Preparation method and application of silk fibroin-based hydrogel

Abstract

The invention discloses a preparation method and application of silk fibroin-based hydrogel, and the preparation method of the silk fibroin-based hydrogel comprises the following steps: step 1, extracting a silk fibroin solution by using silkworm cocoon as a raw material; 2, a preset amount of cross-linking agent is added into the silk fibroin solution, stirring reaction is conducted, then an excessive dopamine and aminomannose mixed solution is added, silk fibroin-based hydrogel is obtained, the molar ratio of the added dopamine to the aminomannose is 1: (0.5-2), probiotics are placed into the silk fibroin-based hydrogel through a micro-fluidic chip, and the hydrogel is prepared through the steps of preparing the silk fibroin-based hydrogel through the micro-fluidic chip. The probiotic microcapsules are obtained. The invention aims to realize that the probiotic microcapsule is used for treating intestinal inflammation, not only can be used for treating enteritis, but also can be used for encapsulating the probiotics from being damaged by gastric acid and gastrointestinal digestive enzyme, and meanwhile, the growth and reproduction of the probiotics are not influenced.

Description

Technical Field

[0001] The invention relates to the technical field of probiotics, and in particular to a preparation method and application of a silk fibroin-based hydrogel. Background Art

[0002] At present, inflammatory bowel disease (IBD) is an intestinal inflammation caused by genetic, environmental and other factors, and the exact cause of IBD has not yet been determined, and tissue damage and pathogen infection are the main causes of intestinal inflammatory reactions, and intestinal flora is one of the main environmental inducers of IBD. The traditional direct oral administration method used to treat enteritis requires continuous administration and is prone to side effects. Among them, the traditional oral probiotics for the treatment of enteritis, due to the influence of adverse external environments such as gastric acid and gastrointestinal digestive enzymes, a large number of probiotics die when they reach the site of intestinal inflammation, which greatly weakens the therapeutic effect; furthermore, the immobilization method can protect probiotics from gastrointestinal discomfort and environmental pressure. The invention patent of the existing patent CN118020941A discloses a probiotic-hydrogel oral delivery system and its preparation method, which can achieve therapeutic effects to a certain extent, but reduces the adhesion and growth of probiotics to the site of action, and thus the therapeutic effect is affected. Therefore, it is urgent to select a drug that can encapsulate probiotics to protect them from damage by gastric acid and gastrointestinal digestive enzymes, while not affecting the growth and reproduction of the probiotics themselves, and can also ensure the therapeutic effect of intestinal inflammation. Summary of the invention

[0003] The main purpose of the present invention is to provide a preparation method and application of silk fibroin-based hydrogel, aiming to solve the technical problem that the existing probiotics-hydrogel oral delivery system cannot guarantee the therapeutic effect.

[0004] To achieve the above object, the present invention provides a method for preparing a silk fibroin-based hydrogel, the method comprising the following steps:

[0005] Step 1, extracting silk fibroin from silkworm cocoons as raw materials, and preparing a corresponding silk fibroin solution;

[0006] Step 2, adding a preset amount of cross-linking agent to the silk fibroin solution and stirring the reaction, and then adding an excess of dopamine and amino mannose mixed solution to obtain a silk fibroin-based hydrogel, wherein the molar ratio of the added dopamine to amino mannose is 1: (0.5-2).

[0007] Optionally, in step 2, the total molar amount of dopamine and amino mannose is 10 times and less than 20 times the molar amount of silk fibroin.

[0008] Optionally, the step 1 specifically includes the following steps:

[0009] Step 11, adding the silkworm cocoons to a sodium carbonate solution and boiling for 30 minutes, then washing the residual sericin with deionized water for multiple times, and finally drying or air-drying overnight to obtain dry degummed silk fibers;

[0010] Step 12, immersing the degummed silk fiber in a 9.3 mol / L lithium bromide solution and placing it in a 50-60° C. oven for 4 hours to dissolve the fiber and obtain a viscous silk solution;

[0011] Step 13, dialyzing the viscous silk solution for multiple times, and finally centrifuging to obtain a purified silk fibroin solution.

[0012] Optionally, in step 2, the cross-linking agent includes N-hydroxysuccinimide NHS and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC solution.

[0013] Optionally, the molar ratio of NHS to EDC is 2:1.

[0014] Optionally, the molar ratio of dopamine to amino mannose is 1:2.

[0015] In addition, in order to achieve the above object, the present invention also provides a method for preparing probiotic microcapsules, the method comprising the following steps:

[0016] Probiotics are placed into silk fibroin-based hydrogel using a microfluidic chip to obtain probiotic microcapsules, wherein the silk fibroin-based hydrogel is prepared by any of the above-mentioned preparation methods for silk fibroin-based hydrogel, and the mobile phase flow rate on the microfluidic chip is controlled to be 12 μL / min, and the stationary phase flow rate is 1 μL / min.

[0017] Optionally, the average diameter of the probiotic microcapsules is 98.258 um.

[0018] In addition, in order to achieve the above object, the present invention also provides a probiotic microcapsule prepared by the above method.

[0019] In addition, in order to achieve the above-mentioned purpose, the present invention also provides the use of the above-mentioned probiotic microcapsules in the fields of food, drug preparation, and health care product preparation.

[0020] Beneficial effects:

[0021] (1) The preparation method of the silk fibroin-based hydrogel of the present invention comprises extracting a silk fibroin solution from silkworm cocoons as raw materials; adding a preset amount of a cross-linking agent to the silk fibroin solution and stirring the solution for reaction, and then adding an excess of a mixed solution of dopamine and aminomannose to obtain a silk fibroin-based hydrogel. The silk fibroin-based hydrogel has a relatively long main chain and a branch chain with multiple functional groups, thereby combining the functions of silk fibroin, dopamine Dopa, and aminomannose Mannose-NH2, which can facilitate the subsequent application of functional probiotics.

[0022] (2) Probiotics are placed in silk fibroin-based hydrogels using a microfluidic chip to obtain probiotic microcapsules, and the prepared microcapsules have a high degree of homogeneity. The microcapsules encapsulate live bacteria, which are protected from erosion by gastric acid and gastrointestinal digestive enzymes, and achieve slow and sustained release of the target protein. In addition, dopamine-modified silk fibroin hydrogels enhance bacterial colonization time, and mannose can target macrophage mannose receptors; probiotic microcapsules have anti-inflammatory, scavenging reactive oxygen species, and enhanced repair capabilities (silk fibroin inhibits inflammation, dopamine and mannose scavenges reactive oxygen species). The new material synthesized by the "one-pot method" using silk fibroin, dopamine and mannose enhances the adhesion of dopamine itself, and also has the function of targeting the intestine. It can be used to encapsulate probiotics to enhance the stress resistance and intestinal colonization ability of probiotics, and improve the anti-inflammatory ability of probiotics.

[0023] (3) The preparation method of the present invention is simple, convenient and low-cost. The silk fibroin-based hydrogel microcapsule SS has a strong anti-inflammatory function, which has been confirmed in vitro and in colitis models. The silk fibroin-based hydrogel microcapsule SS and the probiotic microcapsules formed by SS-encapsulated probiotics are also used in the food field, drug preparation, and health care product preparation fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a schematic diagram of the synthesis principle of a method for preparing a silk fibroin-based hydrogel according to an embodiment of the present invention;

[0025] Figure 2 This is a physical comparison chart of the anti-inflammatory properties of ABTS at different concentrations of S0, S1, S2, and S3 pure material microcapsules; Figure 3 It is a bar graph of the ABTS removal efficiency of pure material microcapsules of S0, S1, S2, and S3;

[0026] Figure 4 This is a physical comparison chart of the anti-inflammatory properties of ABTS at different concentrations of S0, S1, S2, and S3 bacterial microcapsules; Figure 5 It is a bar graph of the ABTS removal efficiency of S0, S1, S2, and S3 bacterial microcapsules;

[0027] Figure 6This is a physical comparison of the anti-inflammatory properties of PTIO at different concentrations of S0, S1, S2, and S3 pure material microcapsules; Figure 7 It is a bar graph of the PTIO removal efficiency of pure material microcapsules of S0, S1, S2, and S3;

[0028] Figure 8 This is a physical comparison chart of the anti-inflammatory properties of PTIO at different concentrations of S0, S1, S2, and S3 bacterial microcapsules; Fig. 9 It is a bar graph of the PTIO removal efficiency of S0, S1, S2, and S3 bacterial-encapsulated microcapsules;

[0029] Fig.10 This is a physical comparison chart of the anti-inflammatory performance of S0, S1, S2, and S3 pure material microcapsules at different concentrations of DPPH; Fig.11 It is a bar graph of the DPPH removal efficiency of pure material microcapsules of S0, S1, S2, and S3;

[0030] Fig.12 This is a physical comparison diagram of the hydroxyl radical scavenging effects of S0, S1, S2, and S3 pure material microcapsules at different concentrations; Fig.13 It is a bar graph of the hydroxyl radical scavenging efficiency of pure material microcapsules of S0, S1, S2, and S3;

[0031] Fig.14 This is a physical comparison diagram of the hydroxyl radical scavenging effects of S0, S1, S2, and S3 bacterial microcapsules at different concentrations; Fig.15 It is a bar graph of the hydroxyl radical scavenging efficiency of S0, S1, S2, and S3 bacterial microcapsules;

[0032] Fig.16 It is a bar graph of the ability of S0, S1, S2, and S3 bacterial microcapsules to inhibit lipid peroxidation;

[0033] Fig.17 This is a physical comparison diagram of the superoxide anion scavenging effects of S0, S1, S2, and S3 bacterial microcapsules at different concentrations;

[0034] Fig.18 It is a bar graph of the superoxide anion scavenging efficiency of S0, S1, S2, and S3 bacterial microcapsules;

[0035] Fig.19 This is a physical comparison chart of the enzyme-like activities of S0, S1, S2, and S3 microcapsules at different concentrations.

[0036] Fig. 20 It is a bar graph of the enzyme-like activity efficiency of S0, S1, S2, and S3 bacterial microcapsules;

[0037] Fig.21 It is a bar graph of the hydrogen peroxide removal efficiency of S0, S1, S2, and S3 bacterial microcapsules;

[0038] Fig. 22 is a microscopic image of the probiotic microcapsule of the present invention;

[0039] Fig.23 is a particle size distribution diagram of the probiotic microcapsule of the present invention;

[0040] Fig.24 Normal growth curves of MYECN strains and probiotic microcapsules MYE@SS;

[0041] Fig.25 This is the growth curve of MYECN strain and probiotic microcapsule MYE@SS after 1.5h of digestion in simulated gastric fluid;

[0042] Fig.26 This is the growth curve of MYECN strain and probiotic microcapsule MYE@SS after digestion in simulated intestinal fluid for corresponding time;

[0043] Fig. 27 The corresponding weight change curves of Control group, DSS group and SS group;

[0044] Fig.28 The pictures and bar graphs of colon length corresponding to the Control group, DSS group, and SS group;

[0045] Fig.29 This is the distribution diagram of spleen weight changes corresponding to the Control group, DSS group, and SS group;

[0046] Fig.30 It is the bar graph of each cell index of blood routine test corresponding to Control group, DSS group and SS group;

[0047] Fig.31 The fecal changes of mice in the Control group, DSS group, and SS group from day 1 to day 16;

[0048] Figure 32-35 The graphs show the changes in respiratory exchange rate, carbon dioxide exhalation, oxygen absorption and energy consumption of experimental mice in different groups after treatment within 24 hours (night and day);

[0049] Fig.36 The graphs show the changes in the total amount of respiratory metabolism indicators during the night and day in the experimental mice after treatment in different groups;

[0050] Fig.37 H&E staining images of the heart, liver, spleen, lung and kidney tissues of experimental mice after treatment in different groups;

[0051] Fig.38 H&E staining images of mouse colon tissues of experimental mice after treatment in different groups;

[0052] Fig.39 The colon histopathological scores of mice after treatment in different groups;

[0053] Fig.40 This is a graph showing the disease activity index scoring results of mice after treatment in different groups.

[0054] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0055] It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0056] The present invention provides a method for preparing a silk fibroin-based hydrogel, the method comprising the following steps:

[0057] Step 1: extracting silk fibroin from silkworm cocoons as raw materials and preparing a corresponding silk fibroin solution. Specifically, the extraction of the silk fibroin solution includes the following:

[0058] Step 11, adding the silk cocoons to a sodium carbonate solution and boiling for 30 minutes, then washing the residual sericin with deionized water for multiple times, and finally drying or air-drying overnight to obtain dry degummed silk fibers, wherein 10g of silk cocoons are added to 3L of sodium carbonate solution, and the 3L of sodium carbonate solution contains 6.36g to 13g of anhydrous sodium carbonate, and drying in an oven at 50-60°C.

[0059] Step 12, immersing the degummed silk fiber in a 9.3 mol / L lithium bromide solution and placing it in a 50-60° C. oven for 4 hours to dissolve the fiber and obtain a viscous silk solution;

[0060] Step 13, dialyze the viscous silk solution for multiple times, preferably, dialyze with deionized water for 3 days, the deionized water is replaced every 3 hours in the first 6 hours, and then every 6 hours in the next 34 hours, and then the solution is centrifuged at 9000 rpm for 20 minutes to remove impurities to obtain purified silk fibroin Silkfibroin, SF, and add a corresponding amount of antibiotics to the silk fibroin SF and store it at 4°C for future use.

[0061] Furthermore, the silk fibroin SF is prepared into a silk fibroin solution with a corresponding mass fraction. Preferably, the mass fraction of the silk fibroin solution is 4%.

[0062] Step 2, adding a preset amount of cross-linking agent to the silk fibroin solution and stirring the reaction, then adding an excess of dopamine Dopa and amino mannose Mannose-NH2 mixed solution to obtain a silk fibroin-based hydrogel, wherein the molar ratio of the added dopamine to amino mannose is 1: (0.5-2), and the cross-linking agent includes N-hydroxysuccinimide NHS and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide EDC solution, and the molar ratio of NHS to EDC is 2: 1, and the total molar amount of dopamine and amino mannose is more than 10 times the molar amount of silk fibroin. Preferably, the molar ratio of dopamine to amino mannose is: dopamine: amino mannose = 1: 1, or dopamine: amino mannose = 1: 2, or dopamine: amino mannose = 2: 1.

[0063] Then the specific reaction principle in this step is as shown below:

[0064]

[0065] It can be seen that the silk fibroin-based hydrogel generated by the above reaction has a long main chain as a new substance and the side chains have multiple functional groups. It also has the functions of silk fibroin, dopamine, and amino mannose, which can be convenient for the subsequent application of functional probiotics. The specific synthesis principle diagram is as follows Figure 1 As shown. Taking the total molar amount of dopamine and aminomannose equal to 10 times the molar amount of silk protein as an example, the optimal ratio of dopamine and aminomannose was determined. The first control group was to react only silk protein with a crosslinker without adding dopamine and aminomannose, which was recorded as S0; the second was to react silk protein with a crosslinker, and add a molar ratio of dopamine: aminomannose of 1:1 to continue the reaction, which was recorded as S1: The third was to react silk protein with a crosslinker, and add a molar ratio of dopamine: aminomannose of 1:2 to continue the reaction, which was recorded as S2; the fourth was to react silk protein with a crosslinker, and add a molar ratio of dopamine: aminomannose of 2:1 to continue the reaction, which was recorded as S3. The four silk protein-based hydrogel microcapsules S0, S1, S2, and S3 were tested for their ability to scavenge reactive oxygen and reactive nitrogen and scavenging hydroxyl radicals, and the following anti-inflammatory index test results were obtained.

[0066] The anti-inflammatory index determination includes ABTS anti-inflammatory performance determination, PTIO anti-inflammatory performance determination, and DPPH anti-inflammatory performance determination. Figure 2 The figure shows the actual comparison of the anti-inflammatory performance of ABTS at different concentrations of S0, S1, S2, and S3 pure material microcapsules. Figure 3 It is a bar chart of the ABTS removal efficiency of pure material microcapsules of S0, S1, S2, and S3. It can be seen from the figure that the removal efficiency of S1, S2, and S3 is significantly higher than that of S0.

[0067] as well as Figure 4 The figure shows the actual comparison of the anti-inflammatory properties of ABTS at different concentrations of S0, S1, S2, and S3 bacterial microcapsules. Figure 5 It is a bar graph of the ABTS removal efficiency of S0, S1, S2, and S3 bacterial microcapsules. It can be seen from the figure that the removal efficiency of S1, S2, and S3 is higher than that of S0, and the removal efficiency of S3 is the best.

[0068] as well as Figure 6 The figure shows the actual comparison of the anti-inflammatory properties of PTIO at different concentrations of S0, S1, S2, and S3 pure material microcapsules. Figure 7 It is a bar chart of the PTIO removal efficiency of pure material microcapsules of S0, S1, S2, and S3. It can be seen from the figure that the removal efficiency of S1, S2, and S3 is higher than that of S0.

[0069] as well as Figure 8 The figure shows the actual comparison of the anti-inflammatory performance of PTIO at different concentrations of S0, S1, S2, and S3 bacterial microcapsules. Fig. 9 It is a bar graph of the PTIO removal efficiency of S0, S1, S2, and S3 bacterial microcapsules. It can be seen from the figure that the removal efficiency of S1, S2, and S3 is higher than that of S0.

[0070] as well as Fig.10 The figure shows the actual comparison of the anti-inflammatory performance of S0, S1, S2, and S3 pure material microcapsules at different concentrations of DPPH. Fig.11 It is a bar graph of the DPPH removal efficiency of pure material microcapsules of S0, S1, S2, and S3. It can be seen from the figure that the removal efficiency of S1, S2, and S3 is significantly higher than that of S0.

[0071] And the scavenging ability of S0, S1, S2 and S3 in pure material microcapsules or bacterial microcapsules on hydroxyl radicals was determined, among which, Fig.12 This is a physical comparison chart of the hydroxyl radical scavenging effects of S0, S1, S2, and S3 pure material microcapsules at different concentrations. Fig.13 The bar graph shows the hydroxyl radical scavenging efficiency of pure material microcapsules of S0, S1, S2, and S3. It can be seen from the graph that when the concentration is 0.5 mg / mL, S0 is greater than S1, S2, and S3, and when the concentration increases to 1 mg / mL and 2 mg / mL, the scavenging efficiency of S2 is higher than S0, S1, and S3. Fig.14 The figure shows the actual comparison of the hydroxyl radical scavenging effects of S0, S1, S2 and S3 bacterial microcapsules at different concentrations. Fig.15 It is a bar graph of the hydroxyl radical scavenging efficiency of S0, S1, S2 and S3 bacterial microcapsules. It can be seen from the figure that the scavenging efficiency of S2 is greater than that of S0, S1 and S3.

[0072] And the ability of S0, S1, S2, and S3 bacterial microcapsules to inhibit lipid peroxidation, scavenge superoxide anions, and enzyme-like activity was determined. Fig.16 The bar graph shows the ability of S0, S1, S2, and S3 microcapsules to inhibit lipid peroxidation. It can be seen from the graph that the removal efficiency of S2 is greater than that of S0, S1, and S3. Fig.17 This is a physical comparison chart of the superoxide anion removal effects of S0, S1, S2, and S3 bacterial microcapsules at different concentrations. Fig.18 The bar graph shows the superoxide anion removal efficiency of S0, S1, S2, and S3 bacterial microcapsules. It can be seen from the figure that the removal efficiency of S1, S2, and S3 is significantly higher than that of S0. Fig.19 This is a physical comparison chart of the enzyme-like activities of S0, S1, S2, and S3 microcapsules at different concentrations. Fig. 20 It is a bar graph of the enzyme activity efficiency of S0, S1, S2 and S3 bacterial microcapsules. It can be seen from the graph that for SOD enzyme activity, the contents of S1, S2 and S3 are significantly higher than S0, for CAT enzyme activity, the contents of S0, S2 and S3 are significantly higher than S1, and for POD enzyme activity, the content of S2 is significantly higher than S0, S1 and S3. Fig.21 The bar graph of hydrogen peroxide removal rates of S0, S1, S2, and S3 bacterial microcapsules shows that the removal efficiency of S3 is lower than that of S0, S1, and S2.

[0073] According to the anti-inflammatory index measurement results obtained above, the entropy weight-TOPSIS comprehensive evaluation method was used to determine the dosage ratio with comprehensive anti-inflammatory effect, as shown in Table 1 , which is a mathematical modeling creation based on the comprehensive evaluation of active microcapsules at different ratios.

[0074] Table 1 - Mathematical modeling to create a data table for comprehensive evaluation of different ratios of live microcapsules

[0075]

[0076]

[0077] According to the mathematical modeling data table in Table 1, the comprehensive evaluation results of probiotic live microcapsules after entropy weight-TOPSIS mathematical modeling in Table 2 were obtained.

[0078] Table 2- Comprehensive evaluation results

[0079] item Positive ideal solution distance D Negative ideal solution distance D- Relative proximity C Sorting results MYE@S0 6.259 4.375 0.411 4 MYE@S1 4.685 5.147 0.523 2 MYE@S2 2.671 7.408 0.735 1 MYE@S3 6.956 4.964 0.416 3

[0080] That is, the molar ratio of dopamine to amino mannose obtained at 1:2 has the best effect, and the silk fibroin-based hydrogel material S2 is named SS.

[0081] Among them, the preparation steps of the probiotic microcapsules are: using a microfluidic chip to place probiotics into different silk fibroin-based hydrogels to obtain probiotic microcapsules, wherein the silk fibroin-based hydrogel is prepared by the preparation method of the silk fibroin-based hydrogel described above. Specifically, 100 μl of bacterial solution was pre-cultured in 100 ml LB overnight, and the bacterial solution was centrifuged at 5000 rpm for 5 min for standby use, and resuspended with 1 ml PBS + 40% sucrose, and the bacterial solution: material = 1:10 was controlled, and the gun tip was blown and mixed after adding as DP, and oil + soybean phosphatidylcholine (1% w / w) was used as CP. Using microfluidic technology, DP: 1 μl / min, CP: 12 μl / min. After centrifugation, the finished product was stored at 4°C for 45 minutes, -20°C for 1 hour, and -80°C overnight, and freeze-dried at -80°C for 14-15 hours. The specific microscopic structure of the probiotic microcapsule is as follows Fig. 22 As shown in the figure, it can be seen that the particle size of the probiotic microcapsules is basically the same, the microcapsule morphology is relatively uniform, and the particle size distribution of the probiotic microcapsules is further determined. Fig.23 As shown, the proportion of those distributed around 98 μm is relatively large, and the average diameter of the probiotic microcapsules finally generated is calculated to be 98.258 μm.

[0082] Taking the MY ECN constructed in our laboratory as an example, the engineered probiotics encapsulated in SS were named MYE@SS. The protective effect of silk fibroin-based hydrogel SS on probiotics was determined.

[0083] Verification 1: MYECN strains and probiotic microcapsules MYE@SS were grown normally, and the OD600 values ​​were recorded from 0 to 15 hours. The specific results are as follows: Fig.24 As shown in the figure, the normal growth of bacteria in MYECN strains and MYE@SS is basically in synchronization, that is, after the MYECN probiotics are encapsulated by silk fibroin-based hydrogel, the growth curve of the probiotics does not change significantly, indicating that the encapsulation of silk fibroin-based hydrogel does not affect the normal growth of probiotics.

[0084] Verification 2, the growth curves of MY ECN strains and probiotic microcapsules MY E@SS were respectively subjected to 1.5 h of digestion in simulated gastric fluid, and the OD600 values ​​from 0 to 15 h were recorded. The specific results are as follows Fig.25 As shown in the figure, the OD600 value of the MYECN strain did not fluctuate much. In the simulated gastric juice, the probiotics hardly grew, while the probiotics encapsulated with the silk fibroin-based hydrogel SS could grow normally. Furthermore, the probiotic microcapsules MYE@SS were basically unaffected after being treated with gastric juice. This shows that the silk fibroin-based hydrogel SS can help probiotics resist adverse external environments, such as the digestive effect of gastric juice.

[0085] Verification 3, the MYECN strain was digested in simulated intestinal fluid for 2h and 4h and then allowed to grow normally, and the probiotic microcapsule MYE@SS was digested in simulated intestinal fluid for 4h and then allowed to grow normally, and the OD600 values ​​in 0-15h were recorded accordingly, and the following were obtained: Fig.25 The growth curve shown in the figure shows that the probiotics digested in simulated intestinal fluid for 4 hours can significantly affect the normal growth of the probiotics, while the probiotics encapsulated in the silk fibroin-based hydrogel SS can still grow normally after being digested in simulated intestinal fluid for 4 hours, indicating that the silk fibroin-based hydrogel SS can protect the probiotics from the effects of long-term digestion in the intestinal fluid.

[0086] Furthermore, the efficacy of silk fibroin-based hydrogel microcapsules was verified by the therapeutic effect on ulcerative colitis in mice.

[0087] Experimental animals

[0088] 24 male C57BL6 / j mice aged 7-8 weeks were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., production license number: SCXK (Xiang) 2016-0002, and were uniformly raised in the local experimental animal center, with a temperature of 23℃±2℃, a humidity of 55%±5%, a 12h day and night cycle lighting, caged, and free access to food and water. All animal experiments were in accordance with the requirements of the local experimental animal ethics review committee.

[0089] Establishment of a mouse model of ulcerative colitis

[0090] Male C57BL6 / j mice aged 7-8 weeks were randomly divided into groups of 8 mice, with a total of 3 groups, namely the Control group, the DSS group, and the SS group. The establishment of the ulcerative colitis model refers to the existing technology. After 7 days of adaptation, the Control group drank water normally, and the other groups drank 3% DSS (36-50kDa) freely for one week. Three days before the first gavage, arabinose at a concentration of 10g / L was added to the drinking water of each group of mice until sampling. After 14 days, each preparation group was gavaged every other day, for a total of 4 times.

[0091] The control group and the DSS group were intragastrically administered with 200 μL PBS (pH 7.4) every other day, and the SS group was intragastrically administered with 100 μL SS microcapsules (1×10 10 cfu / mL), and the body weight of mice was monitored every day during the experiment.

[0092] Recording of mouse biochemical indicators and sample collection: During the experiment, the weight of the mice was recorded daily. After the experiment, the colon length of each mouse was recorded and photographed.

[0093] Pathological analysis: The colon samples were washed twice with saline, immersed in 1 mL of tissue fixative solution containing 4% paraformaldehyde, and the tissues were embedded in paraffin. 10 μm paraffin sections were used for H&E staining observation, and the HE staining results were analyzed and evaluated. HE staining scoring criteria: The total histological score was expressed as E+I, with a total score of 16 points. The specific histological scoring criteria are shown in Table 3.

[0094] Table 3 - DSS colitis histological grading scheme

[0095]

[0096] Detection of inflammatory factors: The colon samples were homogenized and the contents of TNF-α, IL-1β, and IL-10 were determined according to the instructions of the ELISA kit.

[0097] Statistical analysis: Unless otherwise stated, all experimental results were repeated at least three times, and all data were analyzed by GraphPadPrism 8.0. All quantitative data are expressed as mean ± SEM. Significance was assessed using unpaired Student's t-test and one-way and two-way analysis of variance (ANOVA), and p < 0.05 was considered to be significantly different.

[0098] Experimental results:

[0099] 1. Pathological indicators: body weight

[0100] like Fig. 27 As described above, after DSS treatment, the weight loss of the DSS group and the SS group was basically the same, indicating that the mouse ulcerative colitis model was successfully established. After treatment from the 7th day, the silk fibroin-based hydrogel microcapsule SS can reduce the weight loss after DSS treatment, and weight gain began to appear on the 10th day. However, the weight of the DSS group still did not increase significantly, indicating that the silk fibroin-based hydrogel microcapsule SS can treat the weight loss caused by ulcerative colitis.

[0101] 2. Pathological indicators: colon length and spleen weight.

[0102] like Fig.28 The figure shows the colon length of each group of mice after DSS treatment. The length of the colon can reflect the severity of inflammation. The more severe the colitis, the shorter the colon length. It can be seen from the figure that after the treatment with silk fibroin-based hydrogel microcapsules SS, the length of the colon is close to that of the normal group (PBS group). This shows that silk fibroin-based hydrogel microcapsules SS can improve the symptoms of shortened colon in colitis.

[0103] as well as Fig.29The figure shows the spleen weight of each group of mice after DSS treatment. The weight of the mouse spleen can reflect the severity of inflammation to a certain extent. The more severe the colitis, the heavier the spleen. It can be seen from the figure that after treatment, there is a significant difference between the DSS group and the normal group (PBS group), and there is no significant difference between the SS group and the normal group. This shows that SS can significantly reduce the severity of ulcerative colitis in mice caused by DSS.

[0104] 3. Routine blood test

[0105] After collecting blood with an anticoagulant tube, the indicators of each cell in the blood routine test were measured. Fig.30 As shown in the figure, it can be seen that the blood routine indexes of the SS group are close to those of the normal group (PBS group). The increase in Gran% (granulocyte ratio), Mon% (monocyte ratio), Lym% (lymphocyte ratio) and the number of platelets all indicate the occurrence of inflammation, and the blood routine index results of the SS group after treatment are closer to those of the normal group.

[0106] 4. Occult blood test

[0107] The concentration of o-tolidine is generally prepared at 0.5-1g / L. (1% o-tolidine reagent is required, prepared with a mixture of glacial acetic acid and anhydrous ethanol), mix the mouse feces from different experimental groups with 200μL of normal saline. Centrifuge the mixed fecal sample for 10 minutes and take the supernatant. Take 100μL of supernatant and put it in a new EP tube, add 100μL of o-tolidine reagent, and mix well. Add 10μl of hydrogen peroxide and observe the color change. If blue staining appears, it means that there is occult blood in the feces. The darker the color, the more serious the occult blood in the feces.

[0108] By observing the fecal changes of mice in different experimental groups from 1 to 16 days, Fig.31 As shown in the figure, the modeling group (DSS group) is significantly darker than the normal group (PBS group), indicating that the modeling is successful. The treatment group (SS group) has a significant change in color compared to other modeling groups, and as the observation time increases, the color of the blood in the stool gradually becomes lighter. It can be seen that silk fibroin-based hydrogel microcapsules SS can significantly improve the symptoms of fecal occult blood caused by colitis.

[0109] 5. Respiratory metabolism measurement

[0110] Based on the fact that intestinal diseases affect the host's energy metabolism, studies have shown that patients with inflammatory bowel disease (IBD) suffer from weight loss, malnutrition and several other metabolic changes that affect their quality of life, that is, the body weight, respiratory exchange ratio (RER), food intake and body fat content of mice with acute inflammation all decrease. Mice with inflammation will change their metabolism, which can be used as a measurement indicator of response or adaptation to severe colon inflammation. The severity of the disease can then be reflected by the maximum oxygen consumption and carbon dioxide production. Furthermore, by measuring the changes in respiratory exchange rate, energy consumption, oxygen absorption and carbon dioxide exhalation of experimental mice after treatment in different groups within 24 hours (night and day), the specific measurement results are as follows Figure 32-35 As shown, Fig.32 is the change in respiratory exchange rate during 24 hours (night and day) of respiratory metabolism. Fig.33 The amount of carbon dioxide exhaled during respiratory metabolism within 24 hours (night and day), Fig.34 is the amount of oxygen absorbed during respiratory metabolism within 24 hours (night and day), Fig.35 The figure shows the changes in energy consumption during respiratory metabolism within 24 hours (night and day). As can be seen from the figure, the respiratory metabolic indicators of the silk fibroin-based hydrogel microcapsule SS group are closer to the normal group (PBS group), indicating that the silk fibroin-based hydrogel microcapsule SS has the ability to significantly restore the respiratory metabolism of mice with colitis.

[0111] Furthermore, the total changes of respiratory metabolic indicators at night and during the day in the experimental mice after treatment in different groups were measured. Fig.36 As shown, Fig.36 b is the respiratory exchange rate during the day and night, Fig.36 a is the total respiratory exchange rate during the night and day, Fig.36 d is the oxygen absorption during the day and night, Fig.36 c is the total oxygen absorption during the night and day, Fig.36 f is the amount of carbon dioxide exhaled during the day and night, Fig.36 e is the total amount of carbon dioxide exhaled during the night and day, Fig.36 h is the energy consumption during the day and night, Fig.36 a is the total energy consumption during the night and day. It can be seen from the figure that the respiratory metabolism indicators of the silk fibroin-based hydrogel microcapsule SS group are closer to the normal group (PBS group) both during the day and at night. This further indicates that the silk fibroin-based hydrogel microcapsule SS has the ability to significantly restore the respiratory metabolism of mice with colitis.

[0112] 6. In vivo biocompatibility test

[0113] The heart, liver, spleen, lung and kidney tissues were collected for H&E staining in the normal group and all the modeling groups with DSS added to drinking water. Fig.37As shown, the heart, liver, spleen, lung and kidney tissues and cell structures of all groups were normal. And the H&E staining of mouse colon tissue was obtained, as shown Fig.38 As shown in the figure, H&E staining was used to observe the specific morphology of colon tissue sections. Compared with the normal group (PBS group), the colon of mice in the DSS group showed obvious inflammatory reaction, which was manifested by a large number of neutrophil infiltration, crypt dissolution and disappearance, and the colon mucosa and goblet cells were basically completely destroyed, and the colon histopathological score was significantly increased. The SS group restored the integrity of the colon structure to a certain extent.

[0114] And the colon tissues of different groups of mice were evaluated histologically, that is, according to the evaluation criteria in Table 3, Fig.39 The mouse histopathological scores, where the average score of the DSS group was 8, and the colon tissue of SS-treated mice showed significant therapeutic effects, with reduced histopathological scores, regular finger-like crypt structures, intact epithelium, and reduced inflammatory cell infiltration, similar to healthy colons, that is, SS treatment significantly reduced the colon histopathological scores of UC mice. In summary, SS can effectively alleviate the level of colon inflammation and improve colon tissue damage in ulcerative colitis mice. This shows that silk fibroin-based hydrogel microcapsules SS will not cause other tissue lesions and has good biocompatibility.

[0115] 7. Mouse Disease Activity Index (DAI score)

[0116] The disease activity index of mice in different groups was measured, that is, the percentage of weight loss, stool characteristics and fecal occult blood level were measured, wherein the specific standards and scores are shown in Table 4.

[0117] Table 4 Disease Activity Index (DAI) parameters and their related scores

[0118] Fraction Weight loss percentage (%) Stool characteristics Fecal occult blood level 0 constant normal normal 1 1-5 Slightly loose Occult blood positive 2 5-10 No shape Blood stains are obvious 3 10-15 Pasty Visible blood in stool 4 >15

[0119] According to the scoring criteria in Table 4 above, we can get Fig.40 The scoring results shown showed that the DAI scores of SS-treated mice were significantly reduced on day 15 compared with those of PBS- and SS-treated colitis mice, indicating that SS treatment had a therapeutic effect on mice.

[0120] In summary, the ability of silk fibroin-based hydrogel SS to treat ulcerative colitis in mice was determined, and it was found that silk fibroin-based hydrogel SS can significantly shorten the course of ulcerative colitis, reduce the degree of inflammation, and will not cause damage to other tissues and organs, and has good biocompatibility. In addition, in addition to having strong in vitro and in vivo anti-inflammatory abilities, silk fibroin-based hydrogels can also be used to embed probiotics to protect probiotics from the adverse environment of the gastrointestinal tract. Furthermore, the probiotic microcapsules prepared in the present invention can be used to prepare drugs for treating ulcerative colitis.

[0121] In addition, in order to achieve the above-mentioned purpose, the present invention also provides the use of the above-mentioned probiotic microcapsules in the fields of food, drug preparation, and health care product preparation.

[0122] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

Claims

1. A method for preparing a silk fibroin-based hydrogel, characterized in that: The method comprises the following steps: Step 1, extracting silk fibroin from silkworm cocoons as raw materials, and preparing a corresponding silk fibroin solution; Step 2, adding a preset amount of cross-linking agent to the silk fibroin solution and stirring the reaction, and then adding an excess of dopamine and amino mannose mixed solution to obtain a silk fibroin-based hydrogel, wherein the molar ratio of the added dopamine to amino mannose is 1: (0.5-2).

2. The method for preparing the silk fibroin-based hydrogel according to claim 1, characterized in that: In step 2, the total molar amount of dopamine and amino mannose is more than 10 times the molar amount of silk fibroin.

3. The method for preparing the silk fibroin-based hydrogel according to claim 1, characterized in that: The step 1 specifically comprises the following steps: Step 11, adding the silkworm cocoons to a sodium carbonate solution and boiling for 30 minutes, then washing the residual sericin with deionized water for multiple times, and finally drying or air-drying overnight to obtain dry degummed silk fibers; Step 12, immersing the degummed silk fiber in a 9.3 mol / L lithium bromide solution and placing it in a 50-60° C. oven for 4 hours to dissolve the fiber and obtain a viscous silk solution; Step 13, dialyzing the viscous silk solution for multiple times, and then centrifuging to obtain a purified silk fibroin solution.

4. The method for preparing the silk fibroin-based hydrogel according to claim 1, characterized in that: In step 2, the cross-linking agent includes N-hydroxysuccinimide NHS and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC solution.

5. The method for preparing the silk fibroin-based hydrogel according to claim 4, characterized in that: The molar ratio of NHS to EDC is 2:

1.

6. The method for preparing the silk fibroin-based hydrogel according to claim 1, characterized in that: The molar ratio of dopamine to amino mannose is 1:

2.

7. A method for preparing probiotic microcapsules, characterized in that: The method comprises the following steps: Probiotics are placed into a silk fibroin-based hydrogel using a microfluidic chip to obtain probiotic microcapsules, wherein the silk fibroin-based hydrogel is prepared using the preparation method of the silk fibroin-based hydrogel described in any one of claims 1 to 6, and the mobile phase flow rate on the microfluidic chip is controlled to be 12 μl / min and the stationary phase flow rate is 1 μl / min.

8. The method for preparing the probiotic microcapsules according to claim 7, characterized in that: The average diameter of the probiotic microcapsules is 98.258 μm.

9. A probiotic microcapsule prepared according to the method of claim 7 or 8.

10. Use of the probiotic microcapsules described in claim 9 in the fields of food, drug preparation, and health care product preparation.

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