Pediococcus pentosaceus microcapsule and preparation method thereof

Abstract

The invention discloses a pediococcus pentosaceus microcapsule and a preparation method thereof. The composite embedding agent is prepared by an endogenous emulsification method. According to the microcapsule prepared by the invention, the storage stability, the heat resistance and the antibacterial activity of the pentose tablet ball STS-6 are remarkably improved. The mannan oligosaccharide and the sodium alginate are combined for use, the pullulan and the poly gamma-glutamic acid are combined for use, and the schizophyllum commune polysaccharide and the rice bran protein are combined for use for synergistic interaction.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotic product preparation, and particularly relates to a Pediococcus pentosaceus microcapsule and a preparation method thereof. Background Art

[0002] Pediococcus pentosaceus belongs to the genus Pediococcus of the family Streptococcus. It is a Gram-positive bacterium and a lactic acid bacterium. Pediococcus pentosaceus has been approved as a safe strain in my country. It has beneficial functions such as inhibiting foodborne pathogens, regulating intestinal immune function, and lowering cholesterol. The International Dairy Federation (IDF) stipulates that the content of probiotics in food must reach at least 10 7 However, the survival rate of probiotics is affected by many factors, such as environmental stress during processing and storage, the low pH value of gastric acid, and the high bile salt environment. This significantly reduces the vitality of probiotics when they reach the intestine, thus limiting their effectiveness.

[0003] So far, in order to solve the challenges of poor environmental stability and low gastrointestinal activity of probiotics, people have proposed a variety of strategies to improve the tolerance of strains. Among them, microencapsulation can enhance the resistance of probiotics to adverse external environments such as temperature, pH, light, and oxygen, and is also beneficial to the long-term storage stability and tolerance of probiotics in the gastrointestinal environment. In addition, certain bioactive compounds, such as carbohydrates (such as prebiotics), proteins (such as milk protein and plant protein) and other substances (such as omega-3 fatty acids and curcumin), are used as protective or excipient ingredients co-encapsulated with probiotics. At present, prebiotics commonly used in such co-encapsulated microcapsules include fructooligosaccharides, inulin, and galacto-oligosaccharides. Manno-oligosaccharides are oligosaccharides composed of mannose units, which are connected by β-1,4 glycosidic bonds. They have the effect of promoting the growth of probiotics and have attracted widespread attention as a new type of prebiotics. Co-encapsulation with prebiotics has been shown to promote the bioactivity of host bacteria and help improve the survival of probiotics during storage and exposure to gastrointestinal conditions. To date, there have been no reports on the preparation of Pediococcus pentosaceus microcapsules based on mannooligosaccharides. Summary of the Invention

[0004] The purpose of the present invention is to provide a Pediococcus pentosaceus microcapsule and a preparation method thereof.

[0005] The invention discloses a Pediococcus pentosaceus microcapsule, which is prepared by adopting a composite embedding agent and an endogenous emulsification method.

[0006] Preferably, the composite embedding agent comprises 0.8-1.8 wt% mannooligosaccharide, 2-5 wt% sodium alginate, and 0.5-1.2 wt% chitosan, and preferably comprises 1.4 wt% mannooligosaccharide, 3.5 wt% sodium alginate, and 0.8 wt% chitosan.

[0007] Preferably, the composite embedding agent comprises 1-1.5 wt% pullulan and 2-4 wt% poly-gamma-glutamic acid, and preferably comprises 1.2 wt% pullulan and 3 wt% poly-gamma-glutamic acid.

[0008] Preferably, the composite embedding agent comprises 0.5-1.5 wt% of Schizophyllan and 2.5-4.5 wt% of rice bran protein, and preferably comprises 0.5-1.5 wt% of Schizophyllan and 2.5-4.5 wt% of rice bran protein.

[0009] The preparation method of the Pediococcus pentosaceus microcapsules is carried out according to the following steps:

[0010] (1) Take 20-40 mL of Pediococcus pentosaceus suspension and 200-400 mL of embedding agent solution and mix them evenly. Disperse 0.6-1.2 g of CaCO3 powder into the mixed solution. Then, add the suspension to the oil phase at a water-to-oil ratio of 1:2-4 and mechanically stir for 10-20 min to form water-in-oil droplets.

[0011] (2) Add 300-500 μL of glacial acetic acid and continue stirring for 20-40 min, then add 40-80 mL of washing medium, stir slowly and let stand for 1-3 h, remove the upper oil phase by aspiration, and collect the microcapsules by centrifugation;

[0012] (3) Wash with sterile saline 2-4 times to remove the oil phase and unencapsulated bacteria on the surface of the microcapsules and store at 2-8°C.

[0013] The oil phase is soybean oil containing 1 wt% Span-80; the washing medium is acetate solution with a pH of 5.5.

[0014] The preparation method of the Pediococcus pentosaceus suspension is as follows: Pediococcus pentosaceus is inoculated into MRS liquid culture medium, cultured at 37°C for 16-24 hours, centrifuged at 4000-6000 r / min for 8-12 minutes, and the bacterial sludge is washed with sterile physiological saline and resuspended. The number of viable bacteria is 10 9 -10 10 CFU / mL, and stored in a 4°C refrigerator for future use.

[0015] The embedding agent solution comprises 0.8-1.8 wt% mannooligosaccharide solution, 2-5 wt% sodium alginate solution and 0.5-1.2 wt% chitosan solution, and the volume ratio is (0.2-3): (0.2-3):1.

[0016] The embedding agent solution comprises 1-1.5 wt% pullulan solution and 2-4 wt% poly-gamma-glutamic acid solution, and the volume ratio is (0.2-3):1.

[0017] The embedding agent solution comprises 0.5-1.5 wt% schizophyllan solution and 2.5-4.5 wt% rice bran protein solution, and the dosage volume ratio is (0.2-3):1.

[0018] The present invention has the beneficial effects of significantly improving the storage stability, heat resistance, and antibacterial properties of Pentosan STS-6. The combination of mannooligosaccharide and sodium alginate, pullulan and poly-γ-glutamic acid, and schizophyllan and rice bran protein enhances synergy. DETAILED DESCRIPTION

[0019] To facilitate understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0020] Example 1

[0021] A method for preparing Pediococcus pentosaceus microcapsules is carried out according to the following steps:

[0022] (1) 30 mL of Pediococcus pentosaceus suspension and 300 mL of embedding agent solution were mixed evenly, 0.8 g of CaCO3 powder was dispersed in the mixed solution, and then the suspension was added to the oil phase (soybean oil containing 1 wt% Span-80) according to a water-to-oil ratio of 1:3, and mechanically stirred for 15 minutes to form oil-in-water droplets; the embedding agent solution was 1.2 wt% mannooligosaccharide solution, 3 wt% sodium alginate solution, and 0.8 wt% chitosan solution, with a volume ratio of 1:1:1;

[0023] The preparation method of the Pediococcus pentosaceus suspension is as follows: Pediococcus pentosaceus is inoculated into MRS liquid culture medium, cultured at 37°C for 20 hours, centrifuged at 5000 rpm for 10 minutes, and the bacterial sludge is washed with sterile physiological saline and resuspended. The number of viable bacteria is 5×10 9 CFU / mL, stored in a 4°C refrigerator for future use;

[0024] (2) Add 400 μL of glacial acetic acid and continue stirring for 30 min. Then add 60 mL of washing medium (acetate solution, pH 5.5), stir slowly and let it stand for 2 h. Remove the upper oil phase by aspiration and collect the microcapsules by centrifugation.

[0025] (3) Wash the microcapsules three times with sterile saline to remove the oil phase and unencapsulated bacteria on the surface of the microcapsules and store them at 4°C.

[0026] Example 2

[0027] A method for preparing Pediococcus pentosaceus microcapsules is carried out according to the following steps:

[0028] (1) 25 mL of Pediococcus pentosaceus suspension and 240 mL of embedding agent solution were mixed evenly, 0.7 g of CaCO3 powder was dispersed into the mixed solution, and then the suspension was added to the oil phase (1 wt% Span-80 soybean oil) at a water-to-oil ratio of 1:2, and mechanically stirred for 12 minutes to form water-in-oil droplets; the embedding agent solution was 1.2 wt% pullulan solution and 3 wt% poly-gamma-glutamic acid solution, with a volume ratio of 2:1;

[0029] The preparation method of the Pediococcus pentosaceus suspension is as follows: Pediococcus pentosaceus is inoculated into MRS liquid culture medium, cultured at 37°C for 20 hours, centrifuged at 5000 rpm for 10 minutes, and the bacterial sludge is washed with sterile physiological saline and resuspended. The number of viable bacteria is 5×10 9 CFU / mL, stored in a 4°C refrigerator for future use;

[0030] (2) Add 320 μL of glacial acetic acid and continue stirring for 22 min. Then add 45 mL of washing medium (acetate solution, pH 5.5), stir slowly and let it stand for 1.5 h. Remove the upper oil phase by aspiration and collect the microcapsules by centrifugation.

[0031] (3) Wash twice with sterile saline to remove the oil phase and unencapsulated bacteria on the surface of the microcapsules and store at 4°C.

[0032] Example 3

[0033] A method for preparing Pediococcus pentosaceus microcapsules is carried out according to the following steps:

[0034] (1) 40 mL of a Pediococcus pentosaceus suspension and 400 mL of an embedding agent solution were mixed evenly, 1.2 g of CaCO3 powder was dispersed into the mixed solution, and then the suspension was added to an oil phase (1 wt% Span-80 soybean oil) at a water-to-oil ratio of 1:4, and mechanically stirred for 20 min to form water-in-oil droplets; the embedding agent solution was a 1.5 wt% Schizophyllan solution and a 3.5 wt% rice bran protein solution, with a volume ratio of 1:4;

[0035] The preparation method of the Pediococcus pentosaceus suspension is as follows: Pediococcus pentosaceus is inoculated into MRS liquid culture medium, cultured at 37°C for 20 hours, centrifuged at 5000 rpm for 10 minutes, and the bacterial sludge is washed with sterile physiological saline and resuspended. The number of viable bacteria is 5×10 9 CFU / mL, stored in a 4°C refrigerator for future use;

[0036] (2) Add 500 μL of glacial acetic acid and continue stirring for 40 min. Then add 80 mL of washing medium (acetate solution, pH 5.5), stir slowly and let it stand for 3 h. Remove the upper oil phase by aspiration and collect the microcapsules by centrifugation.

[0037] (3) Wash with sterile saline 4 times to remove the oil phase and unencapsulated bacteria on the surface of the microcapsules and store at 6°C.

[0038] Comparative Example 1

[0039] A method for preparing Pediococcus pentosaceus microcapsules is carried out according to the following steps:

[0040] (1) 30 mL of Pediococcus pentosaceus suspension and 300 mL of embedding agent solution were mixed evenly, 0.8 g of CaCO3 powder was dispersed in the mixed solution, and then the suspension was added to the oil phase (soybean oil containing 1 wt% Span-80) at a water-to-oil ratio of 1:3, and mechanically stirred for 15 minutes to form water-in-oil droplets; the embedding agent solution was 4.2 wt% mannooligosaccharide solution and 0.8 wt% chitosan solution, with a volume ratio of 2:1;

[0041] The preparation method of the Pediococcus pentosaceus suspension is as follows: Pediococcus pentosaceus is inoculated into MRS liquid culture medium, cultured at 37°C for 20 hours, centrifuged at 5000 rpm for 10 minutes, and the bacterial sludge is washed with sterile physiological saline and resuspended. The number of viable bacteria is 5×10 9 CFU / mL, stored in a 4°C refrigerator for future use;

[0042] (2) Add 400 μL of glacial acetic acid and continue stirring for 30 min. Then add 60 mL of washing medium (acetate solution, pH 5.5), stir slowly and let it stand for 2 h. Remove the upper oil phase by aspiration and collect the microcapsules by centrifugation.

[0043] (3) Wash the microcapsules three times with sterile saline to remove the oil phase and unencapsulated bacteria on the surface of the microcapsules and store them at 4°C.

[0044] Comparative Example 2

[0045] A method for preparing Pediococcus pentosaceus microcapsules is carried out according to the following steps:

[0046] (1) 30 mL of Pediococcus pentosaceus suspension and 300 mL of embedding agent solution were mixed evenly, 0.8 g of CaCO3 powder was dispersed in the mixed solution, and then the suspension was added to the oil phase (soybean oil containing 1 wt% Span-80) at a water-to-oil ratio of 1:3, and mechanically stirred for 15 minutes to form water-in-oil droplets; the embedding agent solution was 4.2 wt% sodium alginate solution and 0.8 wt% chitosan solution, with a volume ratio of 2:1;

[0047] The preparation method of the Pediococcus pentosaceus suspension is as follows: Pediococcus pentosaceus is inoculated into MRS liquid culture medium, cultured at 37°C for 20 hours, centrifuged at 5000 rpm for 10 minutes, and the bacterial sludge is washed with sterile physiological saline and resuspended. The number of viable bacteria is 5×10 9 CFU / mL, stored in a 4°C refrigerator for future use;

[0048] (2) Add 400 μL of glacial acetic acid and continue stirring for 30 min. Then add 60 mL of washing medium (acetate solution, pH 5.5), stir slowly and let it stand for 2 h. Remove the upper oil phase by aspiration and collect the microcapsules by centrifugation.

[0049] (3) Wash the microcapsules three times with sterile saline to remove the oil phase and unencapsulated bacteria on the surface of the microcapsules and store them at 4°C.

[0050] Comparative Example 3

[0051] A method for preparing Pediococcus pentosaceus microcapsules is carried out according to the following steps:

[0052] (1) 25 mL of a Pediococcus pentosaceus suspension and 240 mL of an embedding agent solution were mixed uniformly, 0.7 g of CaCO3 powder was dispersed into the mixed solution, and then the suspension was added to an oil phase (1 wt% Span-80 soybean oil) at a water-to-oil ratio of 1:2, and mechanically stirred for 12 minutes to form water-in-oil droplets; the embedding agent solution was a 4.2 wt% pullulan solution;

[0053] The preparation method of the Pediococcus pentosaceus suspension is as follows: Pediococcus pentosaceus is inoculated into MRS liquid culture medium, cultured at 37°C for 20 hours, centrifuged at 5000 rpm for 10 minutes, and the bacterial sludge is washed with sterile physiological saline and resuspended. The number of viable bacteria is 5×10 9 CFU / mL, stored in a 4°C refrigerator for future use;

[0054] (2) Add 320 μL of glacial acetic acid and continue stirring for 22 min. Then add 45 mL of washing medium (acetate solution, pH 5.5), stir slowly, and let it stand for 1.5 h. Remove the upper oil phase by aspiration and collect the microcapsules by centrifugation.

[0055] (3) Wash twice with sterile saline to remove the oil phase and unencapsulated bacteria on the surface of the microcapsules and store at 4°C.

[0056] Comparative Example 4

[0057] A method for preparing Pediococcus pentosaceus microcapsules is carried out according to the following steps:

[0058] (1) 25 mL of Pediococcus pentosaceus suspension and 240 mL of embedding agent solution were mixed evenly, 0.7 g of CaCO3 powder was dispersed in the mixed solution, and then the suspension was added to the oil phase (1 wt% Span-80 soybean oil) at a water-to-oil ratio of 1:2, and mechanically stirred for 12 minutes to form water-in-oil droplets; the embedding agent solution was a 4.2 wt% poly-gamma-glutamic acid solution;

[0059] The preparation method of the Pediococcus pentosaceus suspension is as follows: Pediococcus pentosaceus is inoculated into MRS liquid culture medium, cultured at 37°C for 20 hours, centrifuged at 5000 rpm for 10 minutes, and the bacterial sludge is washed with sterile physiological saline and resuspended. The number of viable bacteria is 5×10 9 CFU / mL, stored in a 4°C refrigerator for future use;

[0060] (2) Add 320 μL of glacial acetic acid and continue stirring for 22 min. Then add 45 mL of washing medium (acetate solution, pH 5.5), stir slowly and let it stand for 1.5 h. Remove the upper oil phase by aspiration and collect the microcapsules by centrifugation.

[0061] (3) Wash twice with sterile saline to remove the oil phase and unencapsulated bacteria on the surface of the microcapsules and store at 4°C.

[0062] Comparative Example 5

[0063] A method for preparing Pediococcus pentosaceus microcapsules is carried out according to the following steps:

[0064] (1) 40 mL of a Pediococcus pentosaceus suspension and 400 mL of an embedding agent solution were mixed uniformly, 1.2 g of CaCO3 powder was dispersed into the mixed solution, and then the suspension was added to an oil phase (1 wt% Span-80 soybean oil) at a water-to-oil ratio of 1:4, and mechanically stirred for 20 min to form water-in-oil droplets; the embedding agent solution was a 5 wt% Schizophyllan polysaccharide solution;

[0065] The preparation method of the Pediococcus pentosaceus suspension is as follows: Pediococcus pentosaceus is inoculated into MRS liquid culture medium, cultured at 37°C for 20 hours, centrifuged at 5000 rpm for 10 minutes, and the bacterial sludge is washed with sterile physiological saline and resuspended. The number of viable bacteria is 5×10 9 CFU / mL, stored in a 4°C refrigerator for future use;

[0066] (2) Add 500 μL of glacial acetic acid and continue stirring for 40 min. Then add 80 mL of washing medium (acetate solution, pH 5.5), stir slowly and let it stand for 3 h. Remove the upper oil phase by aspiration and collect the microcapsules by centrifugation.

[0067] (3) Wash with sterile saline 4 times to remove the oil phase and unencapsulated bacteria on the surface of the microcapsules and store at 6°C.

[0068] Comparative Example 6

[0069] A method for preparing Pediococcus pentosaceus microcapsules is carried out according to the following steps:

[0070] (1) 40 mL of a Pediococcus pentosaceus suspension and 400 mL of an embedding agent solution were mixed uniformly, 1.2 g of CaCO3 powder was dispersed into the mixed solution, and then the suspension was added to an oil phase (1 wt% Span-80 soybean oil) at a water-to-oil ratio of 1:4, and mechanically stirred for 20 min to form water-in-oil droplets; the embedding agent solution was a 5 wt% rice bran protein solution;

[0071] The preparation method of the Pediococcus pentosaceus suspension is as follows: Pediococcus pentosaceus is inoculated into MRS liquid culture medium, cultured at 37°C for 20 hours, centrifuged at 5000 rpm for 10 minutes, and the bacterial sludge is washed with sterile physiological saline and resuspended. The number of viable bacteria is 5×10 9 CFU / mL, stored in a 4°C refrigerator for future use;

[0072] (2) Add 500 μL of glacial acetic acid and continue stirring for 40 min. Then add 80 mL of washing medium (acetate solution, pH 5.5), stir slowly and let it stand for 3 h. Remove the upper oil phase by aspiration and collect the microcapsules by centrifugation.

[0073] (3) Wash with sterile saline 4 times to remove the oil phase and unencapsulated bacteria on the surface of the microcapsules and store at 6°C.

[0074] Experimental Example 1: Determination of microcapsule embedding efficiency

[0075] Microcapsule lysis solution: Sodium citrate and NaHCO3 were dissolved in distilled water to prepare a mixture of 0.06 mol / L sodium citrate and 0.2 mol / L NaHCO3. The pH was adjusted to 7.6 with HCl solution. The mixture was sterilized in an autoclave at 115°C for 20 min and then cooled for use.

[0076] Add 1g of microcapsules to 9mL of encapsulation solution and shake in a 37℃ shaker (180r / min) for 30min to encapsulate. Samples were diluted and spread on MRS agar. Incubate inverted in a 37℃ incubator and count. At the same time, calculate the number of viable bacteria in the initial bacterial suspension. The encapsulation efficiency of the microcapsules can be expressed as formula (1):

[0077]

[0078] Where: N, the number of viable bacteria of Pediococcus pentosaceus in 1 mL of cystic fluid; V, the volume of cystic fluid; N0, the number of viable bacteria embedded in 1 mL of bacterial suspension; V0, the volume of bacterial suspension used to prepare microcapsules; M, the total mass of prepared microcapsules; M0, the mass taken from cystic fluid.

[0079] The experimental results were statistically analyzed using SPSS 24.0 software, and the measurement data were analyzed using The data were expressed as mean ± standard deviation. The Kolmogorov-Smirnov test was used to test the normality of the data. For data that met the normal distribution, the t-test was used to compare the mean differences between the two groups. The difference was considered statistically significant when P < 0.05. The results are shown in Tables 1-3:

[0080] Table 1

[0081]

[0082] Note: * represents P < 0.05 compared with the group in Example 1.

[0083] Table 2

[0084]

[0085] Note: * represents P < 0.05 compared with the group in Example 2.

[0086] Table 3

[0087]

[0088]

[0089] Note: * represents P < 0.05 compared with the group in Example 3.

[0090] Experimental Example 2: Determination of thermal stability of microcapsules

[0091] 1g of microcapsules were dispersed in 9mL of sterile PBS buffer (0.01M, pH 7.4), mixed, and heat-treated in a 72℃ water bath for 30min. Samples were taken every 15min to count the number of living cells, and the free bacterial suspension was used as a control. The experimental results were statistically analyzed using SPSS24.0 software, and the quantitative data were analyzed using The data were expressed as mean ± standard deviation. The Kolmogorov-Smirnov test was used to test the normality of the data. For data that met the normal distribution, the t-test was used to compare the mean differences between the two groups. The difference was considered statistically significant when P < 0.05. The results are shown in Tables 4-6:

[0092] Table 4

[0093]

[0094] Note: * represents P < 0.05 compared with the group in Example 1.

[0095] Table 5

[0096]

[0097] Note: * represents P < 0.05 compared with the group in Example 2.

[0098] Table 6

[0099]

[0100] Note: * represents P < 0.05 compared with the group in Example 3.

[0101] Experimental Example 3: Gastric juice tolerance test

[0102] Simulated gastric fluid: Add HCl to a 2 g / L NaCl solution until the pH of the solution reaches 2.0. Add pepsin to the solution and stir thoroughly to dissolve it to ensure that the final concentration of pepsin is 0.3 g / L. Filter and sterilize.

[0103] 1g of microcapsules were dispersed into 9mL of simulated gastric fluid preheated at 37℃, and shaken at 37℃ and 180r / min for 2h. Samples were taken at 0, 30, 60, 90, and 120min, and the number of living cells was calculated. The experimental results were statistically analyzed using SPSS24.0 software. The quantitative data were analyzed using The data were expressed as mean ± standard deviation. The Kolmogorov-Smirnov test was used to test the normality of the data. For data that met the normal distribution, the t-test was used to compare the mean differences between the two groups. The difference was considered statistically significant when P < 0.05. The results are shown in Tables 7-9:

[0104] Table 7

[0105]

[0106] Note: * represents P < 0.05 compared with the group in Example 1.

[0107] Table 8

[0108]

[0109] Note: * represents P < 0.05 compared with the group in Example 2.

[0110] Table 9

[0111]

[0112] Note: * represents P < 0.05 compared with the group in Example 3.

[0113] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A Pediococcus pentosaceus microcapsule, characterized in that: The composite embedding medium was prepared by endogenous emulsification method.

2. The Pediococcus pentosaceus microcapsule according to claim 1, characterized in that The composite embedding agent comprises 0.8-1.8 wt% mannooligosaccharide, 2-5 wt% sodium alginate, and 0.5-1.2 wt% chitosan, and preferably comprises 1.4 wt% mannooligosaccharide, 3.5 wt% sodium alginate, and 0.8 wt% chitosan.

3. The Pediococcus pentosaceus microcapsule according to claim 1, characterized in that The composite embedding agent comprises 1-1.5 wt% pullulan and 2-4 wt% poly-gamma-glutamic acid, and preferably comprises 1.2 wt% pullulan and 3 wt% poly-gamma-glutamic acid.

4. The Pediococcus pentosaceus microcapsule according to claim 1, characterized in that The composite embedding agent comprises 0.5-1.5 wt% of Schizophyllan and 2.5-4.5 wt% of rice bran protein, and preferably comprises 0.5-1.5 wt% of Schizophyllan and 2.5-4.5 wt% of rice bran protein.

5. The method for preparing the Pediococcus pentosaceus microcapsules according to claim 1, characterized in that: Follow these steps: (1) Take 20-40 mL of Pediococcus pentosaceus suspension and 200-400 mL of embedding agent solution and mix them evenly. Disperse 0.6-1.2 g of CaCO3 powder into the mixed solution. Then, add the suspension to the oil phase at a water-to-oil ratio of 1:2-4 and mechanically stir for 10-20 min to form water-in-oil droplets. (2) Add 300-500 μL of glacial acetic acid and continue stirring for 20-40 min, then add 40-80 mL of washing medium, stir slowly and let stand for 1-3 h, remove the upper oil phase by aspiration, and collect the microcapsules by centrifugation; (3) Wash with sterile saline 2-4 times to remove the oil phase and unencapsulated bacteria on the surface of the microcapsules and store at 2-8°C.

6. The method for preparing Pediococcus pentosaceus microcapsules according to claim 5, characterized in that: The oil phase is soybean oil containing 1 wt% Span-80; the washing medium is acetate solution with a pH of 5.

5.

7. The method for preparing Pediococcus pentosaceus microcapsules according to claim 5, characterized in that: The preparation method of the Pediococcus pentosaceus suspension is as follows: Pediococcus pentosaceus is inoculated into MRS liquid culture medium, cultured at 37°C for 16-24 hours, centrifuged at 4000-6000 r / min for 8-12 minutes, and the bacterial sludge is washed with sterile physiological saline and resuspended. The number of viable bacteria is 10 9 -10 10 CFU / mL, and stored in a 4°C refrigerator for future use.

8. The method for preparing Pediococcus pentosaceus microcapsules according to claim 5, characterized in that: The embedding agent solution comprises 0.8-1.8 wt% mannooligosaccharide solution, 2-5 wt% sodium alginate solution and 0.5-1.2 wt% chitosan solution, and the volume ratio is (0.2-3): (0.2-3):

1.

9. The method for preparing Pediococcus pentosaceus microcapsules according to claim 5, characterized in that: The embedding agent solution comprises 1-1.5 wt% pullulan solution and 2-4 wt% poly-gamma-glutamic acid solution, and the volume ratio is (0.2-3):

1.

10. The method for preparing Pediococcus pentosaceus microcapsules according to claim 5, characterized in that: The embedding agent solution comprises 0.5-1.5 wt% schizophyllan solution and 2.5-4.5 wt% rice bran protein solution, and the dosage volume ratio is (0.2-3):1.

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