Preparation method of probiotic microcapsule

Abstract

The invention relates to the technical field of microcapsule preparation, in particular to a preparation method of a probiotic microcapsule. The preparation method comprises the following steps: mixing a probiotic suspension and a tris (2-carboxyethyl) phosphine solution, and carrying out a reduction reaction to obtain a thiolated probiotic suspension; and mixing the thiolated polysaccharide and the thiolated probiotic suspension, and carrying out a covalent ligation reaction to obtain the probiotic microcapsule. According to the preparation method disclosed by the invention, disulfide bonds on the surfaces of the probiotics are reduced into free mercaptan through simple chemical reaction, so that thiolated polysaccharide and the probiotics are covalently bound, and the polysaccharide and the probiotics are more firmly bound. The microcapsule prepared from the modified polysaccharide and the probiotics can also be combined with mucoprotein rich in the mucous layer of the intestinal tract to generate a catalyst-free thiol-disulfide exchange reaction, and colonization is carried out at the mucous layer in the intestinal tract, so that better intestinal disease treatment and probiotic benefits are generated.

Description

Technical Field

[0001] The present invention relates to the technical field of microcapsule preparation, and in particular to a method for preparing probiotic microcapsules. Background Art

[0002] Probiotics are microorganisms that are beneficial to the host. They have the potential to regulate intestinal flora, enhance immunity, and lower cholesterol. They are widely used in bioengineering, agriculture, food safety, and life sciences. Probiotics are living microorganisms, and adequate intake of these microorganisms is beneficial to the host's health.

[0003] To provide health benefits, probiotics must maintain a certain number of live bacteria, generally considered to be at least 10 per gram or per milliliter of product. 7 CFU. However, due to the many factors that affect the vitality of probiotics, such as temperature, pH, gas composition and mechanical force, probiotic preparations are easily inactivated, resulting in reduced efficacy. In order to overcome the impact of the harsh environment of probiotics in the gastrointestinal tract on their vitality, microencapsulation has become an effective method to protect probiotics. Traditional microencapsulation is a process of burying core materials (solids, liquids and gases) in tiny closed microcapsules using materials that can form thin films; existing microencapsulation technologies are mostly multi-bacteria encapsulation, and single-bacteria encapsulation technologies are mostly encapsulated through electrostatic interactions. However, the product structure obtained by the above-mentioned encapsulation method is relatively loose, and there are risks such as easy shedding of the protective film and easy penetration of gastrointestinal digestive fluid, which reduces the survival rate of probiotics and thus reduces their health benefits in the host body. Therefore, how to improve the stability of the combination of the protective film and probiotics has become a technical problem that needs to be solved urgently in this field. Summary of the Invention

[0004] The purpose of the present 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] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

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

[0007] 1) mixing a probiotic suspension and a tris(2-carboxyethyl)phosphine solution, and performing a reduction reaction to obtain a thiolated probiotic suspension;

[0008] 2) After mixing the thiolated polysaccharide and the thiolated probiotic suspension, a covalent linking reaction is performed to obtain probiotic microcapsules.

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

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

[0011] Optionally, the reduction reaction temperature is 20-30° C. and the time is 1 hour.

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

[0013] Optionally, the temperature of the covalent linking reaction is 20-30° C., the time is 1 h, and the rotation speed is 750 rpm / min.

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

[0015] 1) mixing the polysaccharide with a portion of the thiol solution to obtain a modified polysaccharide;

[0016] 2) The modified polysaccharide, the dicyclohexylcarbodiimide solution and another portion of the thiol solution are mixed, the pH thereof is adjusted, and then the mixture is reacted. The reaction product is purified and dried in sequence to obtain the thiolated polysaccharide.

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

[0018] The preparation method of the dicyclohexylcarbodiimide solution comprises the following steps: mixing dicyclohexylcarbodiimide and demineralized water to obtain the dicyclohexylcarbodiimide solution; the concentration of the dicyclohexylcarbodiimide is 125 mmol / L.

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

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

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

[0022] The present invention provides a new method for producing probiotic microcapsules. Through a simple chemical reaction, disulfide bonds on the surface of probiotics are reduced to free thiols, allowing them to spontaneously form disulfide bonds with thiolated polysaccharides for covalent bonding in the absence of a catalyst. Compared to electrostatic forces, covalent bonding is a stronger force, which can make the bond between polysaccharides and probiotics more secure, thereby improving the survival rate of probiotics in the intestine. In addition, the amount of introduced thiols can be adjusted by varying the feed ratio. Microcapsules prepared from these modified polysaccharides and probiotics can also bind to mucins rich in the intestinal mucus layer, undergoing a catalyst-free thiol-disulfide exchange reaction, and colonize the intestinal mucus layer, resulting in better treatment of intestinal diseases and probiotic benefits. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting 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 terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

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

[0028] The raw materials used in the present invention can be obtained commercially or prepared using existing technologies.

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

[0030] 1) mixing a probiotic suspension and a tris(2-carboxyethyl)phosphine solution, and performing a reduction reaction to obtain a thiolated probiotic suspension;

[0031] 2) After mixing the thiolated polysaccharide and the thiolated probiotic suspension, a covalent linking reaction is performed to obtain probiotic microcapsules.

[0032] Since many proteins are rich in cysteine ​​residues and regulate their folding and stability through internal disulfide bonds, disulfide bonds are also present on the surface of probiotics. When these disulfide bonds are reduced under appropriate conditions, they can produce an interface rich in active thiols, which are highly reactive towards various biomolecules and polymers. At the same time, polysaccharides can also be thiolated, so the thiol groups on the surface of probiotics can undergo covalent bonding reactions with the thiol groups on the surface of polysaccharides, forming disulfide bonds to form stable probiotic microcapsules.

[0033] The invention first mixes a probiotic suspension with a tris(2-carboxyethyl)phosphine solution, and performs a reduction reaction to obtain a thiolated probiotic suspension.

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

[0035] The present invention does not specifically limit the preparation method of the probiotic suspension, as long as the activity and concentration of the probiotics in the suspension meet the requirements. In an embodiment of the present invention, the probiotics are first washed twice with deionized water and then resuspended in a phosphate buffer to prepare the probiotic suspension; the phosphate buffer has a pH of 7.4 and a temperature of 0 to 4°C.

[0036] The present invention has no limitation on the types of probiotics, as long as the probiotics can produce effects such as regulating intestinal flora, enhancing body immunity, and lowering cholesterol. In an embodiment of the present invention, the probiotics include Bifidobacterium and E. Coli Nissle 1917; the concentration of the probiotic suspension is 10 9 LogCFU / mL.

[0037] In an embodiment 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 reduction reaction temperature is 20-30° C., preferably 22-28° C., more preferably 24-26° C., and the time is 1 hour.

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

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

[0041] In the present invention, the thiolated polysaccharide and the thiolated probiotic suspension are mixed and then subjected to a covalent linking reaction to obtain probiotic microcapsules.

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

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

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

[0045] In the present invention, the preparation method of the thiolated polysaccharide comprises the following preparation steps:

[0046] 1) mixing the polysaccharide with a portion of the thiol solution to obtain a modified polysaccharide;

[0047] 2) The modified polysaccharide, dicyclohexylcarbodiimide solution and another portion of thiolation are mixed, and the pH thereof is adjusted, followed by reaction. The reaction products are purified and dried in sequence to obtain thiolated polysaccharide.

[0048] The present invention has no particular limitation on the type of polysaccharide, as long as it can be modified to obtain a thiolated polysaccharide by thiolization. In an embodiment of the present invention, the polysaccharide in step 1) is chitosan.

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

[0050] The invention mixes modified polysaccharide, dicyclohexylcarbodiimide solution and another part of thiol solution, adjusts the pH thereof, and then reacts. The reaction products are purified and dried in sequence to obtain thiolated polysaccharide.

[0051] The invention utilizes dicyclohexylcarbodiimide solution to activate the primary amino groups in the modified polysaccharide, and then utilizes thiol solution to react with the activated primary amino groups to obtain thiolated polysaccharide.

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

[0053] In an embodiment of the present invention, the method for purifying the product generated by the reaction is: placing the polymer solution in a cellulose membrane tube, dialyzing it in hydrochloric acid in the dark for 3 days; then dialyzing it in hydrochloric acid containing NaCl twice, and finally dialyzing it in hydrochloric acid for 2 days;

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

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

[0056] In an embodiment of the present invention, the preparation method of the dicyclohexylcarbodiimide solution is as follows: dicyclohexylcarbodiimide and demineralized water are mixed to obtain the dicyclohexylcarbodiimide solution; the concentration of the dicyclohexylcarbodiimide is 125 mmol / L.

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

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

[0059] The technical solutions provided by the present invention are 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) dissolving 500 mg of chitosan in 50 mL of 1% 2-mercaptoacetic acid to obtain a modified polysaccharide;

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

[0063] 3) mixing the modified polysaccharide from step 1) and the dicyclohexylcarbodiimide solution from step 2), adding 500 mg of 1% 2-mercaptoacetic acid, adjusting the pH of the solution to 5, and stirring 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 5 mM HCl for 3 days in the dark; then dialyzed twice in 5 mM hydrochloric acid containing 1% NaCl; to maintain the pH value of the culture medium at 4, the sample was dialyzed again in 5 mM hydrochloric acid for 2 days. After dialysis, the sample was dried at -50°C for 48 hours to obtain the thiolated polysaccharide.

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

[0066] 6) 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 concentration of 10 9 LogCFU / mL of Bifidobacterium suspension.

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

[0068] 8) The thiolated bifidobacteria were washed with PBS, and the prepared thiolated bifidobacteria were suspended in PBS and stored at 4°C.

[0069] 9) 100 mL of the thiolated bifidobacterium prepared in step 8) and 1 g of the thiolated chitosan prepared in step 4) were mixed, and the mixture was shaken at 750 rpm / min at room temperature for 1 hour. After the reaction, the mixture was centrifuged in a centrifuge (6000×g, 5 min) and washed twice with PBS to obtain bifidobacterium microcapsules.

[0070] Comparative Example 1

[0071] Encapsulation of Chitosan and Bifidobacterium:

[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 agar LB medium of Bifidobacterium and culture it in liquid LB medium overnight at 37°C

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

[0075] (4) The bifidobacteria obtained in step (3) and the chitosan obtained in step (1) were mixed and shaken at 750 rpm / min for 1 hour at room temperature. After the reaction, the mixture was centrifuged in a centrifuge (6000×g, 5 min) and washed twice with PBS to obtain bifidobacterium microcapsules.

[0076] Comparative Example 2

[0077] Thiolated chitosan and encapsulation of bifidobacteria:

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

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

[0080] (3) After mixing the modified polysaccharide of step (1) and the dicyclohexylcarbodiimide solution of step (2), 500 mg of 1% 2-mercaptoacetic acid was added and mixed, and after mixing, the pH of the solution was adjusted to 5, and then the mixture was stirred 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 5 mM HCl for 3 days in the dark; then dialyzed twice in 5 mM hydrochloric acid containing 1% NaCl; to maintain the pH value of the culture medium at 4, the sample was dialyzed again in 5 mM hydrochloric acid for 2 days. After dialysis, the sample was dried at -50°C for 48 hours to obtain the thiolated polysaccharide;

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

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

[0084] (7) The bifidobacteria obtained in step (6) and the modified chitosan obtained in step (4) were mixed and shaken at 750 rpm / min for 1 hour at room temperature. After the reaction, the mixture was centrifuged in a centrifuge (6000×g, 5 min) and washed twice with PBS to obtain bifidobacterium microcapsules.

[0085] Comparative Example 3

[0086] Entrapment of Chitosan and Thiolated 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 LB medium and culture it in liquid LB medium at 37°C overnight;

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

[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 thiolated Bifidobacterium;

[0091] (5) The thiolated bifidobacteria were washed with PBS, and the prepared thiolated bifidobacteria were suspended in PBS and stored at 4°C.

[0092] (6) The thiolated bifidobacteria of step (5) and the chitosan of step (1) were mixed, and the mixture was shaken at 750 rpm / min for 1 hour at room temperature. After the reaction, the mixture was centrifuged in a centrifuge (6000×g, 5 min) and washed twice with PBS to obtain bifidobacterium microcapsules.

[0093] Test example

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

[0095] An aqueous solution (0.5%, w / v) of thiolated TSP or TSP (control) in phosphate buffer (pH 0.1, 8 M) was mixed with Ellman's reagent (0.03%, w / v), then mixed with phosphate buffer (pH 8.0, 5 M), incubated at 2°C for 25 hours, 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 thiol groups after modification were 2.5 times that of the unmodified ones.

[0097] (2) Determination of cystication activity

[0098] Since probiotic microcapsules are wrapped with polyelectrolyte wall materials, they need to be released with cystolysis fluid before their activity can be measured. The cystolysis fluid is generally phosphate buffered saline.

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

[0100]

[0101] The number of live bacteria embedded in the microcapsules: After the microcapsule samples were completely dissolved in the cystic fluid, the number of live bacteria in the cystic fluid was measured;

[0102] The original viable count of probiotics: measure the viable count of the bacterial sludge initially added.

[0103]

[0104] Concentration of probiotics embedded in microcapsules: After the microcapsule sample is completely dissolved in the cystic fluid, the concentration of probiotics in the cystic fluid is measured;

[0105] Initial concentration of probiotics: Determine the initial concentration of bacterial sludge.

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

[0107] Table 1 Encapsulation efficiency test results of Bifidobacterium preparation

[0108]

[0109] Note: The letters in the table represent the significance level (p < 0.05)

[0110] (4) Microcapsule gastrointestinal release assay

[0111] 0.1g of the Bifidobacterium microcapsules to be tested were added to 9.9mL of simulated gastric fluid and continuously shaken to mix. At 30, 60, 90, and 120 minutes of incubation, 1mL of the solution was removed and immediately added to 9mL of PBS buffer solution. The solution was stirred and shaken at 37°C for 1 hour to fully disintegrate the Bifidobacterium microcapsules. Subsequently, the microcapsules were inoculated onto LB culture plates through a gradient dilution process for counting. After 120 minutes of simulated gastric fluid incubation, the pH was adjusted to 7.0 using 1M sodium hydroxide, and 10mL of simulated intestinal fluid was added and mixed thoroughly. At 1, 2, 3, and 4 hours of incubation, 1mL of the solution was removed and the survival rate of Bifidobacteria was determined.

[0112] Table 2 Survival test results of Bifidobacterium in simulated gastric fluid

[0113]

[0114] Table 3 Survival test results of Bifidobacterium in simulated intestinal fluid

[0115]

[0116]

[0117] Note: The letters in the table represent the significance level (p < 0.05)

[0118] The results are shown in Tables 2 and 3. The bifidobacterium microcapsules can survive well in the simulated gastrointestinal fluid, and the survival rate of bifidobacteria is very good. After the bifidobacteria and polysaccharides are thiolated, the microcapsules significantly improve the survival rate of bifidobacteria in the gastrointestinal tract, and the protective effect on bifidobacteria is more significant.

[0119] (5) Determination of bile salt resistance of microcapsules

[0120] Bile salts at a concentration of 2% by mass were added to a PBS buffer solution at pH 8. The survival rate of bifidobacteria in the microcapsules was measured every hour at 37°C to investigate the tolerance of the microcapsules to bile salts.

[0121] Table 4 Survival test results of bifidobacteria in bile salts

[0122]

[0123] Note: The letters in the table represent the significance level (p < 0.05)

[0124] The results are shown in Table 4. The bile salt resistance of the thiolated Bifidobacterium microcapsules is significantly improved compared with that of the unmodified Bifidobacterium microcapsules.

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

[0126] Weigh 2 g of bifidobacterium microcapsules into a sterile glass bottle, seal it, put it into an aluminum bag, seal it and store it in a 4°C constant temperature box. Take samples after 90 days to measure the survival rate of bifidobacteria and investigate the long-term stability.

[0127] Table 5 Long-term stability test results of Bifidobacterium microcapsules

[0128]

[0129] Note: The letters in the table represent the significance level (p < 0.05)

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

[0131] (7) Thermal stability determination 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°C, 65°C, and 75°C for 10 minutes. After the heat treatment, add 15 mL of phosphate buffer to the sterile tube, rapidly cool, and measure the survival rate of Bifidobacterium.

[0133] Table 6 Thermal stability test results of Bifidobacterium microcapsules

[0134]

[0135] Note: The letters in the table represent the significance level (p < 0.05)

[0136] The results are shown in Table 6. The survival rate of the probiotic capsules after thiol modification is higher than that of the unmodified probiotic microcapsules, which proves that microencapsulation can effectively resist the thermal environment.

[0137] (8) Determination of ethanol tolerance of microcapsules

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

[0139] Table 7 Ethanol tolerance test results of microcapsules

[0140]

[0141] Note: The letters in the table represent the significance level (p < 0.05)

[0142] The results are shown in Table 7. The survival rate of the thiolated Bifidobacterium capsules was higher than that of the unmodified probiotic microcapsules, which proved that microencapsulation can effectively improve the ethanol resistance of the bacteria.

[0143] 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing probiotic microcapsules, characterized in that: The method comprises the following preparation steps: 1) mixing a probiotic suspension and a tris(2-carboxyethyl)phosphine solution, and performing a reduction reaction to obtain a thiolated probiotic suspension; 2) After mixing the thiolated polysaccharide and the thiolated probiotic suspension, a covalent linking reaction is performed to obtain probiotic microcapsules.

2. The preparation method according to claim 1, characterized in that The probiotics include Bifidobacterium and E. Coli Nissle 1917; 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 tris(2-carboxyethyl)phosphine solution is 0.1-1 mol / L, and the volume ratio of the tris(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 temperature is 20-30° C. and the time is 1 hour.

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

6. The preparation method according to claim 1, characterized in that The temperature of the covalent linking reaction is 20-30° C., the time is 1 h, and the rotation speed is 750 rpm / min.

7. The preparation method according to claim 1, characterized in that The preparation method of the thiolated polysaccharide comprises the following preparation steps: 1) mixing the polysaccharide with a portion of the thiol solution to obtain a modified polysaccharide; 2) The modified polysaccharide, the dicyclohexylcarbodiimide solution and another portion of the thiol solution are mixed, the pH thereof is adjusted, and then the mixture is reacted. The reaction product is purified and dried in sequence to obtain the thiolated polysaccharide.

8. The preparation method according to claim 7, characterized in that The polysaccharide in step 1) includes chitosan; the thiolation solution includes 2-mercaptoacetic acid solution, 3-mercaptopropionic acid, and cysteine ​​solution; the volume concentration of the thiolation solution is 1%; The preparation method of the dicyclohexylcarbodiimide solution comprises the following steps: mixing dicyclohexylcarbodiimide and demineralized water to obtain the dicyclohexylcarbodiimide solution; the concentration of the dicyclohexylcarbodiimide solution is 125 mmol / L.

9. The preparation method according to claim 8, characterized in that The usage ratio of the polysaccharide and a portion of the thiolation solution is 10 mg:1 mL; the mass ratio of the polysaccharide and dicyclohexylcarbodiimide is 20-22:1; and the mass ratio of the other portion of the thiolation solution and dicyclohexylcarbodiimide is 20-22:

1.

10. The preparation method according to claim 7, characterized in that The pH after adjustment is 5; the reaction temperature is 20-30° C., and the reaction time is 2-6 hours.

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