Preparation method of ackermann muciniphile combined prebiotic double-layer microcapsule

Abstract

The invention relates to the technical field of biology, in particular to a double-layer microcapsule of ackermania mucilaginosa and prebiotics and a preparation method thereof.The double-layer microcapsule is prepared from bacteria and mixed gum, the weight ratio of the bacteria to the mixed gum is 1: 5, the bacteria are ackermania mucilaginosa, and the mixed gum comprises sodium alginate, carob gum or xanthan gum; the embedding rate of the microcapsule is 91.00%-96.14%, the survival rate is obviously improved, and the protection capability on strains is improved; under the three storage temperatures, the survival rate of the microcapsule embedded bacterial strain added with the prebiotics is obviously higher than that of the microcapsule without the prebiotics under the same condition.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for preparing Akkermansia muciniphila combined with prebiotics double-layer microcapsules. Background Art

[0002] Microcapsules can encapsulate substances in the body of microcapsules through special methods, with diameters ranging from a few microns to several centimeters. Currently, a variety of materials are used to encapsulate probiotics to help them resist adverse effects from the environment, such as sodium alginate, pectin, xanthan gum, carrageenan and chitosan. Microcapsule wall materials can act as a barrier to prevent probiotics from direct contact with adverse external factors, thereby increasing the stability and activity of probiotics and maintaining their activity during transportation and in the gastrointestinal environment.

[0003] There are many ways to microencapsulate probiotics. Spray drying has the advantages of batch production, low cost and high efficiency, but high temperature conditions and oxygen environment may cause the activity of strains to decrease. The probiotic microcapsules prepared by the emulsification method are small and easy to produce industrially, but the strong shear force of the agitator will cause the diameters of the microcapsules to be different and the sphericity is poor. The extrusion method is the most commonly used technology because it is easy to operate. The probiotics are mixed with a pre-prepared microcapsule wall material colloidal solution for electrostatic adsorption, and then the mixture is dripped into a hardener (such as CaCl2 solution) through a needle to form microcapsules, but the microcapsule particles are large, and the influence of the wall material on the growth of the strain needs to be considered. The materials used have limitations, and alginate microgels are still fragile, especially in acidic environments. When a single wall material is used for microencapsulation, it is difficult to play a protective role under gastric acid conditions, so the current method of using two or more wall materials in combination is considered. Microcapsules prepared by the extrusion method are usually spherical with a diameter of 2-5 mm and a large particle size. However, the large particle size means that the microcapsules are more isolated from the internal substances, which is more conducive to resisting environmental pressure and thus protecting probiotics.

[0004] As the next generation of probiotics, Akkermansia muciniphila must be kept in sufficient quantity during preparation, storage and gastrointestinal tract (GIT) if it is to play its positive role. Therefore, in order to improve the survival ability of probiotics in various stress environments, it is necessary to explore the protection technology of strains. Microencapsulation, resistance induction and the addition of protective agents provide protection for the vitality of strains. Microencapsulation can provide a physical barrier for probiotics to resist adverse external conditions, and the size, shape, color, etc. of the microcapsules are suitable for use in food, do not interfere with the sensory perception of food, and have broad application prospects. At present, many probiotic microcapsules have been used in dairy products, health products and cosmetics industries.

[0005] The inventors found in their research that Akkermansia muciniphila has a strong bile salt tolerance, but its acid resistance is extremely poor and it is easily inactivated by gastric acid. Its oxygen tolerance is also extremely low and it cannot survive in an aerobic environment. Since acid and oxygen have a great impact on its survival rate, this poses a great challenge to its application, subsequent product development and shelf life storage. Therefore, most of the products containing Akkermansia muciniphila currently on the market are dead bacteria products.

[0006] In view of the above technical problems, the present invention provides a double-layer microcapsule of Akkermansia muciniphila combined with prebiotics, in which Akkermansia muciniphila is in a living state and the survival rate of the strain can reach more than 90%. The particle size of the prepared microcapsule is 2.38-2.47 mm; the embedding rate and strain activity are higher. Summary of the invention

[0007] The primary purpose of the present invention is to provide a double-layer microcapsule of Akkermansia muciniphila combined with prebiotics, which is prepared from bacteria and mixed gum, wherein the weight ratio of the bacteria to the mixed gum is 1:5, the bacteria is Akkermansia muciniphila, and the mixed gum includes sodium alginate, carob bean gum or xanthan gum.

[0008] Preferably, the mixed gum further comprises galacto-oligosaccharide.

[0009] Preferably, the mixed glue also includes chitosan.

[0010] The second object of the present invention is to provide a method for preparing Akkermansia muciniphila combined with prebiotics double-layer microcapsules, comprising the following steps:

[0011] (1) The Akkermansia muciniphila suspension was centrifuged, the supernatant was discarded, the precipitate was collected, and it was washed with sterile PBS; the bacterial precipitate was resuspended with a lyophilization protective agent at a ratio of 1:30 to obtain a high-concentration bacterial suspension;

[0012] (2) adding the bacterial suspension obtained in step (1) into the mixed gel at a ratio of 1:5, stirring and mixing;

[0013] (3) uniformly adding the solution obtained in step (2) dropwise to the solidifying liquid, stirring during the adding process, to obtain a single-layer microcapsule;

[0014] (4) discarding the solidified liquid from the solution obtained in step (3), collecting the microcapsules, washing them with sterile water, transferring them to the chitosan solution, and stirring;

[0015] (5) discarding the chitosan solution from the solution obtained in step (4), collecting the microcapsules, washing the unreacted chitosan with sterile water, and freeze-drying to obtain double-layer microcapsules;

[0016] The mixed gum described in step (2) includes sodium alginate, carob bean gum or xanthan gum.

[0017] Preferably, the lyoprotectant in step (1) is trehalose and sucrose in a ratio of 1:1.

[0018] Preferably, the solidifying liquid in step (3) is a calcium chloride solution.

[0019] Preferably, the pH of the sterile PBS in step (1) is 7.0.

[0020] Preferably, the stirring is performed using a magnetic stirrer.

[0021] The third object of the present invention is to provide double-layer microcapsules prepared by the preparation method.

[0022] The beneficial effects of the present invention are as follows: (1) the present invention provides four kinds of double-layer microcapsules of Akkermansia muciniphila CIP 107961 combined with prebiotics, and the particle sizes of the four kinds of microcapsules are 2.38-2.47 mm; the embedding rate of the microcapsules with added prebiotics is higher than that of the microcapsules without added prebiotics;

[0023] (2) The experimental results showed that microencapsulation can significantly improve the activity of the strain in the gastrointestinal fluid environment. The microcapsules containing carob bean gum have a better protective effect on the strain in the gastrointestinal fluid than the microcapsules containing xanthan gum.

[0024] (3) After heat treatment, the survival rate of microencapsulated strains was significantly higher than that of free strains;

[0025] (4) After vacuum freeze-drying, the number of viable bacteria in both microcapsule-embedded strains and free bacteria decreased, but the survival rate of microcapsule-embedded strains was significantly higher than that of free strains, and the addition of prebiotics increased the survival rate of the strains;

[0026] (5) The surface of the microcapsules containing xanthan gum is relatively smooth, with no obvious protrusions and grooves under low-power microscope, and slight wrinkles under high-power microscope; the surface of the microcapsules containing carob bean gum is relatively rough, with large wrinkles and depressions under low-power microscope, and many obvious fine wrinkles can be observed under high-power microscope; the addition of prebiotic oligosaccharides modifies the morphology of the microcapsules and improves the protection ability against strains;

[0027] (6) The number of live bacteria in all microcapsules and freeze-dried bacterial powders decreased significantly after storage at 25°C, while the storage effect was better at 4°C and -20°C. At the three storage temperatures, the survival rate of the microcapsules encapsulated with prebiotics was significantly higher than that of the microcapsules without prebiotics under the same conditions. Comprehensive analysis showed that the microcapsules prepared by combining xanthan gum and prebiotics had the best protection for the vitality of the strains during storage. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In the following figures, A, B, C, and D represent microcapsules with different embedding wall materials, respectively, wherein A is 2% sodium alginate, 1% xanthan gum, and 0.4% chitosan; B is 2% sodium alginate, 1% xanthan gum, 0.4% chitosan, and 1% galacto-oligosaccharide; C is 2% sodium alginate, 1% carob gum, and 0.4% chitosan; D is 2% sodium alginate, 1% carob gum, 0.4% chitosan, and 1% galacto-oligosaccharide; FC represents free Akkermansia muciniphila CIP 107961 strain.

[0029] Figure 1 Microcapsule preparation process flow chart

[0030] Figure 2 Growth curves of Akkermansia muciniphila CIP 107961 in modified BHI medium containing different embedding materials

[0031] Figure 3 The viable bacterial counts of four kinds of microcapsules and free bacteria in simulated gastric fluid Note: The changes in the viable bacterial counts of the four kinds of microcapsules and free bacteria in simulated gastric fluid during the 4-h treatment (at 0, 1, 2, 3, and 4 h, respectively) were measured.

[0032] Figure 4 Survival rate and viable bacterial count change of four microcapsules and free bacteria in simulated gastric fluid Note: The survival rate and logarithmic reduction value of the four microcapsules and free bacteria were measured during the 4-hour treatment in simulated gastric fluid (at 0, 1, 2, 3, and 4 hours, respectively). Lowercase letters indicate that the survival rate of microcapsule-embedded strains and free bacteria is significantly different (p<0.05) when treated for the same time.

[0033] Figure 5 The viable bacterial counts of four microcapsules and free bacteria in simulated intestinal fluid Note: The changes in the viable bacterial counts of four microcapsules and free bacteria in simulated intestinal fluid during 4 hours of treatment (at 0, 1, 2, 3, and 4 hours, respectively) were measured.

[0034] Figure 6Survival rate and viable bacterial count change of four microcapsules and free bacteria in simulated gastric fluid Note: The survival rate and logarithmic reduction value of four microcapsules and free bacteria in simulated intestinal fluid were measured during the 4-hour treatment (at 0, 1, 2, 3, and 4 hours, respectively). Lowercase letters indicate that the survival rate of microcapsule-embedded strains and free bacteria is significantly different (p<0.05) when treated for the same time.

[0035] Figure 7 Note: The changes in the viable bacterial counts of the four microcapsules and free bacteria in simulated gastrointestinal fluid were measured during the 6-h treatment in simulated gastric fluid, of which the first 2 h were the changes in simulated gastric fluid, and 3-6 h were the changes in simulated intestinal fluid.

[0036] Figure 8 The survival rate and viable bacterial count changes of four microcapsules and free bacteria in simulated gastrointestinal fluid Note: The survival rate and logarithmic reduction of four microcapsules and free bacteria in simulated gastric fluid for 6 hours were measured, of which the first 2 hours were the changes in simulated gastric fluid, and 3-6 hours were the changes in simulated intestinal fluid. Lowercase letters indicate that the survival rate of microcapsule-embedded strains and free bacteria is significantly different (p<0.05) when treated for the same time.

[0037] Fig. 9 The survival rate and the change of viable bacteria count of four microcapsules and free bacteria after heat treatment Note: The survival rate and the logarithmic reduction value of the four microcapsules and free bacteria after 50℃ and 60℃ treatment for 10min were measured respectively. Lowercase letters indicate that the survival rate of microcapsule-embedded strains and free bacteria is significantly different (p<0.05) when treated at the same temperature.

[0038] Fig.10 The number of viable bacteria of four microcapsules and free bacteria after vacuum freeze drying

[0039] Fig.11 The survival rate and the change of viable bacteria count of four microcapsules and free bacteria after vacuum freeze drying Note: The survival rate and the logarithmic reduction value of four microcapsules and free bacteria after vacuum freeze drying were measured respectively. Lowercase letters indicate that the survival rate of microcapsule embedded strains and free bacteria after vacuum freeze drying is significantly different (p<0.05).

[0040] Fig.12SEM images of four types of microcapsules encapsulating Akkermansia muciniphila CIP 107961 after vacuum freeze-drying. Notes: A1: overall morphology of microcapsule A (×45); A2: surface morphology of microcapsule A at low magnification (×500); A3: surface morphology of microcapsule A at high magnification (×1500); B1: overall morphology of microcapsule B (×45); B2: surface morphology of microcapsule B at low magnification (×300); B3: surface morphology of microcapsule B at high magnification (×2000); C1: overall morphology of microcapsule C (×50); C2: surface morphology of microcapsule C at low magnification (×300); C3: surface morphology of microcapsule C at high magnification (×2000); D1: overall morphology of microcapsule D (×50); D2: surface morphology of microcapsule D at low magnification (×300); D3: surface morphology of microcapsule D at high magnification (×1800).

[0041] Fig.13 FTIR spectra of four microcapsules and their corresponding wall materials Note: Figure A: FTIR spectra of microcapsules A and B and their corresponding wall materials; Figure B: FTIR spectra of microcapsules C and D and their corresponding wall materials.

[0042] Fig.14 Changes in survival rate and viable count of four microcapsules and free bacteria stored under frozen conditions Note: Lowercase letters indicate significant differences in the survival rate of microcapsule-embedded strains and free bacteria after storage under frozen conditions for the same number of days (p<0.05).

[0043] Fig.15 Changes in survival rate and viable count of four microcapsules and free bacteria under refrigerated conditions Note: Lowercase letters indicate significant differences in the survival rate of microcapsule-embedded strains and free bacteria after storage for the same number of days under refrigerated conditions (p<0.05).

[0044] Fig.16 Changes in survival rate and viable count of four microcapsules and free bacteria stored at room temperature Note: Lowercase letters indicate significant differences in the survival rate of microcapsule-embedded strains and free bacteria after storage for the same number of days at room temperature (p<0.05).

[0045] Fig.17 Survival rates of four microcapsules and free bacteria after 90 days of storage under three conditions DETAILED DESCRIPTION

[0046] The present invention is further described below in conjunction with examples. The following examples are illustrative rather than restrictive, and the protection scope of the present invention cannot be limited by the following examples.

[0047] In the following examples, unless otherwise specified, the methods used are all conventional methods, and the reagents and consumables used can be purchased from the market.

[0048] In the following examples, the formula of each culture medium is as follows:

[0049] Enriched liquid culture medium: BHI (brain infused heart broth) 38.5 g / L; mucin 0.4 g / L; soy peptone 16 g / L; L-threonine 4 g / L; glucose 11.3 g / L; N-acetylglucose 5.5 g / L; L-cysteine ​​hydrochloride 0.5 g / L.

[0050] Improved BHI liquid (solid) culture medium: BHI (brain infused heart broth) 38.5 g / L; mucin 0.4 g / L; KH2PO4 0.4 g / L; Na2HPO4 0.53 g / L; NaCl 0.3 g / L; NaHCO3 0.4 g / L; MgCl2·6H2O 0.1 g / L; L-cysteine ​​hydrochloride 0.5 g / L; vancomycin hydrochloride 6 mg / L; (agar 20 g / L).

[0051] Selective culture medium: BHI (brain infusion broth) 38.5 g / L; L-cysteine ​​hydrochloride 0.5 g / L; mucin 0.4 g / L; agar 20 g / L; vancomycin hydrochloride 6 mg / L; kanamycin sulfate 12 mg / L; gentamicin sulfate 10 mg / L, solid culture medium plus agar 20 g / L.

[0052] It should be noted that the strain used in the following examples is Akkermansia muciniphila CIP 107961, which was purchased from Guangdong Microbiological Culture Collection Center and is numbered GDMCC No. 1.1346. However, the Akkermansia muciniphila CIP 107961 described in the present invention can be replaced by other Akkermansia muciniphila.

[0053] Example 1. Preparation of microcapsules

[0054] 1. Test strains and preparations

[0055] (1) Experimental strains

[0056] Akkermansia muciniphila CIP 107961 was purchased from Guangdong Microbiological Culture Collection Center and is numbered GDMCC No.1.1346 (the numbers of other collection centers are ATCC BAA-835, and all numbers mentioned in the text can have the same meaning).

[0057] (2) Main ingredients of the preparation

[0058] The common preparation ingredients used in this experiment are shown in Table 1.

[0059] Table 1 Common preparation ingredients

[0060]

[0061]

[0062] 2. Experimental Methods

[0063] (1) Strain activation

[0064] Before the experiment, the strain Akkermansia muciniphila CIP 107961 stored in a -80°C ultra-low temperature freezer was inoculated on a solid plate, and a single colony was picked and inoculated into a modified BHI medium, cultured anaerobically at 37°C for 5-7 days, and subcultured 2-3 times until the strain recovered its vitality.

[0065] (2) Expansion of strains

[0066] The activated Akkermansia muciniphila CIP 107961 was inoculated into 50 mL of modified BHI liquid culture medium at a 2% inoculation rate, cultured anaerobically at 37°C for 48 h, and then transferred to 200 mL of fresh culture medium. This step was repeated until it was transferred to 600 mL of modified BHI liquid culture medium and cultured anaerobically at 37°C for 72 h. It can be clearly observed that the color of the culture medium became darker and became uniformly turbid.

[0067] (3) Material affinity determination

[0068] To investigate whether different microcapsule wall materials have an effect on the growth of Akkermansia muciniphila CIP 107961. 2% (w / v) sodium alginate, 1% (w / v) xanthan gum, 1% (w / v) carob bean gum, and 0.4% (w / v) chitosan were added to the modified BHI medium, and the modified BHI medium without additional ingredients was used as a control. The OD of Akkermansia muciniphila CIP 107961 was adjusted. 600 The inoculum was 0.5 (±0.25), and 5% of the inoculum was inoculated into the above culture medium, and cultured anaerobically at 37°C. The OD of the bacterial solution was measured every 24 hours. 600 The growth curve of Akkermansia muciniphila CIP 107961 strain in different culture media was drawn with the culture time as the horizontal axis and the absorbance value measured each time as the vertical axis, and the results were repeated three times.

[0069] (4) Preparation of four types of microcapsules

[0070] Akkermansia muciniphila CIP 107961 combined with prebiotics double-layer microcapsules, the preparation process is shown in Figure 1. The cultured bacterial suspension was centrifuged at 4°C and 8000rpm for 10min, the supernatant was discarded, the precipitate was collected, and washed twice with sterile PBS (pH 7.0). The bacterial precipitate was resuspended to 20mL with a freeze-dried protective agent at a ratio of 1:30 to obtain a bacterial suspension with a higher concentration. At a bacterial-gel ratio of 1:5, 5mL of the resuspended bacterial suspension was added to 25mL of mixed gel A, B, C, and D, respectively, and a magnetic stirrer was used to mix them thoroughly. A 0.45mm sterile syringe was used to uniformly add the bacterial-gel mixture to the solidifying liquid. During the addition process, a magnetic stirrer was used to stir the reaction at a fixed speed for 30-50min to obtain a single-layer microcapsule. The solidifying liquid was discarded, the microcapsules were collected, washed 2-3 times with sterile water, transferred to the chitosan solution, and a magnetic stirrer was used to react at a fixed speed for 40-60min to coat it. The chitosan solution was discarded, the microcapsules were collected, and the unreacted chitosan was washed away with sterile water to obtain double-layer wet capsules (A, B, C, D).

[0071] (5) Determination of particle size and embedding rate

[0072] The particle sizes of 10 microcapsules prepared above were measured using a vernier caliper.

[0073] The determination of microcapsule embedding efficiency was based on Darjani et al.

[210] The method was slightly modified. Weigh 1g of each of the four microcapsules prepared above, add them to 9mL of encapsulation solution, shake for 15-30min, and take samples after the microcapsules are completely lysed. After gradient dilution, take 100μL of the dilution solution and spread it evenly on the modified BHI solid culture medium. After anaerobic culture at 37℃ for 72h, count and calculate the microcapsule embedding rate according to the following formula. The concentrated bacterial solution was diluted with the same ratio as the bacterial-gel ratio (1:5) as a reference indicator, and three parallels were performed for each capsule.

[0074]

[0075] Where: N is the number of viable bacteria embedded in each microcapsule (Lg CFU / g); N0 is the number of viable bacteria after the concentrated bacterial solution was diluted 1:5 when preparing the microcapsules (Lg CFU / mL).

[0076] (6) Activity of the four microcapsules in simulated gastric fluid

[0077] Preparation of simulated gastric fluid (SGF): Measure 100 mL of ultrapure water, adjust its pH to 2.5, add 1 g of pepsin (1%) after sterilization, wait for it to completely dissolve, and filter it with a 0.22 μm microporous filter membrane for use.

[0078] Weigh 1g of each of the four microcapsules, absorb 1mL of free bacterial solution, add 9mL of simulated gastric fluid preheated at 37℃, place in 37℃ anaerobic box, and take samples at 0, 1, 2, 3, and 4h. After discarding the simulated gastric fluid, add 9mL of cyst dissociation solution, and after the cyst is completely dissociated, dilute it 10 times with sterile saline. Pipette 100μL of appropriate gradient dilution on modified BHI solid culture medium, spread evenly, count after anaerobic culture at 37℃ for 72h, and calculate the survival rate of microcapsules in simulated gastric fluid according to the following formula. Each capsule is carried out in three parallels.

[0079]

[0080] Where: N i is the number of viable bacteria in each microcapsule at time i in simulated gastric fluid (Lg CFU / mL); N0 is the number of viable bacteria in each microcapsule at 0h (Lg CFU / mL).

[0081] (7) Activity of the four microcapsules in simulated intestinal fluid

[0082] Preparation of simulated intestinal fluid (SIF): refer to Amakiri et al.

[211] The method was slightly modified. Weigh 0.68g potassium dihydrogen phosphate and 0.3g ox bile salt, add 100mL water to dissolve them, add 1g trypsin (1%) after sterilization, wait until it is completely dissolved, filter with a 0.22μm microporous filter membrane for use.

[0083] Weigh 1 g of each of the four microcapsules, absorb 1 mL of free bacterial solution, add 9 mL of simulated intestinal fluid preheated at 37°C, place in a 37°C anaerobic chamber, and take samples at 0, 1, 2, 3, and 4 h to determine the number of live bacteria using the spreading counting method, which is the same as (6).

[0084] (8) Activity of four microcapsules in continuous simulated gastrointestinal fluid

[0085] Weigh 1g of each of the four microcapsules, absorb 1mL of free bacterial solution, add 9mL of simulated gastric fluid preheated at 37℃, place in a 37℃ anaerobic box, and take samples at 0, 1, and 2 hours to determine the number of live bacteria. After 2 hours of simulated gastric fluid environment, discard the simulated gastric fluid, transfer to 9mL of simulated intestinal fluid, and place in a 37℃ anaerobic box again. Take samples at the next 1, 2, 3, and 4 hours to determine the number of live bacteria. The whole process was reacted for a total of 6 hours, and the coating counting method was used, the method was the same as (6).

[0086] (9) Determination of heat resistance of four microcapsules

[0087] Weigh 1g of each of the four microcapsules, absorb 1mL of free bacterial solution, add 9mL of 0.85% sterile saline, and measure the number of live bacteria after reacting in a water bath at 50℃ and 60℃ for 10min. The coating counting method is the same as (6).

[0088] (10) Activity determination of four microcapsules under vacuum freeze-drying conditions

[0089] Weigh 3g of each of the four microcapsules into a vial and add 2mL of freeze-drying protective agent. Prefreeze at -20℃ for 5h, freeze at -80℃ for 12-24h, and finally place in a vacuum freeze dryer for 24-30h to obtain dry capsules. Dissolve the freeze-dried microcapsules in 0.85% sterile saline and determine the number of viable bacteria. The coating counting method is the same as (6).

[0090] (11) SEM analysis

[0091] Four kinds of freeze-dried microcapsules were taken and adhered to the sample stage of a scanning electron microscope with double-sided tape. Gold was sprayed in a vacuum coating machine to coat the samples with a conductive film. Then, a field emission scanning electron microscope (FE-SEM, SU8010, Hitachi, Japan) was used to select an appropriate magnification for observation.

[0092] (12) FTIR analysis

[0093] Four kinds of freeze-dried microcapsules and various wall material components were mixed with KBr, ground into thin slices, and analyzed using a Fourier transform infrared spectrometer (Vertex 70v, Bruker, Germany) at 2 cm -1 The resolution is 4000-400cm -1 Scan each sample within the range of .

[0094] (13) Storage stability of four microcapsules

[0095] The storage stability of the four freeze-dried microcapsules and freeze-dried bacterial powders was measured at -20°C (simulated freezing), 4°C (simulated refrigeration), and 25°C (simulated room temperature). Samples were taken after 0, 30, 60, and 90 days of storage, and the plate was spread and counted using the same method as 4.3.6.

[0096] (14) Data analysis and processing

[0097] The data were subjected to one-way analysis of variance (ANOVA) using the statistical software IBM SPSS Statistics 26, and the Duncan test was used to analyze the significance of the data. p<0.05 indicated that the difference was significant. Origin 2021 was used to draw the graph.

[0098] 3. Results and Analysis

[0099] (1) Material affinity

[0100] After the microcapsules are ingested by the human body, it takes a certain amount of time to reach the gastrointestinal tract and release the encapsulated probiotics. Therefore, the materials and strains that encapsulate the probiotics will be in the gastrointestinal tract at the same time. It is necessary to explore whether the encapsulation materials themselves will affect the growth of the strains in the intestine. This experiment determined the growth curves of the experimental strains in the modified BHI medium containing four wall materials: sodium alginate, carob bean gum, xanthan gum, and chitosan.

[0101] The results are as follows Figure 2 As shown, Akkermansia muciniphila CIP 107961 grows well in the modified BHI culture containing the four materials, and the growth curve is similar to that in the blank culture medium, all of which grow rapidly from 0 to 48 hours and reach a stable period at around 48 hours. Among them, the initial absorbance of the xanthan gum and chitosan culture media is higher, but the absorbance change values ​​are similar to those of the other culture media, all around 0.572. Therefore, these four materials can all be used as wall materials for encapsulating Akkermansia muciniphila CIP 107961.

[0102] (2) Microcapsule particle size and embedding rate

[0103] The wet capsule particle size, embedding rate and number of live bacteria in the four microcapsules are shown in Table 2. All four microcapsules are uniformly spherical and smooth in surface. Through the standard deviation of particle size, it is found that the morphological uniformity of microcapsules CD containing carob bean gum is better. The particle size of the four wet capsules is 2.38-2.47mm, which is not significantly different (p<0.05), indicating that the addition of prebiotics has no effect on the particle size of wet capsules.

[0104] Table 2 Morphology, particle size, embedding rate and number of viable bacteria of four microcapsules

[0105]

[0106] Note: The lowercase letters in the upper right corner indicate that the four microcapsules have significant differences under the same test index (p<0.05). A, B, C, and D represent microcapsules with different embedding wall materials, where A is 2% sodium alginate, 1% xanthan gum, and 0.4% chitosan; B is 2% sodium alginate, 1% xanthan gum, 0.4% chitosan, and 1% galacto-oligosaccharide; C is 2% sodium alginate, 1% carob bean gum, and 0.4% chitosan; D is 2% sodium alginate, 1% carob bean gum, 0.4% chitosan, and 1% galacto-oligosaccharide.

[0107] The embedding rates of the four microcapsules were all higher than 90.00%, and there were significant differences (p<0.05), indicating that the embedding effect of Akkermansia muciniphila CIP 107961 microcapsules prepared by the four microcapsule wall material combinations was better. Among them, the embedding rate of microcapsule B was significantly higher than that of other microcapsules, reaching 96.14%, followed by microcapsule D at 94.14%, both of which were higher than microcapsules AC without prebiotics. The number of viable bacteria in the bacterial solution before embedding was 9.09±0.059Lg CFU / mL. After embedding, the number of viable bacteria in each microcapsule decreased. This phenomenon may be caused by the dissipation of some bacteria in the solution during the embedding process. However, the number of viable bacteria in microcapsules with prebiotics (B and D) was higher than that in microcapsules without prebiotics (A and C).

[0108] (3) Survival ability in simulated gastric fluid

[0109] The changes in the number of live bacteria and the survival rate of the free strains and the four microcapsule combinations of strains after being treated with simulated gastric fluid (SGF) are shown in Figure 2. Figure 3 and Figure 4 As shown, they all decreased with the extension of time, and there was a significant difference (p<0.5). After 1 hour of gastric juice treatment, the survival rate of the strains embedded in the four microcapsule combinations was higher than 92.00%, while the survival rate of free bacteria was only 61.25%. After 2 hours, the survival rate of the strains embedded in the four microcapsule combinations remained at around 90.00%, while the activity of free bacteria further decreased, and the survival rate decreased to 56.04%. After 3 hours, the strains embedded in microcapsule A and microcapsule B decreased by 1.56 and 1.43Lg CFU / g, respectively, and the survival rate decreased to 81.18% and 83.67%, while microcapsule B and microcapsule D maintained a high activity, with a survival rate higher than 86.00%. After 4 hours of treatment with gastric juice, this change trend was maintained. The survival rates of the four microcapsule-encapsulated strains were 75.47%-85.03%, and the number of live bacteria was 6.25-7.26Lg CFU / g, with the number of live bacteria decreasing by 1.25-2.03Lg CFU / g; while the number of live bacteria of free bacteria decreased by 4.94Lg CFU / mL, with only 4.15Lg CFU / mL surviving, and the survival rate was only 45.67%.

[0110] From the analysis of the figure, it can be seen that in the process of gastric juice treatment, compared with free bacteria, microcapsule embedding can significantly improve the activity and stability of the strain, and reduce the stress of acid and pepsin in the gastric environment on the strain. The activity of the strain embedded in the combination of microcapsules containing carob bean gum is the best, and the survival rate of microcapsules C and D can still reach 85.00% after 4 hours. The protection effect of microcapsule A containing only xanthan gum is the worst, and the survival rate after 4 hours is only 75.47%, but on this basis, microcapsule B with prebiotics can increase the survival rate of the strain to 79.62%, which shows that the addition of prebiotics can increase the protection of the strain.

[0111] (4) Survival ability in simulated intestinal fluid

[0112] The changes in the number of live bacteria and the survival rate of the free strains and the four microcapsule combinations of strains after being treated with simulated intestinal fluid (SIF) are shown in Figure 2. Figure 5 and 6 As shown in the figure, they all decreased with time, and there was a significant difference (p<0.5).

[0113] After 1 hour of treatment with simulated intestinal fluid, the survival rate of free bacteria was only 64.19%, while the number of live bacteria encapsulated by the four microcapsules decreased by 0.63-1.33Lg CFU / g, and the survival rate of the strains was 84.76%-92.49%. After 2 hours, the survival rate of the strains encapsulated by the four microcapsules was higher than 80.00%, and even the survival rate of the strain encapsulated by microcapsule B could reach 87.52%, while the survival rate of free bacteria dropped to 58.69%. After 3 hours, the survival rate of the strains encapsulated by the four microcapsules was still higher than 75%, while the survival rate of free bacteria dropped to 52.99%. After 4 hours of intestinal fluid treatment, the survival rate of the microcapsule-encapsulated strains ranged from 70.84% ​​to 73.43%, and the number of live bacteria was 5.91-6.19 Lg CFU / g, a decrease of 2.21-2.55 Lg CFU / g; while the survival rate of free bacteria was only 49.77%, and the number of live bacteria decreased by 4.66 Lg CFU / mL.

[0114] Depend on Figure 6 It can be seen that, in the process of intestinal fluid treatment, compared with free bacteria, microcapsule embedding can significantly improve the activity and stability of the strain, and reduce the stress of the alkaline environment, trypsin and bile salt in the intestine on the strain. The survival rate of the strain in microcapsules C and D containing carob gum was significantly higher than that in microcapsules A and B containing xanthan gum, which shows that the tolerance of the microcapsule combination containing carob gum to simulated intestinal fluid is higher than that of xanthan gum, which may be caused by carob gum being a neutral gum and xanthan gum being an anionic gum. Strangely, unlike in the simulated gastric fluid environment, the addition of prebiotic oligosaccharides did not significantly improve the survival rate of the strain, and the protective effect of the embedding combination on the strain was similar to that of the strain without prebiotics, and even reduced the survival rate of the strain. This may be because prebiotics are hydrophilic, which makes the microcapsules break down faster in the intestinal fluid, penetrate more intestinal fluid, and reduce the activity of the strain.

[0115] (5) Survival ability in simulated gastrointestinal fluid

[0116] The changes in the number of live bacteria and survival rate of the free strains and the four microcapsule combinations of the strains after being treated with SGF for 2 hours and then transferred to SIF for 4 hours are as follows Figure 7 and 8 shown.

[0117] After 2 hours of SGF reaction, the survival rate of the microcapsule-embedded strains remained at 91.21%-92.43%, and the number of viable bacteria decreased by 0.63-0.77Lg CFU / g; after SIF treatment, the activity of the embedded strains decreased significantly, and the survival rate dropped to 78.32%-86.28% after only 1 hour in the intestinal fluid, among which microcapsules A and B had the largest decrease. After 6 hours of reaction, the survival rates of microcapsules C and D containing carob gum were 71.74% and 70.14%, which were significantly higher than those of microcapsules A and B containing xanthan gum. After 6 hours of SGF digestion, the number of viable bacteria of free bacteria decreased from 9.09Lg CFU / mL to 2.46Lg CFU / mL, a decrease of 6.63Lg CFU / mL, and the survival rate was only 27.07%. However, since microcapsules B and D containing prebiotics have a higher encapsulation rate and a higher initial bacterial content, their viable bacterial counts were 5.92±0.045Lg CFU / g and 6.04±0.016Lg CFU / g after 6 hours of reaction, respectively, which were higher than microcapsules A (5.72±0.041Lg CFU / g) and microcapsule C (6.02±0.015Lg CFU / g) without prebiotics.

[0118] This indicates that microcapsules containing carob bean gum have a stronger protective effect on strains in the gastrointestinal environment than microcapsules containing xanthan gum, and microcapsules with added prebiotics can maintain a similar survival rate to microcapsules without added prebiotics and contain a higher number of viable bacteria.

[0119] (6) Determination of heat resistance

[0120] Probiotics grow best at optimal temperatures, but high temperatures during production may affect their activity. Currently, many studies have found that microencapsulation can improve the thermal stability of probiotics. [213，214] The changes in the number of live bacteria and survival rate of the four microcapsules and free bacteria prepared in this experiment after being treated at 50℃ and 60℃ for 10 minutes are shown in the figure below. Fig. 9 As shown in Figure 2, with the increase of temperature, the number of live bacteria and the survival rate of microcapsule-embedded strains and free strains decreased significantly, and there were significant differences at different temperatures (p<0.05).

[0121] After the free bacteria were treated at 60℃ for 10min, the number of viable bacteria decreased by 4.27±0.099Lg CFU / g, and the survival rate was only 53.08%. The survival rates of the microcapsule-embedded strains after treatment at 50℃ and 60℃ were 86.63%-93.63% and 70.25%-73.32%, respectively; the number of viable bacteria decreased by 0.56-1.14Lg CFU / g and 2.33-2.41Lg CFU / g, respectively. Microcapsules A and B have stronger tolerance to high temperature than microcapsules C and D. Among them, microcapsule B had the highest survival rate after treatment at 50℃ and 60℃, which were 93.63% and 73.32%, respectively; while the survival rates of microcapsules A, C, and D embedded strains after treatment at 60℃ were not significantly different (p<0.05), all around 71.00%.

[0122] (7) Microcapsule morphology and viable bacterial count after vacuum freeze drying

[0123] The particle size of the microcapsules after vacuum freeze drying was measured, and the results are shown in Table 3. It was found that the particle size of all microcapsules was reduced, and there was a significant difference (p<0.05). Among them, the particle size of microcapsule B was the largest, reaching 2.10±0.25mm, indicating that it was less shrunken; the particle size of microcapsules C and microcapsules D after drying was only 1.53±0.10mm, indicating that the microcapsules containing carob bean gum shrank more significantly after freeze drying.

[0124] Table 3 Particle size of microcapsules before and after vacuum freeze drying

[0125]

[0126] Changes in the number of live bacteria and survival rate of microcapsules and free bacteria after vacuum freeze drying Fig.10 and 11 As shown, there were significant differences in their survival rates (P<0.5). The number of viable bacteria in each microcapsule combination and free bacteria decreased after low-temperature treatment. The number of viable bacteria in the microcapsule combination embedded strains decreased by 0.60-1.34Log CFU / g, among which the number of viable bacteria in microcapsule B decreased the least, while the number of free bacteria decreased by 2.02Log CFU / mL. The survival rate of free bacteria was only 77.80%, and the microcapsules B (93.04%) and D (89.56%) with prebiotics were significantly higher than those of microcapsules A (90.25%) and C (83.71%) without prebiotics.

[0127] This shows that microcapsule encapsulation can significantly improve the strain's tolerance to vacuum freeze-drying. The addition of prebiotic oligosaccharides makes the microcapsule cross-linking more dense, effectively reducing the damage to the strain caused by low temperature conditions and improving the protection of the microcapsule on the vitality of the strain.

[0128] (8) SEM analysis

[0129] Scanning electron microscopy of the vacuum freeze-dried microcapsules Fig.12 From the following images, we can see that all capsules are spherical, slightly shrunk compared to the wet capsule state, and the diameter is about 1.5-2.0 mm, indicating that different wall material combinations have no significant effect on the size of the microcapsules after freeze-drying.

[0130] Depend on Fig.12 It can be found that the surface morphology and structure of each microcapsule are quite different. The surfaces of microcapsule A (sodium alginate-xanthan gum) and microcapsule B (sodium alginate-xanthan gum-GOS) are relatively smooth and uniform, with no obvious protrusions and grooves under low magnification, and slight wrinkles under high magnification (1500-2000 times). The surfaces of microcapsules C (sodium alginate-carob bean gum) and microcapsule D (sodium alginate-carob bean gum-GOS) are relatively rough, with large wrinkles and depressions under low magnification, and many obvious fine wrinkles can be observed under high magnification (1800-2000 times). This shows that the microcapsules embedded in xanthan gum have a more uniform structure than the microcapsules embedded in carob bean gum, the degree of cross-linking is denser, and they are more tolerant to the stimulation of freeze-drying. In addition, it also shows that sodium alginate, xanthan gum and GOS have good compatibility. Compared with microcapsule B, microcapsule A is rougher ( Fig.12 , A1, B1); Microcapsule D has a fuller structure than microcapsule C, and the degree of fine wrinkles on the surface is also smaller than that of microcapsule C ( Fig.12 , D3, C3). This shows that the addition of prebiotic GOS provides a certain support for the structure of the microcapsules, modifies and improves the morphology of the microcapsules, making them more complete, and also improves the protection ability against strains.

[0131] (9) FTIR analysis

[0132] FTIR can be used to identify the relationship between the wall material and the microcapsules used to prepare the microcapsules. The FTIR spectra of four microcapsules and wall materials are shown in Figure 2. Fig.13 As shown. Sodium alginate at 3403cm -1 The absorption peak is due to the stretching vibration of -OH, 2938cm -1 The absorption peak of 1611cm -1 and 1419cm -1 There is a stretching vibration of -COOH at 1029cm -1 The absorption peak of COC is due to stretching vibration.

[215] Xanthan gum at 3431cm -1 、2936cm -1 The characteristic absorption peaks at 1618 cm are -OH stretching vibration and CH bond stretching vibration. -1 and 1417cm-1 The absorption peak at 10000 is due to the stretching vibration of -C=O and the asymmetric stretching vibration of -COOH.

[216] . Carob bean gum is also at 3431cm -1 、2934cm -1 and 1031cm －1 The characteristic absorption peak of polysaccharides appears at

[0133] Galacto-oligosaccharides are polyhydroxy compounds with a peak at 1646 cm -1 and 1419cm -1 There are asymmetric and symmetric stretching vibration absorption peaks of -COOH at 1200-900cm -1 and 900-600cm -1 The characteristic absorption peaks in the region gradually decreased after the microcapsules were formed, indicating that hydrogen bonds may have formed between galacto-oligosaccharides and sodium alginate, xanthan gum or carob bean gum.

[217] . Free bacteria at 3370cm -1 、1653-1424cm -1 and 1045-600cm -1 There are characteristic absorption peaks in the region, including the stretching vibration characteristic peak of -NH; the FTIR spectrum of the prepared microcapsules is at 3400cm -1 、1630-1420cm -1 and 1038-600cm -1 There is an absorption peak in the region; this indicates that the functional groups of the free bacteria produce ionic cross-linking with the wall material through chemical and physical effects, thereby being successfully and stably encapsulated in the four microcapsules.

[0134] (10) Storage stability

[0135] Table 4 The number of viable bacteria of four microcapsules and free bacteria stored under three conditions (Lg CFU / g)

[0136]

[0137]

[0138] Note: The lowercase letters in the upper right corner indicate that there are significant differences in the number of viable bacteria of each microcapsule-embedded strain and free bacteria when stored at the same temperature and time (p<0.05). - indicates that no strain was detected.

[0139] The viable counts of the four freeze-dried microcapsules and free bacteria after storage at -20°C (simulated freezing conditions), 4°C (simulated refrigeration conditions) and 25°C (simulated room temperature conditions) for 90 days are shown in Table 4. The results showed that the viable counts of the microcapsule-embedded strains under the three storage conditions were higher than those of the free bacteria, and there were significant differences (p<0.05).

[0140] From Table 4 and Fig.14 It was found that under freezing conditions, the number of live bacteria and the survival rate of microcapsule-embedded strains and free bacteria decreased with the extension of storage time. After 30 days of storage in the freezer (-20±2℃), except for the survival rate of microcapsule C embedded strains of 89.53%, the survival rate of other microcapsule-embedded strains was higher than 93.00%, while the survival rate of free bacteria was only 70.91%. After 60 days of storage, the survival rate of all microcapsule-embedded strains was higher than 86%, among which the survival rates of microcapsules A and B were higher than 90%, while the number of live bacteria of free bacteria decreased by 2.5Lg CFU / mL, and the survival rate was 64.6%. After 90 days, the number of viable bacteria in the free bacteria was only 3.95±0.019Lg CFU / mL, and the survival rate dropped to 55.88%; except for the number of viable bacteria in microcapsule C, which was only 5.55±0.101Lg CFU / g, the number of viable bacteria in the remaining microcapsules was higher than 6.5Lg CFU / g. During the 90-day storage process, the survival rate of the strains embedded in the microcapsules with added probiotics was significantly higher than that without added. After 90 days of storage, the survival rate of the strains embedded in microcapsule B was still as high as 90.84%, higher than microcapsule A (87.72%); the survival rate of microcapsule D was 84.8%, also higher than microcapsule C (79.5%).

[0141] From Table 4 and Fig.15 It was found that under refrigerated conditions, the number of live bacteria and the survival rate of microcapsule-embedded strains and free bacteria decreased with the extension of storage time. After refrigerated storage (4±2℃) for 30 days, except for the survival rate of microcapsule C embedded strains of 86.77%, the survival rate of other microcapsule-embedded strains was higher than 91%, while the survival rate of free bacteria was only 65.32%. After 60 days of storage, the survival rate of all microcapsule-embedded strains was higher than 80.00%, among which the survival rate of microcapsule B embedded strains was as high as 91.32%, while the survival rate of free bacteria dropped to 57.95%. After 90 days of storage, the survival rate of free bacteria dropped to 52.06%, and the number of live bacteria was only 3.68±0.155Lg CFU / mL; the survival rate of the microcapsule-embedded strains with added prebiotics was significantly higher than that without added prebiotics, among which the strain embedded in microcapsule B had the highest survival rate, reaching 89.42%, and the number of live bacteria was 7.27±0.045Lg CFU / g, which only decreased by 0.86Lg CFU / g; the survival rate of microcapsule D was 87.18%, which was also higher than that of microcapsule C (79.19%).

[0142] From Table 4 and Fig.16It was found that under room temperature conditions, the number of live bacteria and the survival rate of microcapsule-embedded strains and free bacteria decreased significantly, and decreased with the extension of storage time. After storage at room temperature (25±2℃) for 30 days, the survival rate of microcapsule-embedded strains was 55.33%-68.92%, and the number of live bacteria was less than 6.0Lg CFU / g, which was lower than the recommended value of probiotics, while the survival rate of free bacteria was only 16.28%. After storage for 60 days, the survival rate of all microcapsule-embedded strains was higher than 50.00%, and the survival rate of microcapsule B was the highest, reaching 65.74%, but no free bacteria were detected. After storage for 90 days, the survival rate of microcapsule-embedded strains was 35.53%-56.90%, and the number of live bacteria decreased by 3.51-4.5Lg CFU / g. Among them, microcapsules B and D with prebiotics provided higher protection for the activity of strains stored at 25℃. This shows that the activity of both free bacteria and embedded strains is difficult to maintain at room temperature, but the addition of prebiotics significantly improves the activity of the strains.

[0143] From Table 4 and Fig.17 Comprehensive analysis found that the number of live bacteria and the survival rate of microcapsule-embedded strains were significantly higher than those of free bacteria during storage (p<0.05), and the survival rate of the same microcapsule was different at different temperatures. The survival rate of strains embedded in microcapsules A and B prepared by xanthan gum was higher under frozen conditions than under refrigerated conditions, the survival rate of strains embedded in microcapsule C prepared by carob bean gum was similar under frozen and refrigerated conditions, and the survival rate of strains embedded in microcapsule D was higher under refrigerated conditions than under frozen conditions. Under room temperature conditions, the survival rate of free bacteria and all microcapsule-embedded strains decreased significantly.

[0144] At the same storage temperature, different microcapsules have different protective abilities against bacterial activity. At frozen and refrigerated storage temperatures, the survival rates of strains embedded in microcapsules A and B prepared with xanthan gum were higher than those in microcapsules C and D prepared with carob gum; at the three storage temperatures, the survival rates of strains embedded in microcapsules B and D with added prebiotics were higher than those in microcapsules A and C with the same wall material but without added prebiotics.

[0145] The survival rates of strain B in microcapsules embedded with a combination of xanthan gum and GOS as wall materials after 90 days of storage under frozen, refrigerated and room temperature conditions were 90.84%, 89.42% and 56.9%, respectively, indicating that this microcapsule combination can provide the best protection for the strain.

[0146] In summary, (1) the particle size of the four microcapsules was 2.38-2.47 mm; the encapsulation rates of microcapsules B and D with added prebiotics were 96.14% and 94.14%, respectively, and the encapsulation rates of microcapsules A and C without added prebiotics were about 91.00%.

[0147] (2) Microencapsulation can significantly improve the activity of strains in the gastrointestinal fluid environment. Microcapsules containing carob bean gum have a better protective effect on strains in the gastrointestinal fluid than microcapsules containing xanthan gum. After 4 hours of SGF or SIF treatment, the survival rate of strains embedded in microcapsules CD was significantly higher than that in microcapsules AB; after 2 hours of SGF treatment and then transferred to SIF treatment for 4 hours, the survival rates of microcapsules C (71.74%) and microcapsules D (70.14%) containing carob bean gum were significantly higher than those of microcapsules A (68.8%) and microcapsule B (67.35%) containing xanthan gum, while the survival rate of free bacteria was only 27.07%.

[0148] (3) After heat treatment, the survival rate of the microcapsule-embedded strain was significantly higher than that of the free strain. After treatment at 60°C for 10 min, the survival rate of the free bacteria was only 53.08%. Microcapsule B had the best protective effect on the strain, with a survival rate of 73.32%, while the survival rates of the strains embedded in microcapsules A, C, and D were all around 71.00%.

[0149] (4) After vacuum freeze-drying, the number of live bacteria in microcapsule-embedded strains and free bacteria decreased, but the survival rate of microcapsule-embedded strains was significantly higher than that of free strains, and the addition of prebiotics increased the survival rate of strains. The survival rates of microcapsule BD embedded strains with prebiotics were 93.04% and 89.56%, which were significantly higher than those of microcapsules A (90.25%) and C (83.71%) without prebiotics under the same conditions; while the survival rate of free bacteria was only 77.80%.

[0150] (5) The surface of microcapsules AB containing xanthan gum is relatively smooth, with no obvious protrusions and grooves under low magnification, and slight wrinkles under high magnification; the surface of microcapsules CD containing carob gum is relatively rough, with large wrinkles and depressions under low magnification, and many obvious fine wrinkles can be observed under high magnification; the addition of prebiotic oligosaccharides modifies the morphology of microcapsules and improves the protection ability of strains. Through FTIR analysis, it is confirmed that free bacteria and wall materials are stably embedded in the four microcapsules through ionic crosslinking reaction.

[0151] (6) The number of live bacteria in all microcapsules and freeze-dried bacterial powders decreased significantly after storage at 25°C, while the storage effect was better at 4°C and -20°C. At the three storage temperatures, the survival rate of the microcapsules encapsulated with prebiotics was significantly higher than that of the microcapsules without prebiotics under the same conditions. Comprehensive analysis showed that microcapsule B prepared by combining xanthan gum and prebiotics had the best protection for the vitality of the strains during storage.

Claims

1. A double-layer microcapsule of Akkermansia muciniphila combined with prebiotics, characterized in that: The invention is prepared from bacteria and mixed gum, wherein the weight ratio of the bacteria to the mixed gum is 1:5, the bacteria is Akkermansia muciniphila, and the mixed gum comprises sodium alginate, carob bean gum or xanthan gum.

2. The double-layer microcapsule according to claim 1, characterized in that The mixed gum also includes galacto-oligosaccharide.

3. The double-layer microcapsule according to claim 1, characterized in that The mixed glue also includes chitosan.

4. A method for preparing Akkermansia muciniphila combined with prebiotics double-layer microcapsules, characterized in that: The steps include: (1) The Akkermansia muciniphila suspension was centrifuged, the supernatant was discarded, the precipitate was collected, and it was washed with sterile PBS; the bacterial precipitate was resuspended with a lyophilization protective agent at a ratio of 1:30 to obtain a high-concentration bacterial suspension; (2) adding the bacterial suspension obtained in step (1) into the mixed gel at a ratio of 1:5, stirring and mixing; (3) uniformly adding the solution obtained in step (2) dropwise to the solidifying liquid, stirring during the adding process, to obtain a single-layer microcapsule; (4) discarding the solidified liquid from the solution obtained in step (3), collecting the microcapsules, washing them with sterile water, transferring them to the chitosan solution, and stirring; (5) discarding the chitosan solution from the solution obtained in step (4), collecting the microcapsules, washing the unreacted chitosan with sterile water, and freeze-drying to obtain double-layer microcapsules; The mixed gum described in step (2) includes sodium alginate, carob bean gum or xanthan gum.

5. The preparation method according to claim 4, characterized in that: The freeze-drying protective agent in step (1) is trehalose and sucrose in a ratio of 1:

1.

6. The preparation method according to claim 4, characterized in that: The solidifying liquid described in step (3) is a calcium chloride solution.

7. The preparation method according to claim 4, characterized in that: The pH of the sterile PBS in step (1) is 7.

0.

8. The preparation method according to claim 4, characterized in that: The stirring was performed using a magnetic stirrer.

9. Double-layer microcapsules prepared by the preparation method according to any one of claims 4 to 8.

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