A broad-spectrum universal nano-coated probiotic based on mussel myoglobin and its preparation method and application

Abstract

The application discloses a kind of broad-spectrum general-purpose nano-coating probiotics based on Mytilus edulis mucin and a preparation method and application thereof.The method accelerates the deposition of Mytilus edulis mucin on the surface of probiotics by means of non-covalent bond, hydrogen bond, cation-pi and other supramolecular interaction modes between Mytilus edulis mucin and bacteria, and realizes the independent coating of probiotics by complexing and coordinating the catechol group in Mytilus edulis mucin with cations and driving it to self-assemble into nanoscale aggregates.The Mytilus edulis mucin nano-coating provided by the application is not only suitable for encapsulating aerobic bacteria, but also has a protective effect on anaerobic bacteria, and can realize the synchronous release of probiotics.More importantly, the addition of cations enhances the adhesion ability of probiotics while improving the density of the coating network structure, resulting in nano-coating probiotics with high stability and biocompatibility.The nano-coating probiotics provided by the application have simple and efficient preparation process, are suitable for large-scale production, and are expected to become an ideal carrier for probiotic delivery.

Description

TECHNICAL FIELD

[0001] The present patent relates to the technical field of probiotics, in particular to a broad-spectrum universal nano-coated probiotic based on mussel adhesive protein and a preparation method and application thereof. BACKGROUND

[0002] Probiotics are microorganisms that are beneficial to the health of the host, which can improve intestinal health by regulating the composition and activity of intestinal microorganisms, participating in nutrient metabolism or immune regulation, etc. However, during the production and processing of probiotics and in vivo transportation, they are easily affected by various harsh factors (high temperature, gastric acid, bile salts, etc.), resulting in a significant decrease in their biological activity, thereby affecting their colonization in the intestinal tract and the exertion of probiotic effects. Therefore, it is very important to efficiently protect and deliver probiotics, prolong their residence time in the intestinal tract, and increase their survival number in the host body.

[0003] Inspired by the self-protection behavior of bacteria in complex environments, researchers encapsulate probiotics in natural biological materials to improve their resistance to extreme environments and endow them with excellent adhesion properties. Common probiotic encapsulation technologies (such as emulsions, gels, microcapsules, etc.) can improve the tolerance of probiotics to harsh environmental stress, but these methods have defects such as high cost, low encapsulation rate, and low probiotic activity. Compared with traditional encapsulation technologies, single-cell encapsulation technology can endow probiotics with a "coat" to resist adverse environments while ensuring normal material exchange between probiotics and the external environment. Therefore, it is necessary to find suitable materials and design a "coat" suitable for most probiotics.

[0004] Mussel adhesive protein (MAP) is a kind of mucin secreted by the byssus gland of mussels, which can firmly adhere to any surface under seawater. The dopamine group is an important component of mussel adhesive protein, and its ortho-phenol group has strong adhesion. It has been widely used as a coating layer for various inorganic and organic surfaces. In addition, the lysine residues in mussel adhesive protein have strong positive charges at the physiological pH of the human body, which can bind to negatively charged cells through electrostatic interactions, enhancing the protection effect on cells. More importantly, when lysine residues and dopamine groups coexist and reach a certain concentration, cation-π interaction can replace the interfacial water interaction, mediating strong adhesion on the cell surface. At the same time, the hydrophobic groups of amino acids in mussel adhesive protein can combine with the lipid bilayer of the cell membrane through hydrophobic interaction and adhere to the cell surface. Therefore, as a natural polymer material, mussel adhesive protein can deposit on the cell surface and interact with it in multiple ways. However, currently, mussel adhesive protein is mainly applied in the fields of medical wound repair and daily cosmetics, and its application in the field of probiotics technology has not been reported.

[0005] As shown in the following patent: Application No. 202110290344.0, Publication No. CN 113197314B, and Invention Name "Layer-by-layer Self-assembly Probiotic Microcapsule and Its Preparation Method", a layer-by-layer self-assembly probiotic microcapsule and its preparation method are disclosed. The method spontaneously adsorbs multiple layers of probiotic wet capsules into the microporous structure of porous starch through electrostatic interaction, obtaining a layer-by-layer self-assembly probiotic microcapsule. Although this method effectively improves the efficiency of probiotic delivery and colonization survival in the intestinal tract, there are gaps between the probiotic microcapsule wall skeletons, which are not suitable for the protection and delivery of anaerobic bacteria, and do not have universal characteristics. In addition, this method requires the introduction of chemical reagents for starch modification, and the preparation process is complex, with high production cost.

[0006] As shown in the following patent: Application No. 202311471775.2, Publication No. CN117297099A, and Invention Name "Probiotic Coating Preparation Method Based on Sodium Alginate and Metal Polyphenol Network", a probiotic coating preparation method based on sodium alginate and metal polyphenol network is disclosed. The method deposits the agglomerates formed by the complexation of polyphenols and metal ions on the surface of probiotics, and forms a coating layer by inducing sodium alginate with calcium ions. The metal polyphenol network coating in this method can self-assemble quickly and has no obvious effect on the normal growth of probiotics. However, the single metal polyphenol network coating has weak protection ability for probiotics during gastrointestinal transportation, resulting in low cell viability. In addition, the combination of polyphenolic substances and salivary proteins can trigger the perception of astringency in the body, and the introduction of metals can accelerate the oxidation of the system, limiting its application prospects.

[0007] The article "Tumor Antigen Oral Delivery System Based on Surface Coated Probiotics" discloses a preparation method of yeast cell membrane coated probiotics. In this method, the yeast cell membrane plays the role of protecting probiotics and antigens and targeting the intestinal lymphatic system, and the probiotics play the role of carrying antigens and immune adjuvants. The results show that compared with uncoated probiotics, yeast cell membrane coating can improve the bioavailability of probiotics, enhance their enrichment in Peyer's patches, and promote the activation and antigen presentation of dendritic cells. Although this method can protect probiotics and antigens from gastrointestinal environmental stimuli, the production process of yeast cell membrane is complex and costly. In addition, the introduction of yeast cell membrane limits the material exchange between probiotics and the external environment, and cannot maximize the health benefits of probiotics.

[0008] Therefore, it is a technical problem that needs to be solved by those skilled in the art to provide a nano-coated probiotic preparation method that has both gastric acid resistance and intestinal adhesion ability, high biocompatibility, good universal characteristics, and is suitable for industrial production. SUMMARY

[0009] In order to overcome the shortcomings of the prior art, the primary object of the present application is to provide the application of mussel adhesive protein in the field of preparation of nano-coated probiotics, and provide a broad-spectrum universal nano-coated probiotics based on mussel adhesive protein, which is coated by mussel adhesive protein, the adhesive protein carrier enhances the tolerance of probiotics to the stomach environment, so that they can reach the intestine unhindered and play their probiotic function.

[0010] Another object of the present application is to provide a preparation method of a broad-spectrum universal nano-coated probiotics based on mussel adhesive protein, which is coated by mussel adhesive protein, prolongs the residence time of probiotics in the intestine, and synchronously releases the probiotics in the intestine to maximize the health benefits of probiotics. The principle of coating probiotics with mussel adhesive protein is that the non-covalent bond, hydrogen bond, cation-π and other supramolecular interaction modes between mussel adhesive protein and bacteria accelerate the deposition of mussel adhesive protein on the surface of probiotics, and further complex coordination of calcium ions and other cations with catechol groups in mussel adhesive protein and drive self-assembly into nanoscale aggregates to achieve independent coating of probiotics and give them the ability to resist adverse external environment.

[0011] This method is not only suitable for encapsulating aerobic bacteria, but also has protective effect on anaerobic bacteria, and can realize synchronous release of probiotics to maximize the health benefits of probiotics.

[0012] To achieve the above object, the specific technical scheme adopted by the present application is:

[0013] 1) Cultivate probiotics to logarithmic growth phase, wash multiple times to ensure no medium residue, remove supernatant, collect bacterial slurry, resuspend the bacterial slurry in sterile ultrapure water, and adjust the cell concentration.

[0014] 2) Resuspend the probiotics obtained in step 1) with a cationic aqueous solution and vortex to disperse.

[0015] 3) Add mussel adhesive protein solution to the solution obtained in step 2) and vortex to obtain a probiotic suspension.

[0016] 4) Adjust the pH of the probiotic suspension obtained in step 3) to 6.0, and wash the solution multiple times with sterile water with a pH of 6.0, and centrifuge to obtain mussel adhesive protein nano-coated probiotics.

[0017] 5) Resuspend the bacterial slurry after centrifugation in step 4) in sterile water with the same pH and store in a 4°C refrigerator.

[0018] Preferably, the probiotics in step 1) are one or more of Lactobacillus paracasei, Bifidobacterium, Lactobacillus plantarum and Lactobacillus.

[0019] Further, the probiotics in step 1) are Lactobacillus plantarum (PL) and Bifidobacterium (BB).

[0020] Preferably, the medium in step 1) is MRS broth medium; the centrifugal speed is 5000 rpm, the centrifugal time is 10 min, and the washing times are 3 times; the probiotic cell concentration is 1×10 10 ~ 1×10 11 CFU / mL.

[0021] Preferably, the cation in step 2) is selected from one or more of Ca 2+ , Mg 2+ , Zn 2+ , and Fe 2+ . Further, the cation in step 2) is selected from Ca 2+ , and the specific cation aqueous solution is a calcium chloride solution.

[0022] The concentration of the cation aqueous solution in step 2) is 25 mM, the vortex speed is 2000-3000 rpm, and the vortex time is 5-10 min.

[0023] The purpose of resuspending the probiotics with the cation aqueous solution in step 2) is to enable the complexation of the cations such as calcium ions with the catechol groups in the mussel adhesive protein and drive the self-assembly of the catechol groups into nanoscale aggregates, so as to realize the independent coating of the probiotics.

[0024] Preferably, the volume ratio of the probiotic bacterial suspension to the cation solution in step 2) is 1:0.4.

[0025] Preferably, the concentration of the mussel adhesive protein solution in step 3) is 2 mg / mL, the vortex speed is 3000 rpm, and the vortex time is 10 min.

[0026] Preferably, the volume ratio of the probiotic bacterial suspension to the mussel adhesive protein solution in step 3) is 1:4.

[0027] Preferably, hydrochloric acid is used to adjust the pH of the probiotic bacterial suspension in step 4), and the concentration of the hydrochloric acid is 0.1M.

[0028] A third object of the present application is to provide the application of the above-mentioned mussel adhesive protein-based broad-spectrum universal nanocoating probiotics, which are specifically used in the food industry, the pharmaceutical field, and the like.

[0029] Compared with the prior art, the present application has the following advantages and benefits:

[0030] (1) The preparation method of the coated probiotics provided by the present application utilizes the non-covalent bond between the mussel adhesive protein and the bacterial body,

[0031] Hydrogen bonding, cation-pi and other supramolecular interaction modes accelerate the deposition of mussels' mucin on the surface of probiotics, and further enable the complexation and coordination of calcium ions with the ortho-diphenol groups in the mussels' mucin and drive the self-assembly of the mussels' mucin into nanoscale agglomerates, so that the probiotics are independently coated, and the ability of resisting external adverse environment is endowed.

[0032] (2) The mussels' mucin adopted in the application has high biocompatibility, the characteristic groups in the molecular structure can interact with the intestinal epithelial mucosa, improve the stability of the coating structure, and endow the probiotics with excellent adhesion performance, significantly prolong the residence time of the probiotics in the intestinal tract, and maximize the health benefits of the probiotics.

[0033] (3) The mussels' mucin coating adopted in the application has good universal characteristics, which is not only suitable for encapsulating aerobic bacteria, but also has a protective effect on anaerobic bacteria, and can realize the synchronous release of probiotics and efficient regulation of intestinal health.

[0034] (4) The preparation method of the nano-coated probiotics provided by the application is simple and low in cost, and is suitable for industrial scale production, which lays a foundation for the industrial application of the mussels' mucin coated probiotics. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 PL@MAP and BB@MAP are electric fluorescence microscope images.

[0036] Figure 2 PL, PL@MAP, BB and BB@MAP are transmission electron microscope images.

[0037] Figure 3 PL, PL@MAP, BB and BB@MAP are growth curve graphs.

[0038] Figure 4 PL, PL@MAP, BB and BB@MAP are Fourier transform infrared spectroscopy graphs.

[0039] Figure 5 PL, PL@MAP, BB and BB@MAP are fluorescence spectroscopy graphs.

[0040] Figure 6 PL, PL@MAP, BB and BB@MAP are potential graphs.

[0041] Figure 7 PL, PL@MAP, BB and BB@MAP are adhesion rate graphs.

[0042] Figure 8 PL, PL@MAP, BB and BB@MAP are in vitro simulated digestion graphs

[0043] Figure 9 Storage stability graph of PL, PL@ MAP, BB and BB@ MAP

[0044] Figure 10 Hydrogen peroxide damage graph of PL, PL@ MAP, BB and BB@ MAP DETAILED DESCRIPTION

[0045] The application will be further described below in conjunction with the embodiments. It is to be noted that the descriptions of these embodiments are used to help understand the application and do not constitute a limitation to the application. In addition, the technical features involved in the various embodiments of the application described below can be combined with each other as long as there is no conflict.

[0046] In the application, the test methods used are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0047] The application provides the following two embodiments:

[0048] Embodiment 1: In this embodiment, the probiotic bacteria are Lactobacillus plantarum, and the cations are calcium ions, and the specific disclosure is as follows:

[0049] 1) Lactobacillus plantarum is cultured to the logarithmic growth phase, centrifuged at 5000 rpm for 10 min, washed 3 times to ensure no medium residue, and the bacterial slurry is collected and resuspended in sterile ultrapure water, and the probiotic bacterial cell concentration is adjusted to 1×10 10 ~ 1×10 11 CFU / mL.

[0050] 2) Mix 25 mM calcium chloride aqueous solution with the bacterial suspension at a ratio of 0.4:1, vortex at 3000 rpm for 10 min.

[0051] 3) Mix 2 mg / mL aqueous solution of mussel myoglobin with the bacterial suspension at a ratio of 4:1, vortex at 3000 rpm for 10 min, and obtain a mussel myoglobin-coated probiotic bacterial suspension.

[0052] 4) The pH of the above-obtained probiotic bacterial suspension is adjusted to 6.0, and the solution is washed multiple times with sterile water with a pH of 6.0, and centrifuged to obtain mussel myoglobin nano-coated probiotic bacteria.

[0053] 5) The bacterial slurry after centrifugation in step 4) is dispersed in the same pH sterile water, and stored in a 4°C refrigerator.

[0054] Example 2: The technical solution in this embodiment is different from that of the above-mentioned Example 1 in that the probiotic bacteria are Bifidobacterium, and the specific disclosure is as follows:

[0055] 1) Culture Bifidobacterium to the logarithmic growth phase, centrifugal speed 5000 rpm, centrifugal time 10 min, wash 3 times to ensure no medium residue, collect the slurry, and suspend the slurry in sterile ultrapure water, adjust the probiotic cell concentration to 1x10 10 ~ 1x10 11 CFU / mL.

[0056] 2) Mix 25 mM calcium chloride aqueous solution with the bacterial suspension at a ratio of 0.4:1, vortex at 3000 rpm for 10 min.

[0057] 3) Mix 2 mg / mL aqueous solution of mussel myoglobin with the bacterial suspension at a ratio of 4:1, vortex at 3000 rpm for 10 min, to obtain a mussel myoglobin-coated probiotic bacterial suspension.

[0058] 4) Adjust the pH of the above-obtained probiotic bacterial suspension to 6.0, and wash the solution with sterile water at pH 6.0 multiple times, and centrifuge to obtain mussel myoglobin nano-coated probiotic bacteria.

[0059] 5) Redisperse the bacterial slurry after centrifugation in step 4) in sterile water of the same pH, and store in a 4°C refrigerator.

[0060] Experimental results

[0061] Fluorescence microscope observation

[0062] An electronic fluorescence microscope (ECLIPSE Ni-E, Nikon Corporation, Japan) was used to observe the cell morphology of the coated bacteria. First, 100 mg of mussel myoglobin was dissolved in 10 mL of 0.1 M carbonate buffer, and 1 mg of FITC was dissolved in 0.5 mL of dimethyl sulfoxide. Second, the FITC solution was slowly added to the mussel myoglobin solution, and dialysis (MWCO 8,000-14,000 Da) was performed for 60 hours to remove free FITC. Finally, the solution was dried at 45°C to obtain FITC-labeled mussel myoglobin. PL@MAP and BB@MAP labeled with FITC were obtained according to Example 1 and Example 2. As shown in Figure 1 , the green fluorescence of FITC co-localizes with PL and BB under bright field, proving that the mussel myoglobin is modified on the surface of the bacteria, i.e., the bacteria are successfully dressed in a nano-coat.

[0063] Transmission electron microscope observation

[0064] The surface morphology of PL, BB, PL@MAP and BB@MAP was observed by TEM (JEM-1400Flash, Tokyo, Janpan). First, 1000 μL of bacterial sample was centrifuged at 5000 rpm for 10 min, the supernatant was removed and 100 μL of sterile water was added, and the mixture was blown into a uniform suspension. Then, 20 μL of bacterial solution was added to a 300-mesh carbon-coated copper grid and waited for 3 min. Finally, filter paper was used to absorb the excess liquid and dried under a baking lamp for 3 min to facilitate observation under TEM. Figure 2 Transmission electron microscopy images show that a clear nanocoating appears around the bacteria, with a thickness of about 100-150 nm, indicating that mussel mucin is successfully wrapped around the bacterial surface.

[0065] Growth curve determination

[0066] PL, BB, PL@MAP and BB@MAP obtained in Example 1 and Example 2 were cultured in MRS liquid

[0067] Dilute the culture medium to an optical density (OD600) between 0.1 and 0.2 and incubate at 37°C. Use a microplate reader (Powerwave XS, Biotek, USA) to monitor bacterial growth at 37°C every 1 h, and plot a growth curve based on the results. Figure 3 It can be seen that the growth of PL@MAP and BB@MAP is similar to that of PL and BB, indicating that the presence of mussel mucin does not affect the normal growth and proliferation of probiotics.

[0068] Infrared spectroscopy analysis

[0069] The interaction between the mussel mucin coating and the probiotics was analyzed using Fourier transform infrared spectroscopy (Nicolet iS10, Thermo Fisher Scientific, USA). PL, BB, PL@MAP obtained in Example 1 and Example 2, and BB@MAP were freeze-dried into powders, mixed with KBr at a ratio of 1:100, ground, and compressed into tablets. Fourier transform infrared spectroscopy was used to obtain wavelengths from 500 to 4000 cm -1 The spectral signal recorded was averaged over 64 scans with a resolution of 4 cm -1 .according to Figure 4 The results show that PL is at 3293cm -1 (OH and NH stretching vibration), 2933 cm -1 (CH stretching vibration), 1652 cm -1 (C=O stretching vibration and amide I) and 1534 cm -1The characteristic peaks of amide I and amide II of PL@MAP shifted to 1650 cm -1 and 1536 cm -1 , indicating that electrostatic interaction promotes the formation of the coating. In addition, the hydroxyl group at 3293 cm -1 The stretching vibration at 3290 cm -1 The characteristic groups of BB@MAP extended from 2935 cm-1 to 2938 cm-1 (CH stretching vibration), confirming that hydrophobic interactions were involved in the construction of the coating.

[0070] Fluorescence spectroscopy analysis

[0071] The surface components of probiotics were analyzed using a fluorescence spectrophotometer (F-4600, Hitachi, Japan). PL, BB, PL@MAP obtained in Example 1 and Example 2, and BB@MAP were placed in a quartz cuvette and placed in a sample cell for detection. The experimental parameters were set as follows: the spectroscopic element was a grating with 150 lines per millimeter, the scanning time was 2 seconds, the scanning range was 300 nm-650 nm, the excitation wavelength was 290 nm, and the excitation was performed every 5 nm. Figure 5 PL and BB exhibit a typical absorption peak at 330 nm, which is associated with the interaction of various amino acids in cells, including L-glutamic acid, D-alanine, and L-cysteine. The fluorescence intensities of PL@MAP and BB@MAP were significantly higher than those of PL and BB, indicating that after coating with mussel mucin, some paired basic amino acid residues in the mussel mucin adhered to the negatively charged cell surface through electrostatic attraction. Furthermore, some cysteine ​​sequences in the mussel mucin were linked to cysteine ​​residues in the cell peptidoglycan layer via disulfide bonds, contributing to improved protein binding stability.

[0072] Potential measurement

[0073] The PL, BB, Example 1 and

[0074] The potential changes of PL@MAP and BB@MAP obtained in Example 2. Figure 6 The experimental results show that PL and BB are negatively charged, which is related to the abundant lipoteichoic acid in the cell wall. After the probiotics bind to the mussel mucin, the amount of negative charge is significantly reduced, indicating that electrostatic interactions occur between the mussel mucin and the BB and PL. In other words, some paired basic amino acid residues in the mussel mucin are deposited on the negatively charged cell surface to form a nanocoating.

[0075] Adhesion rate determination

[0076] Preparation of mucin films: First, 500 mg of mucin was dissolved in 50 mL of purified water and stirred at room temperature for 2 hours. Then, 500 μL of glycerol was added to the solution, and sonication was performed for 2 minutes to remove bubbles. Finally, the solution was poured into a plastic mold (D = 9 cm) and dried in a 60°C oven for 4 hours. The prepared film was removed and stored at 50% humidity and 25°C for 48 hours.

[0077] Adhesion rate calculation: Mucin film was mixed with PL@MAP and BB@MAP suspension (100 μL, 1×10 8 CFU / mL) for 30 min. After three rinses with sterile PBS, the rinse solution was inoculated into MRS medium for counting. The adhesion rate was calculated using the following formula:

[0078] Adhesion rate = (A1-A0) / A1 × 100%

[0079] Where A0 is the number of non-adherent probiotics, and A1 is the number of initial active probiotics.

[0080] like Figure 7 As shown in the data, the adhesion rates of PL@MAP (50.23 ± 2.01%) and BB@MAP (58.26 ± 1.90%) to the mucin film were significantly higher than those of PL (41.57 ± 1.60%) and BB (45.27 ± 1.55%), indicating that a strong interaction occurred between the mussel mucin coating and the mucin film, thereby enhancing the adhesion performance of probiotics and exerting a better colonization effect.

[0081] Digestion stability assay

[0082] Simulate gastric digestion: First, 2 mL of PL, BB, PL@MAP obtained in Example 1 and Example 2, and

[0083] BB@MAP was mixed with 16 mL of a 2 mg / mL NaCl solution and the pH was adjusted to 1.2. Next, 6.4 mg of pepsin and 4 mL of NaCl were added to the mixture, adjusting the pH to 2.0 and initiating simulated gastric digestion. After 2 hours, the pH was adjusted to 7.5, terminating gastric digestion.

[0084] Simulated small intestinal digestion: 5 mg / mL porcine bile salts were dispersed in deionized water and the pH was adjusted to 7.5. Pancreatic enzyme (1.6 mg / mL) was then added and the pH of the system was adjusted to 7.5. Equal amounts of simulated intestinal fluid were mixed with the digestive fluid produced in the above process and digested for 2 h.

[0085] Simulated colon digestion: mix equal amount of PBS buffer with the digestion solution produced in the above process, and digest for 2 h.

[0086] The experimental results are shown in Figure 8 As shown in the table, the viable cell count of BB and PL is basically zero after being subjected to simulated gastric juice and bile salt solution; while the viable cell count of the mussels' mucin coated probiotics remains at a high level. The experimental results show that the mussel mucin based nano coating proposed in the present application has excellent gastrointestinal resistance and universal characteristics.

[0087] Storage stability determination

[0088] Under the storage condition of 4 ℃, the PL, BB, PL@MAP and BB@MAP obtained in the implementation example 1 and the example 2 are stored for 15 days.

[0089] The strain activity after 15 days of storage is explored, and the viable cell count is detected by the plate counting method. Figure 9 The results show that after 15 days of storage at 4 ℃, the viable cell count of PL and BB is 7.23 and 6.85 Log CFU / mL respectively, while the viable cell count of PL@MAP and BB@MAP is 9.02 and 8.25 Log CFU / mL respectively, proving that the mussel mucin as a physical barrier can isolate the external adverse environment and improve the storage stability of probiotics.

[0090] Hydrogen peroxide damage determination

[0091] The PL, BB, PL@MAP and BB@MAP obtained in the implementation example 1 and the example 2 are resuspended in 1 mL of 50 µM H2O2, and further incubated in a 37℃ constant temperature incubator with gentle shaking. 50 µL of the solution is taken out at 15 min, 30 min and 60 min respectively, washed with sterile water, and the viable cell count is detected by the plate counting method. Figure 10 As shown in the table, after 30 min of H2O2 treatment, PL@MAP and BB@MAP are still alive, but the survival rate of PL and BB is zero, proving that the mussel mucin can resist the invasion of H2O2 and improve the survival rate of probiotics.

[0092] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit it. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced by the same, without departing from the spirit and scope of the technical solutions of the present application, which should be covered in the scope of the claims of the present application.

Claims

1. A method for preparing a broad-spectrum universal nano-coated probiotic based on mussel mucin, characterized in that: The specific steps include: 1) Cultivate the probiotics to the logarithmic growth phase, wash several times to ensure no culture medium remains, remove the supernatant, collect the bacterial sludge, resuspend the sludge in sterile ultrapure water, and adjust the cell concentration; 2) resuspending the probiotics obtained in step 1) in a cationic aqueous solution and vortexing to disperse the probiotics; 3) adding the mussel mucin solution to the solution obtained in step 2) and vortexing to obtain a probiotic suspension; 4) adjusting the pH of the probiotic suspension obtained in step 3) to 6.0, washing the suspension multiple times with a sterile water solution having a pH of 6.0, and centrifuging the solution to obtain the mussel mucin nano-coated probiotics; 5) Re-disperse the centrifuged bacterial sludge from step 4) in sterile water of the same pH and store in a refrigerator at 4°C; In step 2), the cation is Ca 2+ .

2. The method for preparing coated probiotics according to claim 1, characterized in that: The probiotics in step 1) are one or more of Lactobacillus paracasei, Bifidobacterium, and Lactobacillus plantarum.

3. The method for preparing coated probiotics according to claim 1, characterized in that: In step 2), the concentration of the cationic aqueous solution is 25 mM, the vortex speed is 2000-3000 rpm, and the vortex time is 5-10 min.

4. The method for preparing coated probiotics according to claim 1, characterized in that: In step 2), the volume ratio of the probiotic suspension to the cationic solution is 1:0.

4.

5. The method for preparing coated probiotics according to claim 1, characterized in that: In step 3), the concentration of the mussel mucin solution is 2 mg / mL, the vortex speed is 2000-3000 rpm, and the vortex time is 5-10 minutes.

6. The method for preparing coated probiotics according to claim 1, characterized in that: In step 3), the volume ratio of the probiotic suspension to the mussel mucin solution is 1:

4.

7. Application of the nano-coated probiotics prepared by the preparation method according to any one of claims 1 to 6 in the food industry.

8. A broad-spectrum universal nano-coated probiotic prepared by the preparation method according to any one of claims 1 to 6.

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