A method for preparing probiotic microcapsules

Abstract

This invention relates to the field of microcapsule preparation technology, and more particularly to a method for preparing probiotic microcapsules. The invention involves mixing a probiotic suspension with a tris(2-carboxyethyl)phosphine solution and performing a reduction reaction to obtain a thiolized probiotic suspension. Then, a thiolized polysaccharide is mixed with the thiolized probiotic suspension and subjected to a covalent bonding reaction to obtain probiotic microcapsules. This invention uses a simple chemical reaction to reduce the disulfide bonds on the surface of probiotics to free thiols, enabling the thiolized polysaccharide and probiotics to covalently bind, resulting in a stronger bond between the polysaccharide and the probiotics. The microcapsules prepared from the modified polysaccharide and probiotics can also bind to the mucin-rich mucus layer in the intestinal lining, undergoing a catalyst-free thiol-disulfide exchange reaction, colonizing the mucus layer in the intestine, and producing better therapeutic effects for intestinal diseases and enhanced probiotic benefits.

Description

Technical Field

[0001] This invention relates to the field of microcapsule preparation technology, and in particular to a method for preparing probiotic microcapsules. Background Technology

[0002] Probiotics are microorganisms that are beneficial to the host. They have effects such as regulating gut microbiota, enhancing immunity, and lowering cholesterol. They are widely used in bioengineering, agriculture, food safety, and life and health fields. Probiotics refer to live microorganisms; ingesting sufficient amounts of these microorganisms is beneficial to the host's health.

[0003] In order to provide health benefits, probiotics must maintain a certain number of live bacteria, generally considered to be at least 10 per gram or milliliter of product. 7 CFU. However, due to numerous factors affecting probiotic viability, such as temperature, pH, gas composition, and mechanical forces, probiotic preparations are highly susceptible to inactivation, leading to reduced efficacy. To overcome the impact of the harsh environment in the gastrointestinal tract on probiotic viability, microencapsulation has become an effective method for protecting probiotics. Traditional microencapsulation involves using materials that can form thin films to encapsulate core materials (solid, liquid, and gas) within tiny, closed microcapsules. Existing microcapsule technologies mostly involve multi-bacterial encapsulation, while single-bacterial encapsulation technologies largely rely on electrostatic interactions to form capsules. However, the product structures obtained by these encapsulation methods are relatively loose, posing risks such as easy detachment of the protective membrane and easy penetration by gastrointestinal digestive fluids, reducing the survival rate of probiotics and thus diminishing their health benefits in the host. Therefore, improving the binding stability between the protective membrane and probiotics has become a pressing technical problem to be solved in this field. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing probiotic microcapsules to improve the stability of the combination of polysaccharides and probiotics, thereby increasing the survival rate of probiotics in the intestine.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for preparing probiotic microcapsules, comprising the following preparation steps:

[0007] 1) Mix the probiotic suspension and tris(2-carboxyethyl)phosphine solution and carry out a reduction reaction to obtain a thiolized probiotic suspension;

[0008] 2) After mixing the thiolated polysaccharide and the thiolated probiotic suspension, a covalent linkage reaction was carried out to obtain probiotic microcapsules.

[0009] Optionally, the probiotics include Bifidobacterium and E. coli Nissle 1917; the concentration of the probiotic suspension is 10. 9LogCFU / mL.

[0010] Optionally, the concentration of the tri(2-carboxyethyl)phosphine solution is 0.1–1 mol / L, and the volume ratio of the tri(2-carboxyethyl)phosphine solution to the probiotic suspension is 1:100.

[0011] Optionally, the reduction reaction is carried out at a temperature of 20–30°C for 1 hour.

[0012] Optionally, the ratio of the thiolated polysaccharide to the thiolated probiotic suspension is 1g:100mL.

[0013] Optionally, the covalent bonding reaction is carried out at a temperature of 20–30°C for 1 hour and at a rotation speed of 750 rpm / min.

[0014] Optionally, the preparation method of the thiolated polysaccharide includes the following preparation steps:

[0015] 1) The polysaccharide was mixed with a portion of the thiolized solution to obtain the modified polysaccharide;

[0016] 2) The modified polysaccharide, dicyclohexylcarbodiimide solution and another part of the thiolation solution were mixed and the pH was adjusted. Then the reaction was carried out. The products generated by the reaction were purified and dried in sequence to obtain the thiolated polysaccharide.

[0017] Optionally, the polysaccharide in step 1) includes chitosan; the thiolation solution includes a 2-mercaptoacetic acid solution, a 3-mercaptopropionic acid solution, and a cysteine ​​solution; the volume concentration of the thiolation solution is 1%.

[0018] The method for preparing the dicyclohexylcarbodiimide solution is as follows: dicyclohexylcarbodiimide is mixed with demineralized water to obtain a dicyclohexylcarbodiimide solution; the concentration of the dicyclohexylcarbodiimide is 125 mmol / L.

[0019] Optionally, the ratio of the polysaccharide to a portion of the thiolation solution is 10 mg: 1 mL; the mass ratio of the polysaccharide to dicyclohexylcarbodiimide is 20–22:1; and the mass ratio of the other portion of the thiolation solution to dicyclohexylcarbodiimide is 20–22:1.

[0020] Optionally, the pH after pH adjustment is 5; the reaction temperature is 20–30°C, and the time is 2–6 h.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention provides a novel method for manufacturing probiotic microcapsules. Through a simple chemical reaction, the disulfide bonds on the surface of probiotics are reduced to free thiols. This allows for the spontaneous formation of disulfide bonds and covalent bonding with thiolized polysaccharides without a catalyst. Compared to electrostatic forces, covalent bonding is a stronger force, resulting in a more robust binding between the polysaccharides and probiotics, thus improving the survival rate of probiotics in the intestine. Furthermore, the amount of thiols introduced can be adjusted by different feed ratios. These modified polysaccharides and probiotic-based microcapsules can also bind to mucin-rich substances in the intestinal mucus layer, undergoing a catalyst-free thiol-disulfide exchange reaction. This allows for colonization of the intestinal mucus layer, leading to better treatment of intestinal diseases and enhanced probiotic benefits. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] All raw materials used in this invention can be obtained commercially or prepared using existing technologies.

[0029] This invention provides a method for preparing probiotic microcapsules, comprising the following preparation steps:

[0030] 1) Mix the probiotic suspension and tris(2-carboxyethyl)phosphine solution and carry out a reduction reaction to obtain a thiolized probiotic suspension;

[0031] 2) After mixing the thiolated polysaccharide and the thiolated probiotic suspension, a covalent linkage reaction was carried out to obtain probiotic microcapsules.

[0032] Because many proteins contain abundant cysteine ​​residues and regulate their folding and stability through internal disulfide bonds, disulfide bonds also exist on the surface of probiotics. When these disulfide bonds are reduced under appropriate conditions, they can generate an interface rich in reactive thiols. This interface is highly reactive to various biomolecules and polymers. Simultaneously, polysaccharides can also undergo thiolation modification, so the thiol groups on the surface of probiotics can covalently bind with the thiol groups on the surface of polysaccharides, forming disulfide bonds to create stable probiotic microcapsules.

[0033] The present invention first mixes a probiotic suspension and a tris(2-carboxyethyl)phosphine solution and carries out a reduction reaction to obtain a thiolized probiotic suspension.

[0034] In this invention, the role of the tri(2-carboxyethyl)phosphine solution is to reduce the disulfide bonds on the surface of probiotics into free thiol groups.

[0035] This invention does not impose any specific limitations on the preparation method of the probiotic suspension, as long as the activity and concentration of the probiotics in the suspension meet the requirements. In the embodiments of this invention, the probiotics are first washed twice with deionized water, and then resuspended in phosphate buffer to prepare the probiotic suspension; the pH value of the phosphate buffer is 7.4, and the temperature of the phosphate buffer is 0-4℃.

[0036] This invention does not limit the types of probiotics, as long as the probiotics can regulate intestinal flora, enhance immunity, and lower cholesterol. In the embodiments of this invention, the probiotics include Bifidobacterium and E. coli Nissle 1917; the concentration of the probiotic suspension is 10. 9 LogCFU / mL.

[0037] In embodiments of the present invention, the concentration of the tris(2-carboxyethyl)phosphine solution is 0.1–1 mol / L, preferably 0.2–0.8 mol / L, and more preferably 0.4–0.6 mol / L; the volume ratio of the tris(2-carboxyethyl)phosphine solution to the probiotic suspension is 1:100.

[0038] In an embodiment of the present invention, the temperature of the reduction reaction is 20-30°C, preferably 22-28°C, and more preferably 24-26°C; the time is 1 hour.

[0039] In an embodiment of the present invention, after the reduction reaction is completed, the thiolized probiotics need to be washed to remove the unreacted tris(2-carboxyethyl)phosphine from the surface.

[0040] In an embodiment of the present invention, the prepared thiolized probiotics were suspended in PBS and stored at 4°C.

[0041] In this invention, thiolated polysaccharides and thiolated probiotic suspensions are mixed and then subjected to a covalent linkage reaction to obtain probiotic microcapsules.

[0042] In an embodiment of the present invention, the ratio of the amount of thiolated polysaccharide to thiolated probiotic suspension is 1g:100mL.

[0043] In an embodiment of the present invention, the temperature of the covalent bonding reaction is 20-30°C, the time is 1 hour, and the rotation speed is 750 rpm / min.

[0044] In an embodiment of the present invention, after the covalent linkage reaction is completed, the product is centrifuged at 6000×g for 5 min and washed twice with PBS to completely remove any unattached probiotics.

[0045] In this invention, the preparation method of the thiolated polysaccharide includes the following preparation steps:

[0046] 1) The polysaccharide was mixed with a partially thiolated solution to obtain the modified polysaccharide;

[0047] 2) The modified polysaccharide, dicyclohexylcarbodiimide solution and another part of thiolation were mixed and the pH was adjusted. Then the reaction was carried out. The products generated by the reaction were purified and dried in sequence to obtain thiolated polysaccharide.

[0048] This invention does not have any particular limitation on the type of polysaccharide, as long as it can be obtained by thiolation modification. In the embodiments of this invention, the polysaccharide in step 1) is chitosan.

[0049] In an embodiment of the present invention, the thiolation solution comprises a 2-mercaptoacetic acid solution, a 3-mercaptopropionic acid solution, and a cysteine ​​solution; the volume concentration of the thiolation solution is 1%.

[0050] In this invention, modified polysaccharide, dicyclohexylcarbodiimide solution and another part of thiolation solution are mixed and the pH is adjusted, and then the reaction is carried out. The products generated by the reaction are purified and dried in sequence to obtain thiolated polysaccharide.

[0051] This invention utilizes a dicyclohexylcarbodiimide solution to activate the primary amino group in the modified polysaccharide, and then uses a thiolation solution to react with the activated primary amino group to obtain a thiolated polysaccharide.

[0052] In an embodiment of the present invention, the modified polysaccharide and dicyclohexylcarbodiimide solution are first mixed, and then another portion of the thiolized solution is added and mixed.

[0053] In an embodiment of the present invention, the method for purifying the product generated by the reaction is as follows: the polymer solution is placed in a Vixin membrane tube and dialyzed in hydrochloric acid in the dark for 3 days; then dialyzed twice in hydrochloric acid containing NaCl, and finally dialyzed in hydrochloric acid for 2 days.

[0054] The cellulose membrane has a molecular cutoff of 12 kDa; the hydrochloric acid concentration is 5 mmol / L; and the sodium chloride concentration is 1%.

[0055] In an embodiment of the present invention, the drying temperature is -50°C and the time is 48 hours.

[0056] In an embodiment of the present invention, the dicyclohexylcarbodiimide solution is prepared by mixing dicyclohexylcarbodiimide and demineralized water to obtain a dicyclohexylcarbodiimide solution; the concentration of the dicyclohexylcarbodiimide is 125 mmol / L.

[0057] In embodiments of the present invention, the ratio of the polysaccharide to the partially thiolated solution is 10 mg: 1 mL; the mass ratio of the polysaccharide to dicyclohexylcarbodiimide is 20–22:1; and the mass ratio of thioglycolic acid to dicyclohexylcarbodiimide is 20–22:1.

[0058] In an embodiment of the present invention, the pH after pH adjustment is 5; the reaction temperature is 20-30°C, and the time is 2-6 hours.

[0059] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0060] Example 1

[0061] 1) Dissolve 500 mg of chitosan in 50 mL of 1% 2-mercaptoacetic acid to obtain modified polysaccharide;

[0062] 2) Dissolve dicyclohexylcarbodiimide in 1 mL of demineralized water, and add water to a final concentration of 125 mmol / L to obtain a dicyclohexylcarbodiimide solution;

[0063] 3) After mixing the modified polysaccharide from step 1) and the dicyclohexylcarbodiimide solution from step 2), add 500 mg of 1% 2-mercaptoacetic acid and mix. After mixing, adjust the pH of the solution to 5, and then stir the mixture at room temperature for 4 hours to obtain a polymer solution.

[0064] 4) The polymer solution was placed in a cellulose membrane tube with a molecular cutoff of 12 kDa and dialyzed in the dark in 5 mM HCl for 3 days; then dialyzed twice in 5 mM hydrochloric acid containing 1% NaCl; in order to maintain the pH of the culture medium at 4, the sample was dialyzed again in 5 mM hydrochloric acid for 2 days. After dialyzing, it was dried at -50℃ for 48 h to obtain thiolized polysaccharide.

[0065] 5) Pick a single colony from the Bifidobacterium agar medium and incubate it overnight at 37°C in liquid LB medium.

[0066] 6) Then wash the Bifidobacteria twice with deionized water and resuspend them in 987.5 μL of ice-cold phosphate-buffered saline (PBS, pH 7.4) to prepare a concentration of 10. 9 Bifidobacterium suspension at LogCFU / mL.

[0067] 7) Mix 12.5 μL of 0.1 mol / L tris(2-carboxyethyl)phosphine and 0.125 μL of the Bifidobacterium suspension from step 6), and react at room temperature for 1 h to obtain thiolized Bifidobacterium.

[0068] 8) Wash the thiolized Bifidobacterium with PBS, suspend the prepared thiolized Bifidobacterium in PBS, and store at 4°C.

[0069] 9) Mix 100 mL of thiolated Bifidobacterium obtained in step 8) with 1 g of thiolized chitosan obtained in step 4), and shake at 750 rpm / min for 1 hour at room temperature. After the reaction is complete, centrifuge at 6000×g for 5 min, wash twice with PBS to obtain Bifidobacterium microcapsules.

[0070] Comparative Example 1

[0071] Encapsulation of chitosan and bifidobacteria:

[0072] (1) Dissolve 500 mg of chitosan in 50 mL of purified water to obtain a sugar solution;

[0073] (2) Pick a single colony from the Bifidobacterium agar medium and incubate it overnight at 37°C in liquid LB medium.

[0074] (3) Then, the Bifidobacteria were washed twice with deionized water and resuspended in 987.5 μL of ice-cold phosphate-buffered saline (PBS, pH 7.4) to prepare a solution with a concentration of 10. 9 Bifidobacterium suspension with LogCFU / mL

[0075] (4) Mix the Bifidobacterium from step (3) and the chitosan from step (1), and react with shaking at 750 rpm / min for 1 hour at room temperature. After the reaction is complete, centrifuge (6000×g, 5min) and wash twice with PBS to obtain Bifidobacterium microcapsules.

[0076] Comparative Example 2

[0077] Encapsulation of thiolized chitosan and Bifidobacteria:

[0078] (1) Dissolve 500 mg of chitosan in 1% 2-mercaptoacetic acid to obtain modified polysaccharide;

[0079] (2) Dissolve dicyclohexylcarbodiimide in 1 mL of demineralized water and add it to a final concentration of 125 mmol / L to obtain a dicyclohexylcarbodiimide solution;

[0080] (3) After mixing the modified polysaccharide from step (1) and the dicyclohexylcarbodiimide solution from step (2), add 500 mg of 1% 2-mercaptoacetic acid and mix. After mixing, adjust the pH of the solution to 5, and then stir the mixture at room temperature for 4 hours to obtain a polymer solution.

[0081] (4) The polymer solution was placed in a cellulose membrane tube with a molecular cutoff of 12 kDa and dialyzed in the dark for 3 days in 5 mM HCl; then dialyzed twice in 5 mM hydrochloric acid containing 1% NaCl; in order to maintain the pH of the culture medium at 4, the sample was dialyzed again in 5 mM hydrochloric acid for 2 days. After dialyzing, it was dried at -50℃ for 48 h to obtain thiolized polysaccharide.

[0082] (5) Pick a single colony from the Bifidobacterium agar medium and incubate it overnight at 37°C in liquid LB medium.

[0083] (6) Then, the Bifidobacteria were washed twice with deionized water and resuspended in 987.5 μL of ice-cold phosphate-buffered saline (PBS, pH 7.4) to prepare a solution with a concentration of 10. 9 Bifidobacterium suspension at LogCFU / mL;

[0084] (7) Mix the Bifidobacterium from step (6) and the modified chitosan from step (4), and shake at 750 rpm / min for 1 hour at room temperature. After the reaction is complete, centrifuge at 6000×g for 5 min and wash twice with PBS to obtain Bifidobacterium microcapsules.

[0085] Comparative Example 3

[0086] Encapsulation of chitosan and thiol-modified Bifidobacterium:

[0087] (1) Dissolve 500 mg of chitosan in 50 mL of purified water to obtain a sugar solution;

[0088] (2) Pick a single colony from the Bifidobacterium agar medium and incubate it overnight at 37°C in liquid LB medium.

[0089] (3) Then, the Bifidobacteria were washed twice with deionized water and resuspended in 987.5 μL of ice-cold phosphate-buffered saline (PBS, pH 7.4) to prepare a solution with a concentration of 10. 9 Bifidobacterium suspension at LogCFU / mL;

[0090] (4) Mix 12.5 μL of 0.1 mol / L tris(2-carboxyethyl)phosphine and 0.125 μL of the Bifidobacterium suspension from step 6) and react at room temperature for 1 h to obtain thiolized Bifidobacterium;

[0091] (5) Wash the thiolized Bifidobacterium with PBS, suspend the prepared thiolized Bifidobacterium in PBS, and store at 4°C.

[0092] (6) Mix the thiolized Bifidobacterium from step (5) with the chitosan from step (1), and shake at 750 rpm / min for 1 hour at room temperature. After the reaction is complete, centrifuge at 6000×g for 5 min and wash twice with PBS to obtain Bifidobacterium microcapsules.

[0093] Test case

[0094] (1) Detection of modified polysaccharides and modified bifidobacteria

[0095] A 0.5% w / v aqueous solution of thiolized TSP or TSP (control) in phosphate buffer (pH 0.1, 8 M) was mixed with Ellman's reagent (0.03%, w / v), and then mixed with phosphate buffer (pH 8.0, 5 M). The mixture was incubated at 2 °C for 25 h, and the absorbance of the reaction mixture at 420 nm was measured. The number of thiol groups / g of the polymer was determined using a calibration curve prepared by reacting a standard solution of L-cysteine ​​with Ellman's reagent, as described above. The results showed that the modified chitosan contained 456 ± 18 μmol of thiol groups per gram.

[0096] The chemically modified Bifidobacteria were subjected to group determination using the same method, and the results showed that the modified thiol groups were 2.5 times that of the unmodified ones.

[0097] (2) Decomposition activity assay

[0098] Because probiotic microcapsules are encapsulated by polyelectrolyte wall materials, the probiotics need to be released with an unpacking solution before their activity is measured. The unpacking solution is usually a phosphate buffer solution.

[0099] (3) Calculation of embedding yield and survival rate

[0100]

[0101] Viable bacteria count embedded in microcapsules: After the microcapsule sample is completely dissolved in the unpacking solution, the viable bacteria count in the unpacking solution is measured.

[0102] Original viable count of probiotics: The number of viable bacteria in the initial added probiotic sludge was determined.

[0103]

[0104] Concentration of probiotics encapsulated in microcapsules: After the microcapsule sample was completely dissolved in the uncapsulation solution, the concentration of probiotics in the uncapsulation solution was measured.

[0105] Initial concentration of probiotics: The initial concentration of the added probiotic sludge was measured.

[0106] The test results are shown in Table 1:

[0107] Table 1. Encapsulation efficiency test results of Bifidobacterium preparations

[0108]

[0109] Note: Letters in the table represent significance levels (p < 0.05).

[0110] (4) Microcapsule gastrointestinal release assay

[0111] 0.1 g of the tested Bifidobacterium microcapsules were added to 9.9 mL of simulated gastric fluid and continuously agitated. At incubation times of 30, 60, 90, and 120 min, 1 mL of the solution was immediately added to 9 mL of PBS buffer and stirred at 37°C for 1 h to allow the Bifidobacterium microcapsules to fully decapsulate. The solution was then serially diluted and inoculated into LB agar plates for counting. After 120 min of culture in simulated gastric fluid, the pH was adjusted to 7.0 using 1 M sodium hydroxide solution. Then, 10 mL of simulated intestinal fluid was added and mixed thoroughly. At incubation times of 1, 2, 3, and 4 h, 1 mL of the solution was collected to determine the Bifidobacterium survival rate.

[0112] Table 2. Survival rate test results of Bifidobacteria in simulated gastric juice.

[0113]

[0114] Table 3. Survival rate test results of Bifidobacteria in simulated intestinal fluid.

[0115]

[0116]

[0117] Note: Letters in the table represent significance levels (p < 0.05).

[0118] The results are shown in Tables 2 and 3. The Bifidobacterium microcapsules survived well in the simulated gastrointestinal fluid, and the survival rate of Bifidobacterium was very good. After the thiolation treatment of Bifidobacterium and polysaccharides, the microcapsules significantly improved the survival rate of Bifidobacterium in the gastrointestinal tract and the protective effect on Bifidobacterium was more significant.

[0119] (5) Determination of bile salt tolerance properties of microcapsules

[0120] Add 2% (w / w) of bile salt to a PBS buffer solution at pH 8, and measure the survival rate of Bifidobacteria in the microcapsules every 1 hour at 37°C to investigate the tolerance of the microcapsules to bile salt.

[0121] Table 4. Survival rate test results of Bifidobacteria in bile salts

[0122]

[0123] Note: Letters in the table represent significance levels (p < 0.05).

[0124] The results, as shown in Table 4, indicate that the bile salt resistance of the thiolated Bifidobacterium microcapsules was significantly improved compared to that of the unmodified Bifidobacterium microcapsules.

[0125] (6) Determination of the storage stability of microcapsules

[0126] Weigh 2g of Bifidobacterium microcapsules into a sterile glass bottle, seal it, and then place it in an aluminum bag sealed in plastic and stored in a constant temperature incubator at 4℃. Take samples after 90 days to measure the survival rate of Bifidobacterium and examine its long-term stability.

[0127] Table 5. Results of long-term stability test of Bifidobacterium microcapsules

[0128]

[0129] Note: Letters in the table represent significance levels (p < 0.05).

[0130] The results are shown in Table 5. The survival rate of the thiol-modified Bifidobacterium capsules was higher than that of the unmodified Bifidobacterium microcapsules, proving that microencapsulation can effectively improve the long-term stability of the strain.

[0131] (7) Determination of the thermal stability of Bifidobacterium microcapsules

[0132] Weigh 0.2 g of Bifidobacterium microcapsules into a sterile EP tube, add 4.8 mL of phosphate buffer, and incubate at 55℃, 65℃, and 75℃ for 10 min each. After heat treatment, add 15 mL of phosphate buffer to the sterile tube, rapidly cool, and determine the survival rate of Bifidobacterium.

[0133] Table 6. Results of thermal stability tests on Bifidobacterium microcapsules

[0134]

[0135] Note: Letters in the table represent significance levels (p < 0.05).

[0136] The results are shown in Table 6. The survival rate of probiotic capsules after thiol modification was higher than that of unmodified probiotic microcapsules, proving that microencapsulation can effectively resist thermal environments.

[0137] (8) Ethanol tolerance test of microcapsules

[0138] Weigh 0.2g of microcapsules into a sterile EP tube, add 4.8mL of phosphate buffer, add 75% ethanol and react for 1h, then determine the probiotic survival rate.

[0139] Table 7 Results of ethanol tolerance test for microcapsules

[0140]

[0141] Note: Letters in the table represent significance levels (p < 0.05).

[0142] The results are shown in Table 7. The survival rate of the thiol-modified Bifidobacterium capsules was higher than that of the unmodified probiotic microcapsules, proving that microencapsulation can effectively improve the ethanol resistance of the strains.

[0143] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing probiotic microcapsules, characterized in that, The preparation steps include the following: 1) Mix the probiotic suspension and tris(2-carboxyethyl)phosphine solution and carry out a reduction reaction to obtain a thiolized probiotic suspension; 2) After mixing the thiolated polysaccharide and the thiolated probiotic suspension, a covalent linkage reaction was carried out to obtain probiotic microcapsules; The probiotic is Bifidobacterium; The preparation method of the thiolized polysaccharide includes the following preparation steps: S1. Chitosan is mixed with a portion of a thiolated solution to obtain a modified polysaccharide; S2. The modified polysaccharide, dicyclohexylcarbodiimide solution and another part of thiolation solution are mixed and the pH is adjusted. Then the reaction is carried out. The products generated by the reaction are purified and dried in sequence to obtain thiolated polysaccharide. The thiolation solution comprises a 2-mercaptoacetic acid solution, a 3-mercaptopropionic acid solution, and a cysteine ​​solution; the volume concentration of the thiolation solution is 1%. The ratio of the polysaccharide to a portion of the thiolation solution is 10 mg: 1 mL; the mass ratio of the polysaccharide to dicyclohexylcarbodiimide is 20–22:1; the mass ratio of the other portion of the thiolation solution to dicyclohexylcarbodiimide is 20–22:

1. The pH after pH adjustment is 5; step S2. The reaction temperature is 20-30℃, and the time is 2-6h.

2. The preparation method according to claim 1, characterized in that, The concentration of the probiotic suspension is 10. 9 LogCFU / mL.

3. The preparation method according to claim 2, characterized in that, The concentration of the tri(2-carboxyethyl)phosphine solution is 0.1–1 mol / L, and the volume ratio of the tri(2-carboxyethyl)phosphine solution to the probiotic suspension is 1:

100.

4. The preparation method according to claim 1, characterized in that, The reduction reaction is carried out at a temperature of 20–30°C for 1 hour.

5. The preparation method according to claim 1, characterized in that, The ratio of the amount of thiolized polysaccharide to the thiolized probiotic suspension is 1g:100mL.

6. The preparation method according to claim 1, characterized in that, In step 2), after the thiolized polysaccharide and the thiolized probiotic suspension are mixed, they are covalently linked at 20-30°C for 1 hour with a stirring rate of 750 rpm / min.

7. The preparation method according to claim 1, characterized in that, The method for preparing the dicyclohexylcarbodiimide solution is as follows: dicyclohexylcarbodiimide is mixed with demineralized water to obtain a dicyclohexylcarbodiimide solution; the concentration of the dicyclohexylcarbodiimide solution is 125 mmol / L.

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