Probiotic biofilm gel microspheres as well as preparation method and application thereof

Abstract

The invention discloses a probiotic biofilm gel microsphere as well as a preparation method and application thereof, and the probiotic biofilm gel microsphere is prepared by taking pectin, sodium alginate, escherichia coli Nissle1917 and iron-sulfur nano-enzyme as raw materials, preparing the raw materials through a microfluidic technology and then culturing the raw materials. The preparation method comprises the following steps: dissolving pectin and sodium alginate in deionized water, sequentially adding iron-sulfur nano-enzyme and Escherichia coli Nissle1917 into the deionized water, uniformly stirring, preparing through a microfluidic technology, and carrying out biological in-situ culture to make probiotics in the solution form a biological membrane. The cultured probiotic biofilm gel microspheres not only have the advantages of high stress resistance, good biocompatibility and the like, but also have good application prospects in the aspects of combined treatment and recovery of inflammatory enteritis and the like.

Description

Technical Field

[0001] This invention relates to probiotic biofilm gel microspheres, their preparation methods, and applications. In particular, it relates to an iron-sulfur nanoenzyme / probiotic biofilm microsphere based on plant pectin encapsulation, its preparation method, its resistance to stress under a series of harsh environments, and its combined application in the treatment of inflammatory bowel disease. It belongs to the fields of microbiology and medical application technology. Background Technology

[0002] Inflammatory bowel disease (IBD) refers to a group of nonspecific chronic inflammatory diseases of the gastrointestinal tract with unknown causes. Currently, traditional treatments for IBD mainly include immunosuppressants such as 5-aminosalicylic acid, glucocorticoids, methotrexate, and thiopurine. However, these drugs have limited efficacy and serious side effects. Studies have found that during the pathogenesis of IBD, the inflammatory sites produce large amounts of reactive oxygen species (ROS), which not only exacerbate tissue damage and intestinal barrier disruption but also lead to increased secretion of pro-inflammatory cytokines. Furthermore, further research has revealed a large number of complex gut microbiota, and a close correlation between gut microbiota dysbiosis and IBD pathogenesis. Clearing ROS and regulating gut microbiota have become potential therapeutic targets for future IBD drugs.

[0003] Pectin and sodium alginate, as plant polysaccharides, not only possess excellent biocompatibility, non-toxicity, and encapsulation properties, but sodium alginate also readily cross-links with metal ions to form hydrogels. Micron-sized pectin-sodium alginate gel microspheres were prepared using microfluidic technology to encapsulate *Escherichia coli* Nissle1917 (EcN) and iron-sulfur nanozymes (GFeSNs). In vitro culture allowed ECN to form a probiotic biofilm within the microspheres. Compared to planktonic ECN and microspheres without a biofilm, the plant pectin-encapsulated probiotic biofilm gel microspheres significantly enhanced the probiotics' and nanoparticles' resistance to the gastrointestinal digestive environment and their ability to colonize the intestines. They also exerted antioxidant and gut microbiota-regulating effects at inflammatory sites, thus providing a new treatment method and expanding the application scope of probiotic biofilms. Summary of the Invention

[0004] Objectives of the Invention: One objective of this invention is to provide a plant pectin-encapsulated iron-sulfur nanoenzyme / probiotic biofilm gel microsphere. Another objective is to provide a method for preparing the probiotic biofilm gel microsphere. A final objective is to provide the application of the probiotic biofilm gel microsphere in a medicament for treating inflammatory bowel disease and / or gastritis.

[0005] Technical solution: In order to solve the above problems, the present invention provides a probiotic biofilm gel microsphere, wherein the probiotic biofilm microsphere comprises microspheres prepared and cultured using pectin, sodium alginate, Escherichia coli Nissle1917 and iron-sulfur nanoenzyme as raw materials through microfluidic technology.

[0006] The preparation method of iron-sulfur nanoenzyme / probiotic biofilm gel microspheres based on plant pectin encapsulation according to the present invention includes the following steps:

[0007] (1) Escherichia coli Nissle1917 was transferred and cultured to the logarithmic growth phase to obtain Escherichia coli Nissle1917 transfer culture for use;

[0008] (2) Pectin and sodium alginate are first dissolved in deionized water, and iron-sulfur nanozyme and Escherichia coli Nissle1917 transfer bacterial solution are added and stirred thoroughly to obtain the aqueous phase;

[0009] (3) Bispan 80 and isopropyl myristate were mixed to prepare an oil phase, and the aqueous phase and oil phase were mixed by microfluidic technology to prepare plant pectin-encapsulated iron-sulfur nanoenzymes / probiotic microspheres.

[0010] (4) The iron-sulfur nanoenzyme / probiotic microspheres coated with plant pectin were solidified in CaCl2 solution, washed, and cultured in liquid LB medium to obtain probiotic biofilm gel microspheres.

[0011] Further, in step (1), *Escherichia coli* Nissle1917 is cultured on LB medium until the OD value reaches 1.0-1.2, and the volume ratio of *E. coli* Nissle1917 to liquid LB medium is 1:100. In step (2), the mass ratio of pectin to sodium alginate is 2:1-1:2, preferably 1:1, and the concentrations of iron-sulfur nanozyme and *E. coli* Nissle1917 in the aqueous phase are 25-100 μg / mL and 10 μg / mL, respectively. 5 ~10 7 CFU / mL, preferably, the concentrations of iron-sulfur nanozyme and Escherichia coli Nissle1917 in the aqueous phase are 50-100 μg / mL and 10 μg / mL, respectively. 7 CFU / mL. Pectin and sodium alginate were dissolved in deionized water and stirred at room temperature for 60-90 min. After adding iron-sulfur nanozyme and Escherichia coli Nissle1917, the mixture was stirred at room temperature for 10-20 min. In step (3), the volume ratio of Span 80 to isopropyl myristate was 1:10-1:40, preferably 1:20. During microfluidic preparation, the volume ratio of the aqueous phase to the oil phase was 1:40-1:100. In step (4), the concentration of the CaCl2 solution was below 750 mM, and the solidification time was 20-60 min. The mixture was cultured in liquid LB medium for 24-48 h, preferably 36 h.

[0012] The preparation and stress resistance assessment of the probiotic biofilm gel microspheres described in this invention are used in drugs to promote intestinal colonization and restore inflammatory bowel disease.

[0013] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0014] (1) All the carriers in this invention are plant polysaccharides, which have good biocompatibility, are non-toxic, have good encapsulation properties, and are easy to cross-link with metal ions to form hydrogels. They can not only effectively protect probiotics and alleviate the stress of harsh external environments, but also provide energy for the growth and reproduction of probiotics.

[0015] (2) The iron-sulfur nanozyme in the microspheres is a synthetically produced nanozyme. Compared with natural nanozymes, it can not only compensate for the poor stability, high price, and allergic reactions of natural reactive oxygen species scavenging enzymes, but also effectively remove excess ROS, achieving a combined anti-inflammatory and probiotic treatment. This study will provide a novel strategy for the treatment of inflammatory bowel disease. Attached Figure Description

[0016] Figure 1 The graph shows the effect of different concentrations of GFeSNs on the viability of Escherichia coli Nissle1917 in Example 3.

[0017] Figure 2 Figure 4 shows the therapeutic effect of different concentrations of GFeSNs on TNBS-induced IBD in mice.

[0018] Figure 3 Electron microscope images of the carrier microspheres prepared in different proportions in Example 5;

[0019] Figure 4 SEM image of the iron-sulfur nanoenzyme / probiotic biofilm gel microspheres encapsulated with plant pectin in Example 5;

[0020] Figure 5 Figure 5 shows the ECN content inside microspheres after culturing ECN-FeS@P / SA in LB medium for different time periods.

[0021] Figure 6 This is a graph showing the survival of the three test substances in simulated gastric fluid in Example 6;

[0022] Figure 7 This is a graph showing the survival of the three test substances in simulated intestinal fluid in Example 6;

[0023] Figure 8 This is a graph showing the survival of the three test substances in Example 7 under simulated high-permeability stress in the ECN.

[0024] Figure 9 This is a graph showing the ECN survival of the three test substances in simulated LEV in Example 7;

[0025] Figure 10 This is a graph showing the survival of ECN in the three test substances in Example 8 after being stored at 4 °C for a period of time;

[0026] Figure 11 This is an H&E staining image of the major organs 15 days after administration of CECN-FeS@P / SA in Example 9;

[0027] Figure 12 This is a figure showing the in vivo therapeutic effect of CECN-FeS@P / SA on IBD mice in Example 10. Detailed Implementation

[0028] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings.

[0029] Example 1

[0030] Preparation of iron sulfide nanozymes GFeSNs: Refer to Example 1 in CN116236504A.

[0031] Example 2

[0032] (1) Resuscitation of Escherichia coli Nissle1917: Take 1 mL of thawed Escherichia coli Nissle1917 (purchased from Zhejiang Baosai Biotechnology Co., Ltd.) and place it in 5 mL of liquid LB medium. Shake overnight at 37 ℃ and 220 rpm. Observe the turbidity to indicate successful resuscitation. Dilute the resuscitation solution tenfold (take 100 μL of the resuscitation solution and add it to 900 μL of PBS buffer to obtain a dilution concentration of 10). -1 Seven gradient dilution solutions were prepared according to this process, each with a concentration of 10... -1 (i.e., a resuscitation solution diluted tenfold), 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 After dilution, it was spread onto LB solid medium and incubated in a 37 ℃ incubator for 15 h. 10 -5 ~10 -7 The presence of stable colonies indicates successful resuscitation of Escherichia coli Nissle1917.

[0033] (2) Inoculation of Escherichia coli Nissle1917: Take 2-3 revived colonies from step (1) and put them into 4 mL of liquid LB medium. Incubate overnight at 37 ℃ and 220 rpm on a shaker.

[0034] (3) Escherichia coli Nissle1917 transfer: Take 50 μL of the inoculation solution from step (2) at a volume ratio of 1:100 and add it to 5 mL of liquid LB medium. Shake at 37 ℃ and 220 rpm for 2-3 h. OD 600 The concentration was 1~1.2, and the Escherichia coli Nissle1917 transfer culture was obtained for later use.

[0035] Example 3

[0036] To determine the optimal dosage of GFaSNs to avoid damaging the ECN and maximize ROS removal, 50 μL of the *Escherichia coli* Nissle 1917 inoculation solution obtained in step (2) of Example 2 was taken and LB liquid medium was added at a volume ratio of 1:100. GFaSNs were then added to achieve concentrations of 50 μg / mL and 100 μg / mL, respectively. OD values ​​were measured every 3 hours using a UV spectrophotometer. 600 The control group (0 μg / mL) was used without the addition of GFeSNs. Results are as follows: Figure 1 As shown, iron-sulfur nanozyme concentrations of 50 μg / mL and 100 μg / mL had almost no effect on ECN activity.

[0037] Example 4

[0038] Three portions of GFeSNs prepared in Example 1 were ultrasonically dissolved in deionized water, with concentrations of 25 μg / mL, 50 μg / mL, and 100 μg / mL, respectively, to obtain three groups of GFeSNs solutions.

[0039] Male BALB / c mice aged 4-6 weeks were acclimatized for one week and then randomly divided into 5 groups: a normal control group (Control), a model group (TNBS), a low-concentration iron-sulfur nanozyme group (LFeS, 25 μg / mL), a medium-concentration iron-sulfur nanozyme group (MFeS, 50 μg / mL), and a high-concentration iron-sulfur nanozyme group (HFeS, 100 μg / mL). The Control group received 0.1 mL of PBS rectally, while the other four groups received the same dose (0.1 mL per mouse) of 2,4,6-trinitrobenzenesulfonic acid (TNBS, 2.5%) rectally. Simultaneously, from day 1 to 5, the Control and TNBS groups received the same dose (0.1 mL) of PBS buffer daily by gavage, while the other three groups received the same dose (0.1 mL) of the three GFeSNs solutions prepared in this embodiment by gavage. Daily changes in mouse body weight, fecal occult blood, and fecal morphology were recorded as DAI scores. Furthermore, mice were sacrificed on day 6, and the colon length of each group was recorded. The results are as follows: Figure 2 As shown, the three treatment groups showed some efficacy in terms of weight loss and increased DAI scores compared to the TNBS group, but there was no significant difference in efficacy between the MFeS and HFeS groups. Taking all factors into consideration, an iron-sulfur nanozyme concentration of 50 μg / mL was chosen as the concentration for subsequent animal experiments.

[0040] Example 5

[0041] (1) Dissolve 0.15 g of pectin and 0.15 g of sodium alginate in 18 mL of deionized water (mass ratio of pectin:sodium alginate = 1:1), stir at room temperature for 60-90 min until the pectin and sodium alginate are completely dissolved in the deionized water.

[0042] (2) Take 1 mg of GFeSNs prepared in Example 1 and dissolve it in 1 mL of deionized water by ultrasonication. Add it to the solution in step (1) and stir at room temperature for 10-20 min until GFeSNs are evenly distributed in the solution.

[0043] (3) Take 5 mL of the Escherichia coli Nissle1917 transfer solution obtained in step (3) of Example 2, centrifuge at 6000 rpm / min for 5 min to obtain the bacterial cells, resuspend the bacterial cells in 1 mL of deionized water and add them to the solution in step (2), stir at room temperature for 10~20 min to obtain the aqueous phase.

[0044] (4) The oil phase is in a volume ratio of Span 80: Isopropyl myristate = 1:20. The water phase and the oil phase are placed in different micro-injection pumps (inner diameter 0.2 mm, outer diameter 0.5 mm) and pumped out at a volume ratio of 1:80 to mix the two phases.

[0045] (5) The material obtained in step (4) is dropped into 750 mM CaCl2 anhydrous ethanol solution and cured for 30 min. It is then washed 2-3 times with deionized water solution to remove the residual oil phase. The iron-sulfur nanoenzyme / probiotic gel microspheres (ECN-FeS@P / SA) are encapsulated in plant pectin.

[0046] (6) After cleaning, the microspheres were added to liquid LB culture medium at a volume ratio of 1:25 and cultured in a constant temperature bacterial incubator at 37 ℃ for 36 h. Fresh culture medium was replaced every 12 h. After 36 h of culture, the probiotic content in the microspheres was the highest, and iron-sulfur nanoenzyme / probiotic biofilm gel microspheres (CECN-FeS@P / SA) encapsulated with plant pectin were obtained.

[0047] Meanwhile, probiotic biofilm gel microspheres with a pectin:sodium alginate mass ratio of 2:1 and 1:2 (the total amount of pectin and sodium alginate is still 0.3g) were prepared according to the above conditions.

[0048] To determine the optimal ratio of pectin and sodium alginate, the morphology and state of the microspheres prepared in step (3) and those cultured in LB liquid medium for 36 h in step (6) were observed using an inverted microscope. The results are as follows: Figure 3 As shown, with the increase of sodium alginate input, the viscosity of the colloid increases. Due to the limitations of the microinjection pump, the synthesized microspheres become smaller and less uniform. When the pectin:sodium alginate mass ratio is 1:1, the microspheres are uniform in size and hardly break down after culturing in liquid LB medium for 36 h.

[0049] SEM scanning analysis was performed on the microspheres cultured in step (6), and the results are as follows: Figure 4 As shown, the probiotics within the microspheres formed stable colonies from single bacterial cells. Finally, to determine the optimal incubation time in LB medium for maximizing the probiotic content within the microspheres, a sample of microspheres was taken every 12 hours, diluted, and plated. The results showed that the probiotic content reached its maximum after 36 hours of incubation, approximately 10.4 CFU / g (see [link to LB medium]). Figure 5 ).

[0050] Example 6

[0051] The ECN-FeS@P / SA and CECN-FeS@P / SA prepared in Example 4 with a pectin:sodium alginate mass ratio of 1:1, and the same bacterial count (10) were compared. 7One mL each of the three analytes (CFU) of planktonic Escherichia coli Nissle1917 (ECN) was added to 10 mL of simulated gastric fluid (prepared by adding 1 g of pepsin to 100 mL of deionized water, shaking well, adding 1 mol / mL dilute hydrochloric acid to pH 1.2, and then filtering through a 0.22 μm membrane) for 2 h. During this period, the three analytes were diluted and plated, and the survival of the probiotics was observed. The results are as follows: Figure 6 As shown.

[0052] Take equal amounts of the three test samples (1 mL each) and add them separately to 10 mL of simulated intestinal fluid (dissolve 6.8 g of KH2PO4 in 500 mL of water, adjust the pH to 6.8 with 0.1 mol / L sodium hydroxide solution; dissolve 10 g of trypsin in an appropriate amount of water, mix the two solutions, dilute with water to 1000 mL, and then filter through a 0.22 μm membrane to obtain simulated intestinal fluid). Incubate for 4 hours. During this period, take the same volume of test sample, dilute and plate, and observe the survival of probiotics. The results are as follows. Figure 7 As shown.

[0053] The results showed that all members of the ECN group died within 1 hour in simulated gastric fluid (see [link]). Figure 6 The survival rate was only 42% after 4 hours in simulated intestinal fluid (see...). Figure 7 In the CECN-FeS@P / SA group, approximately 70% of the probiotics remained viable after 2 hours in gastric fluid, and the survival rate was over 85% after 4 hours in intestinal fluid, significantly higher than the other two groups.

[0054] Example 7

[0055] ECN-FeS@P / SA and CECN-FeS@P / SA, prepared with a pectin:sodium alginate mass ratio of 1:1 as in Example 4, and with the same bacterial count (10... 7 One mL each of planktonic Escherichia coli Nissle1917 (ECN) (CFU) was added to 10 mL of different concentrations of NaCl (5%, 10%, 15%, 20%) and 10 mL of different concentrations of the antibiotic levofloxacin (LEV) (0, 6.25, 12.5, 25, 50 μg / mL), respectively. The solutions were shaken for 4 h and 2 h, respectively, on a shaker (37℃, 220 rpm). The resistance of the three analytes to high osmosis and the antibiotic resistance were then observed using the plate count method. The results are as follows: Figure 8 and Figure 9 As shown in the figure. The results showed that, compared with the planktonic ECN and ECN-FeS@P / SA groups, the CECN-FeS@P / SA group had significantly higher resistance to high permeability and antibiotics, exhibiting the best resistance to high permeability and antibiotics (see Figure 1). Figure 8 and Figure 9 ).

[0056] Example 8

[0057] The ECN-FeS@P / SA and CECN-FeS@P / SA prepared in Example 4 with a pectin:sodium alginate mass ratio of 1:1, and the same bacterial count (10) were compared. 7 1 mL of each of the CFU (Cytotoxic Fumarate) of airborne Escherichia coli Nissle1917 (ECN) was aliquoted, freeze-dried, and stored at 4 °C. One sample was taken weekly, and the viability of the microspheres was observed using a plate count method. Results are as follows: Figure 10 As shown. The results showed that even after being stored in the refrigerator for a month, the survival rate of probiotics in CECN-FeS@P / SA remained as high as 75% (see...). Figure 10 (and far higher than the planktonic ECN and ECN-FeS@P / SA groups).

[0058] Example 9

[0059] Male BALB / c mice aged 4-6 weeks were acclimatized for one week and then randomly divided into a normal control group (Control) and a treatment group (CECN-FeS@P / SA prepared in Example 4), with 5 mice in each group. Mice in both groups were administered 0.1 mL of PBS and CECN-FeS@P / SA by gavage every other day, for a total of 5 times. After 15 days, the mice were euthanized, and serum was collected to detect serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Heart, liver, spleen, lung, kidney, and colon were fixed in 4% paraformaldehyde and stained with Hematoxylin and eosin (H&E). Experimental results are presented by… Figure 11 As shown. Figure 11 The images show the H&E staining of major organs 15 days after administration of CECN-FeS@P / SA in Example 5. (A) shows the serum alanine aminotransferase (ALT) level; (B) shows the serum aspartate aminotransferase (AST) level; and (C) shows the H&E staining of major organs in mice. Compared with the control group, there were no significant changes in liver and kidney function indicators and major organ tissues in the CECN-FeS@P / SA group. The above results indicate that CECN-FeS@P / SA has high biosafety.

[0060] Comparative Example 1: Preparation of iron-sulfur nanoenzymes encapsulated in plant pectin

[0061] The preparation process was the same as in Example 4, except that Escherichia coli Nissle1917 transfer culture was not added to the aqueous phase to obtain plant pectin-encapsulated iron-sulfur nanozyme group (FeS@P / SA).

[0062] Comparative Example 2

[0063] The preparation process was the same as in Example 4, except that GFeSNs were not added to the aqueous phase to obtain a probiotic group encapsulated in plant pectin (ECN@P / SA).

[0064] Example 10

[0065] After a week of acclimatization, 4-6 week old male BALB / c mice were randomly divided into six groups: a normal control group (Control), a model group (TNBS), a plant pectin-encapsulated iron-sulfur nanozyme group (FeS@P / SA prepared in Comparative Example 1), a plant pectin-encapsulated probiotic group (ECN@P / SA prepared in Comparative Example 2), a plant pectin-encapsulated iron-sulfur nanozyme / probiotic group (ECN-FeS@P / SA prepared in Example 4), and a plant pectin-encapsulated iron-sulfur nanozyme / biofilm probiotic group (CECN-FeS@P / SA prepared in Example 4). The Control group was rectally instilled with 0.1 mL of PBS, while the other five groups were rectally instilled with the same dose of 2,4,6-trinitrobenzenesulfonic acid (TNBS, 2.5%). Meanwhile, from day 1 to 5, the Control and DSS groups were administered the same dose of PBS buffer daily by gavage, while the other four groups were administered the corresponding drugs by gavage (FeS@P / SA prepared in Comparative Example 1, ECN@P / SA prepared in Comparative Example 2, ECN-FeS@P / SA prepared in Example 4, and CECN-FeS@P / SA prepared in Example 4). Daily changes in mouse body weight, fecal occult blood, and fecal morphology were recorded as DAI scores. Furthermore, mice were sacrificed on day 6, and colon length was recorded for each group; the results are as follows. Figure 12 As shown.

[0066] Male BALB / c mice aged 4-6 weeks were acclimatized for one week and then randomly divided into six groups. Except for the Control group, the other five groups received free access to 3% (w / v) sodium dextran sulfate (DSS, molecular weight 40 kDa) to induce inflammatory bowel disease for 5 days. Simultaneously, from days 6 to 10, the Control and DSS groups were administered the same dose of PBS buffer daily by gavage, while the other four groups received the corresponding drugs by gavage. Mice were sacrificed on day 11. After slowly washing the colon with pre-cooled PBS, a portion of the colon tissue was excised and fixed in 4% paraformaldehyde for H&E staining. The results are shown below. Figure 12 As shown.

[0067] Figure 12Figure 10 shows the in vivo therapeutic effect of CECN-FeS@P / SA on IBD mice. (A) shows the weight changes of mice in different treatment groups (TNBS induced); (B) shows the DAI score of mice in different treatment groups (TNBS induced); (C) shows the weight changes of mice in different treatment groups (DSS induced); (D) shows the DAI score of mice in different treatment groups (DSS induced); (E) shows the H&E staining of mice in different treatment groups (DSS induced). Figure 12 As shown in (A) and (B), the DAI score of mice in the TNBS group was significantly improved and the colon length was significantly shortened. The CECN-FeS@P / SA group significantly improved the above phenomena and its effect was better than the other three treatment groups. Figure 12 As shown in (C), (D), and (E), the DSS group exhibited significantly improved DAI scores and shortened colon length. Furthermore, H&E staining revealed inflammatory cell infiltration, crypt damage, and disruption of colonic epithelial cell integrity. The CECN-FeS@P / SA group significantly improved these findings, demonstrating superior results compared to the FeS@P / SA, ECN@P / SA, and ECN-FeS@P / SA groups.

[0068] The above experimental results show that the present invention is prepared by encapsulating iron-sulfur nanoenzymes and probiotics with plant polysaccharide gum and sodium alginate as carriers. It has the characteristics of low synthesis cost, significant stress resistance and good biocompatibility. Furthermore, it has been verified in vivo that it can alleviate inflammatory enteritis in mice. It can be preliminarily considered to have great research value and clinical translation potential.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described with reference to preferred embodiments, those skilled in the art should understand that various changes in form and detail can be made without departing from the inventive concept of the present invention, without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A probiotic biofilm gel microsphere, characterized in that, The probiotic biofilm gel microspheres are prepared and cultured using pectin, sodium alginate, Escherichia coli Nissle1917, and iron-sulfur nanozymes as raw materials through microfluidic technology.

2. The method for preparing probiotic biofilm gel microspheres according to claim 1, characterized in that, Includes the following steps: (1) Escherichia coli Nissle1917 was transferred and cultured to the logarithmic growth phase to obtain Escherichia coli Nissle1917 transfer culture for use; (2) Pectin and sodium alginate are first dissolved in deionized water, and iron-sulfur nanozyme and Escherichia coli Nissle1917 transfer bacterial solution are added and stirred thoroughly to obtain the aqueous phase; (3) Bispan 80 and isopropyl myristate were mixed to prepare an oil phase, and the aqueous phase and oil phase were mixed by microfluidic technology to prepare plant pectin-encapsulated iron-sulfur nanoenzymes / probiotic microspheres. (4) The iron-sulfur nanoenzyme / probiotic microspheres coated with plant pectin were solidified in CaCl2 solution, washed, and cultured in liquid LB medium to obtain probiotic biofilm gel microspheres.

3. The preparation method according to claim 2, characterized in that, In step (1), Escherichia coli Nissle1917 was cultured in LB medium until the OD value reached 1.0~1.2, and the volume ratio of Escherichia coli Nissle1917 to liquid LB medium was 1:

100.

4. The preparation method according to claim 2, characterized in that, In step (2), the mass ratio of pectin to sodium alginate is 2:1 to 1:2, and the concentrations of iron-sulfur nanozyme and Escherichia coli Nissle1917 in the aqueous phase are 25 to 100 μg / mL and 10 μg / mL, respectively. 5 ~10 7 CFU / mL.

5. The preparation method according to claim 2, characterized in that, In step (2), when pectin and sodium alginate are dissolved in deionized water, they are stirred at room temperature for 60-90 min. After adding iron-sulfur nanozyme and Escherichia coli Nissle1917, they are stirred at room temperature for 10-20 min.

6. The preparation method according to claim 2, characterized in that, In step (3), the volume ratio of Span 80 to isopropyl myristate is 1:10 to 1:

40.

7. The preparation method according to claim 2, characterized in that, In step (3), the volume ratio of the aqueous phase to the oil phase during microfluidic preparation is 1:40 to 1:

100.

8. The preparation method according to claim 2, characterized in that, In step (4), the concentration of the CaCl2 solution is below 750 mM, and the curing time is 20~60 min.

9. The preparation method according to claim 2, characterized in that, In step (4), the culture is carried out in liquid LB medium for 24-48 hours.

10. The use of the probiotic biofilm gel microspheres of claim 1 in the preparation of a drug for stress resistance assessment to promote intestinal colonization and restore inflammatory bowel disease.

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