Bacterial activity protective coating as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological materials, and discloses a bacterial activity protective coating as well as a preparation method and application thereof. The bacterial activity protective coating is prepared from a methacrylic acid copolymer, a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide compound and N-hydroxysuccinimide, and the bacterial activity protective coating is prepared from the methacrylic acid copolymer, the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide compound and the N-hydroxysuccinimide. According to the preparation method, carboxyl of polymethacrylate is activated through crosslinking of EDC and NHS, covalent linkage is formed with amino on cell walls of probiotics, and a stable and compact pH-responsive protective coating is constructed on the surfaces of bacteria. The coating is insoluble in a gastric acid environment, and can effectively protect probiotics from being damaged by harmful factors such as gastric acid; and the probiotics are released to play a role when the probiotics are dissolved in an intestinal weakly alkaline environment. The survival rate and functionality of the probiotics in gastrointestinal tracts are remarkably improved, and the probiotics have good biocompatibility and have no inhibition effect on growth and proliferation of the probiotics.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and more specifically, to a bacteria active protective coating and a preparation method and application thereof. Background Art

[0002] Probiotics, for example, are prone to inactivation during production and use. Temperature, humidity, oxygen, gastric acid, and bile salts affect the activity and survival of probiotics, from frozen storage of bacteria to processing, storage, and digestion in the body. For example, most probiotics are anaerobic bacteria, and contact with oxygen during production, transportation, and storage will cause the probiotics to die and lose their activity. In addition, when taking probiotics orally, H in gastric acid will + It will cause changes in the surface membrane potential of probiotics, change the surface permeability of probiotics, and thus lead to their death. When probiotics are in the stomach, gastric acid will kill more than 70% of them, and the number of probiotics that finally enter the human intestine is even less than 10 6 The effective standard of CFU / mL (CFU / g) greatly limits the effective oral efficacy of probiotics. Probiotics have weak interactions with intestinal mucus, and combined with digestive tract peristalsis and digestive fluid flushing, it is difficult for probiotics to effectively colonize in the intestine and exert their probiotic effects. Therefore, maintaining the in vitro stability of probiotics and the in vivo intestinal survival rate is an important issue that needs to be studied and solved, and it is also a huge challenge faced in the production and application of probiotics.

[0003] Existing bacterial protection technologies show that delivery carriers can enhance the bacterial activity and intestinal adhesion of probiotics. Currently, oral delivery systems used to achieve intestinal targeted delivery of probiotics generally include microcapsules, hydrogels, oil gels, nanocoatings, emulsions, nanofibers, spores, biofilms, etc.

[0004] Microcapsules are protective carriers with a core-shell structure prepared by spray drying of food macromolecules, which can isolate probiotics from the external environment and achieve controlled release of probiotics. CN115969039A provides a method for preparing probiotic microcapsules based on a W / G / W structure, but the freeze-drying equipment used is expensive, and the processing capacity is small, the process takes a long time, consumes a lot of energy, and the preparation cycle is long, which is also prone to cause certain loss of live bacteria. CN115624180A provides a probiotic microcapsule containing osteopontin and a preparation method thereof, which is strictly limited in conditions and material sources and is not suitable for large-scale preparation, especially sodium alginate, which has low solubility and easily causes high viscosity, limiting the preparation of microcapsules.

[0005] Hydrogel is a three-dimensional network structure with water absorption and expansion formed by hydrophilic macromolecules through physical or chemical cross-linking. Probiotics can be encapsulated inside hydrogel microspheres or inoculated on the surface of microspheres. CN117357468A provides a probiotic gel preparation that can be stored for a long time and its use method and application, but the use scenario of this patent is limited. The prepared gel preparation can only be applied to the skin and mucous membranes and cannot be widely used. CN107714672A provides a vaginal active probiotic gel capsule for women and its preparation method. Although the gelatin used is a relatively simple and cheap raw material, pure gelatin is brittle after drying, has weak molding ability, low ductility, and is easily eroded by bacteria and deteriorated in a humid environment.

[0006] Oil gel is a viscoelastic three-dimensional network structure material containing a non-polar liquid phase, which is formed by adding a gelling agent to liquid oil to self-assemble into a network structure or liquid oil crystals. Oil gel is hydrophobic and emulsifiable. Compared with hydrogel, its physical barrier can better isolate moisture and improve the survival rate of probiotics. Although the emulsion delivery method is easy to operate, it has poor stability and is prone to stratification and precipitation during long-term transportation, resulting in reduced activity of probiotics. In addition, emulsion delivery also faces challenges such as high transportation costs and inconvenient storage. Especially in high temperature or freezing environments, the survival rate of probiotics will be greatly reduced.

[0007] In addition, nanocoatings can form polydopamine nanocoatings through hydrogen bonding, π-π stacking, Michael addition reactions, and Schiff base reactions, encapsulating single living cells and significantly improving the bioavailability of probiotics (more than 30 times that of unencapsulated microorganisms). For example, Liu Jinyao's research group at the Institute of Molecular Medicine, School of Medicine, Shanghai Jiao Tong University, added a small amount of chitosan during the dopamine polymerization process to attach a co-deposited coat of polydopamine and chitosan to the surface of probiotics, protecting the probiotics from erosion by gastric juice and bile, and improving the effect of enteritis treatment. However, the above methods cannot achieve the protection and targeted delivery of probiotics to specific physiological environments. Summary of the invention

[0008] The present invention provides a bacterial active protective coating to overcome the defects of the prior art that the probiotics cannot be protected and targetedly delivered to a specific physiological environment (gastrointestinal tract);

[0009] Another object of the present invention is to provide a method for preparing a bacteria active protective coating;

[0010] Another object of the present invention is to provide an application of a bacteria active protective coating;

[0011] Another object of the present invention is to provide a method for protecting bacteria.

[0012] In order to solve the above technical problems, the technical solution of the present invention is as follows:

[0013] A bacterial active protective coating is prepared from the following components: a methacrylic acid copolymer, a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide compound and N-hydroxysuccinimide (NHS).

[0014] Preferably, a buffer is also included.

[0015] Preferably, the mass ratio of the methacrylic acid copolymer, the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide compound and N-hydroxysuccinimide (NHS) is 1:(8-12):(4-6).

[0016] Preferably, the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide compound includes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0017] Preferably, the methacrylic acid copolymer is a copolymer of methacrylic acid and methyl methacrylate or ethyl acrylate.

[0018] Preferably, the copolymer of methacrylic acid and methyl methacrylate or ethyl acrylate dissolves at pH>5.5.

[0019] Preferably, the molar ratio of methacrylic acid to methyl methacrylate or ethyl acrylate is 1:1.

[0020] Preferably, the copolymer of methacrylic acid and ethyl acrylate includes Eudragit L100-55, and the copolymer of methacrylic acid and methyl methacrylate includes Eudragit L100.

[0021] Eudragit L100-55 used in the present invention is a redispersible powder, which is easy to process and use. Eudragit L100-55 does not dissolve at pH < 5.0, and gradually dissolves at pH > 5.5. Eudragit L100-55 dissolves in an environment of pH > 6. Therefore, both are suitable for controlling the release of drugs in gastric juice and ensuring the targeted release of drugs in the intestine.

[0022] Preferably, the copolymer of methacrylic acid and ethyl acrylate is Eudragit L100-55.

[0023] In an acidic environment, the methacrylic acid portion of the copolymer of methacrylic acid and ethyl acrylate remains non-ionized, resulting in increased hydrophobicity and decreased solubility of the copolymer; ethyl methacrylate can change the sensitivity or range of pH response by adjusting the hydrophilic-hydrophobic balance of the copolymer.

[0024] A bacterial active protective coating comprises the following steps:

[0025] S1, dispersing the methacrylic acid copolymer in a buffer;

[0026] S2, adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide compound and N-hydroxysuccinimide, mixing evenly, and obtaining the bacterial active protective coating after the reaction is completed.

[0027] Furthermore, after the methacrylic acid copolymer is dissolved in a buffer solution, the pH of the buffer solution is 4.0 to 6.5.

[0028] Preferably, the pH is adjusted to 4.5 to 6.0.

[0029] Preferably, the pH is adjusted to 5.0 to 6.0.

[0030] Preferably, the buffer comprises morpholine isosulfonic acid (MES).

[0031] Preferably, the concentration of the morpholine isosulfonic acid is 0.1 mol / L.

[0032] Preferably, the reaction in S2 is carried out at 25 to 40° C. for 1 to 5 hours.

[0033] Preferably, the reaction in S2 is carried out at 37° C. for 2 hours.

[0034] In the cross-linking process, the buffer plays a vital role. The main function of the buffer is to adjust the pH value of the solution. At a pH of 4.5-7.2, it helps the activity of EDC and NHS to reach the best state. EDC and NHS are condensation agents for the amide reaction between carboxyl and amino groups, and they can promote the formation of stable amide bonds between carboxyl and amino groups. The use of the buffer ensures that the cross-linking reaction of EDC and NHS is carried out in a stable and suitable pH environment, thereby improving the efficiency of the cross-linking reaction.

[0035] EDC and NHS can react with the carboxyl functional groups on Eudragit L100-55, thereby achieving effective cross-linking between the two. First, EDC acts as a coupling agent to activate the carboxyl groups on Eudragit L100-55, while NHS can stabilize these activated groups to react with Eudragit L100-55 to form a semi-stable intermediate, preventing the activated Eudragit L100-55 from hydrolyzing in the solution.

[0036] An application of the bacterial active protective coating is used to prepare a coating or carrier with pH responsiveness that can protect the targeted release of active ingredients.

[0037] Preferably, the coating or carrier is applied in fields including agriculture, food, and biomedicine.

[0038] Furthermore, the active ingredients include microorganisms, biomacromolecules, and chemical drugs.

[0039] Preferably, the microorganisms include probiotics, environmental functional bacteria, industrial strains, and pathogenic microorganism carriers; the biological macromolecules include proteins (enzymes, antibodies), nucleic acids (genes, mRNA), and polysaccharides (hyaluronic acid); the chemical drugs include small molecule drugs, targeted drugs, controlled-release drugs, chemical pesticides, and nutritional supplements.

[0040] Furthermore, it is used to prepare a coating or carrier for protecting probiotics and targetedly delivering them to the gastrointestinal tract and other parts where pH changes are obvious.

[0041] The coating of this patent has the potential for wide application, not just in the microbial field. In fact, any application that requires the protection and release of active ingredients under specific environmental conditions may benefit from this technology of pH-responsive materials and covalent bonds. For example, in the field of drug delivery, similar technology can be used to protect and target the delivery of drugs to specific physiological environments, which is of great significance for improving the bioavailability of drugs, reducing side effects, and achieving precision medicine.

[0042] In addition, this technology also has potential application value in other fields such as agriculture, food science, and biotechnology. For example, in the field of plant protection, this technology can be used to protect the active ingredients of pesticides so that they can be released under specific environmental conditions to improve the control effect and reduce pollution to the environment. In the field of food science, this technology can be used to protect and enhance the functional ingredients in food, such as antioxidants and nutrients.

[0043] A bacteria protection method uses the bacteria active protective coating, mixes the bacteria active protective coating with bacteria, and after the reaction is completed, the surface of the bacteria is covered with a dense protective layer.

[0044] Preferably, the preparation of bacterial solution includes the following steps: separating the bacteria by centrifugation, washing with phosphate buffer, and resuspending the washed bacteria with phosphate buffer to form a uniform bacterial solution. The purpose of washing is to remove the culture medium residue and ensure that there is no foreign carboxyl and amino groups to avoid interference with the cross-linking reaction.

[0045] Preferably, the mixture is reacted at 35-40° C. for 20-40 minutes to fully crosslink the methacrylic acid copolymer with the bacterial cell wall. During the reaction, the solution can be stirred upside down to ensure full contact and uniform mixing between the reactants. Such reaction conditions can promote the crosslinking reaction to the greatest extent and improve the crosslinking efficiency.

[0046] Furthermore, after the bacteria active protective coating is mixed with bacteria, the concentration of the methacrylic acid copolymer in the bacteria active protective coating is 0.1-5 mg / mL.

[0047] Further, the bacteria active protective coating and the bacteria are mixed in a volume ratio of 1:1 to 2.

[0048] The bacterial active protective coating is mixed with the bacteria at this volume ratio, which not only realizes the effective linking of the methacrylic acid copolymer to the bacterial cell wall, but also avoids adverse effects on the bacterial activity.

[0049] The present invention activates the copolymer of methacrylic acid and ethyl acrylate to obtain a bacterial active protective coating, and the coating is covalently linked to the probiotic cell wall by chemical bonds to prepare the protective coating. When the probiotics enter the strong acid gastric juice (pH 1.0-4.0) environment of the stomach through oral administration, the coating can effectively protect the probiotics from being killed by the strong acid environment, but when the probiotics pass through the stomach to the intestine, the weak alkaline environment of the intestinal pH between 6.8 and 7.0 can dissolve the coating on the surface of the probiotics, thereby releasing the probiotics to exert their efficacy.

[0050] The present invention adopts the EDC and NHS cross-linking method to successfully covalently link the pH-responsive commercial reagent Eudragit L100-55 to the living cell wall of probiotics. This innovative technology forms an extremely dense protective layer on the surface of probiotics, ensuring that the probiotics are well protected when passing through the gastric acid environment, thereby avoiding the destruction of biological activity by gastric acid. The coating of the present invention not only effectively protects the probiotics from harmful factors such as gastric acid, but also realizes the targeted release of probiotics in the intestine. This technological breakthrough greatly improves the survival rate and functionality of probiotics in the human body, and provides strong support for the application of probiotics in the field of medical health.

[0051] Utilizing the coating of the present invention to coat probiotics, not only can the probiotics be effectively protected from the influence of the external environment, but also the activity of the cells can be maintained during the freeze-drying process. This coating technology not only simplifies the preparation process, but also reduces the production cost, while improving the stability and storage convenience of the probiotics. Specifically, the single-cell coating technology can achieve accurate protection of the probiotics by controlling the thickness and material selection of the coating. The coating material usually has good biocompatibility and stability, and can effectively prevent the direct contact of the probiotics with the external environment, thereby avoiding its activity loss during processing, storage and transportation. In addition, the probiotics after the coating can be stored for a long time by freeze-drying, which not only facilitates transportation and storage, but also can quickly restore the activity of the probiotics when needed, ensuring that it plays the best effect during use.

[0052] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0053] 1. Improve the gastric acid tolerance and survival rate of probiotics. Eudragit L100-55 is used to covalently link bacteria to effectively block the damage of gastric acid to bacteria, ensure their survival through the strong acid environment of the stomach, and significantly improve the gastric acid tolerance of probiotics. Even after being treated in the gastric acid environment for 1 hour, the reduction in the number of colonies was significantly less than that of the control group, showing a strong acid resistance.

[0054] 2. Realize intestinal pH-responsive targeted release. Based on the pH responsiveness of methacrylic acid and ethyl methacrylate copolymer, the coating is intact in the gastric acid environment, and the coating is completely dissolved in the simulated intestinal environment (pH 6.8-7.0), releasing probiotics. The use of the coating of the present invention ensures that the bacteria can be targeted and released into the intestine after passing through the gastric acid environment, achieving the effect of precise delivery and realizing the dual functions of protecting the bacteria in the stomach and releasing them in the intestine.

[0055] 3. Good biocompatibility and no toxic side effects. The bacterial growth curve treated with the coating of the present invention is basically consistent with that of the unused control group, and the growth rate during the culture process is similar, with no statistically significant difference. This shows that the coating used in the present invention has good biocompatibility for bacteria and will not inhibit its growth or affect its activity. This biocompatibility ensures that the coating will not cause any adverse effects on its physiological functions while protecting the probiotics.

[0056] 4. The nano coating evenly covers the surface of probiotics to form a stable protective layer. The EDC and NHS cross-linking activation method is used to covalently connect the coating to the amino groups on the bacterial cell wall to form a stable and dense protective layer. Under a transmission electron microscope, it was observed that a uniform nano protective layer was formed on the surface of the beneficial bacteria, and no damage to the bacterial cell membrane or leakage of contents was found. The uniform thickness of the protective layer and the enhanced image contrast further prove the integrity and stability of the coating.

[0057] 5. Wide application range and potential for industrial application. By adjusting the concentration of Eudragit L100-55 and the addition ratio during application, the coating thickness and protective effect can be flexibly adjusted according to different application requirements. The present invention is not only suitable for the protection and targeted delivery of probiotics, but also can be applied to the protection and release of other active ingredients. In addition, the good material processability and environmental adaptability lay the foundation for its industrial promotion in multiple fields such as food, agriculture and biomedicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 are Fourier transform infrared spectra, A is the infrared spectrum of Eudragit L100-55, B is the infrared spectrum of cross-linking activation of Eudragit L100-55 by EDC and NHS;

[0059] Figure 2 This is the bacterial plate culture colony image after being treated with artificial gastric juice;

[0060] Figure 3 This is a statistical chart of colony counts after artificial gastric juice treatment;

[0061] Figure 4 This is a transmission electron microscope image of bacteria;

[0062] Figure 5 This is the absorbance change curve of the bacterial solution. DETAILED DESCRIPTION

[0063] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0064] Unless otherwise specified, the reagents and materials used in the following examples are commercially available. Eudragit L100-55 used in the present invention was purchased from Rohm, Germany.

[0065] Example 1

[0066] 1. Preparation of bacterial active protective coating

[0067] Eudragit L100-55 was dispersed in morpholine isosulfonic acid buffer (pH 5.5, 0.1 mol / L), 200 mg of 1-ethyl-(3-dimethylaminopropyl)-carbodiimide (EDC) and 100 mg of N-hydroxysuccinimide (NHS) were added in sequence, mixed upside down, and reacted in a 37°C incubator for 2 hours to cross-link and activate the carboxyl groups of Eudragit L100-55 material. After the reaction was completed, a bacterial active protective coating was obtained.

[0068] 2. Preparation of bacterial protective coating

[0069] After centrifugation of the E. coli Nissle 1917 bacterial suspension, the bacteria were separated and washed twice with phosphate buffer. The washed bacteria were resuspended with phosphate buffer to form a uniform bacterial suspension. After the cross-linking activation reaction was completed, the Eudragit L100-55 material solution and the E. coli Nissle 1917 bacterial suspension were mixed at a volume ratio of 1:1 and reacted in a 37°C incubator for 0.5 hours.

[0070] Example 2

[0071] The technical scheme of Example 2 is similar to that of Example 1, except that Eudragit L100-55 is dissolved in morpholinoisosulfonic acid (MES) buffer to prepare 2, 4, and 10 mg / mL solutions, respectively. After the Eudragit L100-55 material solution and the E. coli Nissle 1917 bacterial suspension are mixed at a volume ratio of 1:1, the concentrations of Eudragit L100-55 are 1, 2, and 5 mg / mL, respectively.

[0072] Example 3

[0073] The technical scheme of Example 3 is similar to that of Example 1, except that the Eudragit L100-55 material is dispersed in morpholineethanesulfonic acid (MES) buffer to prepare 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 5 mg / mL, and 10 mg / mL concentration solutions. After the cross-linking activation reaction is completed, the Eudragit L100-55 material liquid is mixed with the E. coli Nissle 1917 bacterial suspension in a volume ratio of 1:2.

[0074] Example 4

[0075] The technical solution of Comparative Example 4 is similar to that of Example 1, except that Eudragit L100-55 is replaced by Eudragit L100.

[0076] Detection Methods

[0077] 1. Infrared spectrum identification

[0078] To verify whether the cross-linking activation of the carboxyl group of Eudragit L100-55 material by EDC and NHS in Example 1 is successful, EDC and NHS can promote the formation of a stable amide bond between the carboxyl group and the amino group. After the cross-linking activation reaction is completed, the sample is freeze-dried and the sample is used for Fourier transform infrared spectrometer to identify the functional group change.

[0079] 2. Colony statistics after artificial gastric juice treatment

[0080] The probiotic E.coli Nissle 1917 cross-linked with Eudragit L100-55 material was cultured on solid culture medium after being treated with artificial gastric juice for different lengths of time.

[0081] 3. Observation of morphological changes

[0082] Probiotics E.coliNissle 1917 were cultured according to standard methods to obtain a sufficient number of live bacteria. Probiotics E.coliNissle 1917 were cross-linked with Eudragit L100-55 material cross-linking solution. Normal probiotics E.coliNissle 1917 and probiotics E.coliNissle 1917 coated with Eudragit L100-55 were fixed, dehydrated, infiltrated, embedded, etc. to prepare samples suitable for transmission electron microscopy (TEM) observation. The samples were observed at high resolution using a transmission electron microscope to record and analyze the nano-coating on the bacterial surface.

[0083] 4. Changes in probiotic growth activity

[0084] To verify whether Eudragit L100-55 would affect the growth activity of probiotics E.coli Nissle1917 after covalent connection within the usage range of 1 mg / mL, to observe whether the protective coating of Eudragit L100-55 had an effect on the growth and proliferation activity of probiotics, and to further analyze whether there was a statistical difference between its growth curve and that of the normal group.

[0085] Results Analysis

[0086] 1. Chemical reaction formula

[0087] EDC is a commonly used coupling agent that can react with carboxyl groups to form active intermediates. EDC is an activator that can further react with the intermediate formed by EDC to form a semi-stable intermediate, thereby improving the efficiency of the cross-linking reaction. The core of the preparation method of the present invention is to use EDC and NHS as cross-linking agents to covalently connect the carboxyl groups on the Eudragit L100-55 material molecules to the amino groups on the bacterial cell wall. This connection method can form a stable and dense protective layer, which effectively protects the probiotics from the adverse external environment.

[0088] In the preparation method of the present invention, the Eudragit L100-55 material is first dissolved in a suitable solvent, then reacted with EDC to form an active intermediate, and further reacted with N-hydroxysulfosuccinimide to generate a more active intermediate to improve the efficiency of the cross-linking reaction. Finally, the probiotic cell wall is fully exposed to the coating solution, and the carboxyl group on the Eudragit L100-55 material is covalently linked to the amino group on the probiotic cell wall by controlling the reaction conditions and reaction time.

[0089]

[0090] 2. Infrared spectrum identification of Eudragit L100-55 cross-linked with EDC / NHS

[0091] like Figure 1 The obtained infrared spectrum shows that Eudragit L100-55 material has a wavelength of 1300-1000 cm -1 The C=O characteristic absorption peak appears, 1260~1000cm -1 There is a CO absorption peak at 1300-1200 cm -1 The carbonyl absorption peak appears at 2900 cm -1 There is a carboxyl absorption peak near 3400-3200 cm -1 OH absorption peak appears, 2986cm after cross-linking -1 The carboxyl absorption peak disappeared. The above results showed that EDC and NHS successfully cross-linked and activated the carboxyl groups on the Eudragit L100-55 material.

[0092] 3. Protective effect on bacteria

[0093] The probiotics cross-linked with Eudragit L100-55 material showed significant protective effects in the artificial gastric fluid environment. Figure 2As shown in the figure, there is a significant difference in the survival rate of bacteria after being treated with artificial gastric juice for different lengths of time. When the probiotics were treated with artificial gastric juice for 0.5h, the colony count of the cross-linked probiotics decreased less than that of the control group without Eudragit L100-55 and Example 4 replaced with Eudragit L100, showing a stronger tolerance. As time goes on, when the treatment time reaches 1h, this protective effect is still significant. Although the colony count of the cross-linked probiotics also decreased, the reduction was significantly less than that of the control group without Eudragit L100-55 ( Figure 2 ), indicating that the cross-linking treatment effectively enhanced the resistance of probiotic E. coli Nissle 1917 to artificial gastric juice.

[0094] This result fully demonstrates the effectiveness of the EDC and NHS cross-linking method in protecting probiotics from adverse factors such as gastric acid. Through covalent bonding, the Eudragit L100-55 material can form a stable protective layer outside the probiotic cell wall, effectively blocking the erosion of bacteria by harmful components in gastric juice. This protective mechanism allows probiotics to maintain a high survival rate after passing through the gastric acid environment, thus providing a strong guarantee for their colonization and function in the intestine.

[0095] also, Figure 3 The data also showed that the survival rate of the cross-linked probiotic E. coli Nissle 1917 was relatively stable at different treatment times. This further verified the stability and reliability of the EDC and NHS cross-linking method, providing strong support for its application in the field of probiotic delivery.

[0096] 4. Morphology observation

[0097] according to Figure 4It can be found that a complete nanocoating was observed on each probiotic E.coliNissle 1917 treated with Eudragit L100-55. This indicates that the Eudragit L100-55 nanomaterial can cover the surface of the probiotic and form a protective layer. The results of transmission electron microscopy showed that after Eudragit L100-55 was connected, the contrast of the probiotic E.coliNissle1917 in the transmission electron microscope image increased significantly. This is due to the increased electron scattering caused by the increase in coating thickness, which improves the contrast of the image. This change not only proves the successful connection of Eudragit L100-55 nanomaterials to the bacterial surface, but also provides an intuitive method to evaluate the thickness and uniformity of the coating. In addition, after connecting Eudragit L100-55, the morphology of the probiotics is normal, the bacterial membrane structure is intact, and the contents have not leaked, indicating that the growth state of the probiotics is good. The above results show that Eudragit L100-55 material with good biocompatibility can form a nano-coating on the surface of probiotics and completely wrap the probiotics, thereby effectively protecting the activity of the probiotics.

[0098] 5. Changes in growth vitality

[0099] The OD value of the bacterial solution in Example 2 at different time periods was detected by an ELISA instrument to draw a growth curve ( Figure 5 ), the growth curves of the probiotics in the 1 mg / mL experimental group and the control group without Eudragit L100-55 were basically the same, with no significant difference. During the culture period, both groups of probiotics grew normally and at similar growth rates. This shows that at a concentration of 1 mg / mL, Eudragit L100-55 has good biocompatibility for the probiotic E.coliNissle 1917, and has no inhibitory effect on the proliferation of probiotics at this concentration. Example 2 There was no significant difference in growth rate between all experimental groups and the control group, indicating that the good biocompatibility of Eudragit L100-55 for the probiotic E.coliNissle 1917 at a covalently linked concentration of 1 to 5 mg / mL was verified.

[0100] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A bacterial active protective coating, characterized in that: The invention comprises the following components: a methacrylic acid copolymer, a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide compound and N-hydroxysuccinimide.

2. A method for preparing the bacterial active protective coating according to claim 1, characterized in that: The following steps are involved: S1, dispersing the methacrylic acid copolymer in a buffer; S2, adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide compound and N-hydroxysuccinimide, mixing evenly, and obtaining the bacterial active protective coating after the reaction is completed; S3. Mix the bacterial active protective coating with the component to be protected to form a coating on the surface.

3. The method for preparing the bacterial active protective coating according to claim 2, characterized in that: After the methacrylic acid copolymer is dissolved in the buffer solution, the pH of the buffer solution is 4.0 to 6.

5.

4. An application of the bacterial active protective coating according to claim 1, characterized in that: Used to prepare pH-responsive coatings or carriers that can protect the targeted release of active ingredients.

5. The use of the bacterial active protective coating according to claim 4, characterized in that: The active ingredients include microorganisms, biomacromolecules, and chemical drugs.

6. The use of the bacterial active protective coating according to claim 4, characterized in that: Used to prepare coatings or carriers for protecting probiotics and targeted delivery to the gastrointestinal tract and other areas with significant pH changes.

7. A method for protecting bacteria, characterized in that: The bacterial active protective coating according to claim 1 is used, the bacterial active protective coating is mixed with bacteria, and after the reaction is completed, the surface of the bacteria is covered with a protective layer.

8. The method for protecting bacteria according to claim 7, characterized in that: After the bacteria active protective coating is mixed with bacteria, the concentration of the methacrylic acid copolymer in the bacteria active protective coating is 0.1-5.0 mg / mL.

9. The method for protecting bacteria according to claim 7, characterized in that: The bacteria active protective coating and the bacteria are mixed in a volume ratio of 1:1 to 2.

10. An active ingredient composition, characterized in that The invention comprises active ingredients and the bacterial active protective coating as claimed in claim 1, wherein the active ingredients comprise microorganisms, biological macromolecules and chemical drugs.

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