Broad-spectrum universal nano-coating probiotics based on mussel mucin as well as preparation method and application of broad-spectrum universal nano-coating probiotics

Abstract

The invention discloses broad-spectrum universal nano-coating probiotics based on mussel mucin as well as a preparation method and application of the broad-spectrum universal nano-coating probiotics. According to the method, the deposition of the mussel mucin on the surfaces of the probiotics is accelerated by virtue of supramolecular action modes such as non-covalent bonds, hydrogen bonds and cations-pi between the mussel mucin and thalli; and positive ions are complexed and coordinated with catechol groups in mussel mucin and are driven to be self-assembled into a nano-scale aggregate, so that independent coating of the probiotics is realized. The mussel mucin nano coating provided by the invention is not only suitable for encapsulating aerobic bacteria and also has a protection effect on anaerobic bacteria, but also can realize synchronous release of probiotics. More importantly, due to the addition of the cations, the adhesion capacity of the probiotics is enhanced while the network structure density of the coating is improved, and the nano-coating probiotics with high stability and biocompatibility are obtained. The nano-coating probiotic provided by the invention is simple and efficient in preparation process, is suitable for large-scale production, and is expected to become an ideal carrier for probiotic delivery.

Description

Technical Field

[0001] This patent relates to the field of probiotic technology, and specifically to a broad-spectrum universal nano-coated probiotic based on mussel mucin, and its preparation method and application. Background Art

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

[0003] Inspired by the self-protection behavior of bacteria in complex environments, researchers encapsulated probiotics in natural biomaterials to improve their resistance to extreme environments and give them 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 technology, single-cell encapsulation technology can give probiotics a cold-proof and warm "coat" attached to the surface of the bacteria, which can resist adverse environments while ensuring normal material exchange between probiotics and the external environment. Therefore, it is very necessary to find suitable materials and design a set of "coats" that meet the needs of most probiotics.

[0004] Mussel Adhesive Protein (MAP) is a mucin secreted by the foot gland of mussels, which can firmly adhere to any surface under seawater. The dopa group is an important component of mussel mucin, and its catechol group has strong adhesion and has been widely used as a coating layer for various inorganic and organic surfaces. In addition, the lysine residues in mussel mucin carry a strong positive charge at the physiological pH value of the human body, and can bind to negatively charged cells through electrostatic interactions, thereby enhancing the protective effect on cells. More importantly, when lysine residues and dopa groups exist at the same time and reach a certain concentration, the cation-π interaction can synergistically replace the interfacial hydration and mediate the strong adhesion of the cell surface. At the same time, the hydrophobic groups of amino acids in mussel mucin can bind to the lipid bilayer of the cell membrane through hydrophobic interactions and adhere to the cell surface. Therefore, as a natural polymer material, mussel mucin can be deposited on the cell surface and interact with it in various ways. However, mussel mucin is currently mainly used in the fields of medical wound repair and daily cosmetics, and its application in the field of probiotic technology has not yet been reported.

[0005] As shown in the following patent: Application number: 202110290344.0, publication number CN 113197314B, invention name "A layer-by-layer self-assembled probiotic microcapsule and its preparation method", discloses a layer-by-layer self-assembled probiotic microcapsule and its preparation method. This method spontaneously adsorbs multi-layer probiotic wet capsules into the microporous structure of porous starch layer by layer through electrostatic interaction to obtain layer-by-layer self-assembled probiotic microcapsules. Although this method effectively improves the efficiency of probiotics in intestinal transport and colonization survival, there are gaps between the capsule wall skeletons of the probiotic microcapsules, which is not suitable for the protection and delivery of anaerobic bacteria and does not have universal characteristics; in addition, this method requires the introduction of chemical reagents for starch modification treatment, the preparation process is complicated, and the production cost is high.

[0006] As shown in the following patent: Application number: 202311471775.2, publication number CN117297099A, invention name "Preparation method of probiotic coating based on sodium alginate and metal polyphenol network", discloses a preparation method of probiotic coating based on sodium alginate and metal polyphenol network. This method deposits aggregates formed by complexation and coordination of polyphenols and metal ions on the surface of probiotics, and induces sodium alginate to form a coating through calcium ions. In this method, the metal polyphenol network coating can self-assemble and form rapidly, and has no obvious effect on the normal growth of probiotics. However, the single metal polyphenol network coating has weak protection for probiotics during gastrointestinal transport, resulting in low cell viability. In addition, polyphenols bind to salivary proteins, which will induce the body's perception of astringency, and the introduction of metals will accelerate the oxidation of the system, limiting the application prospects.

[0007] The article "Oral delivery system of tumor antigens based on surface-coated probiotics" discloses a method for preparing probiotics coated with yeast cell membranes. Among them, the yeast cell membrane plays a role in protecting probiotics and antigens and targeting the intestinal lymphatic system, and the probiotics play a role in carrying antigens and immune adjuvants. The results showed 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 of dendritic cells and antigen presentation. Although this method can protect probiotics and antigens from gastrointestinal environmental stimulation, the production process of yeast cell membranes is complicated and costly. In addition, the introduction of yeast cell membranes limits the exchange of substances between probiotics and the external environment, and cannot maximize the health benefits of probiotics.

[0008] Therefore, it is a technical problem that technicians in this field need to solve urgently to provide a method for preparing nano-coated probiotics that has both gastric acid resistance and intestinal adhesion ability, high biocompatibility and good universal properties, and is suitable for industrial production. Summary of the invention

[0009] In order to overcome the shortcomings of the prior art, the primary purpose of the present invention is to provide an application of mussel mucin in the field of preparing nano-coated probiotics, and to provide a broad-spectrum universal nano-coated probiotic based on mussel mucin. By coating the probiotics with mussel mucin, the mucin carrier enhances the tolerance of the probiotics to the gastric environment, allowing them to reach the intestines unimpeded and exert their probiotic functions.

[0010] Another object of the present invention is to provide a method for preparing a broad-spectrum universal nano-coated probiotic based on mussel mucin, which encapsulates probiotics with mussel mucin, prolongs the retention time of probiotics in the intestine, and releases probiotics synchronously in the intestine, maximizing the health benefits of probiotics. The principle of mussel mucin coating probiotics is that the non-covalent bonds, hydrogen bonds, cation-π and other supramolecular action modes between mussel mucin and bacteria accelerate the deposition of mussel mucin on the surface of probiotics, and further by adding cations such as calcium ions to complex and coordinate with the catechol groups in mussel mucin and drive them to self-assemble into nano-scale aggregates, the probiotics are independently coated and given the ability to resist adverse external environments.

[0011] This method is not only suitable for encapsulating aerobic bacteria, but also has a protective effect on anaerobic bacteria. It can also achieve the simultaneous release of probiotics, maximizing the health benefits of probiotics.

[0012] To achieve the above purpose, the specific technical solution adopted by the present invention is: 1) Cultivate probiotics to the logarithmic growth phase, wash several times to ensure that there is no culture medium residue, remove the supernatant, collect the bacterial sludge, resuspend the bacterial sludge in sterile ultrapure water, and adjust the cell concentration.

[0013] 2) Resuspend the probiotics obtained in step 1) with a cationic aqueous solution and vortex to disperse.

[0014] 3) Add the mussel mucin solution to the solution obtained in step 2) and vortex to obtain a probiotic suspension.

[0015] 4) adjusting the pH of the probiotic suspension obtained in step 3) to 6.0, washing it multiple times with a sterile water solution having a pH of 6.0, and centrifuging to obtain the mussel mucin nano-coated probiotics.

[0016] 5) Re-disperse the centrifuged bacterial sludge in step 4) in sterile water of the same pH and store in a refrigerator at 4°C.

[0017] Preferably, the probiotics in step 1) are one or more of Lactobacillus paracasei, Bifidobacterium, lactic acid bacteria, and Lactobacillus plantarum.

[0018] Furthermore, in step 1), the probiotics are Lactobacillus plantarum (PL) and Bifidobacterium (BB).

[0019] Preferably, in step 1), the culture medium is MRS broth culture medium; the centrifugation speed is 5000 rpm, the centrifugation time is 10 min, the washing times are 3 times; the probiotic cell concentration is 1×10 10 ~ 1×10 11 CFU / mL.

[0020] Preferably, in step 2), the cation is selected as Ca 2+ Mg 2+ 、Zn 2+ , Fe 2+ Furthermore, in step 2), the cation is selected as Ca 2+ , the specific cationic aqueous solution is calcium chloride solution.

[0021] In step 2), the concentration of the cationic aqueous solution is 25 mM, the vortexing speed is 2000-3000 rpm, and the vortexing time is 5-10 min.

[0022] The purpose of resuspending the probiotics in a cationic aqueous solution in step 2 is to enable cations such as calcium ions to complex and coordinate with catechol groups in mussel mucin and drive them to self-assemble into nano-scale aggregates, thereby achieving independent encapsulation of the probiotics.

[0023] Preferably, in step 2), the volume ratio of the probiotic suspension to the cationic solution is 1:0.4.

[0024] Preferably, the concentration of the mussel mucin solution in step 3) is 2 mg / mL, the vortex speed is 3000 rpm, and the vortex time is 10 min; Preferably, in step 3), the volume ratio of the probiotic suspension to the mussel mucin solution is 1:4.

[0025] Preferably, in step 4), hydrochloric acid is used to adjust the pH of the probiotic suspension, and the concentration of the hydrochloric acid is 0.1M.

[0026] The third object of the present invention is to provide the application of the above-mentioned broad-spectrum universal nano-coated probiotics based on mussel mucin, which is specifically applied to the food industry, the pharmaceutical field, etc.

[0027] Compared with the prior art, the beneficial effects and advantages of the present invention are: (1) The method for preparing coated probiotics proposed in the present invention utilizes the non-covalent bonds between mussel mucin and bacteria, Supramolecular action modes such as hydrogen bonding and cation-π accelerate the deposition of mussel mucin on the surface of probiotics, and further enable calcium ions to complex and coordinate with the catechol groups in mussel mucin and drive them to self-assemble into nanoscale aggregates, achieving independent coating of probiotics and giving them the ability to resist adverse external environments.

[0028] (2) The mussel mucin used in the present invention has high biocompatibility. The characteristic groups in the molecular structure can interact with the intestinal epithelial mucosa, thereby improving the stability of the coating structure and giving the probiotics excellent adhesion properties, significantly prolonging the retention time of the probiotics in the intestine, and maximizing the health benefits of the probiotics.

[0029] (3) The mussel mucin coating used in the present invention has good universal properties. It is not only suitable for encapsulating aerobic bacteria, but also has a protective effect on anaerobic bacteria. It can also achieve the simultaneous release of probiotics and efficiently regulate intestinal health.

[0030] (4) The preparation method of the nano-coated probiotics provided by the present invention is simple and low-cost, suitable for industrial-scale production, and lays a foundation for the industrial application of mussel mucin-coated probiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Electric fluorescence microscopy images of PL@MAP and BB@MAP.

[0032] Figure 2 Transmission electron microscopy images of PL, PL@MAP, BB and BB@MAP.

[0033] Figure 3 Growth curves of PL, PL@MAP, BB and BB@MAP.

[0034] Figure 4 Fourier transform infrared spectra of PL, PL@MAP, BB and BB@MAP.

[0035] Figure 5 Fluorescence spectra of PL, PL@MAP, BB and BB@MAP.

[0036] Figure 6 Potential diagrams of PL, PL@ MAP, BB and BB@ MAP.

[0037] Figure 7 The adhesion rate diagram of PL, PL@MAP, BB and BB@MAP.

[0038] Figure 8 In vitro simulated digestion diagram of PL, PL@MAP, BB and BB@MAP Fig. 9 Storage stability diagram of PL, PL@MAP, BB and BB@MAP Fig.10 Hydrogen peroxide damage diagram of PL, PL@MAP, BB and BB@MAP DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with embodiments. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0040] The experimental methods used in the present invention are all conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0041] The present invention provides the following two embodiments: Example 1: In this example, the probiotics are Lactobacillus plantarum, and the cations are calcium ions. The specific disclosure contents are as follows: 1) Cultivate Lactobacillus plantarum to the logarithmic growth phase, centrifuge at 5000 rpm for 10 min, wash three times to ensure that there is no culture medium residue, collect the bacterial sludge, resuspend it in sterile ultrapure water, and adjust the probiotic cell concentration to 1×10 10 ~ 1×10 11 CFU / mL.

[0042] 2) Mix 25 mM calcium chloride aqueous solution and bacterial suspension in a ratio of 0.4:1, vortex at 3000 rpm for 10 min.

[0043] 3) Mix the 2 mg / mL mussel mucin aqueous solution and the bacterial suspension in a ratio of 4:1, vortex at a speed of 3000 rpm and a vortex time of 10 min to obtain a mussel mucin-coated probiotic suspension.

[0044] 4) The pH of the probiotic suspension obtained above was adjusted to 6.0, and washed several times with a sterile water solution having a pH of 6.0, and centrifuged to obtain the mussel mucin nano-coated probiotics.

[0045] 5) Re-disperse the centrifuged bacterial sludge in step 4) in sterile water of the same pH and store in a refrigerator at 4°C.

[0046] Example 2: The difference between the technical solution in this example and the above-mentioned example 1 is that the probiotics are bifidobacteria. The specific disclosure content is as follows: 1) Cultivate Bifidobacterium to the logarithmic growth phase, centrifuge at 5000 rpm for 10 min, wash three times to ensure that there is no culture medium residue, collect the bacterial sludge, resuspend the bacterial sludge in sterile ultrapure water, and adjust the probiotic cell concentration to 1×10 10 ~ 1×10 11 CFU / mL.

[0047] 2) Mix 25 mM calcium chloride aqueous solution and bacterial suspension in a ratio of 0.4:1, vortex at 3000 rpm for 10 min.

[0048] 3) Mix the 2 mg / mL mussel mucin aqueous solution and the bacterial suspension in a ratio of 4:1, vortex at a speed of 3000 rpm and a vortex time of 10 min to obtain a mussel mucin-coated probiotic suspension.

[0049] 4) The pH of the probiotic suspension obtained above was adjusted to 6.0, and washed several times with a sterile water solution having a pH of 6.0, and centrifuged to obtain the mussel mucin nano-coated probiotics.

[0050] 5) Re-disperse the centrifuged bacterial sludge in step 4) in sterile water of the same pH and store in a refrigerator at 4°C.

[0051] Experimental Results Fluorescence microscopy observation The cell morphology of the coated bacteria was observed using an electron fluorescence microscope (ECLIPSE Ni-E, Nikon Corporation, Japan). First, 100 mg of mussel mucin 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. Secondly, the FITC solution was slowly added to the mussel mucin solution and dialyzed (MWCO 8,000-14,000Da) for 60 hours with stirring to remove free FITC. Finally, the solution was dried at 45°C to obtain FITC-labeled mussel mucin. PL@MAP and BB@MAP labeled with FITC were obtained according to Example 1 and Example 2. The results are shown in Figure 2. Figure 1 As shown, the green fluorescence of FITC co-localizes with PL and BB under bright field, proving that mussel mucin is modified on the surface of bacteria, that is, the bacteria are successfully covered with a nanocoat.

[0052] Transmission electron microscopy observation 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, and 100 μL of sterile water was added to the supernatant, and the suspension was blown into a uniform suspension. Secondly, 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 It can be seen from the transmission electron microscopy image that there is an obvious nanocoating around the bacteria, with a thickness of about 100-150 nm, indicating that the mussel mucin is successfully wrapped on the bacterial surface.

[0053] Growth curve assay PL, BB, PL@MAP obtained in Example 1 and Example 2, and BB@MAP were cultured in MRS liquid medium. The cells were diluted to a medium with an optical density (OD600) between 0.1 and 0.2 and incubated at 37°C. The growth of the bacteria at 37°C was monitored at 1-h intervals using an ELISA reader (Powerwave XS, Biotek, USA), and a growth curve was drawn 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.

[0054] Infrared spectroscopy Fourier transform infrared spectroscopy (Nicolet iS10, Thermo Fisher Scientific, USA) was used to analyze the interaction between mussel mucin coating and probiotics. 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 pressed into tablets. Fourier transform infrared spectrometer was used to obtain the 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 -1 The 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 extend from 2935 cm-1 to 2938 cm-1 (CH stretching vibration), which confirms that hydrophobic interactions are involved in the construction of the coating.

[0055] Fluorescence spectroscopy analysis 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 put into a sample pool for detection. The experimental parameters were set as follows: the spectroscopic element was a grating with 150 lines per millimeter, the scanning time was 2s, the scanning range was 300 nm-650nm, the excitation wavelength was 290 nm, and the excitation was performed every 5 nm. Figure 5 It can be seen that PL and BB have typical absorption peaks at 330 nm, which are related to the interaction of multiple amino acids in cells, including L-glutamic acid, D-alanine and L-cysteine. The fluorescence intensity of PL@MAP and BB@MAP is significantly higher than that of PL and BB, which indicates that after coating mussel mucin, some paired basic amino acid residues in mussel mucin adhere to the negatively charged cell surface through electrostatic attraction, and some sequences of cysteine ​​in mussel mucin are connected to cysteine ​​residues in the cell peptidoglycan layer through disulfide bonds, which helps to improve the binding stability of the protein.

[0056] Potentiometric measurement The PL, BB, Example 1 and 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 rich lipoteichoic acid in the cell wall. After the probiotics combined with mussel mucin, the number of negative charges decreased significantly, indicating that electrostatic interactions occurred between mussel mucin and BB and PL, that is, some paired basic amino acid residues in mussel mucin were deposited on the negatively charged cell surface to form a nanocoating.

[0057] Adhesion rate determination Preparation of mucin film: First, 500 mg of mucin was dissolved in 50 mL of pure water and stirred at room temperature for 2 hours. Then, 500 μL of glycerol was added to the solution and ultrasonicated for 2 min to remove bubbles. Finally, the solution was poured into a plastic mold (D = 9 cm) and then dried in an oven at 60 °C for 4 h. The prepared film was taken out and placed at a humidity of 50% and a temperature of 25 °C for 48 h.

[0058] Adhesion rate calculation: Mucin film was incubated with PL@MAP and BB@MAP suspension (100 μL, 1×10 8 CFU / mL) for 30 min. After rinsing three times with sterile PBS, the rinse solution was inoculated into MRS medium for counting. The adhesion rate was calculated by the following formula: Adhesion rate = (A1 -A 0 ) / A 1 ×100 % Among them A 0 is the number of unadhered probiotics, A 1 is the initial number of active probiotics.

[0059] 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 properties of the probiotics and exerting a better colonization effect.

[0060] Digestion stability assay Simulated gastric digestion: First, 2 mL of PL, BB, PL@MAP obtained in Example 1 and Example 2, and BB@MAP was mixed with 16 mL of 2 mg / mL NaCl solution and the pH was adjusted to 1.2. Then, 6.4 mg of pepsin and 4 mL of NaCl solution were added to the mixture and the pH of the system was adjusted to 2.0 to start simulating gastric digestion. After 2 h, the pH was adjusted to 7.5 to terminate gastric digestion.

[0061] Simulated small intestinal digestion: 5 mg / mL porcine bile salts were dispersed in deionized water and the pH was adjusted to 7.5, then pancreatic enzyme (1.6 mg / mL) was added and the pH of the system was adjusted to 7.5. An equal amount of simulated intestinal fluid was mixed with the digestive fluid produced in the above process and digested for 2 h.

[0062] Simulated colon digestion: An equal amount of PBS buffer was mixed with the digestion solution produced in the above process and digested for 2 h.

[0063] The experimental results are as follows Figure 8 As shown in the figure, after being exposed to simulated gastric juice and bile salt solution, the number of live bacteria in BB and PL is basically zero, while the number of probiotic bacteria in the mussel mucin coating remains at a high level. The experimental results show that the nanocoating based on mussel mucin proposed in the present invention has excellent gastrointestinal resistance and universal properties.

[0064] Storage stability test Under 4 °C storage conditions, PL, BB, PL@MAP obtained in Example 1 and Example 2, and BB@MAP The activity of the strains was investigated after 15 days of storage, and the number of viable bacteria was detected by the plate count method. Fig. 9 The result table shows that after 15 days of storage at 4°C, the live bacterial counts of PL and BB were 7.23 and 6.85 Log CFU / mL, while the live bacterial counts of PL@MAP and BB@MAP were 9.02 and 8.25 Log CFU / mL, respectively, proving that mussel mucin can act as a physical barrier to isolate the adverse external environment and improve the storage stability of probiotics.

[0065] Hydrogen peroxide injury assay PL, BB, PL@MAP and BB@MAP obtained in Example 1 and Example 2 were resuspended in 1 mL of 50 μM H 2 O 2 The cells were then incubated in a 37°C incubator with gentle shaking. 50 µL of the solution was taken out at 15 min, 30 min, and 60 min, washed with sterile water, and the number of viable cells was determined by plate counting. Fig.10 As shown, H 2 O 2 After 30 min of treatment, PL@MAP and BB@MAP were still alive, but the survival rate of PL and BB was zero, indicating that mussel mucin can resist H 2 O 2 Invasion, improve the survival rate of probiotics.

[0066] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. Application of mussel mucin in the preparation of nano-coated probiotics.

2. A broad-spectrum universal nano-coated probiotic, It is characterized in that The probiotics are coated with mussel mucin.

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

4. The method for preparing the coated probiotics according to claim 3, It is characterized in that The probiotics in step 1) are one or more of Lactobacillus paracasei, Bifidobacterium, lactic acid bacteria, and Lactobacillus plantarum.

5. The method for preparing the coated probiotics according to claim 3, It is characterized in that Step 2) The cation is Ca 2+ Mg 2+ 、Zn 2+ , Fe 2+ One or more of .

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

7. The method for preparing coated probiotics according to claim 3, It is characterized in that In step 2), the volume ratio of the probiotic suspension to the cationic solution is 1:0.

4.

8. The method for preparing the coated probiotics according to claim 3, It is 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 min.

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

4.

10. Application of the broad-spectrum universal nano-coated probiotics based on mussel mucin prepared according to any of the above claims in the food industry.

Images