Modified recombinant III-type collagen-probiotic microspheres and preparation method thereof

Abstract

The invention relates to the technical field of bioengineering, and particularly provides a modified recombinant III-type collagen-probiotic microsphere and a preparation method thereof. The preparation method comprises the following steps: preparing probiotics-loaded microspheres from modified recombinant III-type collagen through photo-crosslinking based on a microfluidic technology, under the action of shearing force, enabling a fluid continuous phase to wrap a dispersion phase, liquid paraffin and a recombinant III-type collagen probiotic suspension to be in contact shearing to form gel, and under the action of ultraviolet irradiation, synthesizing microspheres with uniform size and good shape, the efficient embedding of the probiotics can be realized. The modified recombinant III-type collagen can effectively protect probiotics from being damaged by harmful factors such as gastric acid and the like, the probiotics are released in intestinal tracts, the survival rate of the probiotics is increased, and the prepared modified recombinant III-type collagen-probiotic microspheres have better cell affinity and better chemical stability, and can be used for preparing the probiotic microspheres. The structural integrity of the microspheres is maintained under different environmental conditions, and the controllable release of the probiotics is realized.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and specifically provides a modified recombinant type III collagen-probiotic microsphere and a preparation method thereof. Background Art

[0002] Probiotics refer to active microorganisms that are beneficial to the human body, usually bacteria or fungi that contribute to good health. These microorganisms can enter the human digestive tract through oral administration and other methods, interact with the intestinal flora, and have a positive impact on the human body. However, after probiotics enter the intestine, they will be affected by the intestinal environment and have a limited survival time. Therefore, it is crucial to increase the survival rate of probiotics in the gastric acid environment and prolong their survival time in the intestine.

[0003] Although probiotic delivery systems have made some progress in improving the survival rate and efficacy of probiotics, there are still some limitations. Therefore, suitable delivery carriers are needed to improve the efficiency and reliability of probiotic delivery systems and better exert the benefits of probiotics.

[0004] Collagen is a common protein in connective tissue, which plays a supporting and structural stabilizing role in the human body. Recombinant type III collagen is highly similar to the structure of the body's own collagen. When loaded with probiotics and delivered, it has better biocompatibility and biodegradability, so it is widely used in the field of bioengineering technology. As raw material, synthetic microspheres have the following advantages as delivery carriers: good biocompatibility, not easy to cause rejection reaction; increase the stability of probiotics; enhance the delivery effect: by adjusting the morphology and structure of microspheres, efficient delivery and targeted release of probiotics can be achieved, and their bioavailability and efficacy can be improved. However, traditional methods for preparing collagen-loaded probiotic microspheres usually rely on emulsification methods, solvent evaporation methods, etc., which are difficult to accurately control the size and morphology of microspheres. Even if physical or chemical cross-linking is used, there are often large differences in the particle size of microspheres, resulting in uneven distribution, affecting their biocompatibility and release performance in the body. Therefore, in view of the limitations of the current probiotic microsphere delivery system, the development of a preparation method for modified recombinant type III collagen-probiotic microspheres and their application in the delivery of probiotics is an urgent problem to be solved. Summary of the invention

[0005] The purpose of the present invention is to provide a modified recombinant type III collagen-probiotic microsphere and a preparation method thereof in view of certain limitations of the current probiotic microsphere delivery system. The obtained modified recombinant type III collagen-probiotic microsphere has good morphology, controllable size and stable structure.

[0006] The method for preparing modified recombinant type III collagen-probiotic microspheres of the present invention specifically comprises the following steps:

[0007] 1) Preparation of dispersed phase solution and continuous phase solution: the probiotics are resuspended in a modified recombinant type III collagen solution with a photoinitiator added thereto to prepare a dispersed phase solution; liquid paraffin is used as the continuous phase.

[0008] The preparation method of modified recombinant type III collagen is as follows: dissolving collagen in a phosphate buffer solution, adding methacrylic anhydride, utilizing the amidation reaction of amino groups (collagen) and carboxyl groups (methacrylic anhydride), and obtaining modified recombinant type III collagen after the reaction is completed.

[0009] Preferably, the mass ratio of recombinant type III collagen to methacrylic anhydride is (1-15):1, and the mass volume ratio of methacrylic anhydride to phosphate buffer solution is (1-10):500.

[0010] The concentration of the collagen solution w / v is 5-20%, and the concentration of the photoinitiator LAP in the collagen solution w / v is 0.1-5%.

[0011] The dosage ratio of modified recombinant III collagen to probiotics is 1 mg: 10^5~10^9 CFU / mL.

[0012] 2) Construction of microfluidic device: Place the syringe containing the dispersed phase solution and the continuous phase solution described in step 1) horizontally on a microinjection pump, and connect the needle to the inlet of the chip using a capillary.

[0013] 3) Preparation of modified recombinant type III collagen-probiotic microspheres: Run the machine to prepare microspheres, collect the microspheres by cross-linking the droplets at the outlet pipe. Wash the excess oil phase on the surface of the collected microspheres, freeze-dry and store for subsequent experiments.

[0014] Preferably, after the dispersed phase and the continuous phase are respectively connected to the microfluidic chip channel, different flow rates of the two phases are set, the dispersed phase flow rate is set to 1 μL / s, and the continuous phase flow rate is set to 1-10 μL / s. At the confluence of the two phases, the dispersed phase is continuously sheared by the continuous phase to form oil-in-water droplets, and the droplets are exposed to UV light (365 nm) for 60 seconds at the outlet and then polymerized into collagen-probiotic microspheres.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] 1. Recombinant type III collagen-probiotic microspheres are prepared by microfluidic technology. The preparation process is simple and the size and shape of the microspheres can be precisely controlled to meet different delivery requirements. Collagen-probiotic microspheres have good physical stability and can maintain their shape and structure. During storage and transportation, it can effectively protect the probiotics loaded inside from the influence of external physical factors.

[0017] 2. The surface of modified recombinant type III collagen microspheres is rich in amine and hydroxyl groups, which are conducive to the attachment and encapsulation of drugs such as probiotics and have high stability. Modified recombinant type III collagen microspheres can effectively protect drug-loaded probiotics from harmful factors such as gastric acid, release them in the intestine, and improve the survival rate and stability of probiotics. Delivering probiotics through collagen microspheres can achieve sustained release of probiotics in the intestine, providing a longer-lasting health care effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the Fourier transform infrared spectrum of the modified recombinant type III collagen obtained in Example 1;

[0019] Figure 2 These are confocal images of modified recombinant type III collagen-probiotic microspheres prepared by different methods obtained in Example 2;

[0020] Figure 3 The fluorescence quantitative results of the modified recombinant type III collagen-probiotic microspheres prepared by different methods obtained in Example 2;

[0021] Figure 4 Microscope images of modified recombinant type III collagen-probiotic microspheres prepared with different photoinitiator contents obtained in Example 3;

[0022] Figure 5 Microscope images of recombinant type III collagen-probiotic microspheres prepared at different collagen concentrations obtained in Example 4;

[0023] Figure 6 Microscope images of recombinant type III collagen-probiotic microspheres prepared at different two-phase flow rate ratios obtained in Example 5;

[0024] Figure 7 The particle size statistics of the recombinant type III collagen-probiotic microspheres prepared with different two-phase flow rate ratios obtained in Example 5;

[0025] Figure 8 This is a SEM image of the recombinant type III collagen-probiotic microspheres obtained in Example 5;

[0026] Fig. 9 This is a fluorescence image of the recombinant type III collagen-probiotic microspheres obtained in Example 6;

[0027] Fig.10 The figure shows the results of the biocompatibility test of recombinant type III collagen-probiotic microspheres with different concentrations on strains;

[0028] Fig.11 The figure shows the results of hemolysis experiment of recombinant type III collagen-probiotic microspheres with different concentrations;

[0029] Fig.12 The figure shows the results of MTT cytotoxicity test of recombinant type III collagen-probiotic microspheres with different concentrations;

[0030] Fig.13 The results of the plate coating at different time points of the simulated in vitro digestion of free probiotics and recombinant type III collagen-probiotic microspheres in Example 9.

[0031] Fig.14 This is a graph showing the live bacterial counts at various time points in the in vitro simulated digestion of free probiotics and recombinant type III collagen-probiotic microspheres in Example 9. DETAILED DESCRIPTION

[0032] The present invention provides a modified recombinant type III collagen-probiotic microsphere and a preparation method thereof. The technical scheme of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0033] Example 1

[0034] The preparation method of modified recombinant type III collagen comprises the following steps:

[0035] Step 1: First, prepare phosphate buffered saline (PBS) at pH 7.4, accurately weigh 5.0g of recombinant type III collagen, add 8mL PBS, and stir to dissolve in a 55°C oil bath. After complete dissolution, add 60μL of methacrylic anhydride, accurately weigh it into a PE tube, and add 2mL PBS to rinse it all into the reaction bottle. The reaction ends after 1h, and the reaction solution is dialyzed against deionized water at 45°C for 3d, during which the water needs to be changed. This is to completely remove low molecular weight impurities with potential cytotoxicity. After the dialysis is completed, filter while hot, take the filtrate, and freeze-dry to obtain a white solid material, which is the modified recombinant type III collagen.

[0036] Step 2: Characterize the modified collagen using Fourier transform infrared spectrometer. Take 1 mg of sample and 100 mg of dry potassium bromide in an agate mortar, mix and grind thoroughly, use a tablet press to press into almost transparent thin slices, put them into the sample holder, and then insert them into the fixed position of the instrument sample chamber for detection. The absorption spectrum of the sample is between 600 and 4000 cm -1 Multiple scans were performed between Figure 1 .

[0037] from Figure 1 As can be seen in the figure, the synthetic modified recombinant type III collagen is at 3338 cm -1 The broad bands on the left and right are the stretching vibration peaks of the -OH group and the NH group, 2925 cm -1 The peak at 1633 cm is the stretching vibration peak of CH. -1 The peak at 1538cm is the stretching vibration peak of amide I with C=O bond, while the peak at 1538cm -1The peak at 1452 cm is the NH bending vibration peak of the amide II band. -1 The peak at 1240 cm is the bending vibration peak of CH. -1 The peak at 1052cm is the CN absorption peak of the amide III band. By comparing the peak shapes of the two, we can find that -1 The peak at 4 is the stretching vibration peak of CNC, which is the acylation reaction between the amino group on collagen and the acid anhydride. The above results show that methacrylic anhydride successfully modified the recombinant type III collagen.

[0038] Example 2

[0039] This example is a comparison of the preparation method of modified recombinant type III collagen-probiotic microspheres, and the steps are as follows:

[0040] Step 1: Take 4 ml of a 10 7 CFU / mL Bacillus subtilis bacterial solution (solvent is phosphate buffer) was centrifuged (5min, 5000rpm), the supernatant was removed and the precipitate was taken out, 20μL PBS was added to break it up, 2μL of dye Hoechst was added, and the culture was placed in a 37°C desktop constant temperature shaker (speed 250rpm) incubated for 30min in a centrifuge (5min, 5000rpm) after which the supernatant was removed and the precipitate was taken out and 4mL PBS was added to break it up.

[0041] Step 2: Prepare collagen-probiotic microspheres by emulsification method, dissolve the modified recombinant type III collagen (preparation method is the same as Example 1) in the probiotic PBS solution described in step 1 above, and the concentration is 10% (w / v). Use a magnetic stirrer to stir for 2h at 300rpm / min to completely dissolve the collagen until a uniform and transparent solution is formed. Prepare liquid paraffin as the oil phase and Span 80 as an emulsifier. The two are mixed evenly in a ratio of oil phase: emulsifier = 9:1 (v / v). At a speed of 3000rpm / min, the above collagen-probiotic solution is added dropwise to the oil phase-emulsifier mixture, and stirring is continued for 30min to form a stable emulsion. Slowly add an appropriate amount of glutaraldehyde as a cross-linking agent (the amount added is 0.5% of the collagen mass) to the emulsion. Stir continuously during the addition process, and after the end, transfer the emulsion to a 37°C constant temperature water bath for reaction for 4h to cause a cross-linking reaction between the collagen molecules to form a microsphere structure. After the reaction is completed, the emulsion is centrifuged at 4000 rpm / min for 15 min, the microspheres will settle at the bottom, the upper liquid is discarded, and the microspheres are cleaned with anhydrous ethanol and deionized water to obtain modified recombinant type III collagen-probiotic microspheres.

[0042] Step 3: Prepare collagen-probiotic microspheres by solvent evaporation method, dissolve the modified recombinant type III collagen (preparation method is the same as Example 1) in the probiotic PBS solution described in step 1 above, with a concentration of 10% (w / v). Use a magnetic stirrer to stir at 300rpm / min for 2h to completely dissolve the collagen until a uniform and transparent solution is formed. Dissolve polyvinyl alcohol (PVA) in dichloromethane, the concentration of PVA is 1% (w / v), and stir it at a speed of 300rpm / min for 2h to completely dissolve it and form a uniform solution. Keep the speed unchanged, add the collagen-probiotic solution dropwise to the above solution, the dropping time is 30min, and the two are fully mixed to form an emulsion. The prepared emulsion was transferred to a rotary evaporator at 50 rpm / min and the temperature was set to 30°C. The organic solvent was evaporated by rotary evaporation. During this process, the collagen solidified to form microspheres. After the solvent was evaporated, the emulsion was centrifuged at 4000 rpm / min for 15 minutes. The microspheres would settle at the bottom. The upper liquid was discarded and washed with anhydrous ethanol and deionized water to obtain modified recombinant type III collagen-probiotic microspheres.

[0043] Step 4: Prepare collagen-probiotic microspheres using microfluidic technology. Dissolve the modified recombinant type III collagen (prepared by the same method as in Example 1) in the probiotic PBS solution described in the above step 1 at a concentration of 10% (w / v). Add 0.5% (w / v) LAP photoinitiator and use a magnetic stirrer to stir at 300 rpm / min for 2 h to completely dissolve the collagen until a uniform and transparent solution is formed.

[0044] A microfluidic platform was built and a dual-channel microfluidic injection pump was prepared. The external phase was a continuous phase, and 5 mL of liquid paraffin was added; the dispersed phase was 5 mL of the above-mentioned modified recombinant type III collagen-probiotic solution. The syringes containing the dispersed phase solution and the continuous phase solution were placed horizontally on the micro-injection pump, and the needles were connected to the inlet of the chip using a capillary. The dispersed phase flow rate was set to 1 μL / s and the continuous phase flow rate to 5 μL / s. At the confluence of the two phases, the dispersed phase was continuously sheared by the continuous phase to form oil-in-water droplets. At the outlet, the droplets were exposed to UV light (365 nm) for 60 seconds and then photocross-linked and polymerized into collagen microspheres, which were then cleaned with anhydrous ethanol and deionized water to obtain modified recombinant type III collagen-probiotic microspheres.

[0045] The loading effects of modified recombinant type III collagen-probiotic microspheres prepared by the above three methods on probiotics were observed under a laser confocal microscope. Figure 2 From the fluorescence images, we can see that the microspheres prepared by the emulsification method and the solvent evaporation method have poor loading effects on probiotics, while the microspheres prepared based on microfluidics technology can effectively load a large number of probiotics. Figure 3 The fluorescence quantitative results and Figure 2 The results are consistent.

[0046] Compared with the emulsification method and the solvent evaporation method, the particle size distribution of microspheres prepared by these two methods is wider, which affects the consistency and stability of the microsphere performance. The microspheres are irregular in shape, which affects the circulation and distribution in the organism. The residual emulsifier and organic solvent also have a great impact on the biocompatibility of the microspheres. In addition, these two methods have poor loading effects on probiotics. The microspheres prepared based on the microfluidic platform are uniform in size, highly dispersible, complete in spherical shape, and smooth in surface, which is conducive to the stable loading of drugs or probiotics.

[0047] Table 1 Comparison of collagen-probiotic microspheres prepared under different processes

[0048]

[0049]

[0050] Example 3

[0051] The preparation steps of modified recombinant type III collagen-probiotic microspheres with different photoinitiator dosages are as follows:

[0052] Step 1: Preparation of bacterial solution. Bacillus subtilis was cultured and activated twice in LB broth medium, each time at 37°C for 48 hours. Then, Bacillus subtilis was gradiently diluted for use.

[0053] Step 2: Preparation of dispersed phase and continuous phase solution. Dissolve 100 mg of modified recombinant type III collagen (prepared in the same manner as in Example 1) in 10 7 In a PBS solution containing 500 CFU / mL Bacillus subtilis and 5 mg photoinitiator (LAP), a modified collagen-PBS solution with a concentration of 10 wt% was prepared as the dispersed phase, and liquid paraffin was used as the continuous phase.

[0054] Step 3: Construction of microfluidic platform. Prepare a dual-channel microfluidic injection pump, with the external phase as the continuous phase, add 5 mL of liquid paraffin as the continuous phase, and the internal phase as the dispersed phase, which is 5 mL of the modified collagen solution containing bacteria in step 2. Place the syringe containing the internal and external phase solutions horizontally on the double pipes, and use a capillary to connect the needle to the inlet of the chip.

[0055] Step 4: Formation of modified recombinant type III collagen-probiotic microspheres. After the dispersed phase and the continuous phase were connected to the microfluidic chip channel respectively, different flow rates of the two phases were set, with the dispersed phase flow rate set at 1 μL / s and the continuous phase flow rate set at 5 μL / s. At the confluence of the two phases, the dispersed phase was continuously sheared by the continuous phase to form oil-in-water droplets. At the outlet, the droplets were exposed to UV light (365 nm) for 60 seconds and then polymerized into modified recombinant type III collagen-probiotic microspheres.

[0056] Step 5: Adjust the dosage parameters of the photoinitiator LAP and conduct parallel experiments. The dosage of LAP in the above steps is 5 mg, and the dosage is adjusted to 1 mg and 3 mg respectively to carry out the experimental process of the above steps 2-4. The formed modified recombinant type III collagen-probiotic microspheres are observed. Figure 4 The results showed that when the amount of LAP was too low, the microspheres had poor morphology, adhered together, and could not form a sphere. Therefore, 5 mg of LAP was selected for subsequent experiments.

[0057] Example 4

[0058] The preparation steps of modified recombinant type III collagen-probiotic microspheres with different modified collagen concentrations are as follows:

[0059] Step 1: Preparation of bacterial solution. Bacillus subtilis was cultured and activated twice in LB broth medium, each time at 37°C for 48 hours. Then, Bacillus subtilis was gradiently diluted for use.

[0060] Step 2: Preparation of dispersed phase and continuous phase solution. Dissolve 100 mg of modified recombinant type III collagen (prepared in the same manner as in Example 1) in 10 7 In a PBS solution containing 500 CFU / mL Bacillus subtilis and 5 mg of photoinitiator (LAP), a modified collagen-PBS solution with a concentration of 5 wt % was prepared as the dispersed phase, and liquid paraffin was used as the continuous phase.

[0061] Step 3: Construction of microfluidic platform. Prepare a dual-channel microfluidic injection pump, with the external phase as the continuous phase, add 5 mL of liquid paraffin as the continuous phase, and the internal phase as the dispersed phase, which is 5 mL of the modified collagen solution containing bacteria in step 2. Place the syringe containing the internal and external phase solutions horizontally on the double pipes, and use a capillary to connect the needle to the inlet of the chip.

[0062] Step 4: Formation of modified recombinant type III collagen-probiotic microspheres. After the dispersed phase and the continuous phase were connected to the microfluidic chip channel respectively, different flow rates of the two phases were set, with the dispersed phase flow rate set at 1 μL / s and the continuous phase flow rate set at 5 μL / s. At the confluence of the two phases, the dispersed phase was continuously sheared by the continuous phase to form oil-in-water droplets. At the outlet, the droplets were exposed to UV light (365 nm) for 60 seconds and then polymerized into modified recombinant type III collagen-probiotic microspheres.

[0063] Step 5: Adjust the concentration of modified recombinant type III collagen and conduct parallel experiments. The concentration of modified recombinant type III collagen in the above steps is 5wt%, and the concentrations are adjusted to 7wt% and 10wt% respectively to conduct the experimental process of the above steps 2-4. By observing the formed modified recombinant type III collagen-probiotic microspheres. Figure 5The results show that when the collagen concentration is 10wt%, modified recombinant type III collagen-probiotic microspheres with uniform size and good dispersibility can be formed. Therefore, the subsequent experiments were carried out when the collagen concentration was 10wt%.

[0064] Table 2 Effects of different concentrations of modified recombinant type III collagen on the formation of probiotic-loaded microspheres

[0065]

[0066] Example 5

[0067] The preparation steps of modified recombinant type III collagen-probiotic microspheres with different flow rate ratios between dispersed phase and continuous phase are as follows:

[0068] Step 1: Preparation of bacterial solution. Bacillus subtilis was cultured and activated twice in LB broth medium, each time at 37°C for 48 hours. Then, the Bacillus subtilis was gradiently diluted for use.

[0069] Step 2: Preparation of dispersed phase and continuous phase solution. Dissolve 100 mg of modified recombinant type III collagen (prepared in the same manner as in Example 1) in 10 7 In a PBS solution containing 500 CFU / mL Bacillus subtilis and 5 mg photoinitiator (LAP), a modified collagen-PBS solution with a concentration of 10 wt% was prepared as the dispersed phase, and liquid paraffin was used as the continuous phase.

[0070] Step 3: Construction of microfluidic platform. Prepare a dual-channel microfluidic injection pump, the outer phase is the continuous phase, add 5mL of liquid paraffin, the inner phase is the dispersed phase, which is 5mL of the collagen solution containing bacteria in step 2, and place the syringe containing the inner and outer phase solutions horizontally on the double pipes, and use a capillary to connect the needle to the inlet of the chip.

[0071] Step 4: Formation of modified recombinant type III collagen-probiotic microspheres. After the dispersed phase and the continuous phase were connected to the microfluidic chip channel respectively, different flow rates of the two phases were set, with the dispersed phase flow rate set at 1 μL / s and the continuous phase flow rate set at 5 μL / s. At the confluence of the two phases, the dispersed phase was continuously sheared by the continuous phase to form oil-in-water droplets. At the outlet, the droplets were exposed to UV light (365 nm) for 60 seconds and then polymerized into modified recombinant type III collagen-probiotic microspheres.

[0072] Step 5: Adjust the flow rate ratio of the dispersed phase and the continuous phase and conduct parallel experiments. In the above steps, change the flow rate ratio of the dispersed phase and the continuous phase to 1:5 μL / s, respectively. Adjust the flow rate ratio to 1:3 μL / s and 1:4 μL / s to carry out the experimental process of the above steps 2-4.

[0073] The collected microspheres were cleaned with anhydrous ethanol and PBS to remove residual oil on the surface, and the size and morphology of the microspheres were observed under a laser confocal microscope under bright field. The clean microspheres were freeze-dried and the morphology was observed under a scanning electron microscope.

[0074] Depend on Figure 6 Confocal images and Figure 7 The particle size statistics show that as the water phase flow rate increases, the particle size of the microspheres gradually decreases. When the flow rate ratio is 1:5 μL / s, modified recombinant type III collagen-probiotic microspheres with a rounder shape and smaller particle size can be formed.

[0075] Table 3 Effect of different flow rate ratios on the formation of probiotic-loaded microspheres

[0076]

[0077] Example 6

[0078] This example is to verify the method of successfully embedding probiotics into modified recombinant type III collagen-probiotic microspheres. The preparation steps are as follows: take 4 ml of a 10 7 CFU / mL Bacillus subtilis bacterial liquid was centrifuged (5min, 5000rpm), the supernatant was removed and the precipitate was taken out, 20μL PBS was added to break it up, 2μL of dye Hoechst was added, and the culture was placed in a 37°C tabletop constant temperature shaker (speed 250rpm) for 30min protected from light with tin foil, and then placed in a centrifuge for centrifugation (5min, 5000rpm), the supernatant was removed and the precipitate was taken out, and 4mL PBS was added to break it up.

[0079] A 10wt% modified recombinant type III collagen solution containing 0.5% photoinitiator was prepared with the above probiotic suspension, and a microfluidic device was built. The flow rate ratio of the dispersed phase and the continuous phase was set to 1:5μL / s, and 5mL of the dispersed phase solution and 5mL of the continuous phase solution were injected into different inlets, respectively. Subsequently, the complex solution was formed at the tip outlet and flowed out and collected in a beaker. The collected droplets were polymerized into modified recombinant type III collagen-probiotic microspheres after being exposed to UV light (365nm) for 60 seconds. The microspheres were washed with deionized water and observed under a laser confocal microscope. By Fig. 9 The confocal image shows that the probiotics were successfully encapsulated into the microspheres.

[0080] Example 7

[0081] This example is to prove the biocompatibility of recombinant type III collagen modified by microsphere wall material with strains, and its preparation steps are as follows:

[0082] Modified recombinant type III collagen was selected as the wall material of the microspheres. In order to verify whether modified recombinant type III collagen has toxic effects on probiotics or inhibits their growth. 1g of modified recombinant type III collagen was dissolved in 1mL of sterile water and mixed with LB broth medium to prepare LB broth medium containing 1%, 3%, 5%, 7%, and 10% of modified recombinant type III collagen. Then 200μL of the first generation Bacillus subtilis culture was added to 10mL of the above five groups of culture media, grown at 37℃ for 12h, and the OD value was measured at 600nm on an ELISA reader to detect its growth activity.

[0083] The results are as follows Fig.10 As shown, there was no significant difference in the survival activity of probiotics in each group in the culture medium containing different concentrations of modified recombinant type III collagen, indicating that the microsphere wall material had no toxic effect on the growth of the strain.

[0084] Example 8

[0085] This example is a method for demonstrating that the modified recombinant type III collagen microspheres have good biocompatibility, and the preparation steps are as follows:

[0086] Step 1: Collect 200 μL of fresh whole blood from healthy C57 mice in an anticoagulation tube, centrifuge at 2000 rpm for 10 min at 4°C, aspirate the supernatant, collect the lower layer of red blood cells, wash 3 times with pre-cooled sterilized PBS (pH = 7.4), and then dilute 5 times as red blood cell stock solution for standby use. Add 1 mL of recombinant type III collagen microsphere-PBS solution loaded with probiotics at concentrations of 0.1, 0.5, 1, 1.5, and 2 mg / mL to each group of centrifuge tubes, and then add 20 μL of red blood cell stock solution to mix with it, use 1 mL of PBS (pH = 7.4) as the negative control group, and 1 mL of deionized water as the positive control, and also add 20 μL of red blood cell stock solution to mix with it. After thorough mixing, the samples were incubated in a 37°C constant temperature incubator for 1 hour, and then centrifuged at 2000 rpm for 10 minutes. Pictures were taken for recording, and then 150 μL of the supernatant from each group was collected using a 96-well plate. The absorbance of the supernatant at 540 nm was detected using an ELISA reader, and the hemolysis rate (%) was calculated using Formula 1.

[0087] Homelysis rate = (OD microspheres - OD PBS ) / (OD H2O -OD PBS )×100% Formula 1

[0088] Step 2: Use MTT kit to detect the effect of probiotic-loaded recombinant type III collagen microspheres on the proliferation of mouse fibroblast L929 cells. L929 cells were evenly plated on a 96-well plate at a density of 8,000 cells / well, and the cells were cultured with complete medium at 37°C and 5% CO. 2 Cultivate in a biochemical incubator until the cells grow in the logarithmic phase. Take the wells with good growth conditions, uniform cell distribution and density for the experiment, prepare recombinant type III collagen microsphere-DMEM solution with concentrations of 0.1, 0.5, 1, 1.5, and 2 mg / mL, and incubate with the cells for 48 hours. After the incubation, aspirate the sample, add 10 μL MTT (5 mg / mL) to each well, and continue incubation for 4 hours. Living cells will convert MTT into visible formazan, discard the supernatant, add 150 μL DMSO to each well to dissolve the formazan, mix on a constant temperature oscillator for 10 minutes, and measure the absorbance at 490 nm on an enzyme reader after the end to analyze the effect of recombinant type III collagen microspheres on cell proliferation.

[0089] Fig.11 The results of the hemolysis experiment showed that the red blood cell hemolysis rate of all concentration groups was less than 5%, indicating that the modified recombinant type III collagen microspheres would not cause hemolysis below 2 mg / mL and had good biocompatibility and safety. Fig.12 The results of the MTT experiment showed that the cell survival rate of the probiotic-loaded collagen microspheres was greater than 80% at all concentrations, and there was no cytotoxicity to L929 cells even at the maximum concentration of 2 mg / mL, indicating that the recombinant type III collagen microspheres had good cell compatibility. In summary, the modified recombinant type III collagen microspheres loaded with probiotics have extremely low risk of hemolysis of red blood cells and have potential clinical application prospects in the delivery of probiotics.

[0090] Example 9

[0091] This example is to verify the in vitro simulated digestion and release process of probiotics after being embedded in modified collagen microspheres. The operation steps are as follows:

[0092] Step 1, preparation of simulated gastric fluid: weigh 0.32g pepsin and 0.2g sodium chloride respectively, add to 100g ultrapure water, stir and mix until completely dissolved, adjust the solution with 1M HCl, slowly add to the solution, and adjust the pH to 2.0. The prepared simulated gastric fluid is sterilized by passing through a 0.22μm filter membrane.

[0093] Step 2, preparation of simulated intestinal fluid: weigh 0.1g trypsin and 0.08g pig bile salt respectively, add them to 100g 0.2M PBS solution, and stir until completely dissolved. The prepared simulated intestinal fluid is sterilized through a 0.22μm filter membrane. Subsequently, the simulated gastric fluid and simulated intestinal fluid are incubated in a 37℃ water bath for 20 minutes to ensure their biological activity.

[0094] 0.2mL of free Bacillus subtilis liquid and 0.2mL of bacteria-loaded microspheres were added to 9.8mL of simulated gastric fluid, mixed thoroughly, and placed in a 37℃ dark shaker at 180rpm for continuous shaking and mixing. When the culture time was 30, 60, 90, and 120min, the samples of each group were taken out and ultrasonically broken with a cell disruptor for 10min to fully release the Bacillus subtilis in the collagen microspheres. The broken solution was diluted 5000 times and the viable bacteria were counted using a plate. After the simulated gastric fluid culture was completed, 10mL of simulated intestinal fluid was added to the other parallel samples and mixed thoroughly, and the samples were placed in a 37℃ dark shaker at 180rpm for continuous shaking and mixing. And when the culture time was 1, 2, 3, and 4h, the samples of each group were taken out, ultrasonically broken with a cell disruptor for 10min, the Bacillus subtilis in the collagen microspheres were fully released, the broken solution was diluted 5000 times, and the viable bacteria were counted using a plate. The above counting method was used to determine the survival rate of probiotics in simulated digestion in vitro, and the survival rate was compared with that of unencapsulated probiotics that had undergone the same treatment.

[0095] like Fig.13 The results showed that Bacillus subtilis had a low tolerance to gastrointestinal fluid. After 1 hour of in vitro simulated gastric fluid digestion, the survival rate of probiotics was only 15.15%, while the probiotics embedded in collagen microspheres had significantly improved tolerance to gastrointestinal fluid, with a survival rate of 79.30%. After 4 hours of in vitro simulated digestion, no live bacteria were detected in the free probiotic group, while the Bacillus subtilis in the collagen microspheres still retained a considerable number of live bacteria after 6 hours of in vitro simulated digestion, with a survival rate of 53.28%. The encapsulation of bacteria in collagen microspheres significantly improved the survival rate of probiotics. The modified recombinant type III collagen can form a protective film around the probiotics. Its three-dimensional mesh structure can reduce the damage of probiotics in gastrointestinal fluid digestion. The collagen membrane can buffer the direct impact of gastric acid on probiotics and reduce the erosion of gastric acid on the cell structure of probiotics. At the same time, it can neutralize gastric acid, absorb bile salts in the intestines, reduce its damage to the activity of probiotics, and collagen microspheres help probiotics maintain a relatively stable internal environmental temperature and improve the tolerance of probiotics in gastrointestinal fluid. In addition, recombinant type III collagen microspheres can control the slow release of probiotics in gastrointestinal fluid, achieve precise release according to different environments, and ensure that probiotics have the opportunity to play a role in the entire gastrointestinal tract.

[0096] The results of the above examples show that the present invention uses methacrylic anhydride-modified collagen as a raw material to synthesize microspheres as a carrier for delivering probiotics, and its advantages are that collagen has good biocompatibility, biodegradability, multifunctionality, stability and enhanced probiotic delivery effect. At the same time, the size-controlled microspheres prepared based on microfluidic technology have the advantages of precise and controllable size, high efficiency and rapidity, good consistency, strong scalability, multifunctionality, good micro-operation environment and reduced resource waste. Compared with the use of probiotics alone, embedding probiotics into microspheres has better living conditions, stability, controlled dosage and targeting, which helps to improve the bioavailability and effect of probiotics, thereby better exerting their probiotic effects and therapeutic effects on gastrointestinal diseases.

[0097] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. Therefore, the protection scope of this patent application shall be based on the content of the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A modified recombinant type III collagen-probiotic microsphere, characterized in that: The modified recombinant type III collagen-probiotic microspheres are probiotic-loaded microspheres prepared by photo-crosslinking of modified recombinant type III collagen based on microfluidics technology.

2. A method for preparing the modified recombinant type III collagen-probiotic microspheres as claimed in claim 1, characterized in that: The preparation method steps are as follows: (1) Preparation of dispersed phase solution and continuous phase solution: Probiotics are resuspended in a modified recombinant type III collagen solution with a photoinitiator added thereto to prepare a dispersed phase solution; liquid paraffin is used as the continuous phase; (2) Construction of a microfluidic device: Place the syringe containing the dispersed phase solution and the continuous phase solution described in step (1) horizontally on a microinjection pump, and connect the needle to the inlet of the chip using a capillary tube; (3) Preparation of modified recombinant type III collagen-probiotic microspheres: The machine was run to prepare microspheres, and the droplets collected in the outlet pipe were exposed to UV light and polymerized into microspheres. The collected microspheres were cleaned of excess oil phase on the surface and freeze-dried for storage.

3. The method for preparing the modified recombinant type III collagen-probiotic microspheres according to claim 2, characterized in that: In step (1), the preparation method of the modified recombinant type III collagen is as follows: the recombinant type III collagen is dissolved in a phosphate buffer solution, methacrylic anhydride is added, and an amidation reaction is carried out using the amino group in the recombinant type III collagen and the carboxyl group in the methacrylic anhydride to obtain the modified recombinant type III collagen.

4. The method for preparing modified recombinant type III collagen-probiotic microspheres according to claim 3, characterized in that: The mass ratio of the recombinant type III collagen to methacrylic anhydride is 1 to 15:1, and the mass volume ratio of methacrylic anhydride to phosphate buffer solution is 1 to 10:

500.

5. The method for preparing modified recombinant type III collagen-probiotic microspheres according to claim 2, characterized in that: In step (1), the concentration of the modified recombinant type III collagen solution is 5-20% w / v; the photoinitiator is LAP, and its concentration in the collagen solution is 0.1-5% w / v.

6. The method for preparing modified recombinant type III collagen-probiotic microspheres according to claim 2, characterized in that: In step (1), the dosage ratio of the modified recombinant III collagen to the probiotics is 1 mg: 10^5~10^9 CFU / mL.

7. The method for preparing modified recombinant type III collagen-probiotic microspheres according to claim 2, characterized in that: In step (3), after the dispersed phase and the continuous phase are respectively connected to the microfluidic chip channel, the water phase flow rate is set to 1 μL / s and the oil phase flow rate is set to 1-10 μL / s. At the confluence of the two phases, the dispersed phase is continuously sheared by the continuous phase to form oil-in-water droplets.

8. The method for preparing modified recombinant type III collagen-probiotic microspheres according to claim 2, characterized in that: In step (3), the droplets at the outlet are exposed to 365 nm UV light for 60 seconds and then polymerized into probiotic-loaded microspheres.

Images