogenized for 10 min to obtain short-chain fatty acid mixtures with a mass ratio of 5:2:

3. Then, 100 μL of the enriched bacterial culture medium of different gradients was spread on modified MacConkey agar plates containing 0.4 wt% porcine bile salts and 10 mM short-chain fatty acid mixtures. The plates were spread evenly with a spreader and then inverted in a 37°C incubator for aerobic incubation for 24 h. S3: Dissolve mucin in 0.1M carbonate buffer at pH 9, add FITC (fiber sulfide), with a mass ratio of FITC to mucin of 1:50, stir at 4℃ in the dark for 8 hours, dialyze to remove free FITC, and freeze dry to obtain FITC-labeled mucin. After culture, observe modified MacConkey agar plates, scrape the pink colonies growing on the plates with a sterile inoculation loop, transfer them to EP tubes containing 1ml of sterile physiological saline, vortex for 1min to prepare a bacterial suspension, weigh 38g of EMB agar powder and add it to 1L of pure water, heat and stir until completely dissolved, then add 1g of FITC-labeled mucin, mix well and put it into an autoclave for autoclaving at 121℃ for 15min. After sterilization, cool the culture medium to 50℃, pour EMB agar into sterile petri dishes in the operating table, and allow it to cool and solidify naturally to obtain fluorescently indicated EMB agar medium. S4: Use an inoculation loop to pick up the bacterial suspension, streak it onto fluorescent indicator EMB agar medium, and incubate it in a 37°C incubator for 24 hours. After the incubation is complete, observe it under a UV lamp. Use a sterile inoculation loop to scrape off colonies with a purplish-black metallic luster and a fluorescent quenching zone around them, and transfer them to sterile physiological saline. S5: Genomic DNA was extracted from the colonies obtained in S5 using a bacterial genomic DNA extraction kit and detected using a multiplex PCR system. The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min, followed by 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 35 cycles, and finally 72℃ final extension for 10 min. S6: The PCR products were detected by agarose gel electrophoresis. Strains that were negative for all virulence genes stx1, stx2, eaeA, elt, and est, and positive for functional genes colicin, mucV, and fimH were selected as candidate strains of enteroprobiotic Escherichia coli. 38g of nutrient agar powder was weighed and added to 1L of pure water. The mixture was heated and stirred until completely dissolved. The mixture was sterilized at 121℃ for 15min. After cooling to 45℃, the mixture was poured into plates. The candidate strains of enteroprobiotic Escherichia coli were streaked onto nutrient agar plates and incubated at 37℃ for 24h. The streaking was repeated 3 times until a single colony with consistent morphology was obtained. The single colony was scraped off and transferred to a cryovial containing 15% glycerol. The colonies were stored at -80℃ for long-term storage to obtain enteroprobiotic Escherichia coli.

3. The method for isolating beneficial intestinal Escherichia coli according to claim 2, characterized in that, The modified M9 basal culture medium described in step S1 includes 12.8g Na2HPO4·7H2O, 3g KH2PO4, 0.5g NaCl, 1g NH4Cl, 0.24g MgSO4, 0.011g CaCl4 and 5g mucin, diluted to 1L with distilled water, and has a pH of 7.

2.

4. The method for isolating beneficial intestinal Escherichia coli according to claim 2, characterized in that, The PCR reaction system described in step S5 includes 12.5 μL 2×Taq PCR Master Mix, 2 μL virulence gene primer mixture, 2 μL functional gene primer mixture, 2 μL template DNA, and sterile double-distilled water to a final volume of 25 μL. The virulence gene primer mixture includes stx1 primer, stx2 primer, eaeA primer, elt primer, and est primer. The functional gene primer mixture includes colicin primer, mucV primer, and fimH primer.

5. A compound freeze-dried powder of intestinal probiotic Escherichia coli, characterized in that, It uses beneficial Escherichia coli in the gut as the active ingredient.

6. The method for preparing a compound lyophilized powder of intestinal probiotic Escherichia coli according to claim 5, characterized in that, Includes the following steps: A1: Inoculate probiotic Escherichia coli into seed culture medium and culture at 37℃ with shaking for 12 h to obtain seed liquid. Then, inoculate the seed liquid into fermentation medium, where the mass ratio of seed liquid to fermentation medium is 1:

20. High-density fermentation culture is carried out at 37℃ for 24 h. During the fermentation process, the pH is controlled at 7.2 and the dissolved oxygen concentration is 30%. A2: After fermentation, centrifuge the fermentation broth at 8000 rpm for 10 min at 4℃, and collect the cell precipitate and fermentation supernatant separately. Wash the cell precipitate three times with sterile physiological saline, resuspend it in a cryoprotectant, and adjust the bacterial concentration to 1×10⁻⁶. 10 -1×10 11 CFU / ml was used to obtain a bacterial suspension; A3: Add the bacterial suspension and fermentation supernatant to the reaction vessel, followed by the addition of fructooligosaccharides. The volume ratio of bacterial suspension, fermentation supernatant, and fructooligosaccharides is 10:10:

1. Set the stirrer speed to 300 rpm and stir for 30 minutes. After stirring and mixing, dispense the mixture into freeze-drying trays and perform vacuum freeze-drying. The vacuum freeze-drying process includes: pre-freezing at -40℃ for 3 hours, primary drying at -20℃ for 24 hours, and desorption drying at 20℃ for 8 hours. After vacuum freeze-drying, the intestinal probiotic Escherichia coli compound freeze-dried powder is obtained.

7. The method for preparing a compound freeze-dried powder of intestinal probiotic Escherichia coli according to claim 6, characterized in that, The fermentation medium described in step A1 comprises 15 g / L tryptone, 10 g / L yeast extract, 5 g / L glucose, 2 g / L K2HPO4, 0.5 g / L MgSO4·7H2O, 0.05 g / L MnSO4·H2O, and 1 mL / L Tween-80, with a pH of 7.

2.

8. The method for preparing a compound lyophilized powder of intestinal probiotic Escherichia coli according to claim 6, characterized in that, The protective agent described in step A2 consists of the following components by weight percentage: 10% skim milk powder, 5% trehalose, 1% monosodium glutamate, 1% glycerol and 83% sterile distilled water.

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