Double-layer coating probiotic compound as well as preparation method and application thereof

Abstract

The invention relates to a double-layer coating probiotic compound and a preparation method and application thereof, and belongs to the technical field of probiotics, the double-layer coating probiotic compound comprises a probiotic group, a metal-polyphenol nano coating for coating the probiotic group and an HMW-HA coating for coating the metal-polyphenol nano coating, wherein the probiotic group is prepared from bifidobacterium animalis subsp. Lactis WKB148, lactobacillus acidophilus LA18, lactobacillus rhamnosus LRa66, lactobacillus casei LC89 and phytobacterium plantarum Lp18; the probiotic compound not only can effectively reduce the influence of a radiation injury environment on the probiotics and significantly improve the survival ability and delivery efficiency of the probiotics in the radiation injury environment, but also can prolong the retention time of intestinal tracts and significantly improve the targeted delivery and long-acting colonization of the probiotics in the radiation injury intestinal tracts, and meanwhile, the probiotic compound can be used for improving the bioavailability of the intestinal tracts. And the probiotics can also synergistically treat the radioactive intestinal injury, so that the treatment effect of the probiotics in the treatment of the radioactive intestinal injury is effectively improved.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotics, and particularly relates to a double-layer coated probiotic complex, a preparation method thereof, and uses thereof. Background Art

[0002] Probiotics generally refer to a class of beneficial active microorganisms that colonize the human body and change the composition of the flora in a certain part of the host. When ingested in sufficient amounts, they can have a positive impact on the intestinal microecological balance of the host, and thus be beneficial to the health of the host. For example, probiotics can inhibit the growth and reproduction of harmful bacteria by competing with harmful bacteria for attachment sites and nutrients on intestinal epithelial cells, thereby maintaining the balance of the intestinal flora and playing a role in regulating the balance of the intestinal flora; also, probiotics can ferment substances such as carbohydrates in the intestine to produce beneficial metabolites such as short-chain fatty acids and vitamins, providing nutrition for the human body, and at the same time regulating the intestinal pH to create an environment unfavorable for the growth of harmful bacteria; probiotics can interact with the intestinal immune system, stimulate the activity of immune cells, enhance the body's immune defense ability, and help the human body better resist diseases.

[0003] Although probiotics have been widely studied for improving intestinal health, they work by regulating the balance of flora, promoting intestinal epithelial repair, and reducing the release of inflammatory factors. However, the survival rate of naked probiotics is low under the action of gastric acid, bile salts and digestive enzymes, and it is difficult for them to reach the small intestine or colon to work. Therefore, some probiotic preparations coated with a protective layer are disclosed in the prior art, which are usually coated with a protective layer by traditional encapsulation technology (such as PLGA, sodium alginate, etc.). Although it is beneficial to enhance the gastric acid tolerance and antibiotic resistance of probiotics, this structure is also easily degraded in gastric acid, especially in the case of complex or harsh intestinal environment. The survival rate of existing probiotics is very low and the intestinal colonization ability is also poor; for example, radiation intestinal injury (RIII) is a common side effect of cancer patients receiving abdominal and pelvic radiotherapy. Amifostine is the only radiation protection drug approved by the Food and Drug Administration for radiotherapy, but its severe side effects limit its use in preventing dry mouth in patients with head and neck tumors. At present, there is a lack of effective and safe treatment strategies for RIII in routine clinical practice, highlighting the urgent need to develop effective treatments to alleviate RIII. RIII manifests as intestinal inflammation, barrier function damage, and imbalance of microbiota. The oxidative stress environment caused by radiotherapy will further reduce the activity of probiotics because, first of all, radiation causes dual damage to the intestinal microbiota; radiation damage not only leads to imbalance of intestinal microbiota, but also induces mucosal oxidative stress and inflammation. Traditional probiotic preparations are difficult to deal with these two pathological mechanisms at the same time; secondly, there is a lack of targeted delivery systems; existing delivery technologies do not combine specific markers of radiation-damaged intestines (such as highly reactive oxygen environments), and cannot achieve precise drug release or microbiota regulation, resulting in low survival rates of existing probiotic preparations in radiation environments, and difficulty in precise colonization in the intestine, and thus cannot play a good role in the treatment of radiation-induced intestinal damage. Therefore, how to effectively improve the therapeutic effect of radiation-induced intestinal damage and effectively reduce side effects during treatment has become a key issue in current research and needs to be solved urgently. Summary of the invention

[0004] The first aspect of the present invention is to solve the above-mentioned technical problems and provide a double-layer coated probiotic complex with better tolerance in the gastrointestinal tract, which can not only effectively reduce the impact of the radiation damage environment on the probiotics and significantly improve the survival ability of the probiotics in the radiation damage environment, but also increase the intestinal retention time and significantly improve the colonization ability of the probiotics in the radiation-damaged intestine, thereby effectively improving the therapeutic effect of probiotics in the treatment of radiation intestinal damage.

[0005] A double-layer coated probiotic complex, comprising a probiotic group, a metal-polyphenol nano-coating that coats the probiotic group, and an HMW-HA coating that coats the metal-polyphenol nano-coating, wherein the probiotic group comprises Bifidobacterium animalis subsp. lactis WKB148, Lactiplantibacillus plantarum Lp18, and at least one Lactobacillus, and the molecular weight of the HMW-HA coating is greater than or equal to 1.8 MDa. In this solution, by configuring the probiotic group, making the probiotic group include a variety of probiotics, and adopting a double-layer coating structure with a metal-polyphenol nano-coating as the inner layer and an HMW-HA coating as the outer layer, through the synergistic cooperation of these three aspects, it can play a synergistic enhancement role in the actual treatment process. It can not only significantly improve the survival rate and delivery efficiency of probiotics, achieve targeted delivery and long-term colonization effects, realize the precise release of probiotics, but also can achieve multi-functional synergistic treatment of radiation-induced intestinal injury, thereby effectively improving the treatment effect of probiotics in the treatment of radiation and intestinal injury; in addition, it can also effectively reduce side effects during the treatment process.

[0006] Preferably, the Lactobacillus in the probiotic group includes Lactobacillus acidophilus LA18, Lactobacillus rhamnosus LRa66, and Lactobacillus casei LC89; the metal-polyphenol nano-coating is a TA-Fe 3+ nano-coating or a TA-Al 3+ nano-coating or a TA-Ga 3+ nano-coating.

[0007] The second aspect of the present invention aims to solve the problem of low-cost and large-scale preparation of the double-layer coated probiotic complex, and provides a preparation method for the double-layer coated probiotic complex, including the following steps: Step S1, shake the bacteria to obtain a bacterial solution, and the bacterial solution contains a probiotic group, and the probiotic group comprises Bifidobacterium animalis subsp. lactis WKB148, Lactiplantibacillus plantarum Lp18, and at least one Lactobacillus; Step S2, coat a metal-polyphenol nano-coating on the probiotic group; Step S3, coat a hyaluronic acid coating with a molecular weight greater than or equal to 1.8 MDa on the metal-polyphenol nano-coating to obtain a double-layer coated probiotic complex. The inside of the double-layer coated probiotic complex is the probiotic group, the probiotic group is coated with a metal-polyphenol nano-coating, and the metal-polyphenol nano-coating is coated with an HMW-HA coating. The preparation method provided by this solution, on the one hand, belongs to a non-genetic modification path, which can effectively avoid genetic engineering risks and simplify the production process, thereby facilitating cost reduction; on the other hand, this method uses room temperature self-assembly and electrostatic layer coating technology, which not only has a simple process and significantly reduced costs, but also can process 1 kg of bacterial powder in a single batch, with a yield of over 85%, thus effectively solving the complex problems of existing MPN coating sol-gel processes, electro-triggered assembly processes, etc., and facilitating low-cost, high-yield, and large-scale industrial production.

[0008] Preferably, the Lactobacillus in the probiotic group includes Lactobacillus acidophilus LA18, Lactobacillus rhamnosus LRa66, and Lactobacillus casei LC89.

[0009] To solve the problem of successfully coating a metal-polyphenol nano-coating outside the probiotic group, further, step S2 includes: S21. Take an appropriate amount of the bacterial solution and centrifuge it on a centrifuge to obtain a precipitate, and repeatedly wash the precipitate with pure water; S22. Add solution A to the precipitate and mix evenly to obtain a first solution, where solution A uses tannic acid; S23. Add solution B to the first solution, solution B is a metal solution, and the metal solution contains trivalent metal ion X 3+ ; After observing that the solution turns black, place the centrifuge tube on a vortex and shake it to obtain a second solution; S24. Centrifuge and wash the second solution with pure water until the supernatant becomes clear, and collect the obtained black product to obtain Mix@TA-X 3+ complex; The inside of the Mix@TA-X 3+ complex is the probiotic group, and the outside is coated with a metal-polyphenol nano-coating. Adopting such steps is beneficial to successfully coating a metal-polyphenol nano-coating outside the probiotic group.

[0010] Preferably, in S23, the trivalent metal ion X in the metal solution 3+ is Fe 3+ , and the obtained product in S24 is Mix@TA-Fe 3+ complex; or, in S23, the trivalent metal ion X in the metal solution 3+ is Al 3+ , and the obtained product in S24 is Mix@TA-Al 3+ complex; or, in S23, the trivalent metal ion X in the metal solution 3+ is Ga 3+ , and the obtained product in S24 is Mix@TA-Ga 3+ complex.

[0011] Preferably, the volume ratio of solution A to the bacterial solution in S21 is 1:2; the volume ratio of solution B to the bacterial solution in S21 is 1:2. This is beneficial to improving the success rate of coating.

[0012] Preferably, the volume ratio of solution A to solution B is 1:1. This is beneficial to improving the success rate of coating.

[0013] In the third aspect of the present invention, the problem of improving the coating success rate and coating uniformity of the metal-polyphenol nano-coating is to be solved. Further, the concentration ratio of solution A to solution B is 3:1. In this solution, by controlling the concentration ratio of solution A to solution B to 3:1, not only can the successful coating of the metal-polyphenol nano-coating be ensured, and the problem of improving the coating success rate of the metal-polyphenol nano-coating can be solved, but also a more uniform and better coating effect can be obtained, effectively preventing problems such as overcoating resulting in too thick a coating and coating caking. This is not only beneficial to further improving the treatment effect but also can reduce the difficulty of subsequent coating.

[0014] Preferably, the concentration of solution A is 0.6 mg / mL; the concentration of solution B is 0.2 mg / mL.

[0015] To solve the problem of improving the success rate of coating the HMW-HA coating, further, step S3 includes S31. Take the synthesized Mix@TA-X 3+ centrifuge the complex to obtain a precipitate; S32. Add water to the precipitate to dilute the precipitate; then add the diluted precipitate to the HA solution and mix evenly to obtain a third solution; S33. Place the third solution on a stirrer and stir; S34. Centrifuge and wash the third solution again, and finally obtain the product Mix@TA-X 3+ / HA complex. By adopting such steps, it is beneficial to successfully coat the HMW-HA coating outside the metal-polyphenol nano-coating and is beneficial to obtaining a double-layer coating probiotic complex with better size, better effect, and higher success rate.

[0016] In the fourth aspect of the present invention, the problem of improving the success rate of obtaining a smaller-sized nano-scale double-layer coating probiotic complex is to be solved. Further, in S32, the volume ratio of the water added to the precipitate to the added HA solution is 1:1. In this solution, by precisely controlling the volume of water added to the precipitate and the volume of the HA solution, and making the volume ratio of the water added to the precipitate to the HA solution 1:1, not only can a smaller-sized nano-scale double-layer coating probiotic complex be ensured, and the problem of improving the success rate of obtaining a nano-scale double-layer coating probiotic complex can be solved, but also the HMW-HA coating is uniform, which is not only convenient for delivery and digestion but also beneficial to improving the bioavailability and achieving a better treatment effect.

[0017] Preferably, the water added to the precipitate is 1 mL; the dosage of the HA solution is 1 mL; the concentration of the HA solution is 1.0 mg / mL.

[0018] Furthermore, the use of the above double-layer coated probiotic complex in the preparation of drugs for radiation enteritis. It can not only effectively reduce the impact of the radiation-damaged environment on probiotics, significantly improve the survival ability of probiotics in the radiation-damaged environment, but also increase the intestinal retention time and significantly improve the colonization ability of probiotics in the irradiated intestine, thereby effectively improving the therapeutic effect of probiotics in the treatment of radiation-induced intestinal injury.

[0019] Furthermore, the use of the above double-layer coated probiotic complex in the preparation of drugs for obesity intervention.

[0020] Compared with the prior art, a double-layer coated probiotic complex provided by the present invention has the following beneficial effects: (1) Significantly improving the survival rate and delivery efficiency of probiotics: enabling the survival rate of probiotics in the probiotic complex to reach 95.2% in simulated gastric acid (pH 2.0) (less than 10% in the uncoated group), solving the problem of low survival rate of traditional probiotic preparations caused by gastric acid damage, especially significantly improving the survival ability of probiotics in the radiation-damaged environment.

[0021] (2) Targeted delivery and long-term colonization: The HMW-HA coating in the probiotic complex can bind to the intestinal mucus layer through the CD44 receptor, with an adhesion rate of 92% (only 65% for the single-layer MPN), and the colonization time is extended to 2 weeks, thereby increasing the intestinal retention time and significantly improving the colonization ability of probiotics in the irradiated intestine. At the same time, the pH / ROS dual-responsive characteristics of the double-layer coating (TA / Fe 3+ dissociating in the high-ROS environment in the intestine) achieve the precise release of probiotics, and further effectively improve the therapeutic effect of probiotics in the treatment of radiation and intestinal injury.

[0022] (3) Achieving multi-functional synergistic treatment of radiation-induced intestinal injury: ROS scavenging and anti-inflammatory repair; in a radiation-induced injury mouse model, the coated probiotics reduce the intestinal ROS level by 67%, reduce inflammatory factors (IL-6, TNF-α) by 50%, and increase the crypt cell regeneration rate by 2.3 times. Mechanism: The inner layer TA directly scavenges free radicals through phenolic hydroxyl groups; the outer layer HMW-HA inhibits the NF-κB pathway and blocks the inflammatory signal transduction. Microbiota-mucosa dual repair: Bifidobacterium animalis subsp. lactis WKB148 promotes the expression of intestinal barrier protein (ZO-1), and glutathione secreted by Lactiplantibacillus plantarum Lp18 and HMW-HA synergistically repair mucosal damage, so that the probiotic complex can not only be applied to the radiation-induced intestinal injury environment, but also has the ability to actively repair and regulate the intestinal oxidative stress and inflammatory microenvironment. Description of the Drawings

[0023] Figure 1 It is a transmission electron micrograph of the complex obtained in step S2 in Comparative Experiment 1.

[0024] Figure 2 It is the transmission electron microscopy image of the complex obtained in step S2 of Comparative Experiment 2.

[0025] Figure 3 It is the transmission electron microscopy image of the complex obtained in step S2 of Example 1 of the present invention.

[0026] Figure 4 It is the transmission electron microscopy image of the complex obtained in step S2 of Comparative Experiment 3.

[0027] Figure 5 It is the transmission electron microscopy image of the complex obtained in step S2 of Comparative Experiment 4.

[0028] Figure 6 It is the transmission electron microscopy image of the complex obtained in step S32 of Example 1 of the present invention.

[0029] Figure 7 It is the transmission electron microscopy image of the complex obtained in step S32 of Comparative Experiment 5.

[0030] Figure 8 It is the transmission electron microscopy image of the complex obtained in step S32 of Comparative Experiment 6.

[0031] Figure 9 It is the transmission electron microscopy image of the complex obtained in step S32 of Comparative Experiment 7.

[0032] Figure 10 It is the transmission electron microscopy image of the complex obtained in step S32 of Comparative Experiment 8.

[0033] Figure 11 It is the schematic structural diagram of the double-layer coated probiotic complex prepared in Example 1 of the present invention.

[0034] Figure 12 It is the schematic diagram when treating radiation enteritis with the probiotic complex prepared in Example 1 of the present invention.

[0035] Figure 13 It is the photo comparison of the bacterial colonies formed by each group on the agar plate.

[0036] Figure 14 In it, (A) is the in vivo imaging map of the digestive tract of the first group of mice; (B) is the in vivo imaging map of the digestive tract of the second group of mice; (C) is the in vivo imaging map of the digestive tract of the third group of mice; (D) is the in vivo imaging map of the digestive tract of the fourth group of mice.

[0037] Figure 15 It is the colony count of the Mix group, Mix@TA-Fe group and Mix@TA-Fe / HA group at different times.

[0038] Figure 16 Schematic diagram of the viability assay for live / dead cells in each group.

[0039] Figure 17 Shows the preventive and therapeutic effects of the double-coated drug in the radiation enteritis model, where: A is the experimental flow chart, different components of the drug were fed, abdominal radiotherapy at 6.5 Gy was performed on the 7th day, and the mice were sacrificed on the 10th day to obtain small intestine tissues; B is the survival rate up to 30 days; C is the clinical score of mice in each experimental group; D is the length of the small intestine tissue of mice on the 10th day; E is the daily body weight change of mice in each group 3 days after radiotherapy, and the data was normalized to the percentage of the body weight on the 0th day (n = 6, error bars represent ±SD relative to the mean); F is the length of the colon tissue of mice on the 10th day; G is the number of white blood cells in the peripheral blood of mice on the 10th day; H is the number of red blood cells in the peripheral blood of mice on the 10th day; I is the concentration of the anti-inflammatory factor TGF-β on the 10th day (n = 6, error bars represent ±SD relative to the mean); J is the concentration of the pro-inflammatory factor IL-1β on the 10th day (n = 6, error bars represent ±SD relative to the mean); K is the concentration of the pro-inflammatory factor TNF-α on the 10th day (n = 6, error bars represent ±SD relative to the mean); L is the concentration of the anti-inflammatory factor IL-10 on the 10th day (n = 6, error bars represent ±SD relative to the mean); M is the concentration of the pro-inflammatory factor IL-6 on the 10th day (n = 6, error bars represent ±SD relative to the mean).

[0040] Figure 18 In (A), it reveals the microscopic tissue changes in the intestine from a pathological perspective. The colon sections of rats in each group on the 10th day were stained with H&E for analysis, scale bar: 200 μm; (B) shows the pathological immunohistochemical evaluation of intestinal stem cells (Lgr5 cells) and intestinal proliferation (Ki67). Staining images of Lgr5 cells, cell proliferation (Ki67), goblet cells secreting mucus (PAS), and crypt Paneth cells secreting lysosomal enzymes (lysosomal enzyme Lysozyme) in the small intestine tissues of 6 animals in each group are presented.

[0041] Figure 19 In it, A is the quantitative comparison chart of Lgr5 cells / crypts in the small intestine tissue; B is the quantitative comparison chart of Ki67 cells / crypts in the small intestine tissue; C is the quantitative comparison chart of goblet cells (PAS) / villi in the small intestine tissue; D is the quantitative comparison chart of crypts / peripheral length in the small intestine tissue; E is the quantitative comparison chart of lysosomes (Lysozyme) / crypts in the small intestine tissue; F is the quantitative comparison chart of E-cadherin in the small intestine tissue; G is the quantitative comparison chart of occludin in the small intestine tissue; H is the quantitative comparison chart of zonula occludens-1 (ZO-1) in the small intestine tissue.

[0042] Figure 20The middle is a semi-quantitative analysis of the expression of ZO-1, Occludin and E-cadherin (reflecting intestinal barrier integrity and function) in the small intestine of each group by immunofluorescence staining.

[0043] Figure 21 The figure is the body weight change curve of each group of animals in Example 4.

[0044] Figure 22 It is the Lee index of each group of experimental animals in Example 4.

[0045] Figure 23 A is the quantitative correspondence diagram of serum triglyceride (TG) among the main blood lipid indicators after treatment; B is the quantitative comparison diagram of total cholesterol (TCHO) among the main blood lipid indicators after treatment; C is the quantitative comparison diagram of low-density lipoprotein (LDL) among the main blood lipid indicators after treatment; D is the quantitative comparison diagram of high-density lipoprotein (HDL) among the main blood lipid indicators after treatment.

[0046] Explanation of the marks in the figure: 1. Double-layer coating probiotic complex; 11. Probiotic group; 12. Tannic acid (TA); 13. X 3+ ; 14. High molecular weight hyaluronic acid coating (HMW-HA coating); 2. Stomach; 3. Intestine; 4. Radiation source. DETAILED DESCRIPTION

[0047] Example 1

[0048] This embodiment provides an engineering method for preparing a double-layer coating probiotic complex, including step S1, shaking the bacteria to obtain a bacterial solution, the bacterial solution contains a probiotic group, the probiotic group includes animal Bifidobacterium lactis subspecies WKB148, plant Lactobacillus Lp18 and at least one lactobacillus, etc. During implementation, the type and number of lactobacillus can be determined according to actual needs and set aside.

[0049] In this embodiment, WecMix01 probiotic freeze-dried powder is used, which contains animal Bifidobacterium lactis subspecies WKB148, Lactobacillus acidophilus LA18, Lactobacillus rhamnosus LRa66, Lactobacillus casei LC89 and Lactobacillus plantarum Lp18. The probiotic freeze-dried powder is stored at -20 degrees to improve its survival rate and form particles suitable for coating. Specifically, during implementation, the optimized freeze-drying process (using lactose / trehalose as a protective agent and low-temperature spray drying) is preferably used to preserve the probiotic freeze-dried powder, so that the survival rate of live bacteria is ≥90%, and the number of live bacteria decreases ≤1 log CFU / g after storage for 6 months, overcoming the defect of fast activity loss of traditional freeze-dried powder.

[0050] Due to the use of WecMix01 probiotic freeze-dried powder, the probiotic group in the bacterial solution prepared in this step simultaneously includes Lactobacillus animalis subsp. WKB148, Lactobacillus acidophilus LA18, Lactobacillus rhamnosus LRa66, Lactobacillus casei LC89, and Lactiplantibacillus plantarum Lp18. These five probiotics achieve complementary functions of antioxidant, anti-inflammatory, and flora regulation, and can play a synergistic role in the treatment of radiation-induced intestinal injury, thereby achieving a more significant therapeutic effect.

[0051] Step S2: Coating the probiotic group with a metal-polyphenol nanocoating. This is to protect the probiotics from surviving in the gastric acid and bile salt environment, improve the intestinal targeting delivery ability; endow the probiotics with antioxidant ability and increase their survival rate in the radiation injury environment.

[0052] This step specifically includes: S21, taking 1 mL of the bacterial solution and centrifuging it on a centrifuge. During centrifugation, the rotation speed of the centrifuge can be 6000 rpm, the centrifugation time can be preferably controlled at 5 - 10 min, and the centrifugation temperature can be preferably controlled at 4°C; after centrifugation, a precipitate (actually a precipitate of a probiotic group mixed with multiple probiotics) is obtained, and then the precipitate is washed 3 times with pure water.

[0053] S22: Adding a certain amount of solution A to the precipitate and mixing evenly to obtain a first solution. In this embodiment, solution A uses tannic acid (TA) 12, and the dosage of solution A is: 500 μL of solution A is added per milliliter of the bacterial solution, that is, the volume ratio of solution A to the bacterial solution in step S21 is 1:2. In practice, the concentration of solution A can be determined according to actual needs. As an example, in this step, the concentration of solution A is 0.6 mg / mL.

[0054] S23: Adding a certain amount of solution B to the first solution. Solution B is a metal solution, and the metal solution contains trivalent metal ions (for convenience, in this embodiment, X 3+ 13 represents trivalent metal ions). In practice, the metal solution can be determined according to actual needs. For example, the trivalent metal ion X 3+ 13 in the metal solution can be Fe 3+ 、Al 3+ 、Ga 3+ etc. As an example, in this embodiment, solution B uses FeCl3 solution, which contains Fe 3+. In this embodiment, the dosage of solution B is: 500 μL of solution B is added to each milliliter of the bacterial solution, that is, the volume ratio of solution B to the bacterial solution in step S21 is 1:2. During implementation, the concentration of solution B can be determined according to actual requirements. By way of example, in this step, the concentration of solution B is 0.2 mg / mL; that is, in this embodiment, the volume ratio of the dosage of solution A to the dosage of solution B is 1:1, and the concentration ratio of solution A to solution B is 3:1 (that is, the concentration ratio of TA to Fe is 3:1), which can not only coat the probiotic group with a metal-polyphenol nanocoating to obtain a very good coating effect, as Figure 3 shown, but also effectively improve the coating success rate of the metal-polyphenol nanocoating. After adding solution B to the first solution, it is observed that the solution instantly turns black. At this time, place the centrifuge tube on a vortex and shake for 1 min to fully mix it with the precipitate to obtain a second solution.

[0055] S24. Centrifuge and wash the second solution with pure water until the supernatant becomes clear, and collect the obtained black product to obtain the Mix@TA-X 3+ complex. The inside of the Mix@TA-X 3+ complex is the probiotic group, and the outside is coated with a metal-polyphenol nanocoating. In this embodiment, the trivalent metal ion X 3+ used is Fe 3+ , and the obtained is the Mix@TA-Fe 3+ complex; in other embodiments, the trivalent metal ion X 3+ can be Al 3+ , and at this time, what is obtained in this step is the Mix@TA-Al 3+ complex; the trivalent metal ion X 3+ can also be Ga 3+ , and at this time, the obtained is the Mix@TA-Ga 3+ complex.

[0056] In this step, during centrifugation, the rotation speed of the centrifuge can be 6000 rpm, the centrifugation time is preferably controlled at 5 min, and the centrifugation temperature is preferably controlled at 4 °C. Finally, according to the ratio of replenishing 1 milliliter of water per milliliter of the bacterial solution, re-fix the volume back to the previous concentration. For example, the concentration of solution A is 0.6 mg / mL, and the concentration of solution B is 0.2 mg / mL. Through the metal-polyphenol self-assembly technology, a nanocoating is formed on the surface of the probiotics by using the complexation of TA 12 and X 3+ 13. X 3+ 13 forms a stable TA-X 3+ complex by interacting with the phenolic hydroxyl groups of TA 12 and coats the probiotic group in a weakly acidic environment (pH~5.0).

[0057] To improve the coating success rate and coating uniformity of the metal-polyphenol nanocoating in this step, it was found during implementation that the concentration ratio of solution A to solution B needs to be strictly controlled. Because according to previous experiments, only by strictly controlling the concentration ratio of solution A to solution B can the successful coating of the metal-polyphenol nanocoating be ensured and a more uniform coating effect be obtained. Specifically, in the previous comparative experiments, comparative experiment 1, comparative experiment 2, comparative experiment 3, and comparative experiment 4 were carried out simultaneously by the method of controlling variables. Among them, the difference between step S2 in comparative experiment 1 and the above-mentioned step S2 in this embodiment is that the concentration ratio of solution A to solution B is 1:1 instead of 3:1, and other experimental conditions are the same as above. The transmission electron micrograph of the complex obtained in step S2 of comparative example 1 is as Figure 1 shown. In the figure, hardly any protruding iron ions can be seen and they are very discrete, indicating that the metal-polyphenol nanocoating was not successfully coated.

[0058] The difference between step S2 in comparative experiment 2 and the above-mentioned step S2 in this embodiment is that the concentration ratio of solution A to solution B is 2:1, and other experimental conditions are the same as above. The transmission electron micrograph of the complex obtained in step S2 of comparative experiment 2 is as Figure 2 shown. In the figure, there are more protruding iron ions attached to the surface of the probiotic group, but the internal probiotic group is not effectively covered and the coating is very discrete, indicating that the metal-polyphenol nanocoating outside the probiotic group was not successfully coated.

[0059] The difference between step S2 in comparative experiment 3 and the above-mentioned step S2 in this embodiment is that the concentration ratio of solution A to solution B is 4:1, and other experimental conditions are the same as above. The transmission electron micrograph of the complex obtained in step S2 of comparative experiment 3 is as Figure 4 shown. In the figure, there are too many protruding iron ions attached to the surface of the probiotic group, making the metal-polyphenol nanocoating outside the probiotic group too thick and showing signs of agglomeration, resulting in an uneven coating. During the treatment process, the internal probiotics are not easily released, thus causing the metal-polyphenol nanocoating outside the probiotic group to not achieve the expected effect and also affecting the subsequent coating process, increasing the difficulty of subsequent coating.

[0060] The difference between step S2 in comparative experiment 4 and the above-mentioned step S2 in this embodiment is that the concentration ratio of solution A to solution B is 5:1, and other experimental conditions are the same as above. Since the concentration of solution A is too high, there is too much TA and solution A is too viscous. The transmission electron micrograph of the complex obtained in step S2 of comparative experiment 4 is as Figure 5 shown. In the figure, it is obvious that the metal-polyphenol nanocoating outside the probiotic group has agglomerated and the coating is very uneven, not only resulting in coating failure, but also unable to achieve a better treatment effect, and seriously affecting the subsequent coating process, increasing the difficulty of subsequent coating.

[0061] By comparison, by controlling the concentration ratio of solution A to solution B to 3:1, not only can the successful coating of the metal-polyphenol nanocoating be ensured, the problem of improving the coating success rate of the metal-polyphenol nanocoating can be solved, but also a more uniform and better coating effect can be obtained, effectively preventing problems such as overcoating resulting in too thick a coating and coating caking. This is not only beneficial to further improving the treatment effect, but also can reduce the difficulty of subsequent coating.

[0062] Step S3: Coating a high molecular weight hyaluronic acid coating (HMW-HA coating) 14 on the metal-polyphenol nanocoating. The molecular weight of the high molecular weight hyaluronic acid coating is greater than or equal to 1.8 MDa. Characteristics of the HMW-HA coating 14: pH-responsive release: Stable under gastric acid (pH < 3.0) in the stomach 2, and gradually degrades in the small intestine (pH 6.5 - 7.5), as Figure 12 shown (in the figure, 3 is the intestine and 4 is the radiation source), releasing the probiotic group 11. At the same time, the HMW-HA coating has an antioxidant effect. Specifically, the complex of TA 12 and X 3+ 13 can neutralize free radicals and reduce the impact of radioactive damage on probiotics. In addition, the HMW-HA coating can also form a protective barrier: improving the tolerance of probiotics to bile salts and digestive enzymes. This step specifically includes S31: Centrifuging the synthesized Mix@TA-X 3+ complex to remove impurities and supernatant, and obtaining a precipitate.

[0063] S32: Adding a certain amount of water to the precipitate to dilute the precipitate, obtaining the Mix@TA-X 3+ complex, and then adding the diluted Mix@TA-X 3+ complex to a certain amount of HA solution and mixing evenly to obtain a third solution; In implementation, the amount of water added is equal to the amount of HA solution added. For example, when the amount of water added to the precipitate is y mL, correspondingly, y mL of HA solution is added; As an example, in this embodiment, the amount of water added is 1 mL, and correspondingly, the dosage of the HA solution is also 1 mL, that is, the volume ratio of the water added to the precipitate to the HA solution added is 1:1. In implementation, the concentration of the HA solution can be determined according to actual needs, and the concentration of the HA solution can preferably be controlled at 0.6 - 1.5 mg / mL. For example, in this step, the concentration of the HA solution is 1.0 mg / mL, and the volume ratio of the water added to the precipitate to the HA solution added is 1:1. The transmission electron micrograph of the obtained double-layer coated probiotic complex is as Figure 6As shown in the figure, it can be seen that the HMW-HA coating completely wraps the metal-polyphenol nano-coating. The coating is uniform and very thin, making the volume of the entire double-layer coating probiotic complex small enough to reach the nanoscale for transportation and digestion, which is beneficial to improving bioavailability.

[0064] To improve the success rate of obtaining a smaller-sized nano-scale double-layer coating probiotic complex, it was found during implementation that: the amount of water and the amount of HA solution added in step S32 have a direct impact on whether a double-layer coating probiotic complex with better size, better effect, and higher success rate can be obtained. Corresponding comparative experiments were conducted to explore this, specifically, comparative experiments 5, 6, 7, and 8 were carried out simultaneously by the method of controlling variables. Among them, the difference between step S32 in comparative experiment 5 and the above-mentioned step S32 in this embodiment is that: the volume ratio of water added to the precipitated HA solution is 3:1, and other experimental conditions are the same as those in this embodiment. The transmission electron micrograph of the complex obtained in step S32 of comparative experiment 5 is as Figure 7 shown, and no hyaluronic acid coating is seen, indicating that the HMW-HA coating was not successfully coated.

[0065] The difference between step S32 in comparative experiment 6 and the above-mentioned step S32 in this embodiment is that: the volume ratio of water added to the precipitated HA solution is 2:1, and other experimental conditions are the same as those in this embodiment. The transmission electron micrograph of the complex obtained in step S32 of comparative experiment 6 is as Figure 8 shown. It can be seen that there is a hyaluronic acid coating in some local areas and no hyaluronic acid coating in other local areas. The hyaluronic acid coating does not completely wrap the metal-polyphenol nano-coating, indicating that the HMW-HA coating was not successfully coated.

[0066] The difference between step S32 in comparative experiment 7 and the above-mentioned step S32 in this embodiment is that: the volume ratio of water added to the HA solution is 1:2, and other experimental conditions are the same as those in this embodiment. The transmission electron micrograph of the complex obtained in step S32 of comparative experiment 7 is as Figure 9 shown. It can be seen that the hyaluronic acid coating completely wraps the metal-polyphenol nano-coating, and it is obvious that the thickness of the hyaluronic acid coating is relatively thick, making the volume of the entire composite too large, which is not conducive to transportation and digestion and affects bioavailability.

[0067] The difference between step S32 in comparative experiment 8 and the above-mentioned step S32 in this embodiment is that: the volume ratio of water added to the precipitated HA solution is 1:3, and other experimental conditions are the same as those in this embodiment. The transmission electron micrograph of the complex obtained in step S32 of comparative experiment 8 is as Figure 10As shown, although the visible hyaluronic acid coating completely wraps the metal-polyphenol nano-coating, the transparency of the entire composite is relatively high, indicating that the coating thickness of the hyaluronic acid coating is too thick, making the volume of the entire composite too large. This not only makes it difficult to obtain a smaller-sized nano-scale double-layer coated probiotic composite, which is not convenient for delivery and digestion, but also significantly reduces the bioavailability.

[0068] By comparison, by controlling the volume ratio of the water added to the precipitate to the HA solution added to 1:1, not only can a smaller-sized nano-scale double-layer coated probiotic composite be ensured, which can solve the problem of improving the success rate of obtaining the nano-scale double-layer coated probiotic composite, but also the HMW-HA coating is uniform, which is not only convenient for delivery and digestion, but also conducive to improving the bioavailability and achieving better therapeutic effects.

[0069] S33. Place the third solution on a stirrer and stir. The stirring time can preferably be controlled at 6 - 12 h. For example, the third solution can be stirred on the stirrer for 8 h, 9 h, or 10 h, etc. Since the HA solution is relatively viscous, continuously stirring the third solution in this step is conducive to a wider range of coating of the inner layer coating.

[0070] S34. Centrifuge and wash the third solution again, and finally obtain the product Mix@TA-X 3+ / HA composite, where Mix@TA-X 3+ / HA composite has a double-layer coating structure. As Figure 11 shown, the inside is probiotic group 11, and the probiotic group 11 is coated with a metal-polyphenol nano-coating (in this embodiment, it is a TA-X 3+ complex layer, which can also be written as: MPN(TA / X 3+ ))). The metal-polyphenol nano-coating is the inner layer, and the metal-polyphenol nano-coating is coated with an HMW-HA coating 14. The HMW-HA coating (hyaluronic acid coating) is the outer layer. In this step, when centrifuging, the rotation speed of the centrifuge can be 6000 rpm, the centrifugation time can preferably be controlled at 5 min, and the centrifugation temperature can preferably be 4°C.

[0071] It can be understood that when the trivalent metal ion X 3+ in solution B 3+ is Fe 3+ in this step, the obtained product is Mix@TA-Fe Figure 11 / HA composite, as 3+ shown; in implementation, when the trivalent metal ion X 3+ in solution B 3+ is Al 3+ in this step, the obtained product is Mix@TA-Al3+ At this time, what is obtained in this step is Mix@TA-Ga 3+ / HA complex.

[0072] In practice, to determine whether the HMW-HA coating is successfully coated, it can be achieved by measuring the particle size of the probiotic group. For example, detecting the probiotic complex prepared in this example: (1) Coating morphology and particle size characterization: Transmission electron microscopy (TEM) shows that a uniform nanolayer is formed on the surface of the probiotics after coating. (2) Dynamic light scattering (DLS) results: The particle size before coating is 1.2 ± 0.3 μm, and the particle size increases to 1.8 ± 0.2 μm after double coating, indicating successful coating and the coating is very thin. (3) Zeta potential measurement shows that the zeta potential changes from -15.2 mV to -22.4 mV after coating, indicating that the HA solution is successfully adsorbed on the outer side of the metal-polyphenol nanocoating. The structure of the double-layer coated probiotic complex 1 prepared is as Figure 11 shown. In addition, in the comparative experiment, the Mix@TA-Fe 3+ / HA complex prepared by the above method still maintains a survival rate of more than 85% after storage at room temperature (25°C) for 30 days, which is much higher than that of the uncoated group (50%).

[0073] The structure of the double-layer coated probiotic complex 1 prepared in this example is as Figure 11 shown. The synergistic cooperation between the inner layer MPN (TA / Fe 3+ ) and the outer layer HMW-HA can solve the integrated problems of protection, targeting, and repair, enabling the double-layer coated probiotic complex 1 to effectively improve the therapeutic effect of probiotics in the treatment of radioactive intestinal injury, mainly reflected in: (1) It can significantly improve the survival rate and delivery efficiency of probiotics; the double-layer coating (inner layer TA / Fe 3+ , outer layer HMW-HA) protects the survival rate of probiotics up to 95.2% in simulated gastric acid (pH 2.0) (less than 10% in the uncoated group); it solves the problem of low survival rate caused by gastric acid damage in traditional preparations. Mechanism: The TA / Fe 3+ network forms a dense barrier under acidic conditions to block the penetration of pepsin; at the same time, the outer layer HMW-HA can further reduce the erosion of bile salts on the bacteria.

[0074] (2) Targeted delivery and long-term colonization: Intestinal adhesion and responsive release; HMW-HA binds to the intestinal mucus layer through the CD44 receptor, and the adhesion rate reaches 92% (only 65% for single-layer MPN), as Figure 12 and Figure 14 shown, and the colonization time is extended to 2 weeks. Mechanism: The pH / ROS dual-responsive characteristics of the double-layer coating (TA / Fe 3+Dissociated in the high-ROS environment of the intestine) to achieve precise release of probiotics. Specifically, in a comparative study using Mix@TA-Fe 3+ and Mix@TA-Fe 3+ / HA, four groups of mice fed under the same conditions were selected. The first group of mice was not irradiated (i.e., this group of mice did not have radiation enteritis) and was fed the Mix@TA-Fe 3+ complex. After 6 hours, in vivo imaging of the digestive tract of this group of mice was performed, and the in vivo imaging of the digestive tract of the mice shown in (A) in Figure 14 was obtained; the second group of mice was irradiated (i.e., this group of mice had radiation enteritis) and was fed the Mix@TA-Fe 3+ complex. After 6 hours, in vivo imaging of the digestive tract of this group of mice was performed, and the in vivo imaging of the digestive tract of the mice shown in (B) in Figure 14 was obtained; similarly, the third group of mice was not irradiated (i.e., this group of mice did not have radiation enteritis) and was fed the Mix@TA-Fe 3+ / HA complex. After 6 hours, in vivo imaging of the digestive tract of this group of mice was performed, and the in vivo imaging of the digestive tract of the mice shown in (C) in Figure 14 was obtained; the fourth group of mice was irradiated (i.e., this group of mice had radiation enteritis) and was fed the Mix@TA-Fe 3+ / HA complex. After 6 hours, in vivo imaging of the digestive tract of this group of mice was performed, and the in vivo imaging of the digestive tract of the mice shown in (D) in Figure 14 was obtained; the other experimental conditions of the four groups of mice were the same and will not be elaborated here. When mice have radiation enteritis, the CD44 receptors in the digestive tract of the mice will increase, and the double-layer coated probiotic complex provided in this example can recognize the CD44 receptors and finally stay at the place of the CD44 receptors, achieving the effect of effectively enhancing inflammatory targeted delivery and long-term colonization.

[0075] As can be seen from Figure 14 , after 6 hours, the fluorescence signal intensity of the fourth group of mice was significantly higher than that of other groups, indicating that the polyphenol-coated variant enhanced inflammatory targeted delivery through electrostatic interaction. However, the HA-functionalized system showed a 1.6-fold fluorescence signal in the inflamed area, highlighting the importance of CD44 receptor-mediated targeting. This different retention pattern indicates that the dual targeting strategy combining charge attraction and receptor-mediated recognition significantly enhances site-specific adhesion. Immunohistochemistry and co-localization studies confirmed the overexpression of CD44 receptors in inflammatory lesions and showed that Mix@TA-Fe 3+ / HA preferentially accumulates in these CD44-rich regions, verifying the dual targeting strategy. That is, Mix@TA-Fe in this example 3+ / HA can effectively target and deliver RII through cooperative electrostatic interactions and the involvement of CD44 receptors. This dual-targeting strategy is a feasible approach to enhancing the efficacy of probiotics against RIII.

[0076] (3) Achieving multifunctional synergistic therapy for radiation-induced intestinal injury: ROS scavenging and anti-inflammatory repair; In a mouse model of radiation injury, the coated probiotics reduced the intestinal ROS level by 67%, decreased inflammatory factors (IL-6, TNF-α) by 50%, and increased the crypt cell regeneration rate by 2.3 times. Mechanism: The inner layer TA 12 directly scavenges free radicals through phenolic hydroxyl groups; the outer layer HMW-HA inhibits the NF-κB pathway and blocks inflammatory signal transduction. Microbiota-mucosa dual repair: Bifidobacterium animalis WKB148 promotes the expression of intestinal barrier protein (ZO-1), and glutathione secreted by Lactiplantibacillus plantarum Lp18 and HMW-HA synergistically repair mucosal damage, enabling this probiotic complex to not only be applicable to the radiation-induced intestinal injury environment but also have the ability to actively repair and regulate the intestinal oxidative stress and inflammatory microenvironment.

[0077] The preparation of the double-layer coated probiotic complex by the engineering method provided in this embodiment can effectively avoid the risks of genetic engineering and simplify the production process, belonging to a non-genetic modification path: specifically, through material combination (TA / Fe 3+ and HMW-HA) to replace the overexpression of SOD / CAT in genetically engineered bacteria, avoiding biosafety disputes, and at the same time having ROS scavenging ability comparable to that of the engineered bacteria AAEcN (the DPPH scavenging rate is increased by 3.8 times). In specific implementation, this method uses room-temperature self-assembly and electrostatic layer-by-layer coating (LbL) technology, which can process 1 kg of bacterial powder in a single batch with a yield ≥ 85%, thus effectively solving the problem of the complex process of the MPN coating sol-gel method and being conducive to large-scale production.

[0078] Example 2: In vitro gastrointestinal pH release test

[0079] I. Experimental conditions: Simulated gastrointestinal fluids (SGF, pH 2.0; SIF, pH 7.0) were used to evaluate the pH-dependent release characteristics of the Mix@TA-Fe 3+ / HA complex.

[0080] II. Experimental Procedure: Four groups were set up, namely the natural Mix group, the Mix + SGF group (where SGF represents simulated gastrointestinal fluid), the Mix@TA-Fe + SGF group, and the Mix@TA-Fe / HA + SGF group. Among them, the natural Mix group was the untreated control group (Con): The natural Mix group used Mix probiotics, which is a mixed preparation containing multiple probiotic strains. As an example, the probiotic strains contained in Mix can include Bifidobacterium animalis subsp. lactis WKB148, Lactiplantibacillus plantarum Lp18, Lactobacillus acidophilus LA18, Lactobacillus rhamnosus LRa66, and Lactobacillus casei LC89. In the Mix + SGF group, the number and types of strains contained in Mix were the same as those in the untreated control group (Con). In the Mix@TA-Fe + SGF group, the Mix probiotics were used inside Mix@TA-Fe, and the number and types of strains contained in the Mix probiotics were the same as those in the untreated control group (Con). In the Mix@TA-Fe / HA + SGF group, the Mix probiotics were used inside Mix@TA-Fe / HA, and the number and types of strains contained in the Mix probiotics were the same as those in the untreated control group (Con). The above groups were incubated in different pH environments, and the total incubation time was 2 h. The survival rate of probiotics and the dissociation of the coating in each group were detected during or 2 h after the incubation.

[0081] III. Experimental Results: At pH = 2.0, the TA-Fe 3+ coating in the Mix@TA-Fe 3+ / HA complex was stable, and the survival rate of probiotics > 90%. At pH 7.0, the TA-Fe 3+ coating in the Mix@TA-Fe 3+ / HA complex dissociated, and the survival rate of probiotics was 85%. This indicates that the TA-Fe 3+ coating has excellent gastric acid protection and small intestine-targeted release characteristics.

[0082] Specifically, as Figure 13 shown in: Photos of bacterial colonies formed on agar plates in the Mix + SGF group, Mix@TA-Fe + SGF group, and Mix@TA-Fe / HA + SGF group after incubating with simulated gastric fluid SGF (pH = 2) supplemented with pepsin for 1 hour, encapsulating the in vitro resistance of bacteria to environmental attacks. The uncoated natural Mix group was used as a control, and the dilution time for each plate was 5.5×10 3 . Figure 15It was as follows: After incubation with pepsin-containing SGF (pH = 2) at 37 °C and 200 rpm at a predetermined time point, the number of colonies in the natural Mix group, Mix@TA-Fe+SGF group, and Mix@TA-Fe / HA+SGF group was determined by plate counting. CFU was expressed as colony-forming units. During implementation, the data were expressed as mean ± standard deviation (n = 3), and statistical analysis was performed using Student's t-test (i.e., t-test), where * p < 0.05, ** p < 0.01, *** p < 0.001.

[0083] As Figure 13 and Figure 15 shown, compared with uncoated Mix, the tolerance of coated Mix to SGF was significantly enhanced. After exposure to SGF for 1 h, the survival rate of Mix@TA-Fe / HA was approximately 6 times that of uncoated Mix. As the culture time extended from 1 h to 2 h, only a small fraction of uncoated Mix cells survived successfully. In contrast, a large number of viable bacteria could still be seen in the Mix@TA-Fe and Mix@TA-Fe / HA groups, and the number of viable bacteria in the Mix@TA-Fe / HA group was significantly higher than that in the Mix@TA-Fe group.

[0084] Figure 16 Schematic diagram of the live / dead cell viability experiment. Representative confocal images of the natural Mix group, Mix+SGF group, Mix@TA-Fe group, and Mix@TA-Fe / HA group stained with SYTO 9 (green, viable bacteria) and PI (red, dead bacteria) before and after treatment with pepsin-containing SGF (pH = 2) for 1 h. It can be seen from Figure 16 this that the gastric environment with low pH is more lethal to uncoated Mix, and almost no live cells were observed after SGF incubation. In contrast, a large number of viable coated cells were observed, indicating that surface coating protected Mix from acidic conditions.

[0085] Example 3: Verification of a mouse model of radiation-induced intestinal injury

[0086] I. Experimental conditions: Animal model construction: C57BL / 6J mice (6 - 8 weeks old, n = 30) were given whole abdominal irradiation (6.5 Gy) to establish a radiation-induced intestinal injury model.

[0087] II. Experimental procedure: After modeling, the mice were randomly divided into five groups, namely Group A, B, C, D, and E (n = 6 for each group), and survival experiments were conducted. Among them, Group A: untreated control group (Control), referred to as the control group for short. Group B: radiation + PBS group (R + PBS), referred to as Group R for short. Group C: radiation + Mix uncoated group (R + Mix). Group D: radiation + Mix@TA-Fe group (R + Mix@TA-Fe). Group E: radiation + Mix@TA-Fe / HA group (R + Mix@TA-Fe / HA). Each mouse in the experimental groups was orally administered 0.02 g of the designated strain powder (i.e., Mix, Mix@TA-Fe, and Mix@TA-Fe / HA were fed respectively corresponding to the grouping), containing 600 billion colony-forming units (CFU), and dissolved in phosphate buffer (PBS), and continuously orally administered for 7 days before receiving 6.5 Gy whole abdominal irradiation (WAI). Mice in other groups except the control group were irradiated as described above. Within 3 days after irradiation, the body weight changes and other physical signs of the mice were monitored, and clinical scores were recorded; small intestine tissues, colon, plasma, heart, liver, spleen, kidney, pancreas, and feces were collected 3 days after irradiation, and the specific procedures are shown in Figure 17 Figure A in

[0088] III. Evaluation indicators: Body weight changes, intestinal tissue pathological score (H&E staining), inflammatory factors (TNF-α, IL-6, IL-1β), anti-inflammatory factors (IL-10, TGF-β) detection, intestinal barrier proteins (ZO-1, Occludin), and immunofluorescence of intestinal epithelial tight junctions (E-cadherin).

[0089] IV. Experimental results: 1. Body weight changes: As shown in C and E of Figure 17 , the body weight of the mice in Group R decreased significantly, while the body weight of the R + Mix@TA-Fe / HA group remained stable (p < 0.01), with a higher survival rate and lower clinical score. Figure 17 As shown in D and F of

[0090] , there was no obvious shortening of the small intestine length and colon length after dissection. Figure 17 Figure G - M in

[0091] 2. Inflammatory level: Blood routine showed that there was no obvious increase in white blood cells in the R + Mix@TA-Fe / HA group. ELISA experiments showed that the expressions of pro-inflammatory factors TNF-α, IL-6, and IL-1β in the R + Mix@TA-Fe / HA group were significantly decreased (p < 0.05), and the anti-inflammatory factors IL-10 and TGF-β in the R + Mix@TA-Fe / HA group were increased, which was significantly better than that of the common probiotic group. For details, see Figure 18as shown in (A) therein; Figure 18 In (B), the small intestine tissues of 6 animals in each group were stained with Lgr5, cell proliferation (Ki67), goblet cells (PAS), and crypt Paneth cells (lysosomal enzyme Lysozyme) to evaluate intestinal barrier damage; scale bar: 100 μm; from Figure 18 It can be seen from (B) therein that R+Mix@TA-Fe / HA can promote cell proliferation, reduce crypt cell damage, and effectively reduce intestinal injury.

[0092] 4. Figure 19 A-H in therein are quantitative analyses, which can reflect the integrity of the intestinal mucosal barrier. Barrier proteins: The expression levels of ZO-1, Occludin, and E-cadherin in the R+Mix@TA-Fe / HA group were restored, and the integrity of the intestinal barrier was enhanced. For details, see Figure 19 F-H in therein and Figure 20 . In this example, the data are expressed as mean ± standard deviation (n = 5), and Student's t-test (i.e., t-test) was used for statistical analysis. Among them, * represents P < 0.05, ** represents P 0.01, *** represents P < 0.001, and **** represents P < 0.0001.

[0093] Example 4: Anti-obesity

[0094] Obesity is a chronic metabolic disease caused by the imbalance between energy intake and consumption. Existing obesity intervention methods mainly include lifestyle management (diet control and exercise), drug treatment, and surgical intervention. However, these methods all have certain limitations, such as poor compliance, drug side effects, and the invasiveness of surgery. In recent years, the role of the gut microbiota in the occurrence and development of obesity has received extensive attention. Studies have found that there are significant differences in the gut microbiota composition between obese individuals and normal-weight individuals, manifested as an imbalance in the Bacteroidetes / Firmicutes ratio (a decrease in the Bacteroidetes / Firmicutes ratio), disorders in the metabolism of short-chain fatty acids (SCFAs), and an increase in the microbiota that promotes fat absorption (such as certain strains of the phylum Firmicutes). Probiotics, as a key intervention means for regulating the gut microbiota, have been proven to improve the host's metabolic status, promote energy consumption, and reduce obesity-related inflammation. Although the current probiotic products on the market can regulate the gut microbiota to a certain extent, firstly, due to their low survival rate in the gastrointestinal environment, some strains cannot reach the intestine smoothly to play their roles, resulting in poor actual clinical effects; secondly, probiotics are challenged by the gastrointestinal environment. When passing through the gastrointestinal tract, probiotics will be damaged by factors such as gastric acid, bile salts, and digestive enzymes, leading to a significant decrease in the number of viable bacteria, thus affecting their colonization and function in the intestine. Although the existing probiotic protection coating technology is beneficial to improving the acid tolerance of probiotics, traditional polymer embedding may affect the activity and release pattern of probiotics, and low-molecular-weight hyaluronic acid has poor stability in the intestine and is difficult to provide long-term protection (that is, it is difficult to achieve the dual functions of acid protection and promoting intestinal colonization at the same time), resulting in a significant reduction in the anti-obesity effect of existing probiotic preparations. The probiotic complex prepared in Example 1 above effectively overcomes the defects of traditional probiotic preparations and has the following remarkable technical effects: 1. It can significantly improve the gastrointestinal survival rate of probiotics, enabling them to effectively reach the intestine; 2. It can effectively enhance the intestinal colonization ability of probiotics, improving their persistence and action duration; thus, it can effectively enhance the anti-obesity efficacy of probiotics and optimize the gut microbiota regulation effect. In this example, the efficacy of the probiotic complex prepared by the present invention in anti-obesity was verified through mouse experiments, specifically the effect of this probiotic complex on high-fat diet-induced obese mice.

[0095] I. Establishment of animal model: Experimental subjects: 8-week-old C57BL / 6 male mice (n = 24). Feeding protocol: After 1 week of adaptive feeding for all mice, they were randomly divided into 4 groups (n = 6): 1. HFD + PBS control group (HFD); 2. HFD + uncoated probiotic group (HFD+Mix); 3. HFD + single-coated probiotic group (HFD+Mix@TA-Fe); 4. HFD + double-coated probiotic group (HFD+Mix@TA-Fe / HA). Induction of obesity by high-fat diet: Mice were fed a high-fat diet (HFD, 60% calories from fat) for 2 weeks to induce obesity, and then intervened after 2 weeks; the probiotic complex (1*10 9 CFU / mL) was gavaged once a week for 4 weeks, and samples were taken for analysis at the 9th week.

[0096] II. Key detection indicators: 1. Body weight and fat accumulation: The body weight of mice was measured weekly, and the weights of adipose tissues (white adipose tissue, brown adipose tissue) were measured at the end of the experiment. 2. Lee's index (LEE'S INDEX = (body weight * 1000)^(1 / 3) / body length (cm)). 3. Blood lipid metabolism (TG, TCHO, LDL, HDL).

[0097] III. Experimental results: As can be seen from Figure 21 , the double-coated probiotic complex can significantly reduce fat production. As Figure 22 shows the Lee's index of the four groups of mice = (body weight * 1000)^(1 / 3) / body length (cm), it can be seen from the figure that the double-coated probiotic complex can reduce mouse obesity. As Figure 23 shows the changes in the main blood lipid indexes of mice after treatment: serum triglyceride (TG), total cholesterol (TCHO), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) respectively. As can be seen from Figure 23 , the double-coated probiotic group can relieve liver damage.

[0098] In summary: 1. This double-coated probiotic complex can more significantly inhibit the weight gain induced by high-fat diet, reduce fat accumulation, and improve insulin sensitivity. 2. The gastric acid tolerance experiment shows ( Figure 13 the colony counts of different drugs in simulated gastric juice), and this double-coated probiotic complex significantly improves the gastrointestinal survival rate of probiotics (the survival rate increases by more than 5 times in the pH 2.0 environment). 3. Compared with the control group, the Lee's index and body mass index of mice treated with Mix@TA-Fe / HA also improved. At the same time, various main lipid biochemical indexes, including triglyceride (TG), total cholesterol (TCHO), low-density lipoprotein (LDL), and high-density lipoprotein (HDL), as Figure 23As shown, all of these indicators tend to normalize after treatment, and the indicators reflecting liver function and fat synthesis are the lowest, with the best effects.

Claims

1. A double-layer coated probiotic complex, characterized in that, It includes a probiotic group, a metal-polyphenol nanocoating that coats the probiotic group, and an HMW-HA coating that coats the metal-polyphenol nanocoating. The probiotic group contains Bifidobacterium animalis subsp. lactis WKB148, Lactiplantibacillus plantarum Lp18, and at least one Lactobacillus, and the molecular weight of the HMW-HA coating is greater than or equal to 1.8 MDa.

2. The double-layer coated probiotic complex according to claim 1, wherein, The Lactobacillus in the probiotic group includes Lactobacillus acidophilus LA18, Lactobacillus rhamnosus LRa66, and Lactobacillus casei LC89; The metal-polyphenol nanocoating is TA-Fe 3+ nanocoating or TA-Al 3+ nanocoating or TA-Ga 3+ nanocoating.

3. A method for preparing the double-layer coated probiotic complex described in claim 1, characterized in that, It includes the following steps Step S1: Shake the bacteria to obtain a bacterial solution. The bacterial solution contains a probiotic group, and the probiotic group contains Bifidobacterium animalis subsp. lactis WKB148, Lactobacillus acidophilus LA18, and at least one Lactobacillus; Step S2: Coat a metal-polyphenol nanocoating outside the probiotic group; Step S3: Coat a hyaluronic acid coating with a molecular weight greater than or equal to 1.8 MDa outside the metal-polyphenol nanocoating to obtain a double-coated probiotic complex. The inside of the double-coated probiotic complex is the probiotic group, the probiotic group is coated with a metal-polyphenol nanocoating, and the metal-polyphenol nanocoating is coated with an HMW-HA coating.

4. The preparation method of the double-layer coated probiotic complex according to claim 3, characterized in that, The step S2 includes: S21: Take an appropriate amount of the bacterial solution and centrifuge it on a centrifuge to obtain a precipitate, and repeatedly wash the precipitate with pure water; S22: Add solution A to the precipitate and mix evenly to obtain a first solution, where solution A uses tannic acid; S23. Add solution B to the first solution. Solution B is a metal solution, and the metal solution contains trivalent metal ion X 3+ ; After observing that the solution turns black, place the centrifuge tube on a vortex and shake it to obtain a second solution; S24. Centrifuge and wash the second solution with pure water until the supernatant becomes clear, and collect the obtained black product to obtain the Mix@TA-X 3+ complex, Mix@TA-X 3+ The interior of the complex is a probiotic group, and the exterior is coated with a metal-polyphenol nano-coating.

5. The preparation method of the double-layer coated probiotic complex according to claim 4, characterized in that, In the step S2, the concentration ratio of solution A to solution B is 3:

1.

6. The preparation method of the double-layer coated probiotic complex according to claim 5, characterized in that, The volume ratio of solution A to solution B is 1:1; And / or, the volume ratio of solution A to the bacterial solution in S21 is 1:2; the volume ratio of solution B to the bacterial solution in S21 is 1:

2.

7. The preparation method of the double-layer coated probiotic complex according to claim 5, wherein In S23, the trivalent metal ion X in the metal solution 3+ is Fe 3+ , and what is obtained in S24 is the Mix@TA-Fe 3+ complex; Or, in S23, the trivalent metal ion X in the metal solution 3+ is Al 3+ , and what is obtained in S24 is Mix@TA-Al 3+ complex; Or, in S23, the trivalent metal ion X in the metal solution 3+ is Ga 3+ , and what is obtained in S24 is Mix@TA-Ga 3+ complex; Or, the concentration of solution A is 0.6 mg / mL; the concentration of solution B is 0.2 mg / mL; Or, the Lactobacillus in the probiotic group includes Lactobacillus acidophilus LA18, Lactobacillus rhamnosus LRa66, and Lactobacillus casei LC89.

8. The preparation method of the double-layer coated probiotic complex according to claim 4, characterized in that, The step S3 includes: S31. Taking the synthesized Mix@TA-X 3+ Centrifuging the complex to obtain a precipitate; S32: Add water to the precipitate to dilute the precipitate; then add the diluted precipitate to the HA solution and mix evenly to obtain a third solution; S33: Stir the third solution on a stirrer; S34. Centrifuge and wash the third solution again, and finally obtain the product Mix@TA-X 3+ / HA complex 9. The preparation method of the double-layer coated probiotic complex according to claim 8, wherein, In S32, the volume ratio of the water added to the precipitate to the added HA solution is 1:

1.

10. Use of a double-coated probiotic complex as described in any one of claims 1-2 in the preparation of a drug for radiation enteritis or in the preparation of a drug for obesity intervention.

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