Preparation Method of Mercapto-Modified Chitosan Microspheres and Their Application in Probiotic Entrapment
The preparation of thiol-modified chitosan microspheres through microfluidic control technology has solved the problems of uneven particle size of probiotics embedded in the prior art and the use of chemical reagents, and achieved efficient embedding and stable transportation of probiotics.
Patent Information
- Application Number
- CN202310578781.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-05-22
- Publication Date
- 2025-07-11
- Estimated Expiration
- 2043-05-22
AI Technical Summary
The prior art is difficult to prepare thiol-modified chitosan microspheres with controllable particle size and morphology and stable structure for the embedding of probiotics through microfluidic control technology, and traditional methods have problems with uneven size distribution and the use of chemical reagents.
The method of preparing thiol-modified chitosan microspheres using microfluidic control technology is used to prepare thiol-modified chitosan microspheres with uniform particle size by introducing thiol-modified chitosan on the chitosan molecular chain, activate carboxyl groups using EDC and NHS, and react with cysteine to form thiol-modified chitosan. Combined with microfluidic chip technology, thiol-modified chitosan-probiotic microspheres with uniform particle size are prepared to form a water-in-oil emulsion and cross-link to fix probiotics.
The efficient embedding of probiotics is achieved, the vitality of probiotics in the gastrointestinal tract is improved, and the sufficient number of probiotics reaches the intestinal target site, and the preparation process is stable and controllable, with uniform particle size distribution.
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Figure CN116675785B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of the preparation of modified chitosan microspheres and probiotic encapsulation, and more particularly to a method for preparing mercapto-modified chitosan microspheres and their application in probiotic encapsulation. Background Art
[0002] Probiotics play a crucial role in maintaining the health of the host, mainly including lactic acid bacteria, bifidobacteria and enterococci. It is reported that the viability of probiotics in the food matrix should be greater than 10 6 CFU / g to obtain an ideal probiotic effect. Intake of sufficient probiotics can maintain the balance of the intestinal flora, inhibit the growth of pathogenic bacteria, and regulate the intestinal immune system. The above health benefits of probiotics are based on the highly active state of probiotics entering the host body. However, adverse factors such as low pH value and oxygen may affect the viability of probiotics during processing, storage and gastrointestinal (Gastrointestinal tract, GIT) transportation. Therefore, it is crucial to protect probiotics and transport highly active probiotics to the intestine.
[0003] The construction of a probiotic delivery system is an effective strategy to protect probiotics during storage and gastrointestinal transit, which can protect probiotics from the harsh gastrointestinal environment and ensure that a sufficient number of highly active probiotics reach the intestinal target site. Using natural-derived or synthetic polymers to construct an oral delivery system for probiotics can increase their viability in the GIT. Polysaccharides such as chitosan (CS) have become widely used pH-sensitive carrier matrices due to their biodegradability and controlled release ability. Although various techniques such as extrusion, atomization and emulsification can be used to fabricate probiotic delivery systems, they have some inherent challenges, such as the use of toxic chemical reagents and relatively uneven size distribution. Therefore, strict size distribution and accurate efficacy evaluation are crucial for the downstream application of food functional factors.
[0004] More importantly, controlling the size and particle size distribution of the carrier has an important impact on its practical application as an encapsulation matrix for probiotic delivery systems. Compared with the previous methods for constructing delivery systems such as emulsification-solidification, simple coacervation and complex coacervation, the emerging microfluidic technology is an effective method for preparing monodisperse particles with controllable size and morphology. It has significant advantages such as stable production, simple process, narrow particle size distribution and strong process repeatability.
[0005] Therefore, how to provide a method for preparing mercapto-modified chitosan microspheres based on microfluidic technology and applying them to probiotic encapsulation is an urgent problem to be solved by those skilled in the art. Summary of the Invention
[0006] In view of this, the present invention provides a method for preparing thiol-modified chitosan microspheres based on microfluidic technology, which can prepare monodisperse thiol-modified chitosan microspheres with controllable particle size and morphology and stable structure, and can be used for the embedding of probiotics.
[0007] In order to achieve the above object, the present invention adopts the following technical solution:
[0008] A method for preparing thiol-modified chitosan microspheres comprises the following steps:
[0009] 1) dissolving cysteine hydrochloride monohydrate in a 1% HCl aqueous solution at a ratio of m:v of 50:1, and stirring at room temperature until fully dissolved to obtain a Cys solution;
[0010] 2) adding NHS and EDC to the Cys solution obtained in step 1) with stirring, so that the final molar mass ratio of -COOH:EDC:NHS is 1:1:1 to 1:3:5, and activating for 2 hours at room temperature with stirring;
[0011] 3) adding chitosan to a 1-2% hydrochloric acid aqueous solution at a final concentration of 0.7% to 2% w / v, stirring at room temperature until fully dissolved, to obtain a chitosan solution;
[0012] 4) Add the Cys solution obtained in step 2) to the chitosan solution obtained in step 3), adjust the pH to 5-6 after the chitosan solution is fully dissolved, and 2 The reaction was carried out under protection for 4-24 hours to obtain thiol-modified chitosan polymer CS Cys ;
[0013] 5) CS obtained in step 4) Cys The chitosan was purified by dialyzing with ultrapure water for 2 days and freeze-dried for future use to obtain thiol-modified chitosan.
[0014] Since there are free amino and hydroxyl groups on the chitosan molecular chain, the chitosan molecular chain can be modified by reacting with carboxyl groups. In the present invention, EDC and NHS are carboxyl activators, which promote the condensation reaction between the amino group of chitosan and the carboxyl group of cysteine to generate polymers. The free thiol groups on the thiol-modified chitosan chain can interact with the cysteine residues on the surface of the bacteria to form disulfide bonds to fix more probiotics, thereby achieving a better probiotic embedding effect.
[0015] Preferably, the molar mass ratio of the above-mentioned -COOH:EDC:NHS is 1:1:1.
[0016] Preferably, the pH value of the reaction system is 5.5.
[0017] Preferably, the reaction time is 12 h.
[0018] Preferably, in step 4), the mass ratio of Cys to CS is 1:1 to 1:6.
[0019] Preferably, the mass ratios of cysteine hydrochloride monohydrate to chitosan are 1:1, 1:2, 1:4, and 1:6, respectively.
[0020] As an invention concept identical to the above technical solution, the present invention also claims protection for a thiol-modified chitosan, which is prepared by the method described above.
[0021] As an invention concept identical to the above technical solution, the present invention also claims protection for the application of the thiol-modified chitosan prepared by the above preparation method in the preparation of thiol-modified chitosan-probiotic microspheres based on microfluidic technology.
[0022] As an invention concept identical to the above technical solution, the present invention also claims protection for a method for encapsulating probiotics by microfluidic technology. The thiol-modified chitosan prepared by the above preparation method is used for encapsulation to obtain thiol-modified chitosan-probiotic microspheres. Compared with the previous encapsulation methods, the emerging microfluidic method can prepare monodisperse particles and has significant advantages such as controllable size and morphology, stable yield, and strong repeatability.
[0023] Preferably, the specific process includes the following steps:
[0024] 1) Preparation of the dispersed phase solution and the continuous phase solution: Resuspend bacteria with a 1% CS Cys1:1 (1:1 represents the mass ratio of Cys to CS is 1:1) solution to prepare the dispersed phase solution; Mix 0.5 - 1 g of Span80 with 15 - 30 g of paraffin oil to obtain the continuous phase;
[0025] 2) Preparation of the microfluidic chip: Prepare a polymethyl methacrylate (PMMA) chip;
[0026] 3) Preparation of thiol-modified chitosan-probiotic microspheres: Pump the dispersed phase and the continuous phase described in step 1) into the channels of the PMMA chip evenly, and wash the product to obtain thiol-modified chitosan-probiotic microspheres with uniform particle size.
[0027] Preferably, in step 1), the mass-volume ratio of the thiol-modified chitosan dissolved in a 1% acetic acid aqueous solution is 0.01.
[0028] Preferably, in step 2), the PMMA chip is designed as a two-layer structure. Among them, there are 3 inlets on the top layer and 1 outlet on the bottom layer. The diameters of each inlet and 1 outlet are both 2 mm, the width of the middle channel is 1 mm, and the width of the central flow focusing junction is reduced to 0.5 mm. After that, the two layers are sealed with glue and clamped with a fixture. Finally, a Teflon tube is sealed to the corresponding inlet on the fixture for connection to a pump.
[0029] Preferably, in step 3), after the dispersed phase and the continuous phase are respectively and uniformly pumped into the channels of the PMMA chip, the flow rates of the dispersed phase and the continuous phase are adjusted to 1:5 by a microfluidic instrument. At the flow aggregation node of the chip, the dispersed phase is sheared by the shear stress of the continuous phase to form a water-in-oil emulsion template. A curing solution is placed at the end of the chip outlet to collect the emulsion template, and a crosslinking agent is added along with slight horizontal oscillation. Wait until the emulsion template turns blue.
[0030] Preferably, the crosslinking agent is 1 wt% genipin, the crosslinking time is 6 h, and the curing collection solution is a genipin / glycerol-aqueous solution; the diameter of the thiol-modified chitosan-probiotic microspheres is 200 - 400 μm.
[0031] Through the above technical solutions, compared with the prior art, the present invention discloses a method for preparing thiol-modified chitosan microspheres for probiotic embedding based on microfluidic technology. Using this method, thiol-modified chitosan microspheres with uniform particle size and stable structure can be prepared, and efficient embedding of probiotics can be achieved. Description of the Drawings
[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.
[0033] Figure 1 The drawings are Fourier transform infrared spectra of chitosan, chitosan-cysteine polymer, and cysteine;
[0034] Figure 2 The drawings are schematic diagrams of the adhesion rate results of thiol-modified chitosan films to probiotics;
[0035] Figure 3 The drawings are schematic diagrams of the microfluidic chip design structure;
[0036] Figure 4 The drawings are schematic diagrams of the embedding results of CS / FL-276.1 and CS Cys1：1 / FL-276.1;
[0037] Figure 5 The attached drawings are schematic diagrams of "water-in-oil" droplets and microspheres of CS / FL-276.1 and CS Cys1：1 / FL-276.1;
[0038] Figure 6 The attached drawings are schematic diagrams of "water-in-oil" droplets and microspheres of CS / FL-276.1 and CS Cys1：1 / FL-276.1 particle size distribution diagram of microspheres; Detailed implementation mode
[0039] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the attached drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
[0040] The probiotic strain Bifidobacterium bifidum FL-276.1 (FL-276.1) used in the examples comes from the Functional Dairy and Probiotic Engineering Laboratory of Ocean University of China.
[0041] Example 1 Preparation of mercapto-modified chitosan
[0042] 1. Preparation of chitosan solution
[0043] Chitosan was dissolved in 1% HCl aqueous solution, and the final concentration of chitosan was 1%.
[0044] 2. Preparation of chitosan-cysteine polymer
[0045] 500 mg of L-Cysteine hydrochloride monohydrate (Cys) (-COOH: ca. 2.847 mmol) was dissolved in 10 mL of 1% HCl aqueous solution and stirred at room temperature until completely dissolved. NHS and EDC (-COOH: EDC: NHS molar mass ratio is 1:1:1 to 1:3:5) were added to the obtained Cys solution and stirred. The carboxyl group of Cys was activated at room temperature for 2 h. After the obtained chitosan solution was fully dissolved, the pH was adjusted to 5-6, and the reaction was carried out at room temperature under N2 protection for 4-24 h to obtain the mercapto-modified chitosan polymer. The mass ratios of cysteine hydrochloride monohydrate to chitosan were 1:1, 1:2, 1:4, and 1:6 respectively.
[0046] 3. Characterization of chitosan-cysteine polymer
[0047] The chitosan-cysteine polymer was characterized by Fourier transform infrared spectroscopy. Approximately 1 mg of the sample was mixed with 100 - 200 mg of dry potassium bromide in an agate mortar and ground thoroughly. After being pressed into an almost transparent thin film using a tablet press, it was placed on a sample holder and then inserted into a fixed position in the sample chamber of the instrument for detection. The absorption spectrum of the sample was recorded at a resolution of 4 cm -1 between 500 and 4000 cm -1 , and a total of 64 scans were performed. The results are shown in Figure 1 .
[0048] Example 2 Adhesion Analysis of Probiotic-Thiol Modified Chitosan Films
[0049] 1. Preparation of Thiol-Modified Chitosan Films
[0050] 1.5% (w / v) CS or CS Cys was dispersed in 1% (v / v) aqueous acetic acid solution to prepare a film-forming solution. Then, 25% (w / w) glycerol of CS / CS Cys was added to the solution, and it was stirred for 1 h under N 2 protection until a homogeneous solution was obtained. The film-forming solution was sonicated for 30 min to degas. The solution was placed in a silicone mold (5×5 cm 2 ) and dried in an oven at 60 °C for 6 h.
[0051] 2. Preparation of Bacterial Suspension
[0052] FL-276.1 was cultured and activated twice in MRS medium broth at 37 °C for 48 h. Subsequently, FL-276.1 was serially diluted for later use.
[0053] 3. Quantitative Analysis of the Adhesion of Probiotic-Thiol Modified Chitosan Films
[0054] The FL-276.1 bacterial suspension (10 8 CFU / mL) was pre-oxygenated for 10 min, and then incubated with CS and CS Cys polymer films at 37 °C for 30 min. After that, the remaining FL-276.1 solution was poured onto MRS agar and cultured at 37 °C for 2 d to determine the amount of non-adherent bacteria. The following formula was used to calculate the adhesion rate of FL-276.1 on CS and CS Cys films. See Figure 2 ;
[0055]
[0056] Example 4 Preparation of Monodisperse Thiol-Modified Chitosan-Probiotic Microspheres by Microfluidic Technology
[0057] 1. Preparation of Microfluidic Chips
[0058] A polymethyl methacrylate (PMMA) chip with dimensions of 50 mm × 50 mm × 2 mm was fabricated using a micro-machining tool. As Figure 3 shown, the microfluidic chip is designed as a two-layer structure. Among them, there are 3 inlets on the top layer and 1 outlet on the bottom layer. The diameters of both the inlets and the outlet are 2 mm, the channel width between the inlets and the outlet is 1 mm, and the width of the central flow focusing junction is reduced to 0.5 mm. Then, the two layers are sealed with glue and clamped with a fixture. Finally, Teflon tubes are sealed to the corresponding inlets on the fixture for connection to the pump.
[0059] 2. Preparation of the dispersed phase and the continuous phase
[0060] 1% (w / v) CS Cys1：1 was dissolved in 1% (v / v) aqueous acetic acid solution (pH 5.2 - 6) to form a CS Cys1：1 solution. FL-276.1 (about 10 8 CFU / mL) was resuspended with the CS Cys1：1 solution to obtain the dispersed phase of probiotics; Span 80 (0.75 g) was mixed with paraffin oil (20 g) to obtain the continuous phase.
[0061] 3. Microsphere formation
[0062] The dispersed phase and the continuous phase were respectively and uniformly pumped into the corresponding chip channels. At the central flow aggregation junction of the microfluidic chip, a water-in-oil template was formed.
[0063] Then, by dropping them into a curing bath and gently magnetically stirring, 1 wt% genipin as a cross-linking agent was added for cross-linking for 6 h to obtain thiol-modified chitosan-probiotic microspheres with uniform particle sizes. Finally, the microspheres were thoroughly washed successively with isopropanol and a large amount of deionized water to remove the residual impurities on the surface of the microspheres; the curing collection solution was a genipin / glycerol-aqueous solution; the diameter of the thiol-modified chitosan-probiotic microspheres was 200 - 400 μm.
[0064] 4. Entrapment efficiency analysis
[0065] The entrapment efficiency of two groups of microspheres for FL-276.1 was determined by the plate pour method. Briefly, CS / FL-276.1 or CS Cys1：1 / FL-276.1 was resuspended with phosphate buffer and mechanically disrupted for 30 min. Finally, the released FL-276.1 was plated on MRS agar and cultured under anaerobic conditions at 37 °C for 48 h, and the entrapment efficiency was calculated according to the following formula.
[0066]
[0067] As Figure 4 can be seen, using CS Cys1：1The entrapment rate of FL-276.1 is significantly higher than that of CS for FL-276.1.
[0068] 5. Microsphere Morphology Analysis
[0069] The morphologies of the four kinds of microspheres were observed by ordinary optical microscope and scanning electron microscope, and the results are as Figure 5 shown. After the microspheres were solidified, the morphology of the chitosan microspheres entrapping bacteria and the thiol-modified chitosan microspheres presented uniform spherical shapes, with smooth surfaces and uniform particle sizes.
[0070] 6. Microsphere Particle Size Analysis
[0071] The size distributions of the different microspheres produced were measured by laser scattering. The microspheres were suspended in deionized water and completely homogenized, and the results are as Figure 6 .
[0072] In this specification, each embodiment is described in a progressive manner. The key point of each embodiment is to illustrate the differences from other embodiments. The same or similar parts among the embodiments can be referred to each other.
[0073] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. Use of thiol-modified chitosan in the preparation of thiol-modified chitosan-probiotic microspheres, wherein, Preparation method of mercapto-modified chitosan, comprising the following steps: 1) Dissolve cysteine hydrochloride monohydrate in 1% HCl aqueous solution according to the ratio of m:v of 50:1, and stir at room temperature until fully dissolved to obtain Cys solution; 2) Stir and add NHS and EDC to the Cys solution obtained in step 1) so that the final molar mass ratio of -COOH:EDC:NHS is 1:1:1 to 1:3:5, and activate at room temperature for 2 h by stirring; 3) Add chitosan to 1-2% hydrochloric acid aqueous solution according to the ratio of final concentration w / v of 0.7% - 2%, and stir at room temperature until fully dissolved to obtain chitosan solution; 4) Add the Cys solution obtained in step 2) to the chitosan solution obtained in step 3). After it is fully dissolved, adjust the pH to 5-6, and react for 4-24 h under the protection of N2 at room temperature to obtain the thiol-modified chitosan polymer CS Cys ; 5) For the CS obtained in step 4) Cys Purify it by dialysis with ultrapure water for 2 days, freeze-dry and store for later use to obtain thiol-modified chitosan; Preparation of mercapto-modified chitosan-probiotic microspheres, the specific process comprising the following steps: 1) Preparation of the dispersed phase solution and the continuous phase solution: Resuspend bacteria with 1% CS Cys solution to prepare the dispersed phase solution; Mix 0.5 - 1 g of Span80 with 15 - 30 g of paraffin oil to obtain the continuous phase; 2) Preparation of microfluidic chip: Prepare a polymethyl methacrylate (PMMA) chip; 3) Preparation of mercapto-modified chitosan-probiotic microspheres: Uniformly pump the dispersed phase and the continuous phase described in step 1) into the channels of the PMMA chip respectively, and wash the product to obtain mercapto-modified chitosan-probiotic microspheres with uniform particle size.
2. The application according to claim 1, characterized in that, In step 4), the mass ratio of Cys to CS is 1:1 to 1:
6.
3. A method for encapsulating probiotics, characterized in that, Using the 1% CS solution for preparing the mercapto-modified chitosan-probiotic microspheres in claim 1, the specific process comprises the following steps: Cys The solution, and the specific process includes the following steps: 1) Preparation of the dispersed phase solution and the continuous phase solution: Resuspend bacteria with 1% CS Cys solution to prepare the dispersed phase solution; Mix 0.5 - 1 g of Span 80 with 15 - 30 g of paraffin oil to obtain the continuous phase; 2) Preparation of microfluidic chip: Prepare a polymethyl methacrylate (PMMA) chip; 3) Preparation of mercapto-modified chitosan-probiotic microspheres: Uniformly pump the dispersed phase and the continuous phase described in step 1) into the channels of the PMMA chip respectively, and wash the product to obtain mercapto-modified chitosan-probiotic microspheres with uniform particle size.
4. The method for embedding probiotics according to claim 3, wherein, In step 2), the PMMA chip is designed as a two-layer structure. Among them, there are 3 inlets on the top layer, 1 outlet on the bottom layer. The diameters of the 3 inlets and 1 outlet are all 2 mm, the width of the middle channel is 1 mm, and the width of the central flow focusing junction is reduced to 0.5 mm. Then, the two layers are sealed with glue and clamped with a clamp. Finally, a Teflon tube is sealed to the corresponding inlet on the clamp for connection with the pump.
5. A method for embedding probiotics according to claim 4, characterized in that, After the dispersed phase and the continuous phase are respectively and uniformly pumped into the channels of the PMMA chip in step 3), adjust the flow rates of the dispersed phase and the continuous phase to 1:5 by a microfluidic instrument. At the flow focusing junction of the chip, the dispersed phase is sheared by the shear stress of the continuous phase to form a water-in-oil emulsion template. Place a curing solution at the end of the chip outlet to collect the emulsion template and add a cross-linking agent with slight horizontal shaking. Wait until the "water-in-oil" droplets turn into blue solid microspheres.
6. The method for embedding probiotics according to claim 5, characterized in that , the cross-linking agent is 1 wt% genipin, and the cross-linking time is 6 h.
7. A method for embedding probiotics according to claim 5, characterized in that, The curing solution is a genipin / glycerol-aqueous solution; the diameter of the mercapto-modified chitosan-probiotic microspheres is 200 - 400 μm.