A high-temperature-resistant and acid-resistant lactic acid bacterial microcapsule based on multi-layer coating and a preparation method thereof

Abstract

The application discloses a kind of high-temperature-resistant acid-resistant lactic acid bacteria microcapsules based on multilayer coating and a preparation method thereof, and is mainly related to the technical field of lactic acid bacteria.Compared with the prior art, the high-temperature-resistant acid-resistant lactic acid bacteria microcapsules of the multilayer coating of the application solve the problem of easy inactivation of lactic acid bacteria in high-temperature and acidic environment through the design of multilayer coating structure.The first layer of physical isolation is obtained by preparing an emulsion with lactic acid bacteria and olive oil with olive oil as the continuous phase, reducing the direct contact between the bacterial body and the outside world during subsequent processing.The outer layer wall material is made of a protective agent and sodium alginate, the protective agent is made of natural polysaccharide, citrus cellulose and tapioca starch;Citrus cellulose provides dense film formation, tapioca starch forms a gel to fill the gap when heated, and natural polysaccharide is one of inulin, pullulan and carboxymethyl chitosan.Finally, the high-temperature-resistant acid-resistant lactic acid bacteria microcapsules with good high-temperature resistance and acid resistance are obtained.

Description

Technical Field

[0001] This invention relates to the field of lactic acid bacteria technology, and in particular to a multi-layer coated, heat-resistant and acid-resistant lactic acid bacteria microcapsule and its preparation method. Background Technology

[0002] Lactic acid bacteria are widely used in feed, food, and health products due to their acid-producing properties and good safety profile. Improving the heat resistance of lactic acid bacteria allows strains to withstand processes such as spray drying, pasteurization, or short-term high-temperature heat shock during production without loss of activity. Conversely, improving acid resistance directly affects the survival rate of probiotics in the gastrointestinal tract and the storage stability of products such as fermented milk, preventing post-acidification during low-temperature storage from affecting probiotic activity.

[0003] High-temperature and acid-resistant lactic acid bacteria microcapsules are protective structures formed by encapsulating lactic acid bacteria in functional wall materials using microencapsulation technology. Existing technologies have evolved from early single wall material coating to multi-layer composite wall materials. The wall materials can be compounded with materials such as sodium alginate, β-cyclodextrin, gum arabic, and silica, and the lactic acid bacteria can be encapsulated by methods such as extrusion, emulsification gelation, and fluidized bed coating.

[0004] CN120549171A discloses a lactic acid bacteria microcapsule, its preparation method, and its application. First, sodium alginate, modified montmorillonite, and sterile water are thoroughly mixed. After the system cools, lactic acid bacteria seed culture is added and stirred until homogeneous, forming a uniform emulsion. This emulsion is then sprayed into a calcium chloride solution using an extrusion method, followed by stirring and solidification to obtain microcapsules. Finally, the obtained microcapsules are cultured in a lactic acid bacteria liquid culture medium to obtain the lactic acid bacteria microcapsules. This invention achieves effective protection of lactic acid bacteria through a single coating process combined with a subsequent culture step, improving the wet and heat stability, gastrointestinal resistance, and release effect of lactic acid bacteria in the gastrointestinal tract.

[0005] CN119034631A discloses a method for preparing high-survival-rate double-layer lactic acid bacteria microcapsules. This invention uses hyaluronic acid and hydroxyethyl cellulose as the first layer wall material, reacting them in a calcium chloride solution to generate a single-layer microcapsule. Cross-linking achieves complete encapsulation of the lactic acid bacteria. Pectin, amino acids, starch, and sodium alginate are used as the second layer wall material to further encapsulate the single-layer microcapsules, forming a double-layer wall structure. By specifically adjusting and optimizing the composition of the two wall materials, the survival time and survival rate of the lactic acid bacteria are improved.

[0006] The above-mentioned invention patents do not simultaneously improve the high temperature resistance and acid resistance of lactic acid bacteria microcapsules. Summary of the Invention

[0007] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to simultaneously improve the high temperature resistance and acid resistance of lactic acid bacteria microcapsules.

[0008] To achieve the above objectives, the present invention provides a high-temperature and acid-resistant lactic acid bacteria microcapsule based on multi-layer coating and its preparation method.

[0009] The method for preparing the heat-resistant and acid-resistant lactic acid bacteria microcapsules based on multi-layer coating includes the following steps, in parts by weight:

[0010] (1) Spread the lactic acid bacteria on MRS agar medium and incubate at 35.5-36.5℃ for 18-24 hours. Take a single colony and inoculate it into MRS liquid medium. Incubate at 35.5-36.5℃ for 14-16 hours. Centrifuge at 3-5℃ and 8000-10000r / min for 8-10 min. Discard the supernatant. Wash with 8-12 portions of water 2-4 times and collect to obtain a lactic acid bacteria suspension.

[0011] (2) Add 6-10 parts of lactic acid bacteria suspension to a mixture of 6-10 parts olive oil and 0.1-0.3 parts Tween 80 and stir for 10-20 minutes to obtain lactic acid bacteria emulsion;

[0012] (3) Add 0.5-1.5 parts of natural polysaccharide and 0.5-1.5 parts of citrus cellulose to 8-12 parts of water, heat to 75-85℃, add 2-4 parts of cassava starch and stir for 0.5-1 hours to obtain a protective agent. Add 1-3 parts of sodium alginate, 0.4-0.8 parts of nano Fe3O4 and 8-12 parts of the protective agent to 8-12 parts of water and stir for 15-25 minutes to obtain a wall material solution.

[0013] (4) Add 12-20 parts of lactic acid bacteria emulsion to 25-35 parts of wall material solution and mix for 10-20 minutes. Then, add it to 35-45 parts of 4-6 wt% sodium tripolyphosphate aqueous solution using a syringe. Add hydrochloric acid aqueous solution to adjust the pH to acidic. Stir slowly for 10-14 hours to carry out ionic cross-linking. Filter, wash the filter cake with water 1-3 times, and freeze dry to obtain the multi-layer coated high temperature and acid resistant lactic acid bacteria microcapsules.

[0014] The lactic acid bacteria in step (1) are Lactobacillus plantarum.

[0015] The concentration of the lactic acid bacteria suspension in step (1) is .

[0016] The natural polysaccharide mentioned in step (3) is one or more of inulin, pullulan, and carboxymethyl chitosan.

[0017] In step (4), the sodium tripolyphosphate aqueous solution is a 4-6 wt% sodium tripolyphosphate aqueous solution.

[0018] The hydrochloric acid aqueous solution in step (4) is a 1-2 wt% hydrochloric acid aqueous solution.

[0019] In step (4), adjusting the pH to acidity means adjusting the pH to 4.5-5.0.

[0020] The multi-layered, heat- and acid-resistant lactic acid bacteria microcapsules of this invention address the problem of lactic acid bacteria's easy inactivation in high-temperature and acidic environments through a multi-layered coating structure design. The selection of each layer's structure and raw materials works synergistically to stabilize bacterial activity. First, probiotics and olive oil are made into an emulsion with olive oil as the continuous phase. Olive oil, as an oil phase carrier, can encapsulate the bacteria to form a physically isolated inner layer, reducing direct contact between the bacteria and the external environment during subsequent processing. Tween 80 acts as an emulsifier, allowing the lactic acid bacteria to be evenly dispersed in the oil phase, avoiding bacterial aggregation that would lead to uneven coating. The outer wall material uses a protective agent and sodium alginate as raw materials. The protective agent is made from natural polysaccharides, citrus cellulose, and tapioca starch. Citrus cellulose itself has good film-forming properties and mechanical strength. As a microcapsule shell material, it can form a dense porous network. As a protective agent, it helps to improve the integrity and film density of the capsule in the wall material. Tapioca starch forms a dense gel during heating, filling the gaps between cellulose and natural polysaccharides and reducing the penetration channels of heat and gastric acid. The natural polysaccharide is one of inulin, pullulan, and carboxymethyl chitosan. Carboxymethyl chitosan has cationic properties and can combine with the anionic groups of citrus cellulose to make the outer structure containing the protective agent more dense. Inulin and pullulan adhere tightly to other components through intermolecular hydrogen bonds, which helps to improve the structural stability of the protective agent. Sodium alginate possesses excellent biocompatibility and film-forming ability. The metal ions of nano-Fe3O4 can undergo ionic cross-linking with the carboxyl groups of sodium alginate, improving the mechanical strength of the wall material. The phosphate anions of sodium tripolyphosphate and the protonated amino cations of carboxymethyl chitosan form secondary ionic cross-linking through electrostatic interaction, promoting the interaction between sodium alginate and the surface active groups of the protective layer, resulting in high-temperature and acid-resistant multilayer coated microcapsules of lactic acid bacteria with good high-temperature and acid resistance.

[0021] The beneficial effects of this invention are:

[0022] Compared with existing technologies, the multi-layered coated high-temperature and acid-resistant lactic acid bacteria microcapsules of the present invention solve the problem of easy inactivation of lactic acid bacteria in high-temperature and acidic environments through a multi-layered coating structure design. The inner layer is an emulsion made of lactic acid bacteria and olive oil, with olive oil as the continuous phase, which provides the first layer of physical isolation, reducing the direct contact between the bacteria and the outside environment during subsequent processing. The outer wall material is made from a protective agent and sodium alginate. The protective agent is made from natural polysaccharides, citrus cellulose, and tapioca starch. Citrus cellulose provides a dense film, tapioca starch forms a gel upon heating to fill the gaps, and the natural polysaccharide is one of inulin, pullulan, and carboxymethyl chitosan. The addition of nano-Fe3O4 and sodium tripolyphosphate acts as a cross-linking agent to promote the interaction between sodium alginate and the surface active groups of the protective agent layer, ultimately obtaining high-temperature and acid-resistant multi-layered coated high-temperature and acid-resistant lactic acid bacteria microcapsules. Detailed Implementation

[0023] The parameters of the specific chemical substances used in the examples are from the following sources:

[0024] Lactobacillus plantarum: A commercially available product, purchased from the China Industrial Microbial Culture Collection Center, strain number CICC25034.

[0025] Citrus cellulose: Manufacturer: Jiangsu Caiwei Biotechnology Co., Ltd., Model: CB24656166.

[0026] All water used in the examples was sterile water. Example 1

[0027] A method for preparing heat-resistant and acid-resistant lactic acid bacteria microcapsules based on multi-layer coating includes the following steps:

[0028] (1) Spread *Lactobacillus plantarum* on MRS agar medium and incubate at 36°C for 20 hours. Inoculate a single colony into MRS liquid medium and incubate at 36°C for 15 hours. Centrifuge at 4°C and 9000 r / min for 9 minutes, discard the supernatant, wash three times with 10 g of water, and collect the resulting lactic acid bacteria suspension with a concentration of [missing value]. ;

[0029] (2) Add 8g of lactic acid bacteria suspension to a mixture of 8g olive oil and 0.2g Tween 80 and stir for 15 minutes to obtain lactic acid bacteria emulsion;

[0030] (3) Add 1g inulin and 1g citrus cellulose to 10g water, heat to 80℃, add 3g tapioca starch and stir for 0.5 hours to obtain a protective agent. Add 2g sodium alginate, 0.6g 20nm nano Fe3O4 and 10g protective agent to 10g water and stir for 20 minutes to obtain a wall material solution.

[0031] (4) 16g of lactic acid bacteria emulsion was dropped into 30g of wall material solution and mixed and stirred for 15 minutes. Then, it was dropped into 40g of 5wt% sodium tripolyphosphate aqueous solution through a syringe. 1.5wt% hydrochloric acid aqueous solution was added to adjust the pH to 4.8. The mixture was slowly stirred for 12 hours to carry out ionic cross-linking. The mixture was filtered, the filter cake was washed twice with water, and then freeze-dried to obtain the multi-layer coated high temperature and acid resistant lactic acid bacteria microcapsules. Example 2

[0032] A method for preparing heat-resistant and acid-resistant lactic acid bacteria microcapsules based on multi-layer coating includes the following steps:

[0033] (1) Spread *Lactobacillus plantarum* on MRS agar medium and incubate at 36°C for 20 hours. Inoculate a single colony into MRS liquid medium and incubate at 36°C for 15 hours. Centrifuge at 4°C and 9000 r / min for 9 minutes, discard the supernatant, wash three times with 10 g of water, and collect the resulting lactic acid bacteria suspension with a concentration of [missing value]. ;

[0034] (2) Add 8g of lactic acid bacteria suspension to a mixture of 8g olive oil and 0.2g Tween 80 and stir for 15 minutes to obtain lactic acid bacteria emulsion;

[0035] (3) Add 1g pullulan polysaccharide and 1g citrus cellulose to 10g water, heat to 80℃, add 3g tapioca starch and stir for 0.5 hours to obtain a protective agent. Add 2g sodium alginate, 0.6g 20nm nano Fe3O4 and 10g protective agent to 10g water and stir for 20 minutes to obtain a wall material solution.

[0036] (4) 16g of lactic acid bacteria emulsion was dropped into 30g of wall material solution and mixed and stirred for 15 minutes. Then, it was dropped into 40g of 5wt% sodium tripolyphosphate aqueous solution through a syringe. 1.5wt% hydrochloric acid aqueous solution was added to adjust the pH to 4.8. The mixture was slowly stirred for 12 hours to carry out ionic cross-linking. The mixture was filtered, the filter cake was washed twice with water, and then freeze-dried to obtain the multi-layer coated high temperature and acid resistant lactic acid bacteria microcapsules. Example 3

[0037] A method for preparing heat-resistant and acid-resistant lactic acid bacteria microcapsules based on multi-layer coating includes the following steps:

[0038] (1) Spread *Lactobacillus plantarum* on MRS agar medium and incubate at 36°C for 20 hours. Inoculate a single colony into MRS liquid medium and incubate at 36°C for 15 hours. Centrifuge at 4°C and 9000 r / min for 9 minutes, discard the supernatant, wash three times with 10 g of water, and collect the resulting lactic acid bacteria suspension with a concentration of [missing value]. ;

[0039] (2) Add 8g of lactic acid bacteria suspension to a mixture of 8g olive oil and 0.2g Tween 80 and stir for 15 minutes to obtain lactic acid bacteria emulsion;

[0040] (3) Add 1g of carboxymethyl chitosan and 1g of citrus cellulose to 10g of water, heat to 80℃, add 3g of tapioca starch and stir for 0.5 hours to obtain a protective agent. Add 2g of sodium alginate, 0.6g of 20nm nano Fe3O4 and 10g of protective agent to 10g of water and stir for 20 minutes to obtain a wall material solution.

[0041] (4) 16g of lactic acid bacteria emulsion was dropped into 30g of wall material solution and mixed and stirred for 15 minutes. Then, it was dropped into 40g of 5wt% sodium tripolyphosphate aqueous solution through a syringe. 1.5wt% hydrochloric acid aqueous solution was added to adjust the pH to 4.8. The mixture was slowly stirred for 12 hours to carry out ionic cross-linking. The mixture was filtered, the filter cake was washed twice with water, and then freeze-dried to obtain the multi-layer coated high temperature and acid resistant lactic acid bacteria microcapsules. Example 4

[0042] A method for preparing heat-resistant and acid-resistant lactic acid bacteria microcapsules based on multi-layer coating includes the following steps:

[0043] (1) Spread *Lactobacillus plantarum* on MRS agar medium and incubate at 36°C for 20 hours. Inoculate a single colony into MRS liquid medium and incubate at 36°C for 15 hours. Centrifuge at 4°C and 9000 r / min for 9 minutes, discard the supernatant, wash three times with 10 g of water, and collect the resulting lactic acid bacteria suspension with a concentration of [missing value]. ;

[0044] (2) Add 8g of lactic acid bacteria suspension to a mixture of 8g olive oil and 0.2g Tween 80 and stir for 15 minutes to obtain lactic acid bacteria emulsion;

[0045] (3) Add 2g of carboxymethyl chitosan to 10g of water, heat to 80℃, add 3g of cassava starch and stir for 0.5 hours to obtain a protective agent. Add 2g of sodium alginate, 0.6g of 20nm nano Fe3O4 and 10g of protective agent to 10g of water and stir for 20 minutes to obtain a wall material solution.

[0046] (4) 16g of lactic acid bacteria emulsion was dropped into 30g of wall material solution and mixed and stirred for 15 minutes. Then, it was dropped into 40g of 5wt% sodium tripolyphosphate aqueous solution through a syringe. 1.5wt% hydrochloric acid aqueous solution was added to adjust the pH to 4.8. The mixture was slowly stirred for 12 hours to carry out ionic cross-linking. The mixture was filtered, the filter cake was washed twice with water, and then freeze-dried to obtain the multi-layer coated high temperature and acid resistant lactic acid bacteria microcapsules. Example 5

[0047] A method for preparing heat-resistant and acid-resistant lactic acid bacteria microcapsules based on multi-layer coating includes the following steps:

[0048] (1) Spread *Lactobacillus plantarum* on MRS agar medium and incubate at 36°C for 20 hours. Inoculate a single colony into MRS liquid medium and incubate at 36°C for 15 hours. Centrifuge at 4°C and 9000 r / min for 9 minutes, discard the supernatant, wash three times with 10 g of water, and collect the resulting lactic acid bacteria suspension with a concentration of [missing value]. ;

[0049] (2) Add 8g of lactic acid bacteria suspension to a mixture of 8g olive oil and 0.2g Tween 80 and stir for 15 minutes to obtain lactic acid bacteria emulsion;

[0050] (3) Add 2g of citrus cellulose to 10g of water, heat to 80℃, add 3g of tapioca starch and stir for 0.5 hours to obtain a protective agent. Add 2g of sodium alginate, 0.6g of 20nm nano Fe3O4 and 10g of protective agent to 10g of water and stir for 20 minutes to obtain a wall material solution.

[0051] (4) 16g of lactic acid bacteria emulsion was dropped into 30g of wall material solution and mixed and stirred for 15 minutes. Then, it was dropped into 40g of 5wt% sodium tripolyphosphate aqueous solution through a syringe. 1.5wt% hydrochloric acid aqueous solution was added to adjust the pH to 4.8. The mixture was slowly stirred for 12 hours to carry out ionic cross-linking. The mixture was filtered, the filter cake was washed twice with water, and then freeze-dried to obtain the multi-layer coated high temperature and acid resistant lactic acid bacteria microcapsules.

[0052] Comparative Example 1

[0053] A method for preparing heat-resistant and acid-resistant lactic acid bacteria microcapsules based on multi-layer coating includes the following steps:

[0054] (1) Spread *Lactobacillus plantarum* on MRS agar medium and incubate at 36°C for 20 hours. Inoculate a single colony into MRS liquid medium and incubate at 36°C for 15 hours. Centrifuge at 4°C and 9000 r / min for 9 minutes, discard the supernatant, wash three times with 10 g of water, and collect the resulting lactic acid bacteria suspension with a concentration of [missing value]. ;

[0055] (2) Add 8g of lactic acid bacteria suspension to a mixture of 8g olive oil and 0.2g Tween 80 and stir for 15 minutes to obtain lactic acid bacteria emulsion;

[0056] (3) Add 1g of carboxymethyl chitosan and 1g of citrus cellulose to 10g of water to obtain a protective agent. Add 2g of sodium alginate, 0.6g of 20nm nano Fe3O4 and 10g of protective agent to 10g of water and stir for 20 minutes to obtain a wall material solution.

[0057] (4) 16g of lactic acid bacteria emulsion was dropped into 30g of wall material solution and mixed and stirred for 15 minutes. Then, it was dropped into 40g of 5wt% sodium tripolyphosphate aqueous solution through a syringe. 1.5wt% hydrochloric acid aqueous solution was added to adjust the pH to 4.8. The mixture was slowly stirred for 12 hours to carry out ionic cross-linking. The mixture was filtered, the filter cake was washed twice with water, and then freeze-dried to obtain the multi-layer coated high temperature and acid resistant lactic acid bacteria microcapsules.

[0058] Comparative Example 2

[0059] A method for preparing heat-resistant and acid-resistant lactic acid bacteria microcapsules based on multi-layer coating includes the following steps:

[0060] (1) Spread *Lactobacillus plantarum* on MRS agar medium and incubate at 36°C for 20 hours. Inoculate a single colony into MRS liquid medium and incubate at 36°C for 15 hours. Centrifuge at 4°C and 9000 r / min for 9 minutes, discard the supernatant, wash three times with 10 g of water, and collect the resulting lactic acid bacteria suspension with a concentration of [missing value]. ;

[0061] (2) Add 8g of lactic acid bacteria suspension to a mixture of 8g olive oil and 0.2g Tween 80 and stir for 15 minutes to obtain lactic acid bacteria emulsion;

[0062] (3) Add 2g of carboxymethyl chitosan to 10g of water to obtain a protective agent. Add 2g of sodium alginate, 0.6g of 20nm nano Fe3O4 and 10g of protective agent to 10g of water and stir for 20 minutes to obtain a wall material solution.

[0063] (4) 16g of lactic acid bacteria emulsion was dropped into 30g of wall material solution and mixed and stirred for 15 minutes. Then, it was dropped into 40g of 5wt% sodium tripolyphosphate aqueous solution through a syringe. 1.5wt% hydrochloric acid aqueous solution was added to adjust the pH to 4.8. The mixture was slowly stirred for 12 hours to carry out ionic cross-linking. The mixture was filtered, the filter cake was washed twice with water, and then freeze-dried to obtain the multi-layer coated high temperature and acid resistant lactic acid bacteria microcapsules.

[0064] Comparative Example 3

[0065] A method for preparing heat-resistant and acid-resistant lactic acid bacteria microcapsules based on multi-layer coating includes the following steps:

[0066] (1) Spread *Lactobacillus plantarum* on MRS agar medium and incubate at 36°C for 20 hours. Inoculate a single colony into MRS liquid medium and incubate at 36°C for 15 hours. Centrifuge at 4°C and 9000 r / min for 9 minutes, discard the supernatant, wash three times with 10 g of water, and collect the resulting lactic acid bacteria suspension with a concentration of [missing value]. ;

[0067] (2) Add 8g of lactic acid bacteria suspension to a mixture of 8g olive oil and 0.2g Tween 80 and stir for 15 minutes to obtain lactic acid bacteria emulsion;

[0068] (3) Add 2g of citrus cellulose to 10g of water to obtain a protective agent. Add 2g of sodium alginate, 0.6g of 20nm nano Fe3O4 and 10g of protective agent to 20g of water and stir for 10 minutes to obtain a wall material solution.

[0069] (4) 16g of lactic acid bacteria emulsion was dropped into 30g of wall material solution and mixed and stirred for 15 minutes. Then, it was dropped into 40g of 5wt% sodium tripolyphosphate aqueous solution through a syringe. 1.5wt% hydrochloric acid aqueous solution was added to adjust the pH to 4.8. The mixture was slowly stirred for 12 hours to carry out ionic cross-linking. The mixture was filtered, the filter cake was washed twice with water, and then freeze-dried to obtain the multi-layer coated high temperature and acid resistant lactic acid bacteria microcapsules.

[0070] Comparative Example 4

[0071] A method for preparing heat-resistant and acid-resistant lactic acid bacteria microcapsules based on multi-layer coating includes the following steps:

[0072] (1) Spread *Lactobacillus plantarum* on MRS agar medium and incubate at 36°C for 20 hours. Inoculate a single colony into MRS liquid medium and incubate at 36°C for 15 hours. Centrifuge at 4°C and 9000 r / min for 9 minutes, discard the supernatant, wash three times with 10 g of water, and collect the resulting lactic acid bacteria suspension with a concentration of [missing value]. ;

[0073] (2) Add 8g of lactic acid bacteria suspension to a mixture of 8g olive oil and 0.2g Tween 80 and stir for 15 minutes to obtain lactic acid bacteria emulsion;

[0074] (3) Add 2g of cassava starch to 10g of water, heat to 80℃ and stir for 0.5 hours to obtain a protective agent. Add 2g of sodium alginate, 0.6g of 20nm nano Fe3O4 and 10g of protective agent to 10g of water and stir for 20 minutes to obtain a wall material solution.

[0075] (4) 16g of lactic acid bacteria emulsion was dropped into 30g of wall material solution and mixed and stirred for 15 minutes. Then, it was dropped into 40g of 5wt% sodium tripolyphosphate aqueous solution through a syringe. 1.5wt% hydrochloric acid aqueous solution was added to adjust the pH to 4.8. The mixture was slowly stirred for 12 hours to carry out ionic cross-linking. The mixture was filtered, the filter cake was washed twice with water, and then freeze-dried to obtain the multi-layer coated high temperature and acid resistant lactic acid bacteria microcapsules.

[0076] Comparative Example 5

[0077] A method for preparing heat-resistant and acid-resistant lactic acid bacteria microcapsules based on multi-layer coating includes the following steps:

[0078] (1) Spread *Lactobacillus plantarum* on MRS agar medium and incubate at 36°C for 20 hours. Inoculate a single colony into MRS liquid medium and incubate at 36°C for 15 hours. Centrifuge at 4°C and 9000 r / min for 9 minutes, discard the supernatant, wash three times with 10 g of water, and collect the resulting lactic acid bacteria suspension with a concentration of [missing value]. ;

[0079] (2) Add 8g of lactic acid bacteria suspension to a mixture of 8g olive oil and 0.2g Tween 80 and stir for 15 minutes to obtain lactic acid bacteria emulsion;

[0080] (3) Add 2g sodium alginate and 0.6g 20nm nano Fe3O4 to 20g water and stir for 20 minutes to obtain a wall material solution;

[0081] (4) 16g of lactic acid bacteria emulsion was dropped into 30g of wall material solution and mixed and stirred for 15 minutes. Then, it was dropped into 40g of 5wt% sodium tripolyphosphate aqueous solution through a syringe. 1.5wt% hydrochloric acid aqueous solution was added to adjust the pH to 4.8. The mixture was slowly stirred for 12 hours to carry out ionic cross-linking. The mixture was filtered, the filter cake was washed twice with water, and then freeze-dried to obtain the multi-layer coated high temperature and acid resistant lactic acid bacteria microcapsules.

[0082] Test Example 1

[0083] The multi-layered, heat-resistant, and acid-resistant lactic acid bacteria microcapsules prepared by the methods in Examples 1-5 and Comparative Examples 1-5 were used as test subjects and subjected to wet heat treatment at 80°C and 15% relative humidity for 90 seconds. 0.6g of citric acid and 2.5g of disodium hydrogen phosphate were dissolved in 100mL of water to obtain a decoating solution. 1g of each of the treated multi-layered, heat-resistant, and acid-resistant lactic acid bacteria microcapsules from Examples 1-5 and Comparative Examples 1-5 were added to 9mL of the decoating solution and stirred for 0.5 hours. After centrifugation, the decoated samples were obtained, and viable cell counts were determined according to Appendix B of the national standard GB 7300.502-2023 "Feed Additives Part 5: Microorganisms Lactobacillus plantarum".

[0084] Dissolve 8.5g of sodium chloride in 1000mL of water by stirring for 0.5 hours, then autoclave at 121℃ for 15 minutes to obtain physiological saline. Dilute the samples from the examples and comparative examples at a ratio of 1:30 to 1g of physiological saline and homogenize for 5 minutes to obtain lactic acid bacteria dilutions. Pour each lactic acid bacteria dilution from the examples and comparative examples into sterile Petri dishes. Take 15mL of MRS medium melted and cooled to 45-50℃ and pour it into the Petri dishes, shake well and let it solidify to prepare agar plates for the corresponding media of Examples 1-5 and Comparative Examples 1-5. Incubate upside down at 36℃ for 24 hours and then count the colonies, i.e., the average number of colonies after treatment at 80℃ and 15wt% humidity. The colony counting method is based on the national standard GB / T 13093-2023 "Determination of Total Bacterial Count in Feed". The average values ​​of the data are summarized in Table 1.

[0085]

[0086] The multi-layer coating structure can slow down the heat conduction rate and improve the heat resistance of microcapsules. The difference in colony count among the various examples and comparative examples in Test Example 1 is mainly due to the synergistic effect of the components of each layer and the structural stability.

[0087] In Examples 1-3, a protective agent was prepared by combining natural polysaccharides, citrus cellulose, and tapioca starch. Citrus cellulose forms a dense porous network within the protective agent system, enhancing the thermal barrier effect by reducing heat penetration into the lactic acid bacteria in the core. Tapioca starch forms a dense structure during heating, filling the gaps between components and reducing heat penetration. It also works in concert with the natural polysaccharides and citrus cellulose to construct a thermal barrier. The difference between Examples 1-3 lies in the use of inulin, pullulan, and carboxymethyl chitosan as natural polysaccharides. Inulin, as a soluble dietary fiber, forms synergistic hydrogen bonds with tapioca starch and citrus cellulose, further densifying the layered structure. Pullulan binds to citrus cellulose and tapioca starch through intermolecular hydrogen bonds, enhancing the density of the thermal barrier and extending the heat transfer path. Carboxymethyl chitosan, with its cationic properties, cross-links with the anionic groups of citrus cellulose, effectively blocking heat conduction. The rigidity of its molecular chains enhances the thermal barrier's resistance to heat deformation, preventing the coating structure from expanding and rupturing at high temperatures, thus avoiding a decrease in the survival rate of viable bacteria. Examples 4 and 5 use only a single component with added carboxymethyl chitosan or citrus cellulose combined with tapioca starch as a protective agent. The lack of citrus cellulose will reduce the density, and the lack of carboxymethyl chitosan will prevent the natural polysaccharide from achieving ionic cross-linking, thus reducing the density. Therefore, the high temperature resistance of Examples 4-5 is lower than that of Examples 1-3.

[0088] Comparative Example 5 did not add the protective agent of the present invention, relying solely on the protection of its layered coating structure. Its high-temperature resistance was lower than other examples and comparative examples, resulting in a reduced number of viable bacteria. Comparative Example 1 used a composition of carboxymethyl chitosan and citrus cellulose as the protective agent, while Comparative Examples 2-4 used single-component carboxymethyl chitosan, citrus cellulose, and tapioca starch as protective agents, respectively. Lacking synergistic effects with other components, their heat barrier structure was loose and had high porosity compared to other examples, allowing for rapid heat penetration. After high-temperature treatment, the number of viable bacteria was significantly lower than in other examples.

[0089] Test Example 2

[0090] The multi-layered, heat- and acid-resistant lactic acid bacteria microcapsules prepared by the methods in Examples 1-5 and Comparative Examples 1-5 were used as test subjects. They were added to 10 mL of simulated gastric fluid at a ratio of 1:10 and left to stand for 2 hours. The simulated gastric fluid was manufactured by Nanjing Yixun Biotechnology Co., Ltd., and its model number is YXZC009.

[0091] 0.6 g of citric acid and 2.5 g of disodium hydrogen phosphate were dissolved in 100 mL of water to obtain a decoated solution. 1 g of the multi-layered, heat-resistant, and acid-resistant lactic acid bacteria microcapsules from Examples 1-5 and Comparative Examples 1-5 were added to 9 mL of the decoated solution and stirred for 0.5 hours. After centrifugation, the decoated samples were obtained, and viable cell counts were performed. The colony counting method was based on the national standard GB / T 13093-2023 "Determination of Total Bacterial Count in Feed". Three samples were prepared for each example and comparative example group, and the average values ​​were taken and summarized as shown in Table 2.

[0092]

[0093] The difference between Examples 1-3 lies in the addition of inulin, pullulan, and carboxymethyl chitosan as natural polysaccharides in the protective agent, respectively. In Example 3, the cationic carboxymethyl chitosan can form a cross-linking structure with the anionic groups of citrus cellulose, improving the stability of the wall material and reducing gastric acid penetration. The gel structure of tapioca starch fills the gaps between components, further blocking the damage of the simulated gastric acid environment to the Lactobacillus nucleus. In Examples 1-2, inulin and pullulan lack cationic components and cannot form ionic cross-links with citrus cellulose, resulting in weaker wall material density and protective effect. Examples 4-5 use only carboxymethyl chitosan and citrus cellulose combined with tapioca starch, resulting in insufficient mechanical strength and integrity, and reduced acid resistance protection. Comparative Example 5 did not add any protective agent, making the stability of the outer wall material of the multi-layered coating structure lower than other examples. The stimulation of simulated gastric juice on the Lactobacillus nucleus leads to a reduction in the number of viable bacteria, resulting in lower acid resistance protection than other examples.

[0094] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for preparing a high-temperature and acid-resistant lactic acid bacterial microcapsule based on a multi-layer coating, characterized by, Includes the following steps, in parts by weight: (1) Spread the lactic acid bacteria on MRS agar medium and incubate at 35.5-36.5℃ for 18-24 hours. Take a single colony and inoculate it into MRS liquid medium. Incubate at 35.5-36.5℃ for 14-16 hours. Centrifuge at 3-5℃ and 8000-10000r / min for 8-10 min. Discard the supernatant. Wash with 8-12 portions of water 2-4 times and collect to obtain a lactic acid bacteria suspension. (2) Add 6-10 parts of lactic acid bacteria suspension to a mixture of 6-10 parts olive oil and 0.1-0.3 parts Tween 80 and stir for 10-20 minutes to obtain lactic acid bacteria emulsion; (3) Add 0.5-1.5 parts of natural polysaccharide and 0.5-1.5 parts of citrus cellulose to 8-12 parts of water, heat to 75-85℃, add 2-4 parts of cassava starch and stir for 0.5-1 hours to obtain a protective agent. Add 1-3 parts of sodium alginate, 0.4-0.8 parts of nano Fe3O4 and 8-12 parts of the protective agent to 8-12 parts of water and stir for 15-25 minutes to obtain a wall material solution. (4) Add 12-20 parts of lactic acid bacteria emulsion to 25-35 parts of wall material solution and mix for 10-20 minutes. Then, add it to 35-45 parts of 4-6 wt% sodium tripolyphosphate aqueous solution using a syringe. Add hydrochloric acid aqueous solution to adjust the pH to acidic. Stir slowly for 10-14 hours to carry out ionic cross-linking. Filter, wash the filter cake with water 1-3 times, and freeze dry to obtain the multi-layer coated high temperature and acid resistant lactic acid bacteria microcapsules.

2. The method for preparing multi-layer coating-based high-temperature-resistant acid-resistant lactic acid bacteria microcapsules according to claim 1, characterized in that, The lactic acid bacteria in step (1) are Lactobacillus plantarum.

3. The method for preparing heat-resistant and acid-resistant lactic acid bacteria microcapsules based on multi-layer coating as described in claim 1, characterized in that, The concentration of the lactic acid bacteria bacterial suspension in step (1) is .

4. The method for preparing multi-layer coating-based high-temperature-resistant acid-resistant lactic acid bacteria microcapsules according to claim 1, characterized in that, The natural polysaccharide mentioned in step (3) is one or more of inulin, pullulan, and carboxymethyl chitosan.

5. The method for preparing multi-layer coating-based high-temperature-resistant acid-resistant lactic acid bacteria microcapsules according to claim 1, characterized in that, In step (4), the sodium tripolyphosphate aqueous solution is a 4-6 wt% sodium tripolyphosphate aqueous solution.

6. The method for preparing multi-layer coating-based high-temperature-resistant acid-resistant lactic acid bacteria microcapsules according to claim 1, characterized in that, The hydrochloric acid aqueous solution in step (4) is a 1-2 wt% hydrochloric acid aqueous solution.

7. The method for preparing multi-layer coating-based high-temperature-resistant acid-resistant lactic acid bacteria microcapsules according to claim 1, characterized by, In step (4), adjusting the pH to acidity means adjusting the pH to 4.5-5.

0.

8. A high temperature and acid resistant lactic acid bacterial microcapsule based on multi-layer coating, characterized by, It is prepared by the preparation method according to any one of claims 1-7.

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