Probiotic escherichia coli with stress resistance and application thereof

Abstract

The invention discloses probiotic escherichia coli with stress resistance and application of the probiotic escherichia coli, and belongs to the technical field of microorganisms. The probiotic escherichia coli provided by the invention is named as the probiotic escherichia coli (Escherichia coli Nissle1917) EcN223-YTP, the probiotic escherichia coli is preserved in China Center for Type Culture Collection in Wuhan on August 16, 2024, and the preservation number is CCTCC NO: M 20241801. Compared with the existing EcN strain, the probiotic escherichia coli EcN223-YTP provided by the invention has stronger growth activity, still keeps rapid growth in a stable period, can rapidly increase the viable count, and obviously shortens the fermentation period. The strain has stronger stress resistance characteristics including high temperature resistance, low temperature resistance, low pH, high bile salt, low oxygen, bacteriophage infection and the like, so that the strain has more stable activity and longer shelf life, is easier to preserve and is suitable for development and application.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a probiotic Escherichia coli with stress resistance and application thereof. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance the understanding of the overall background of the invention and should not be necessarily regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Probiotic Escherichia coli Nissle 1917 (EcN) has shown great potential in medical and industrial applications due to its ability to improve intestinal health, regulate flora balance, and reduce inflammatory responses. Studies have shown that EcN can improve the immune defense function of the intestinal mucosa through its probiotic properties, thereby promoting intestinal health. The probiotic function of EcN is associated with its regulation of intestinal microorganisms and the immune response caused by cytokines in the intestine. EcN plays an important immune function in the intestine by regulating the expression of antimicrobial peptides, increasing the secretion of immunoglobulin A (IgA) and mucins, and promoting anti-inflammatory immune responses. EcN can also play an important role in enhancing the tight junctions of porcine intestinal epithelial cells and improving the mucosal barrier by upregulating the expression of tight junction proteins zonula occludens and promoting the upregulation and redistribution of tight junction proteins. Oral administration of EcN can significantly reduce the diarrhea rate of weaned piglets and competitively hinder the invasion of Salmonella, and can also effectively prevent the infection of toxin-producing Escherichia coli. In addition, EcN has the ability to efficiently colonize tumors and can be quickly cleared in normal tissues, so it has attracted much attention in cancer bacterial therapy. At the same time, EcN also has the potential to be used as a live bacterial vaccine carrier, especially in the treatment of diseases related to intestinal health, showing significant application prospects.

[0004] However, the existing EcN has poor stress resistance and a low survival rate in harsh environments (high and low temperatures, low pH, high bile salts, phage infection, etc.). Therefore, it is an urgent problem to develop probiotic Escherichia coli with excellent stress resistance to improve its survival rate and colonization ability in harsh environments. Summary of the invention

[0005] In view of this, the present invention provides a probiotic Escherichia coli with stress resistance and its application. The probiotic Escherichia coli provided by the present invention has stronger stress resistance than the existing EcN strain, so that it has more stable activity and longer shelf life, is easier to preserve and is suitable for development and application.

[0006] In the first aspect, the present invention provides a probiotic Escherichia coli, named probiotic Escherichia coli (Escherichiacoli Nissle 1917) EcN223-YTP, which was preserved in the China Type Culture Collection, Wuhan on August 16, 2024, with the preservation number of CCTCC NO: M 20241801.

[0007] The probiotic Escherichia coli EcN223-YTP described in the present invention is isolated from the intestinal contents of yaks in the Qinghai-Tibet Plateau and collected from healthy yaks in Nagqu, Tibet. It is Gram-negative and is identified as Escherichia coli Nissle 1917 (EcN) by comparing the 16S rRNA gene sequence with the NCBI database. The genome of the EcN223-YTP strain is a circular genome with a size of 5048102 bp, a GC content of 50.58%, and 4772 encoded functional genes.

[0008] In a second aspect, the present invention provides a probiotic agent, the raw materials of which include the probiotic Escherichia coli described in the first aspect.

[0009] Furthermore, the dosage form of the bacterial agent includes liquid, powder and granule, which is not limited in the present invention; the active ingredients may include only the probiotic Escherichia coli described in the present invention, or may include other probiotics and / or prebiotics with physiological activity, which is also not specifically limited in the present invention.

[0010] In a third aspect, the present invention provides a fermented product produced by fermentation using the probiotic Escherichia coli provided in the first aspect or the probiotic agent provided in the second aspect as a starter.

[0011] The fermented product is a fermented food or feed. The fermented food is a fermented dairy product, a fermented bean product or a fermented fruit and vegetable product. The fermented dairy product includes but is not limited to yogurt, sour cream, and cheese. The fermented bean product includes but is not limited to soy milk, fermented black beans, and bean paste. The fruits and vegetables in the fermented fruit and vegetable product include but are not limited to cucumbers, carrots, beets, celery, and cabbage.

[0012] In a fourth aspect, the present invention provides use of the probiotic Escherichia coli provided in the first aspect or the probiotic agent provided in the second aspect in the preparation of a medicament for inhibiting gastrointestinal pathogenic bacteria.

[0013] The probiotic Escherichia coli provided by the present invention can exert a co-aggregation effect with a variety of gastrointestinal pathogenic bacteria, reduce the number of pathogenic bacteria in the intestine, change the flora structure in the intestine, and improve the intestinal microenvironment; the pathogenic bacteria include one or more of Escherichia coli, Staphylococcus aureus, Salmonella paratyphi A or Helicobacter pylori, and have a significant inhibitory effect on Helicobacter pylori in particular.

[0014] In a fifth aspect, the present invention provides use of the probiotic Escherichia coli provided in the first aspect or the probiotic agent provided in the second aspect in the preparation of an anti-cancer adjuvant drug.

[0015] Furthermore, the anticancer adjuvant drug includes an anti-tongue squamous cell carcinoma adjuvant drug, an anti-gastric cancer adjuvant drug, an anti-small intestinal cancer adjuvant drug or an anti-colon cancer adjuvant drug. The probiotic E. coli EcN223-YTP provided by the present invention has a cell adhesion rate of more than 60% to SCC-9 (human tongue squamous cell carcinoma cells), HGC-27 (human gastric cancer cells), HIC (human small intestinal cancer cells) and Caco-2 (human colorectal adenocarcinoma cells).

[0016] In a sixth aspect, the present invention further provides a pharmaceutical preparation comprising an effective dose of the probiotic Escherichia coli described in the first aspect and a pharmaceutically acceptable excipient.

[0017] The pharmaceutically acceptable excipients include one or more of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents.

[0018] In some specific embodiments of the present invention, the dosage form of the pharmaceutical preparation is granules, capsules, tablets, pills or oral liquids.

[0019] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0020] The probiotic Escherichia coli EcN223-YTP provided by the present invention has stronger growth activity than the existing EcN strain, and still maintains rapid growth during the stable period, can quickly increase the number of viable bacteria, and significantly shorten the fermentation cycle. The strain has stronger stress resistance, including resistance to high temperature, low temperature, low pH, high bile salt, low oxygen, phage infection, etc., and therefore has more stable activity and a longer shelf life, is easier to store and is suitable for development and application. In addition, the strain has a clear genetic background and is suitable for genetic improvement. It is expected that its stress resistance and probiotic ability will be further improved by genetic engineering means, and it has broad prospects in the development and application of intestinal probiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings constituting part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute an improper limitation of the present invention. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0022] Figure 1 is a Gram staining result diagram of EcN223-YTP of Example 1 of the present invention;

[0023] Figure 2is a graph showing the measurement results of the growth activity of EcN223-YTP in Example 2 of the present invention;

[0024] Figure 3 This is a diagram showing the acid resistance evaluation results of EcN223-YTP in Example 3 of the present invention;

[0025] Figure 4 This is a diagram showing the bile salt tolerance evaluation results of EcN223-YTP in Example 3 of the present invention;

[0026] Figure 5 It is a diagram showing the results of the high temperature and low temperature resistance evaluation of EcN223-YTP in Example 4 of the present invention, wherein a, 42°C; b, 20°C; c, 4°C;

[0027] Figure 6 This is a diagram showing the instantaneous high temperature resistance evaluation result of EcN223-YTP in Example 4 of the present invention;

[0028] Figure 7 This is a diagram showing the results of the evaluation of the hypoxia tolerance of EcN223-YTP in Example 5 of the present invention; wherein a, 1% O2; b, 0.5% O2;

[0029] Figure 8 This is a diagram showing the results of the identification of the ability of EcN223-YTP to resist phage infection in Example 6 of the present invention;

[0030] Fig. 9 This is a diagram showing the results of the identification of the surface hydrophobicity and agglutination ability of EcN223-YTP in Example 7 of the present invention;

[0031] Fig.10 This is a diagram showing the results of the self-aggregation ability identification of EcN223-YTP in Example 7 of the present invention;

[0032] Fig.11 This is a diagram showing the results of the identification of the coagulation ability of EcN223-YTP in Example 7 of the present invention;

[0033] Fig.12 This is a diagram showing the results of identifying the cancer cell adhesion ability of EcN223-YTP in Example 8 of the present invention. DETAILED DESCRIPTION

[0034] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0035] The technical solution of the present invention is further described below in conjunction with specific examples. Unless otherwise specified, the sources of the raw materials and reagents used in the following examples can be obtained from commercial sources. In the following examples, the EcN strain was purchased from the German Microbial Culture Collection (numbered DSM 6601).

[0036] Example 1 Isolation and identification of probiotic Escherichia coli EcN223-YTP

[0037] (1) Preliminary isolation of probiotic Escherichia coli EcN223-YTP

[0038] Weigh 2 g of the intestinal contents of Tibetan yaks and add them to a sterile PBS solution and shake to mix. Then, place the sample in a water bath at 80°C for 15 min. Then, perform a gradient dilution, with three replicates for each gradient. Take 10 -5 , 10 -6 and 10 -7 After the gradient dilution is shaken and mixed, 200 μL is evenly spread on LB medium and inverted in a 37°C incubator for overnight culture. Use an inoculation loop to pick a single clone colony for purification culture. Repeat this 2-3 times. When the single colony morphology is consistent, use Gram staining solution to stain and identify the isolated strain. A Gram-negative strain was obtained. Its morphological characteristics are: Gram-negative, medium-sized, blunt-ended, mostly single scattered bacilli, such as Figure 1 Save for later use.

[0039] (2) 16S rDNA identification and comparison:

[0040] Prepare 1mL of overnight cultured bacteria, and use a bacterial DNA extraction kit to extract the DNA of this Gram-negative strain. Use the extracted genomic DNA as a template and use the universal primers (27F and 1492R) of the bacterial 16S rRNA gene for PCR amplification. Send the PCR product to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing, and perform a BLAST comparison with the NCBI database to determine the strain species. The results show that the probiotic Escherichia coli EcN223-YTP belongs to the genus Escherichia coli, and has the closest evolutionary relationship with Escherichia coli Nissle1917 (EcN). Its 16S rDNA sequence is shown in SEQ ID NO.1.

[0041] (3) Whole genome sequencing:

[0042] The whole genome sequencing of the probiotic E. coli EcN223-YTP was carried out, and the strain genome was measured to be a circular genome with a size of 5048102bp, a GC content of 50.58%, and 4772 functional genes. Compared with the genome information of the existing EcN strains published by NCBI, there are differences in genome size (EcN: 5,058,102; EcN223-YTP: 5,048,102), the number of coding genes (EcN: 5,045; EcN223-YTP: 4,772), the number of coded proteins (EcN: 23,910; EcN223-YTP: 22,766), the number of secreted proteins (EcN: 309; EcN223-YTP: 391), and cryptic plasmids (EcN: 2; EcN223-YTP: None).

[0043] Example 2 Results of determination of growth activity of probiotic Escherichia coli EcN223-YTP

[0044] The growth curve of Escherichia coli was drawn by turbidimetry, with the culture time (0-24 h) as the horizontal axis and the corresponding absorbance value (OD) at 600 nm of the culture solution of LB liquid medium as the horizontal axis. 600nm ) is used as the vertical axis to plot the growth curves of EcN and EcN223-YTP.

[0045] The growth curve of probiotic E. coli EcN223-YTP is as follows Figure 2 As shown, it can be seen that compared with the existing EcN strain, the EcN223-YTP strain has stronger growth activity: it shows a rapid upward trend 4 hours after inoculation, indicating that it has entered the logarithmic growth stage, gradually enters the stable period after 10 hours of cultivation, and gradually shows a slow downward trend after 18 hours. This trend is more obvious after 24 hours, and gradually enters the decay stage. Compared with EcN, EcN223-YTP still maintains rapid growth during the stable period, can quickly increase the number of viable bacteria, and significantly shorten the fermentation cycle.

[0046] Example 3 Identification of the acid and bile resistance of probiotic Escherichia coli EcN223-YTP

[0047] EcN and EcN223-YTP bacterial cultures were cultured in LB liquid medium and the concentration of the bacterial cultures was adjusted to approximately 1.0 × 10 8CFU / mL. Take equal volumes of EcN223-YTP and EcN bacterial solution and place them in simulated gastric fluid with pH 1.5, 2.0 and 2.5 respectively (take dilute hydrochloric acid with a concentration of 1 mol / L, add water to dilute, adjust the pH to 1.5. Add 1g of pepsin to every 100ml of liquid, mix well, filter with a 0.2μm sterile filter for standby use), and use pH 7.0 simulated gastric fluid as a control. Take equal volumes of EcN223-YTP and EcN bacterial solution and place them in artificial intestinal fluid with pig bile salt concentrations of 0, 0.30%, 0.60% and 0.90% respectively (take 6.8g of KH2PO4 and add 500ml of water to dissolve, adjust the pH to 6.8 with 0.4% (w / w) NaOH. Add 1g of trypsin to every 100ml of liquid, mix well, filter with a 0.2μm sterile filter for standby use), with 3 replicates per group. The gastric acid group and bile salt group were placed in a constant temperature oscillator at 37℃ and 200r / min for 3h and 24h respectively. The percentage of the total number of bacteria after treatment to that before treatment was the EcN survival rate.

[0048] The pH in the stomach of animals is generally between 2.0 and 3.5. Changes in feed can cause the pH of gastric acid to fluctuate between 1.5 and 5.0. Figure 3 The results of the acid resistance test showed that the survival rates of EcN223-YTP and EcN decreased with the decrease of the pH of simulated gastric fluid, but they still maintained a certain survival. Compared with the existing EcN strain, EcN223-YTP has stronger acid resistance and gastric ability. After being treated in simulated gastric fluid with pH 1.5, 2.0 and 2.5 for 3 hours, the survival rate of EcN223-YTP was significantly higher than that of the existing EcN strain. Even in an acidic environment with a pH as low as 1.5, its survival rate can still be higher than 20%.

[0049] In addition to being able to withstand the stomach acid environment, EcN also needs to withstand the high osmotic pressure environment formed by bile salts in the intestine. The bile salt content in the intestine of animals generally fluctuates between 0.03% and 0.50%. Figure 4 The test results show that the probiotic E. coli EcN223-YTP has stronger bile salt tolerance and hyperosmotic environment adaptability than the existing EcN strain. After being treated in artificial intestinal fluid with different bile salt concentrations (0.30%, 0.60% and 0.90%) for 24 hours, the survival rate of EcN223-YTP is significantly higher than that of the existing EcN strain. Even at a high bile salt concentration of 0.9%, the 24-hour survival rate can still be maintained at around 30%. From the comparison of the above results, it can be seen that EcN223-YTP has stronger acid and bile salt resistance than EcN, and has stronger tolerance to pepsin and pancreatic enzymes.

[0050] Example 4 Identification of the ability of probiotic Escherichia coli EcN223-YTP to resist high and low temperatures

[0051] EcN and EcN223-YTP bacterial cultures were cultured in LB liquid medium and the concentration of the bacterial cultures was adjusted to approximately 1.0 × 10 8 CFU / mL, used to determine the growth curve of E. coli under different temperature stresses. Turbidimetry was used to determine the growth curve under different environmental factor stresses. 600nm =0.5, 50 μL of bacterial solution was placed in 50 mL of culture medium for constant temperature shaking culture at a speed of 150 r / min. The temperature stress conditions were 42°C, 20°C and 4°C, and the absorbance was measured every 2 hours. Figure 5 The results showed that compared with the existing EcN strain, EcN223-YTP has stronger adaptability to high and low temperatures. After being treated in a mild high temperature environment (42°C) and a low temperature environment (4°C and 20°C) for 12 hours, its growth ability was higher than that of the existing EcN strain.

[0052] Subsequently, an instantaneous high temperature stress experiment was carried out. Four 9 mL portions of sterile saline were taken into test tubes and preheated to 80°C. Then 1 mL of bacterial solution was added to each test tube. The cells were kept warm for 30, 40, 50 and 60 seconds, and then cooled rapidly. The bacterial solutions of the different insulation time groups were diluted by a 10-fold gradient. 100 μL of the bacterial solution with the best dilution concentration was evenly spread on LB solid culture medium, and the culture was placed in a 37°C incubator for 24 hours. The colonies were counted and the survival rate was calculated. Each group was repeated twice. Figure 6 The results showed that compared with the existing EcN strain, EcN223-YTP had a higher survival rate in an instantaneous high temperature environment (80°C). After being kept at 80°C for 30s, 40s, 50s and 60s, the survival rate of EcN223-YTP was significantly higher than that of the existing EcN strain.

[0053] Example 5 Identification of hypoxia tolerance of probiotic Escherichia coli EcN223-YTP

[0054] The oxygen concentration of the ambient atmosphere is (O2, ~21%), the oxygen concentration of mammalian tissues to function is about 2%-10%, and the oxygen concentration of the microenvironment of human tumors is 0.3% to 4.2%. Therefore, hypoxia tolerance is of great significance to the efficacy of probiotics.

[0055] In this experiment, the hypoxic condition was simulated by the pro Ox-C-chamber system (C-Chamber, C-347, Biospherix, New York, NY) to achieve the control of hypoxic conditions, mixed with 5% CO2 and balanced with N2. The culture was carried out at a constant temperature (37°C) and a shaking speed of 150 r / min. Figure 7The results showed that compared with the existing EcN strain, EcN223-YTP has a stronger tolerance to hypoxia stress. Under oxygen-limited conditions (the oxygen concentration in the hypoxic chamber was set at 1% and 0.5%), the growth activity of EcN223-YTP was significantly higher than that of the existing EcN strain.

[0056] Example 6 Identification of the anti-phage infection ability of probiotic Escherichia coli EcN223-YTP

[0057] Bacteriophages are specific viruses that infect the base cells of industrial fermentation. Due to their widespread existence and difficulty in eradication, they have greatly affected fermentation production. The ability to resist bacteriophages also seriously affects the fermentation and application of probiotics.

[0058] Single colonies of EcN223-YTP and EcN strains were picked and cultured in 5 mL LB liquid medium at 37°C and 200 r / min overnight. The presence of phages was detected using the double-layer plate method, and the E. coli phages used included T7, T1, JMPW2, EEP, T4, and Lamda. Figure 8 The experimental results showed that compared with the existing EcN strain, EcN223-YTP has a stronger ability to resist phage infection. Under the infection of different Escherichia coli phages (T7, T1, JMPW2, EEP, T4, Lamda), the number and size of plaques on the EcN223-YTP plate were significantly lower than those of the existing EcN strain.

[0059] Example 7 Identification of Surface Hydrophobicity and Agglutination Ability of Probiotic Escherichia coli EcN223-YTP

[0060] The first step in the process of probiotic colonization in the gastrointestinal tract of humans or animals is adhesion to host tissues. Cell hydrophobicity determines the ability of bacteria to adhere, which is the key to whether probiotics can reproduce in the intestines of animals. In addition, the ability of bacteria to self-aggregate has an important influence on the adhesion of bacteria to intestinal cells, while co-aggregation eliminates the colonization of gastrointestinal pathogens by preventing pathogens from attaching to host tissues.

[0061] The standard for the classification of bacteria's hydrophobicity is that hydrophobicity below 20% is non-hydrophobic, 20% to 50% is moderately hydrophobic, and above 50% is highly hydrophobic. Probiotics with higher hydrophobicity also have higher adhesion in the intestine, that is, the hydrophobicity of the bacterial surface is proportional to its adhesion to the intestinal epithelial cells. 40% hydrophobicity is the minimum value required for probiotic strains. Fig. 9 The results showed that the hydrophobicity of EcN223-YTP reached 83%, which was 8.2% higher than that of EcN (P<0.05).

[0062] Self-aggregation of strains refers to the phenomenon that the same bacteria aggregate with each other to form multicellular clusters. The aggregation of probiotics can form a barrier to prevent the colonization and infection of pathogenic bacteria. Studies have shown that strains with higher self-aggregation ability also have high adhesion, and self-aggregation can enable probiotics to form steric hindrances to hinder the colonization of pathogens in the intestine. The standard for the classification of bacteria for self-aggregation ability is that self-aggregation between 16% and 35% is low self-aggregation ability, between 36% and 50% is medium self-aggregation ability, and above 50% is high self-aggregation ability. Fig.10 The results showed that the self-aggregation of EcN223-YTP and Ec increased with time, and the self-aggregation of strain EcN223-YTP reached 71.3% at 24h, which was 15.13% higher than that of EcN (P<0.05).

[0063] Probiotics can reduce the number of pathogenic bacteria in the intestines, change the flora structure in the intestines, and improve the intestinal microenvironment through co-aggregation with pathogenic bacteria. Fig.11 The results showed that EcN223-YTP and EcN had strong coaggregation ability against a variety of pathogens, including Escherichia coli (E.coli CMCC 44102), Staphylococcus aureus (S.aureus ATCC25923), Salmonella paratyphi AATCC9150 and Helicobacter pylori (H.pylori SS1). The coaggregation rate of EcN223-YTP strain with Helicobacter pylori reached 77.33%, which was nearly 17% higher than that of EcN strain, indicating that EcN223-YTP may have a stronger inhibitory effect on Helicobacter pylori.

[0064] Example 8 Identification of the Cancer Cell Adhesion Ability of Probiotic Escherichia coli EcN223-YTP

[0065] Probiotic Escherichia coli Nissle 1917 (EcN) has high tumor-targeting colonization ability and has attracted much attention in cancer bacterial therapy. Adhesion ability is one of the prerequisites for the interaction between EcN and cancer cells. We determined the difference in the adhesion ability of EcN223-YTP and EcN to different cancer cells by co-culture of bacteria and cells combined with colony counting method. SCC-9 (human tongue squamous cell carcinoma cells, epithelial, adherent), EC109 (human esophageal carcinoma cells, epithelial, adherent), HGC-27 (human gastric carcinoma cells, epithelial, adherent), HIC (human small intestinal carcinoma cells, epithelial, adherent), Caco-2 (human colorectal adenocarcinoma cells (colorectal cancer), similar in structure and function to differentiated small intestinal epithelial cells, epithelial, adherent), MCF-7 (human breast cancer cells, epithelial, adherent), A549 (human non-small cell lung cancer cells, epithelial, adherent) were subcultured and plated in 12-well plates. After the cells grew to 80% to 90%, they were set aside. EcN bacterial solution was cultured in LB liquid medium, and the concentration of the bacterial solution was adjusted to about 1.0×10 8 CFU / mL, used for cell adhesion performance detection. Fig.12 The results showed that after incubation of EcN223-YTP or EcN with cells for 2 hours, they had a certain adhesion ability to various cancer cells, and the adhesion ability of EcN223-YTP was higher than that of EcN strain. The adhesion rate of EcN223-YTP to human tongue squamous cell carcinoma cell SCC-9, human gastric cancer cell HGC-27, human small intestine cancer cell HIC, and human colorectal adenocarcinoma cell Caco-2 was higher than 60%, among which the adhesion rate of EcN223-YTP exceeded 70% for human gastric cancer cell HGC-27 and human colorectal adenocarcinoma cell Caco-2.

[0066] Example 9 Digestive enzyme encoding genes and digestive enzyme activities in the EcN223-YTP genome

[0067] Humans and mammals lack a complete enzyme production system, and the gastrointestinal microbial flora can secrete a variety of enzymes related to food nutrient digestion, such as proteases, cellulases, amylases, lipases, phytases, β-glucanases, etc. Based on the whole genome sequencing of the EcN223-YTP strain that has been resolved, we found 34 protease-encoding genes, 2 amylase-encoding genes, 3 lipase-encoding genes, 2 cellulase-encoding genes, 2 phytases-encoding genes, 3 lactase-encoding genes, and 3 β-glucosidase-encoding genes among the 4772 genes encoded by it.

[0068] Then we used the Folin phenol method to determine the protease activity, the 3,5-dinitrosalicylic acid colorimetric method to determine the amylase activity, the p-nitrophenol palmitate (p-NPP) method to determine the lipase activity, the sodium carboxymethylcellulose (CMC-Na) method to determine the endoglucanase activity in the cellulase, the vanadium-ammonium molybdate method to determine the phytase activity, the o-nitrophenol-β-D-galactoside (ONPG) method to determine the lactase activity, and the p-nitrophenol-β-D-glucoside method to determine the β-glucosidase activity. The results are shown in Table 1. It can be seen that EcN223-YTP can secrete proteases and lipases, and produce amylases, cellulases, phytases, and β-glucosidases.

[0069] Table 1 Digestive enzyme activity and number of coding genes of EcN223-YTP

[0070] Digestive enzyme types EcN223-YTP digestive enzyme activity (U / mL) Number of coding genes Protease 4.88±0.31 34 Amylase 1.37±0.11 2 Lipase 2.41±0.17 3 Cellulase 0.77±0.06 2 Phytase 3.97±0.21 1 Lactase 0.91±0.03 3 β-Glucosidase 1 3

[0071] Example 10 Preparation of probiotic powder and stability testing

[0072] EcN and EcN223-YTP bacterial cultures were cultured in LB liquid medium and the concentration of the bacterial cultures was adjusted to approximately 1.0 × 10 8 CFU / mL, sucrose, sodium L-ascorbate and skim milk were used as freeze-dried protective agents to prepare freeze-dried bacterial powders of EcN and EcN223-YTP, respectively. The survival rate of bacteria in the bacterial powder was determined by the plate count method.

[0073] The prepared bacterial powder was sealed and stored at 4°C and room temperature respectively. On the 90th day, the number of live bacteria in EcN and EcN223-YTP bacterial powders was determined by plate counting method, and the survival rate of live bacteria in the bacterial powder was calculated by comparing with the concentration of live bacteria at the beginning of bacterial powder preparation. The results of bacterial powder stability test: After 90 days of storage at 4°C, the survival rate of the existing EcN strain was about 83.5±4.6%, and the survival rate of the EcN223-YTP strain was above 92.2±2.3%; after 90 days of storage at room temperature, the survival rate of the existing EcN strain was about 63.8±5.1%, while the survival rate of the EcN223-YTP strain could still be maintained at above 80.3±3.7%, indicating that the EcN223-YTP strain with stress resistance has better bacterial powder stability and can maintain a longer shelf life and biological activity.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A probiotic Escherichia coli, named probiotic Escherichia coli (Escherichia coli Nissle 1917) EcN223-YTP, was deposited in the China Type Culture Collection, Wuhan on August 16, 2024, with the collection number CCTCCNO: M 20241801.

2. A probiotic agent, characterized in that: The raw materials of the probiotic agent include the probiotic Escherichia coli described in claim 1.

3. A fermented product, characterized in that The fermented product is a product produced by fermentation using the probiotic Escherichia coli described in claim 1 or the probiotic agent described in claim 2 as a starter.

4. The fermented product according to claim 3, characterized in that The fermented product is fermented food or feed.

5. The fermented product according to claim 3, characterized in that The fermented food is a fermented dairy product, a fermented bean product or a fermented fruit and vegetable product.

6. Use of the probiotic Escherichia coli according to claim 1 or the probiotic agent according to claim 2 in the preparation of a medicament for inhibiting gastrointestinal pathogenic bacteria.

7. The use according to claim 6, characterized in that The pathogenic bacteria include one or more of Escherichia coli, Staphylococcus aureus, Salmonella paratyphi A or Helicobacter pylori.

8. Use of the probiotic Escherichia coli according to claim 1 or the probiotic agent according to claim 2 in the preparation of an anti-cancer auxiliary drug.

9. The use according to claim 8, characterized in that The anti-cancer auxiliary drug includes an anti-tongue squamous cell carcinoma auxiliary drug, an anti-gastric cancer auxiliary drug, an anti-small intestine cancer auxiliary drug or an anti-large intestine cancer auxiliary drug.

10. A pharmaceutical preparation, characterized in that The invention comprises an effective dose of the probiotic Escherichia coli according to claim 1 and a pharmaceutically acceptable auxiliary material.

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