Biological coating coated probiotics as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological materials, and particularly relates to biological coating coated probiotics as well as a preparation method and application thereof. The biological coating in the biological coating coated probiotics is mussel mucin and sodium alginate, the final concentration of the mussel mucin in a coating system is 0.5-2 mg / mL, and the final concentration of the sodium alginate is 10-20 mg / mL. The biological coating coated probiotics disclosed by the invention has a wide application prospect in treatment of intestinal diseases such as intestinal colitis and the like. By targeting the inflammation part and enhancing the adhesion of the probiotics, the bioavailability of the probiotics can be effectively improved, the anti-inflammatory effect is synergistically exerted, and the intestinal microecological balance is maintained, so that a safe and effective new way for treating intestinal diseases is provided.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and specifically relates to a biological coating (mussel mucin and sodium alginate) coated probiotics, and a preparation method and application thereof. Background Art

[0002] Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is an immune-mediated disease characterized by chronic intestinal inflammation, with an increasing incidence worldwide. Traditional medical strategies for the treatment of IBD mainly use anti-inflammatory drugs (aminosalicylates, corticosteroids), immunosuppressants, and antibiotics. To a certain extent, these strategies can inhibit pathogen infection and regulate immune responses. However, these drugs often face problems such as poor stability, poor pharmacokinetics, and side effects related to systemic drug exposure, which limits their efficacy and safety for long-term use.

[0003] Probiotics attach to intestinal epithelial cells, compete with pathogenic bacteria for nutrients, and continuously produce key biomacromolecules, so probiotic therapy is a promising approach for the treatment of IBD. These probiotics can increase intestinal mucus secretion, enhance mucosal barriers, improve antioxidant capacity, inhibit pro-inflammatory cytokine secretion, promote anti-inflammatory cytokine release, and regulate the intestinal immune system by increasing immunoglobulins and defensins in immune responses. However, due to the limitations of gastric acid, bile acid, and digestive enzymes after oral administration, the bioavailability and therapeutic efficiency of probiotics are low.

[0004] Dosage forms of oral probiotics, including liquids, capsules, etc., have been widely used to protect the vitality of probiotics. However, these dosage forms are still insufficient to protect oral probiotics from gastrointestinal environmental damage and improve their survival rate. In recent years, probiotic surface coatings have become a common strategy for regulating probiotic functions. However, most probiotics are anaerobic bacteria and lack antioxidant defense mechanisms to protect them from oxidation. Therefore, there are few studies on universal coatings for anaerobic probiotics, and the development of anaerobic probiotic bio-coatings is imminent. Summary of the invention

[0005] The purpose of the present invention is to provide a biological coating (mussel mucin and sodium alginate) coated probiotics and a preparation method and application thereof.

[0006] To achieve the above object, the technical solution adopted by the present invention is: A bio-coated probiotic, wherein the bio-coating in the bio-coated probiotic contains mussel mucin and sodium alginate, wherein the final concentration of mussel mucin in the coating system is 0.5-2 mg / mL, and the final concentration of sodium alginate is 10-20 mg / mL.

[0007] The probiotic is Bifidobacterium longum BNCC185354.

[0008] A method for preparing the biological coating-coated probiotics: 1) In k + In the buffer, mussel mucin was mixed with probiotics cultured to the logarithmic growth phase, vortexed thoroughly, incubated in an anaerobic environment, and centrifuged; 2) Adding sodium alginate solution to the above centrifuged precipitate and vortexing, and obtaining the probiotics coated with mussel mucin and sodium alginate bio-coating after centrifugation.

[0009] The probiotics cultured to the logarithmic growth phase are Bifidobacterium longum BNCC185354, which is cultured to the logarithmic growth phase in a culture medium, the culture medium is removed, and then washed multiple times with a phosphate buffer solution with a pH of 7.4 to obtain the probiotics for standby use.

[0010] The culture medium is a MRS broth culture medium containing cysteine, wherein the cysteine ​​concentration is 0.4-0.6 g / L.

[0011] In step 1), the probiotics were cultured in MRS medium until the logarithmic growth phase, 1 mL of the bacterial solution was taken, and centrifuged at 6000 rpm for 5 min to obtain a probiotic precipitate. + In the buffer solution, mussel mucin and probiotic precipitate are mixed to make the final concentration of mussel mucin in the system be 0.5-2 mg / mL. After mixing, the mixture is vortexed for 5-10 minutes, and then incubated in an anaerobic environment for 5-10 minutes. After incubation, the mixture is centrifuged to collect the precipitate.

[0012] The k + The buffer concentration is 0.2M, pH=5.5.

[0013] In the step 2), sodium alginate solution is added to the above centrifuged precipitate and vortexed for 5-10 minutes, and then centrifuged to obtain the probiotics coated with the mussel mucin and sodium alginate bio-coating; wherein the final concentration of sodium alginate in the system is 10-20 mg / mL.

[0014] An application of the biological coating coated probiotics, and an application of the coated probiotics in the preparation of drugs for preventing and treating colitis in mice.

[0015] A method for increasing the number of probiotic colonization, using a system containing mussel mucin and sodium alginate as a coating to coat the probiotics, thereby increasing the number of probiotic colonization.

[0016] Principle of the present invention: The oxidized and unoxidized dopa groups in the mussel mucin molecules will self-crosslink on the surface of the probiotics to form a continuous coating. At the same time, the mussel mucin contains a large amount of positively charged lysine, which will adsorb the negatively charged sodium alginate. When the probiotics reach the intestine, the negatively charged sodium alginate will cause the probiotics to quickly gather at the site of inflammation. The adhesion properties of the mussel mucin make the probiotics adhere to the surface of the intestinal epithelial cells. The probiotics inhibit the growth of pathogens, maintain the intestinal inherent flora, and maintain the balance of the intestinal flora. In addition, the two biomaterials and probiotics can play a synergistic anti-inflammatory role and maintain intestinal health. The present invention is also applicable to various anaerobic probiotics to strengthen the treatment of intestinal diseases.

[0017] Advantages of the present invention: 1. The method of the present invention can form a mussel mucin and sodium alginate biological coating on the surface of probiotics, target the inflammatory site, and improve the colonization of probiotics by enhancing adhesion, thereby improving the bioavailability of probiotics and achieving synergistic treatment.

[0018] 2. This method is simple, rapid and widely applicable. The coating formation is completed within 30 minutes. In addition, this technology is applicable to various anaerobic probiotics. The materials used (mussel mucin, sodium alginate) are all of natural origin, with good biocompatibility and safety, reducing the potential risk of side effects and suitable for long-term use. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Transmission electron microscope images of BL, BL@Map and BL@Map@Alg in Example 1 of the present invention.

[0020] Figure 2 The colony counts of BL, BL@Map and BL@Map@Alg in Example 2 of the present invention after incubation in artificial gastric fluid for 1 h and 2 h.

[0021] Figure 3 Transmission electron microscope images of BL, BL@Map and BL@Map@Alg in Example 2 of the present invention after incubation in artificial gastric fluid for 1 h and 2 h.

[0022] Figure 4 The colony counts of BL, BL@Map and BL@Map@Alg in Example 2 of the present invention after incubation in artificial intestinal fluid for 2 h and 4 h.

[0023] Figure 5 Transmission electron microscope images of BL, BL@Map and BL@Map@Alg in Example 2 of the present invention after incubation in artificial intestinal fluid for 2h and 4h.

[0024] Figure 6It is the average radiation luminescence efficiency of BL, BL@Map and BL@Map@Alg after incubation with colon in Example 3 of the present invention.

[0025] Figure 7 Schematic diagram of the experimental design of BL@Map@Alg in treating DSS-induced acute colitis in mice in Example 4 of the present invention.

[0026] Figure 8 This is the weight change curve of mice in Example 4 of the present invention from day 0 to day 12.

[0027] Fig. 9 These are representative pictures of the colon of mice in different treatment groups in Example 4 of the present invention.

[0028] Fig.10 The contents of TNF-α, IL-6 and IL-1β in the colon tissues of mice in different treatment groups in Example 4 of the present invention.

[0029] Fig.11 These are H&E staining images of colon tissues of mice in different treatment groups in Example 4 of the present invention.

[0030] Fig.12 These are pictures of in situ fluorescent hybridization of colon tissues of mice in different treatment groups in Example 5 of the present invention.

[0031] Fig.13 This is the standard curve of BL concentration and CT value in Example 5 of the present invention.

[0032] Fig.14 The number of BL in different treatment groups determined by RT-qPCR in Example 5 of the present invention. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0034] Most traditional coating designs are targeted at aerobic probiotics, and cannot effectively protect the activity of anaerobic probiotics and promote their colonization. The present invention provides a specific functionalized coating material for anaerobic probiotics to improve their stability and colonization rate, which is a scientific problem that needs to be solved urgently in current research. The present invention uses oxidized and unoxidized dopa groups in mussel mucin molecules to self-crosslink on the surface of probiotics to form a continuous coating. At the same time, mussel mucin contains a large amount of positively charged lysine, which will adsorb negatively charged sodium alginate. The bio-coating-coated probiotics of the present invention have broad application prospects in the treatment of intestinal diseases such as intestinal colitis. By targeting the inflammatory site and enhancing the adhesion of probiotics, the bioavailability of probiotics can be effectively improved, the anti-inflammatory effect can be synergistically exerted, and the balance of intestinal microecology can be maintained, thereby providing a safe and effective new way to treat intestinal diseases.

[0035] The probiotics in the following examples are Bifidobacterium longum BNCC185354, which were purchased from Beina Biotechnology.

[0036] Example 1: Characterization of probiotics in mussel mucin and sodium alginate bio-coatings Bifidobacterium longum BNCC185354 ( Bifidobacterium longum (BL) was cultured in MRS medium until the logarithmic growth phase, 1 mL of bacterial solution was taken, centrifuged at 6000 rpm for 5 min, and washed three times with phosphate buffer solution (PBS, pH = 7.4), and the precipitate was collected to obtain BL bacteria. + In a buffer solution (0.2M, pH=5.5), mussel mucin was mixed with BL precipitate to make the final concentration of mussel mucin (Map) in the system 2mg / mL. After mixing, it was fully vortexed for 10min, and then incubated in an anaerobic environment for 10min. After incubation, it was centrifuged at 6000rpm for 5min, and the precipitate was collected to obtain BL@Map. Subsequently, 500μL, 10mg / mL sodium alginate (Alg) solution was added and vortexed for 10min, and then centrifuged at 6000rpm for 5min, and the precipitate was collected to obtain mussel mucin and sodium alginate bio-coated probiotics (ie, BL@Map@Alg).

[0037] BL (bacterial solution cultured to the logarithmic growth phase), BL@Map, and BL@Map@Alg were dropped onto a copper grid, and the excess liquid was removed after 10 min. After drying at 37 °C for 12 h, the morphology was observed using a transmission electron microscope (TEM). Figure 1 As shown, TEM results showed that an additional bright outer film appeared on the surface of BL@Map@Alg compared with BL, indicating that a coating was formed on the surface of the probiotics.

[0038] Example 2: In vitro evaluation of the protective effect of mussel mucin and sodium alginate bio-coating on probiotics BL, BL@Map and BL@Map@Alg were cultured in artificial gastric fluid (SGF, pH 2.0) at 37°C in an anaerobic environment for 1 h and 2 h, respectively. Appropriate amounts of each bacterial solution were taken for gradient dilution and spread on plates. After culturing at 37°C for 72 h, the dilution gradient of 10 -5 Count the plates. Figure 2As shown in the figure, after 1 hour of anaerobic culture in SGF, significant differences appeared on the plates of different treatment groups. There was almost no live bacterial growth on the plate of the BL group, while the BL@Map group and BL@Map@Alg group showed a certain survival ability compared with the BL group. It is worth noting that after 2 hours of anaerobic culture in SGF, a large amount of live bacterial growth can still be observed on the plate of the BL@Map@Alg group, a small amount of live bacterial growth in the BL@Map group, and almost no surviving colonies in the BL group. This shows that BL@Map@Alg has a good protective effect within 2 hours, protecting the probiotics from the gastric acid environment.

[0039] In addition, after culturing BL, BL@Map and BL@Map@Alg in SGF for 1h and 2h, appropriate amount of bacterial solution was taken, centrifuged at 6000rpm for 5min to obtain bacterial cells, and then dropped on a copper grid. After 10min, the excess liquid was aspirated and dried at 37℃ for 12h. The morphology of BL, BL@Map and BL@Map@Alg after different culturing time in SGF was observed using transmission electron microscopy (TEM). Figure 3 As shown, TEM results show that after 1 hour of culture in SGF, the BL bacteria showed obvious shrinkage and significant morphological changes. After 2 hours of culture in SGF, the BL bacteria showed more serious decomposition and their complete morphology was almost unrecognizable. In contrast, under the same conditions, the BL@Map group showed a certain degree of morphological changes, but they were relatively mild. In particular, in the BL@Map@Alg group, although there was some slight loss of the coating, the overall bacterial morphology was still relatively intact, showing a good protective effect within 2 hours. This shows that BL@Map@Alg can effectively maintain the bacterial structure and slow down the damage of the gastric fluid environment to probiotics.

[0040] BL, BL@Map and BL@Map@Alg were cultured in artificial intestinal fluid (SIF, pH 6.8) at 37°C in an anaerobic environment for 2 h and 4 h, respectively. Appropriate amounts of bacterial cultures were diluted and spread on plates. After culturing at 37°C for 72 h, plate counts were performed. Figure 4 As shown, within 4 h of culture, the number of BL@Map, BL@Map@Alg had no significant difference with BL.

[0041] In addition, after culturing BL, BL@Map and BL@Map@Alg in SIF for 2h and 4h, appropriate amount of bacterial solution was taken, centrifuged at 6000rpm for 5min to obtain bacterial cells, and then dropped on a copper grid. After 10min, the excess liquid was aspirated and dried at 37℃ for 12h. The morphology of BL, BL@Map and BL@Map@Alg after SIF culture for different time periods was observed using a transmission electron microscope (TEM). Figure 5As shown, the TEM results show that the coating gradually dissolves over time, and the morphologies of BL, BL@Map and BL@Map@Alg are similar with no significant differences. It can be seen that the coating form of the present invention, compared with the coating form of the prior art, can ensure the survival rate and activity of probiotics in the gastrointestinal environment, and can proliferate in the intestine, thereby improving its bioavailability.

[0042] Example 3: Evaluation of intestinal adhesion effects of BL, BL@Map and BL@Map@Alg Bifidobacterium longum BL was cultured in MRS medium until the logarithmic growth phase. 1 mL of bacterial solution was taken and centrifuged at 6000 rpm for 5 min. The solution was washed three times with phosphate buffer solution (PBS, pH = 7.4) and the precipitate was collected to obtain BL bacteria. Subsequently, 100 μL of 100 μg / mL fluorescein isothiocyanate (FITC) was added and washed three times with phosphate buffer solution (PBS, pH = 7.4). FITC-labeled BL@Map and BL@Map@Alg were then prepared according to the method in Example 1 above.

[0043] Fresh colons of mice were taken and FITC-labeled BL, BL@Map and BL@Map@Alg (2×10 8 ) and the colon were incubated for 2 h. Finally, each colon was rinsed twice with PBS and observed using an in vivo optical imaging system (IVIS Spectrum, PerkinElmer). The average radiant luminescence efficiency was analyzed using Living Image software. Figure 6 As shown in the figure, compared with the BL group, the fluorescence intensity of BL@Map and BL@Map@Alg was significantly improved, the average radiation efficiency of BL@Map increased by 9.5 times, and the average radiation efficiency of BL@Map@Alg also increased by 5 times. This result shows that the Map coating significantly enhances the adhesion potential of BL, can more effectively bind to colon tissue, and increase its colonization ability. However, since the adhesion of sodium alginate (Alg) is lower than that of mussel mucin (Map), the adhesion effect of BL@Map@Alg is slightly lower than that of BL@Map, but still shows a significant improvement compared with the BL group. Overall, this shows that Map plays a key role in enhancing the adhesion of BL, and although the presence of Alg has some influence, it can still protect probiotics to a certain extent and promote their colonization.

[0044] Example 4: BL@Map@Alg treats DSS-induced acute colitis in mice Thirty C57BL / 6 mice were randomly divided into five groups, with six mice in each group. The five groups were named control group, DSS (dextran sulfate sodium) group, BL group, BL@Map group and BL@Map@Alg group. Figure 7 As shown, BL@Map and BL@Map@Alg were prepared according to the description in Example 1.

[0045] The mice in each group were treated as follows: (1) Control group: free drinking of deionized water; (2) DSS group: free drinking of deionized water containing 3% (w / v) DSS from day 0 to day 7; (3) BL group: free drinking of deionized water containing 3% (w / v) DSS from day 0 to day 7, and oral gavage of 2×10 8 CFU BL / mouse / day; (4) BL@Map group: drinking deionized water containing 3% (w / v) DSS on days 0-7, and gavage of 2×10 8 CFU BL@Map / mouse / day; (5) BL@Map@Alg group: drinking deionized water containing 3% (w / v) DSS on days 0-7, and gavage of 2×10 8 CFU BL@Map@Alg / mouse / day. All mice were killed on the 12th day, and the weight change of each mouse was recorded every day, and the fecal viscosity and fecal occult blood were observed.

[0046] like Figure 8 As shown in the figure, the weight of mice in the DSS-treated group decreased significantly from the 3rd day to the 12th day, indicating that the colitis animal model in mice was successfully established. The mice drinking DSS had different degrees of diarrhea and blood in their stools, and their weight began to decrease on the third day. The weight of mice in the DSS group, BL group, and BL@Map group decreased by 27.4%, 22.4%, and 22.6% on the 7th day, respectively, while the treatment in the BL@Map@Alg group had the most significant effect on reducing the weight loss of mice, which was only 15.7%. After stopping drinking DSS, the weight of mice in the BL group, BL@Map group, and BL@Map@Alg group all recovered.

[0047] After each mouse was sacrificed, the length of the colon was measured. Fig. 9 As shown in the figure, the colon length of mice treated with DSS was significantly shortened. The colon length of the BL@Map@Alg group was the longest compared with the BL and BL@Map groups. It is worth noting that the food intake and motility of mice treated with BL@Map@Alg were most significantly restored, indicating that BL@Map@Alg can delay the development of colitis in mice through a preventive effect.

[0048] A section of colon was taken and homogenized (10% w / v) in pre-cooled PBS (10.0 mM, pH 7.2-7.4) using an electric homogenizer. The colon homogenate was centrifuged at 5000×g for 10 min at 4°C, and the supernatant was diluted 2-fold and used for enzyme-linked immunosorbent assay to determine the levels of tumor necrosis factor (TNF-α) and interleukins (IL-6, IL-1β) in colon tissue.

[0049] like Fig.10 As shown in the figure, the concentrations of TNF-α, IL-6, and IL-1β in mice treated with BL@Map@Alg were the lowest among all treated mice, decreasing by 482.9pg / mL, 54.4pg / mL, and 46.6pg / mL, respectively, compared with the DSS group. This indicates that the inflammatory response induced by DSS mice was largely alleviated.

[0050] The colon of each mouse was fixed in 4% paraformaldehyde for 48 h and then embedded in paraffin. The tissue was cut into 4 μm sections using a paraffin slicer and stained with hematoxylin-eosin (H&E). Fig.11 As shown in the figure, the intestinal structure of the DSS group was severely abnormal, with large-area ulcers in the mucosal layer, erosion and shedding of epithelial cells, a significant decrease in the number of crypts and goblet cells, a large number of inflammatory cells in the mucosal layer, and damage spreading to the submucosal layer. Compared with the DSS group, the intestinal damage in the BL group was alleviated, the number of crypts and goblet cells increased, and a small number of mucosal epithelial cells fell off. The degree of damage in the BL@Map group was further alleviated, no obvious degeneration and shedding of epithelial cells was observed, the number of crypts and goblet cells in the mucosal layer increased further, and a small amount of inflammatory cell infiltration was observed. In the BL@Map@Alg group, the intestinal structure was normal, the epithelial cells were closely arranged, the number of crypts in the mucosal layer was large, the goblet cells were abundant, and only a small amount of inflammatory cells infiltrated. The above results once again indicate that BL@Map@Alg has a significant therapeutic effect in DSS-induced colitis in mice.

[0051] Example 5: Evaluation of colonization effect of BL@Map@Alg Paraffin-embedded colon tissue sections (4 μm) were dewaxed with xylene 3 times, 5 min / time; dehydrated with anhydrous ethanol, 90% ethanol, and 70% ethanol for 5 min in sequence; and washed with 1×PBS 2 times, 5 min / time. Solution A in the bacterial fluorescence in situ hybridization detection kit was added to the sample and allowed to stand at 37°C for 15 min. Solution A was removed, and solution B was added, and allowed to stand at 37°C for 20 min. Solution B was removed, washed with 1×PBS, and dehydrated with 70% ethanol, 90% ethanol, and anhydrous ethanol in sequence and air-dried. BL probe was fluorescently labeled with Texas Red dye, and the BL probe was diluted to 10 ng / μL in the hybridization buffer. 10 μL of probe solution was added to each sample, and a coverslip was placed on the sample, and the sample was sealed with rubber glue. After denaturation at 75°C for 10 min, hybridization was immediately performed at 37°C for 24 h. The rubber glue was removed, and the slides were placed in washing buffer (preheated to 37°C) and washed for 30 min; the specimens were washed with ultrapure water for 2 min, and then dried at room temperature for 20 min. DAPI solution was added and coverslips were placed. Fluorescence images were obtained using a laser scanning confocal microscope (Zeiss LSM880, Germany). Fig.12 As shown, both BL@Map and BL@Map@Alg can increase the colonization number of BL, but the BL@Map@Alg bio-coating significantly increases the colonization number of BL.

[0052] For further quantification, primers were designed based on the gyrA gene on BL (yrA-F: 5'-CAAGGGCGACACGATTCTCT-3'; gyrA-R: 5'-TGTTCACATTCTCGCCGGAT-3'), RT-qPCR, and Qingke Biotechnology was commissioned to establish a standard curve between CT value and BL concentration ( Fig.13 ), then, the colon RNA of each group of mice was extracted, reverse transcribed into DNA, and the CT value was determined by the absolute quantitative method of RT-qPCR. The PCR reaction procedure was: 94℃, 2-3min; 94℃, 10-20s; 55-60℃, 10-20s; 72℃, 20-30s; 40 cycles. The results are shown in Fig.14 The results showed that the colonization rate of BL increased by 6.2 times after coating. These results suggest that bio-coatings can help probiotics better colonize and survive in the intestine, thus exerting long-term beneficial effects on the intestine.

[0053] It can be seen that the oral microecological preparation in which the mussel mucin and sodium alginate biological coating provided in the present application encapsulates a single probiotic bacterial body greatly improves the bioavailability of probiotics. This simple, fast and efficient coating strategy is expected to become a general strategy for anaerobic probiotics and is of great significance.

[0054] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should be included in the protection scope of the present invention.

Claims

1. A probiotic coated with a biological coating, characterized in that: The biological coating in the biological coating-coated probiotics is mussel mucin and sodium alginate, wherein the final concentration of mussel mucin is 0.5-2 mg / mL, and the final concentration of sodium alginate is 10-20 mg / mL.

2. The bio-coated probiotic according to claim 1, characterized in that: The probiotic is Bifidobacterium longum BNCC185354.

3. A method for preparing the biological coating-coated probiotics according to claim 1, characterized in that: 1) In k + In a buffer, mussel mucin is mixed with probiotics cultured to the logarithmic growth phase, vortexed thoroughly, incubated in an anaerobic environment, and centrifuged to obtain a precipitate; 2) adding sodium alginate solution to the above precipitate and vortexing, and obtaining the probiotics coated with mussel mucin and sodium alginate bio-coating after centrifugation.

4. The method for preparing probiotics coated with a biological coating according to claim 3, characterized in that: The operation steps of culturing the probiotics to the logarithmic growth phase are as follows: culturing Bifidobacterium longum BNCC185354 in a culture medium to the logarithmic growth phase, removing the culture medium, and then washing multiple times with a phosphate buffer solution with a pH of 7.4 to obtain the probiotics for standby use.

5. The method for preparing probiotics coated with a biological coating according to claim 4, characterized in that: The culture medium is a MRS broth culture medium containing cysteine, wherein the cysteine ​​concentration is 0.4-0.6 g / L.

6. The method for preparing probiotics coated with a biological coating according to claim 3, characterized in that: In the step 1), the probiotics were cultured in MRS medium until the logarithmic growth phase, 1 mL of the bacterial solution was taken, and centrifuged at 6000 rpm for 5 min to obtain a probiotic precipitate, and then, 1 mL of k + In the buffer solution, mussel mucin and probiotic precipitate are mixed to make the final concentration of mussel mucin in the system be 0.5-2 mg / mL. After mixing, the mixture is vortexed for 5-10 minutes, and then incubated in an anaerobic environment for 5-10 minutes. After incubation, the mixture is centrifuged to collect the precipitate.

7. The method for preparing probiotics coated with a biological coating according to claim 6, characterized in that: The k + The buffer concentration is 0.2M, pH=5.

5.

8. The method for preparing probiotics coated with a biological coating according to claim 3, characterized in that: The vortex time in step 2) is 5-10 min, and the final concentration of sodium alginate in the system is 10-20 mg / mL.

9. An application of the biological coating coated probiotics according to claim 1, characterized in that: The application of the biological coating-coated probiotics in the preparation of medicines for preventing and treating colitis in mice.

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