Liquid microcapsule systems, their preparation methods and applications

Abstract

This invention provides a liquid microcapsule system, its preparation method, and its application. The microcapsule system mainly consists of a stable emulsion formed by uniformly dispersing liquid microcapsules. By mass, the liquid microcapsules mainly consist of 0.5-1.2 parts microbial sludge, 1-3 parts enzymatically hydrolyzed soybean protein, 2-8 parts concentrated phospholipids, 8-10 parts soybean oil, 2-10 parts maltodextrin, 0.8-1.2 parts glycerol, and 6-25 parts water. This invention also provides a method for preparing the above-mentioned microcapsule system, which includes vacuum emulsifying the above raw materials. This invention further provides an application of the above-mentioned liquid microcapsules in the processing of microbial products such as health products, food, probiotic preparations, or probiotic feed. The above-mentioned liquid microcapsule system has the characteristics of high survival rate, good heat resistance, good sustained-release properties, and low cost. Furthermore, the preparation method is simple, and the coating after encapsulation increases the survival rate of the microorganisms in processing applications, thereby reducing application and processing costs.

Description

Technical Field

[0001] This invention belongs to the field of microbial additive application and processing, specifically relating to a liquid microcapsule system, its preparation method, and its application. Background Art

[0002] Microbial feed additives are feed additives approved by the Ministry of Agriculture and Rural Affairs of the People's Republic of China. They exert beneficial effects by improving the ecological balance of the intestinal flora in animals, thereby improving animal health, disease resistance, and digestive capacity. They are an effective means to address issues such as rampant diseases, drug resistance, decreased immunity, reduced survival rates, and decreased farming efficiency. Furthermore, microbial preparations have advantages that other drugs cannot replace, namely, the effects of "treating disease when sick, preventing disease when not sick, and maintaining health when healthy." Even healthy animals can use them to improve their health, and they have a bidirectional regulatory effect on diarrhea and constipation.

[0003] Existing microbial feed additives are mainly in powder or granule form. Their production steps primarily include: strain extraction, strain expansion and propagation, finished product dehydration, and mixing and packaging (Sun Xiaoming. Production and Application of Microbial Feed Additives [J]. Animal Husbandry and Feed, 2013, No. 04: pp. 45-46). Among these steps, the microorganisms cultured in the fermenter are processed into powder or granules after the finished product dehydration step. Therefore, in the microbial feed processing, microbial feed additives are prepared by mixing powder or granules with other raw materials. This leads to the following problems: 1. The production process of microbial additives requires drying equipment, resulting in high production costs; 2. The distribution of microbial feed additives is uneven; 3. Microorganisms exhibit poor acid and heat resistance in the intestines, resulting in low survival rates and poor colonization abilities.

[0004] To address this, Chinese invention patent application CN 113832047 A discloses a TGase-crosslinked modified *Lactobacillus reuteri* microcapsule and its preparation method. The microcapsule primarily uses concentrated *Lactobacillus reuteri* bacterial solution as the core material and fish skin gelatin and maltodextrin as the wall material. The wall material is prepared by TGase crosslinking modification. The *Lactobacillus reuteri* is *Lactobacillus reuteri* NCUF203.1. The preparation method disclosed in this patent application is simple to operate, has low energy consumption, and a high viable bacterial survival rate, providing a good method for preparing *Lactobacillus reuteri* microcapsules by spray drying and reducing the production cost of *Lactobacillus reuteri* preparations. The microcapsules obtained by this invention encapsulate significantly more viable *Lactobacillus reuteri* than microcapsules prepared with uncrosslinked fish skin gelatin, and exhibit good resistance to gastric acid, enteric solubility, and storage stability.

[0005] However, although the aforementioned patent application discloses that Lactobacillus reuteri microcapsules still require drying during the preparation process, their application in feed or food processing still suffers from uneven distribution and high production costs. Furthermore, it is difficult to solve the problem that microorganisms are sensitive to adverse factors such as high temperature and pressure, and are easily affected and inactivated during processing and storage. Summary of the Invention

[0006] In view of this, the purpose of this invention is to prepare a liquid microcapsule system. This liquid microcapsule system is prepared by emulsification under vacuum using microbial sludge, enzymatically hydrolyzed soybean protein, concentrated phospholipids, soybean oil, maltodextrin, and glycerol as raw materials. This results in a high survival rate and encapsulation rate of the microorganisms in the liquid microcapsule system. This not only improves the stability and colonization ability of microorganisms in the intestine, but also allows for subsequent processing of feed by spraying after combination, avoiding the problem of low survival rate of microorganisms caused by external factors such as pressure during the processing of microbial products. This reduces the production cost of the liquid microcapsule system and the processing of microbial products.

[0007] Another objective of this invention is to provide a method for preparing a liquid microcapsule system. This method mainly involves mixing raw materials under vacuum and combining them with an emulsification method to obtain the liquid microcapsule system. The preparation method is simple and low in cost.

[0008] Another objective of this invention is to provide an application of a liquid microcapsule system, which is added to microbial-containing products such as health products, food, probiotic agents, and probiotic feeds using a post-coating process. This allows for a more uniform distribution of the microbial-containing product and improves the survival rate of microorganisms in the product. Consequently, the amount of microcapsule system added to the microbial-containing product can be reduced, thereby lowering costs.

[0009] To achieve the above objectives, the present invention protects the following technical solutions:

[0010] A liquid microcapsule system, mainly composed of a stable emulsion formed by uniformly dispersing liquid microcapsules, wherein, by mass parts, the liquid microcapsule system mainly consists of 0.5-1.2 parts of microbial sludge, 1-3 parts of enzymatically hydrolyzed soybean protein, 2-8 parts of concentrated phospholipids, 8-10 parts of soybean oil, 2-10 parts of maltodextrin, 0.8-1.2 parts of glycerol, and 6-25 parts of water.

[0011] Preferably, the particle size distribution of the liquid microcapsules is in the range of 1500-3000 nm, such as 1500 nm, 1600 nm, 1700 nm, 1800 nm, 1900 nm, 2000 nm, 2100 nm, 2200 nm, 2300 nm, 2400 nm, 2500 nm, 2800 nm, 3000 nm, etc.; more preferably, the particle size distribution of the liquid microcapsules is in the range of 1800-2400 mm. The mass percentage of the concentrated phospholipid in the liquid microcapsule system is preferably 5%-20%. The mass percentage of the maltodextrin in the liquid microcapsule system is preferably 5%-25%.

[0012] Preferably, based on 40 parts by weight, the liquid microcapsule system comprises 1 part microbial slurry, 2 parts enzymatically hydrolyzed soybean protein, 3-7 parts concentrated phospholipids, 10 parts soybean oil, 2-10 parts maltodextrin, 1 part glycerol, and the remainder being water. The microbial slurry is preferably a probiotic slurry, such as one or more combinations of Bacillus spp., lactic acid bacteria, yeast, Clostridium butyricum, and Bifidobacterium spp.; more preferably, the microbial slurry is Lactobacillus reuteri slurry.

[0013] The concentrated phospholipid is preferably a liquid plant-based concentrated phospholipid, and more preferably a liquid soybean-based concentrated phospholipid.

[0014] Enzymatically hydrolyzed soybean protein is preferably enzymatically hydrolyzed yellow soybean protein, enzymatically hydrolyzed black soybean protein, enzymatically hydrolyzed red soybean protein, or enzymatically hydrolyzed mung bean protein.

[0015] A method for preparing a liquid microcapsule system involves mixing and emulsifying 0.5-1.2 parts by weight of microbial sludge, 1-3 parts by weight of enzymatically hydrolyzed soybean protein, 2-8 parts by weight of concentrated phospholipids, 8-10 parts by weight of soybean oil, 2-10 parts by weight of maltodextrin, 0.8-1.2 parts by weight of glycerol, and 6-25 parts by weight of water under vacuum conditions to form multiple uniformly dispersed liquid microcapsules, thereby obtaining a stable emulsion.

[0016] The preparation method described above includes the following steps:

[0017] To prepare the oil phase substance, concentrated phospholipids and soybean oil were uniformly mixed at 40℃~60℃ to obtain the oil phase substance.

[0018] To prepare an aqueous phase substance, maltodextrin, glycerol, and a portion of water are mixed evenly to obtain the aqueous phase substance.

[0019] To prepare the capsule system, first mix the microbial sludge, enzymatically hydrolyzed soybean protein, and the remaining water evenly, then add the oil phase and water phase substances for emulsification to form a stable emulsion.

[0020] Based on the above, the steps for preparing the capsule system include: first, mixing the microbial sludge, enzymatically hydrolyzed soybean protein, and the remaining water under vacuum for 15-45 minutes to form a mixed bacterial solution; then, adding the oil phase and aqueous phase substances to the mixed bacterial solution, and emulsifying at room temperature under vacuum for 60-120 minutes to form a stable emulsion, thereby obtaining the liquid microcapsule system.

[0021] Preferably, in the preparation process of the liquid microcapsule system, each step involves stirring and mixing at a speed of 500–1000 r / min.

[0022] The application of a liquid microcapsule system in the processing of microbial products such as health supplements, food, probiotic preparations, or probiotic feed. Specifically, the liquid microcapsule system is used as an additive in health supplements, food, and probiotic feed.

[0023] Based on the above applications, the liquid microcapsule system is applied to the processing of health products, food, probiotic agents, or probiotic feed using a post-coating process.

[0024] Preferably, the temperature of the post-coating process is room temperature, and the coating pressure of the coating process is a coating machine gauge pressure of less than or equal to 0.7 MPa (7 Bar). The preferred gauge pressure of the coating machine in the post-coating process is 0.2–0.6 MPa.

[0025] Therefore, the liquid microcapsule system provided by this invention is a stable milky white liquid formed by emulsification of microbial sludge, soybean protein, concentrated phospholipids, soybean oil, maltodextrin, glycerol, and water under vacuum. In the above microcapsule system, soybean protein mainly serves as a microbial protectant; maltodextrin mainly serves as a microbial protectant and emulsifier; and concentrated phospholipids mainly serve as an emulsifying stabilizer in the above liquid microcapsule system. Its hydrophobic groups and protein hydrophobic groups can better encapsulate soybean oil droplets, and the hydrophilic groups of concentrated phospholipids and proteins can better extend into the aqueous phase mainly composed of maltodextrin, glycerol, and water, thereby forming a stable emulsion structure. This results in a better encapsulation effect for the liquid microcapsules, and the bacterial activity in the liquid microcapsules can reach 10%. 9 The encapsulation efficiency reaches 99.11% (CFU / mL). The liquid microcapsules exhibit good heat resistance and sustained-release properties, improving the tolerance of *Lactobacillus reuteri* to adverse factors such as digestive enzymes. Therefore, the above-mentioned liquid microcapsule system is easily digestible, readily available, and can be widely applied in probiotic health products, food, pharmaceuticals, and feed.

[0026] The method for preparing the above-mentioned liquid microcapsule system provided by this invention first combines bacterial sludge with enzymatically hydrolyzed soybean protein, and then emulsifies it with the prepared oil phase and aqueous phase under vacuum. Compared with existing methods for preparing probiotic coating materials, this method is simpler, lower in cost, and more effective. Furthermore, the raw materials used in the liquid microcapsules prepared by the method provided by this invention are readily available and commonly used in food, and the prepared liquid microcapsules are stable and easy to store.

[0027] Therefore, the liquid microcapsule bacteria provided by this invention have the characteristics of high survival rate, good heat resistance, good sustained release, and low cost. Moreover, the preparation method is simple, and the coating after coating increases the number of bacteria that survive in processing applications, thereby reducing the application and processing costs. It also solves the problem that existing microorganisms are sensitive to adverse factors such as high temperature and pressure, and are easily affected and inactivated during processing and storage. Attached Figure Description

[0028] Figure 1 These are photographs of phospholipids, with the left image showing a modified phospholipid and the right image showing a concentrated phospholipid.

[0029] Figure 2 The figures show the effect of phospholipids on the liquid microcapsules of Lactobacillus reuteri. Figure A shows the encapsulation efficiency of ESI and EAI, Figure B shows the potential, Figure C shows the average particle size, Figure D shows the particle size distribution, and Figures E1-E4 show microscopic observations. Significant differences are found between data with different lowercase letters in the superscripts of Figures A and C. P < 0.05).

[0030] Figure 3 The images show the appearance of Lactobacillus reuteri liquid microcapsules. Figure A shows the original liquid microcapsules prepared directly. Figure B shows the liquid microcapsules diluted 10 times. Figure C shows the liquid microcapsules diluted 100 times. Figures D and E show the microscopic observation images.

[0031] Figure 4 The figures show the effect of maltodextrin on Lactobacillus reuteri liquid microcapsules. Figure A shows the encapsulation efficiency of ESI and EAI, Figure B shows the potential, Figure C shows the average particle size, Figure D shows the particle size distribution, and Figures E1-E4 show the microscopic observations. Significant differences are found between data with different lowercase letters in the subscripts of Figures A and C. P < 0.05).

[0032] Figure 5 The graph shows the effect of emulsification time on Lactobacillus reuteri liquid microcapsules. Figure A shows the ESI and EAI encapsulation rates; Figure B shows the electrochemical potential; Figure C shows the average particle size; Figure D shows the particle size distribution; and Figures E1-E4 show microscopic observations. Significant differences are found between data with different lowercase letters in the superscripts of Figures A and C. P< 0.05).

[0033] Figure 6 This is a graph showing the effect of temperature on Lactobacillus reuteri microcapsules. Figures A, B, and C show the effect on the total bacterial count in the culture, and Figures E1-E4 show microscopic observations. In Figures AC, the data with different lowercase letters in the superscript labels show significant differences. P <0.05).

[0034] Figure 7 Microscopic images of Lactobacillus reuteri microcapsules in gastrointestinal fluid at different digestion times: 40x microscopic images (A1-A5) and 100x microscopic images (C1-C5) during gastric digestion; 40x microscopic images (B1-B5) and 100x microscopic images (D1-D5) during intestinal digestion.

[0035] Figure 8 The images shown are photographs and microscopic images of the Lactobacillus reuteri liquid microcapsule system before and after spraying processing, as provided in the embodiments of the present invention.

[0036] Figure 9 A bar chart showing the total number of bacterial colonies in the Lactobacillus reuteri liquid microcapsule system before and after spraying processing, as provided in an embodiment of the present invention.

[0037] Figure 10 The graph shows the effect of different spraying pressures on the activity of the Lactobacillus reuteri liquid microcapsule system provided in the embodiments of the present invention. Detailed Implementation

[0038] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0039] The liquid microcapsule system provided by this invention is mainly composed of bacterial sludge, enzymatically hydrolyzed soybean protein, concentrated phospholipids, soybean oil, maltodextrin, glycerol and water as raw materials, which are then vacuum emulsified to form a uniform and stable emulsion.

[0040] The present invention also provides a method for preparing the above-mentioned liquid microcapsule system, comprising:

[0041] To prepare the oil phase substance, concentrated phospholipids and soybean oil are gently heated and mixed evenly to obtain the oil phase substance.

[0042] To prepare an aqueous phase substance, maltodextrin and glycerol were mixed evenly to obtain the aqueous phase substance.

[0043] To prepare the capsule system, firstly, microbial sludge and enzymatically hydrolyzed soybean protein are mixed under vacuum for 15–45 min to form a mixed bacterial solution; then, the oil phase and aqueous phase are added to the mixed bacterial solution, and emulsified at room temperature under vacuum for 60–120 min to form a stable emulsion, thus obtaining the liquid microcapsule system.

[0044] The main reagents and materials used in the following embodiments of the present invention are shown in Table 1.

[0045] Table 1 Main Reagents and Materials

[0046] Main materials and reagents Manufacturer MT476913 Lactobacillus reuteri Henan Provincial Engineering Laboratory for Preservation and Propagation of Industrial Microbial Strains (Zhengzhou, Henan) soy Zhengzhou Yonghui Park Mall Supermarket Procurement Phospholipids Zhengzhou Siwei Technology Co., Ltd. Golden Dragon Soybean Oil Yihai Kerry Grain & Oil Industry Co., Ltd. maltodextrin Solarbio Biotechnology Co., Ltd. Pepsin (10000 NFU / mg) Sangon Biotech (Shanghai) Co., Ltd. Lipase (activity 20000 U / g) Solarbio Biotechnology Co., Ltd. Trypsin (10000 NFU / mg) Sangon Biotech (Shanghai) Co., Ltd.

[0047] The main solutions and substances used in the following embodiments of the present invention are shown below:

[0048] 1) MRS liquid culture medium: peptone 10 g, beef extract 10 g, yeast extract 5 g, glucose 20 g, sodium acetate 5 g, Tween-80 1 ml, magnesium sulfate 0.2 g, manganese sulfate 0.05 g, dipotassium hydrogen phosphate 2 g, C6H 14 2 g N2O7. 1000 ml deionized water, adjust pH to 6.2-6.4 with hydrochloric acid, and sterilize at 121℃ for 20 min.

[0049] 2) MRS solid medium: Add 18 g of agar to the MRS liquid medium prepared in 1) above, and sterilize at 121℃ for 20 min.

[0050] 3) The preparation method of the bacterial sludge includes: bacterial activation: add 1 mL of *Lactobacillus reuteri* inoculum to 100 mL of MRS liquid medium and incubate at 37.0℃ for 14 h for the first activation; add 3 mL of the first-activated *Lactobacillus reuteri* bacterial solution to 100 mL of MRS liquid medium and incubate at 37.0℃ for 14 h for the second activation; take 4 mL of the second-activated *Lactobacillus reuteri* bacterial solution, inoculate it into 100 mL of MRS liquid medium, incubate at 37.0℃ for 14 h, centrifuge at 5000 r / min for 10 min to collect the precipitate to obtain *Lactobacillus reuteri* bacterial sludge, with a viable count of 10. 10 CFU / mL.

[0051] 4) Methods for preparing enzymatically hydrolyzed soybean protein include:

[0052] Preparation of soybean protein isolate (SPI): Soybeans were cleaned to remove stones and other impurities, pulverized and passed through an 80-mesh sieve, and defatted with petroleum ether to remove fat. Defatted soybean powder was mixed with distilled water at a ratio of 1:10 (w / w) and stirred for 1 h. The pH was adjusted to 8.0 with 2 mol / L NaOH, and the mixture was placed in a 50℃ water bath for 1 h. After centrifugation at 5000 r / min for 30 min, the supernatant was collected and the pH was adjusted to 4.8 with 2 mol / L HCl. The mixture was stored at 4℃ overnight, and the next day it was centrifuged at 5000 r / min for 50 min. The precipitate was collected, mixed thoroughly with distilled water, and the pH was adjusted to 7.0. After freeze-drying, SPI was obtained.

[0053] Preparation of enzymatic hydrolysate of soybean protein isolate (ESP): Prepare a 3% SPI solution, adjust the pH to 2.0, stir at 37℃ for 10 min, add pepsin (enzyme to substrate ratio 1:100) to carry out enzymatic hydrolysis, keep the pH constant, stop the reaction after 2 h, adjust the pH to 7.0 to inactivate the enzyme, freeze dry to obtain enzymatic hydrolysate of soybean protein.

[0054] 5) Preparation of simulated gastric and intestinal juices

[0055] To simulate gastric juice, weigh 10 g of sodium chloride, dissolve it in distilled water, adjust the pH to 1.5 with 1.5 mol / L HCl, transfer it to a 500 mL volumetric flask, make up to volume, and add 0.2 g of pepsin. Prepare fresh before use.

[0056] To simulate intestinal fluid, weigh 6.8 g of dipotassium hydrogen phosphate, dissolve it in distilled water, adjust the pH to 7.4 with 1 mol / L NaOH, transfer it to a 1000 mL volumetric flask, make up to volume, and add 2 g of trypsin and 2 g of lipase. Prepare fresh before use.

[0057] The raw material ratio, preparation process parameters, and usage method of the liquid microcapsule system provided by this invention have a significant impact on its processing performance. The following uses Lactobacillus reuteri as an example to further explain and illustrate this invention.

[0058] The liquid microcapsule systems provided in the following embodiments of the present invention are all 40 g, and are mainly prepared through the following steps:

[0059] To prepare the oil phase substance, 3-7 g of concentrated phospholipids and 10 g of soybean oil were stirred evenly at 40℃~60℃ and 1000 r / min to obtain the oil phase substance.

[0060] To prepare the aqueous phase substance, first dissolve 2-10 g of maltodextrin in a portion of water to form a maltodextrin solution, then mix it evenly with 1 g of glycerol to obtain the aqueous phase substance.

[0061] To prepare the capsule system, 2 g of soybean protein and the remaining water were first mixed in a flat-bottomed flask to form a soybean protein solution. Then, 1 g of Lactobacillus reuteri sludge was added and the mixture was stirred under vacuum at 1000 r / min for 20 min. Next, the oil phase and aqueous phase were added and emulsified at room temperature under vacuum (-0.1 MPa) to form Lactobacillus reuteri liquid microcapsules, resulting in a stable emulsion: liquid microcapsule system of 40 g.

[0062] The performance testing methods used in the various embodiments of this invention are as follows:

[0063] (1) Determination of Emulsifying Activity Index (EAI) and Emulsifying Stability Index (ESI)

[0064] Lactobacillus reuteri liquid microcapsules were diluted 1000 times with 0.10% sodium dodecyl sulfate. Using 0.10% sodium dodecyl sulfate as a blank, the absorbance was measured at 500 nm, and the absorbance values ​​at 0 min and 30 min were recorded as A0 and A30, respectively. The absorbance ranged from 0.2 to 0.8. The emulsification stability index (ESI) and emulsification activity index (EAI) were calculated.

[0065] ,

[0066] In the formula: T is a constant 2.303, N is the dilution factor, ρ is the protein concentration of the emulsion, and φ is the oil mass fraction.

[0067] (2) Particle size and zeta potential determination

[0068] The Lactobacillus reuteri liquid microcapsule sample was diluted 1000 times with distilled water. One mL of the sample was then placed at 25°C, and the particle size and zeta potential of the microcapsules were measured using a Malvern particle size analyzer and a Zeta potential analyzer.

[0069] (3) Determination of embedding rate

[0070] The Lactobacillus reuteri microcapsule system sample was diluted 100 times and then the colony count was N0. The sample was then diluted appropriately and centrifuged to collect the supernatant. The colony count was then performed using the plate count method to count N.

[0071] Encapsulation ratio (ME): .

[0072] (4) Heat resistance test

[0073] 1 g of Lactobacillus reuteri liquid microcapsules were diluted 10-fold with distilled water and treated at 70℃, 85℃, and 100℃ for 30 s, 45 s, and 60 s, respectively. Viable bacteria were counted using the serial dilution plate method, with each treatment performed in triplicate. A Lactobacillus reuteri solution was used as a control.

[0074] (5) In vitro simulated digestion test

[0075] Static digestion was performed by adding simulated gastric juice and Lactobacillus reuteri liquid microcapsules to Erlenmeyer flasks and incubating in a 37°C water bath with shaking at 110 rpm for 2 h. Samples were taken every 30 min and relevant indicators were measured immediately. Simulated intestinal juice was added immediately after gastric juice sampling, and the sampling time and method for intestinal juice were the same as for simulated gastric juice. Comparative experiments were also conducted simultaneously.

[0076] (6) Microscopic observation

[0077] Lactobacillus reuteri liquid microcapsules were diluted 10 times and observed under an optical microscope. The samples were then coated and fixed, stained with Gram stain, crystal violet for 1 min, iodine solution for 1 min, 95% ethanol for 30 s for decolorization, and tsartan for 1 min. The samples were then photographed at 16×40 and 16×100 (oil immersion).

[0078] 1. The effect of concentrated phospholipids on liquid microcapsule systems

[0079] Phospholipids are natural surfactants with both hydrophilic and lipophilic groups, and can be used as emulsifiers, stabilizers, adhesives, and lubricants. They are safe, inexpensive, and readily available. However, modified phospholipids, such as... Figure 1 As shown in the left image, it easily separates into layers and turns yellowish at room temperature; while concentrated phospholipids, such as Figure 1 As shown in the right figure, at room temperature, it is a homogeneous, non-stratified solution. Therefore, this invention selects concentrated phospholipids as an emulsion stabilizer to ensure that the liquid microcapsule system provided by this invention is a stable emulsion in which liquid microcapsules are uniformly dispersed. Under the same conditions, the amount of concentrated phospholipids added has a significant impact on the performance of the liquid microcapsule system.

[0080] Experimental conditions: 4 g maltodextrin, 3 g, 4 g, 5 g, 6 g, and 7 g concentrated phospholipids respectively, 1 g Lactobacillus reuteri, 2 g enzymatically hydrolyzed soybean protein, 10 g soybean oil, 1 g glycerol, and the remainder was water; the corresponding liquid microcapsule systems were prepared according to the above formulations in each example, and the stirring and emulsification time for each liquid microcapsule system during preparation was 90 min. The performance test results of each Lactobacillus reuteri liquid microcapsule are as follows... Figure 2 As shown.

[0081] from Figure 2As can be seen, with the increase of concentrated phospholipid concentration, emulsification stability, emulsification activity, and encapsulation efficiency all show very significant changes, first increasing and then decreasing. P <0.05). When the concentrated phospholipid content was 6 g, the emulsification stability, emulsification activity, and encapsulation efficiency were all at their highest. With increasing concentrated phospholipid content, the emulsification stability and encapsulation efficiency of this system continuously improved. P <0.05%, E1 cells were more densely packed and numerous than E2 cells, and E3 cells were larger than E4 cells. A significant decrease was observed when 7 g of concentrated phospholipids was added ( P <0.05). Since concentrated phospholipids are an emulsifier, both the protein and concentrated phospholipids in the liquid microcapsule system are amphiphilic. The hydrophobic groups of phospholipids and the hydrophobic groups of proteins can better encapsulate oil droplets, while the hydrophilic groups of phospholipids and the hydrophilic groups of proteins can better extend into the aqueous phase, thus forming a stable emulsion structure and reducing the interfacial tension between water and oil. However, when the concentrated phospholipids reach a certain concentration, the binding between the protein and the concentrated phospholipids becomes saturated, and the emulsifying performance of the emulsion tends to level off, even reducing stability. Excessive addition of concentrated phospholipids leads to the formation of droplets of different sizes in the composite system, resulting in uneven droplet distribution and easy migration and flow. As the concentrated phospholipid content increases, the particle size first decreases and then increases. There are two main peaks at 3 g, 5 g, and 7 g of concentrated phospholipid content. The peak is relatively low and wide at 4 g, while it is high and narrow at 6 g, indicating that the droplet distribution is relatively uniform.

[0082] Among them, when the amount of concentrated phospholipid added is 6 g, the resulting Lactobacillus reuteri liquid microcapsules are as follows: Figure 3 As shown. The Lactobacillus reuteri liquid microcapsules are made by encapsulating Lactobacillus reuteri with wall materials such as phospholipids and maltodextrin, as... Figure 3 As shown in Figure (A), the microcapsule is a milky white liquid. The microcapsule in Figure (A), diluted 10 times, becomes the white liquid shown in Figure (B). Under a 40x microscope, it appears as uniformly sized spherical particles (D), and under a 100x oil immersion microscope, it appears as bacterial aggregates (E). This demonstrates that the liquid microencapsulation encapsulates the bacterial cells, and the wall material forms a barrier around the cells, isolating them from the external environment and enhancing their stability and activity. Figure (C) is a photograph of the microcapsule diluted 100 times in Figure (A), primarily used to determine various performance indicators of the *Lactobacillus reuteri* liquid microcapsules.

[0083] 2. The effect of maltodextrin on liquid microcapsule systems

[0084] All other things being equal, the amount of maltodextrin added has a significant impact on the performance of the liquid microcapsule system.

[0085] Experimental conditions: Maltodextrin was added in the following quantities: 2 g, 4 g, 6 g, 8 g, and 10 g; concentrated phospholipids: 6 g; *Lactobacillus reuteri*: 1 g; enzymatically hydrolyzed soybean protein: 2 g; soybean oil: 10 g; glycerol: 1 g; and the remainder was water. The calculated concentrations of maltodextrin in the entire liquid microcapsule system were 5%, 10%, 15%, 20%, and 25%, respectively. The corresponding liquid microcapsule systems were prepared according to the above formulations in each example, and the stirring and emulsification time for each liquid microcapsule system during preparation was 90 min. The performance test results of each *Lactobacillus reuteri* liquid microcapsule are as follows: Figure 4 As shown.

[0086] from Figure 4 It can be seen that: with the increase of maltodextrin concentration, both EAI and ESI increase. P <0.05), when the maltodextrin concentration increased to 15%, both EAI and ESI decreased with increasing maltodextrin concentration. P <0.05%. The encapsulation efficiency first increased and then decreased with increasing maltodextrin concentration. P <0.05), the highest encapsulation efficiency was achieved when the maltodextrin concentration reached 15%; E1 cells were more densely packed and numerous than E2 cells, while E3 cells aggregated more than E4 cells. When the maltodextrin concentration reached 20% and 25%, the strong interaction between maltodextrin and the emulsifier phospholipids may have affected the stability of the microcapsules, thus impacting the encapsulation effect. The potential first decreased and then increased in absolute value with increasing maltodextrin concentration. P <0.05%, with the absolute value being smallest at a maltodextrin concentration of 15%. There was no significant difference in particle size between 5% and 10% maltodextrin concentrations. P <0.05%, when the maltodextrin concentration reaches 15%, the particle size decreases ( P <0.05%, and then the particle size gradually increased with increasing concentration. P <0.05); the average particle size distribution peaks at 5% and 10% concentrations are narrow and low, the peaks at 20% and 25% concentrations are both low and relatively wide, and the peak at 15% concentration is the highest with one narrow peak, indicating that when the maltodextrin concentration is 15%, that is, when the maltodextrin addition amount is 6 g, the distribution of Lactobacillus reuteri liquid microcapsule emulsion droplets is relatively uniform.

[0087] 3. Effect of emulsification time on liquid microcapsule system

[0088] Under otherwise identical conditions, the emulsification time of the liquid microcapsule system has a significant impact on its performance.

[0089] Experimental conditions: 6 g maltodextrin, 6 g concentrated phospholipids, 1 g Lactobacillus reuteri, 2 g enzymatically hydrolyzed soybean protein, 10 g soybean oil, 1 g glycerol, and the remainder was water. The above raw materials were emulsified under vacuum at room temperature for 30 min, 60 min, 90 min, 120 min, and 150 min, respectively, to prepare different liquid microcapsule systems. The performance test results of these Lactobacillus reuteri liquid microcapsule systems are as follows: Figure 5 As shown.

[0090] from Figure 5 As can be seen, EAI and ESI first gradually increase with increasing emulsification time. P <0.05), reaching its maximum at 90 min emulsification time, and no significant change in EAI and ESI when emulsification time was further increased. P <0.05%. The encapsulation efficiency first increased and then decreased with increasing emulsification time. P <0.05%, the encapsulation efficiency was highest at 90 min when the emulsification time gradually increased from 30 to 90 min, and decreased when the emulsification time exceeded 90 min. P (<0.05), Figure E1 shows a denser and more numerous spheroids than Figure E2, and Figure E3 shows larger bacterial aggregates than Figure E4. This may be due to prolonged magnetic stirring, which disrupted the encapsulation system, broke the interaction between maltodextrin and the emulsifier phospholipids, reduced emulsion stability, and caused emulsion demulsification. The increase in free bacteria in the emulsion further reduced the encapsulation efficiency. Particle size initially decreased and then increased with increasing emulsification time. The average particle size peak at 30 min was narrow and low, while the peak at 60 min was higher and wider than those at 90 min and 150 min. The two peaks at 120 min indicate uneven particle size distribution, while the sharp and narrow peak at 90 min suggests a more uniform emulsion distribution.

[0091] This embodiment provides a liquid microcapsule system comprising 6 g maltodextrin, 6 g concentrated phospholipids, 1 g Lactobacillus reuteri, 2 g enzymatically hydrolyzed soybean protein, 10 g soybean oil, 1 g glycerol, and the remainder being water. The above ingredients are emulsified under vacuum at room temperature for 90 min to obtain the system. Testing showed that the viability of this liquid microcapsule system reached 10%. 9 The encapsulation efficiency reached 99.11% (CFU / mL). The results of the heat resistance test, in vitro digestion test, and processing application test of this liquid microcapsule system are as follows: Figure 6-10 As shown.

[0092] (1) Results of heat resistance test

[0093] from Figure 6 It can be seen that at 70℃, the total number of colonies in the control group decreased significantly with the increase of treatment time. P<0.05%, and the total bacterial count in the microcapsule group also decreased significantly with increasing treatment time. P <0.05), and the total bacterial count in the control group and the microcapsule group was not significantly different. The control group consisted of bacterial suspension containing MRS medium, which provided some protection for the bacteria. At 85℃, there were no significant changes in either the control group or the microcapsule group with increasing treatment time, but the total bacterial count in the microcapsule group was significantly higher than that in the control group, indicating that the coating material has certain heat resistance. At 100℃, there were no significant changes in either the control group or the microcapsule group with increasing treatment time, but the number of surviving bacteria in the bacterial suspension control group was significantly higher than the total bacterial count in the microcapsule group at each treatment time interval, indicating that the maltodextrin coating material has excellent heat resistance.

[0094] (2) Results of in vitro digestion test

[0095] from Figure 7 (A1-A5) shows that the microcapsules did not undergo significant changes during simulated gastric digestion. P >0.05), and there was no significant change after increasing intestinal fluid ( P >0.05); the control bacterial culture group showed a significant decrease in size immediately after the addition of gastric juice ( P <0.05, and there were no significant changes in gastric and intestinal fluids afterwards. P <0.05); This indicates that both can successfully pass through gastric juice to intestinal juice and be lysed. Because the embedding wall material has a certain protective effect on Lactobacillus reuteri, it avoids the destruction of Lactobacillus reuteri by the strong acid environment. When the microcapsules reach the intestinal juice, the alkaline nature of the intestinal juice neutralizes the acidity of the gastric juice, resulting in an increase in the pH of the solution, which is more suitable for the growth of Lactobacillus reuteri. Therefore, the total number of bacterial colonies in the intestinal juice is greater than that in the gastric juice. In the control group, the bacterial solution contains liquid culture medium, which is also a nutrient and provides nutrients for Lactobacillus reuteri. The increased pH in the intestinal juice also promotes bacterial growth and reproduction, resulting in a higher total number of bacterial colonies in the intestinal juice than in the gastric juice.

[0096] from Figure 7 (B1-B5) shows that: with increasing digestion time, both EAI and ESI gradually decrease, and the changes are not significant after reaching the intestinal fluid level. P >0.05), after being added to gastric juice, the pH decreased rapidly. The microcapsules were in a strongly acidic environment, which reduced their stability, thus causing both EAI and ESI to gradually decrease.

[0097] from Figure 7 From (C1-C5) and (D1-D5), we can conclude that the particle size decreases in gastric juice with increasing digestion time. P <0.05%, no significant change in intestinal fluid; microcapsules lysed in the small intestine and then underwent secondary emulsification, resulting in uniform size; throughout digestion, the absolute value of the potential gradually decreased with increasing digestion time.P <0.05).

[0098] The viable bacterial count in the microcapsule group after 2 hours of static gastric juice digestion was 1.26 × 10⁻⁶. 7 CFU / mL, the viable bacterial count of the microcapsules after 2 hours of intestinal digestion was 2.16 × 10⁻⁶. 8 CFU / mL, after 4 hours of digestion with gastrointestinal fluid, the viable bacterial count of the microcapsules was 1.85 × 10⁻⁶. 8 CFU / mL; this indicates that the liquid microcapsules have a sustained-release effect in the intestine.

[0099] (3) The effect of pressure parameters during processing on the liquid microcapsule system

[0100] Existing microbial agents are susceptible to interference from factors such as pressure during processing and application, which significantly affects their activity. In the liquid microcapsule system provided in this invention, the post-coating process significantly impacts the encapsulation performance of the liquid microcapsules, as illustrated by the following experiments.

[0101] 1) Experimental conditions: The above liquid microcapsules were diluted 100 times. Photos and total bacterial counts of the *Lactobacillus reuteri* liquid microcapsule system before and after spraying were taken using a spray gun with a gauge pressure of 3 bar. Figure 8 and Figure 9 As shown.

[0102] from Figure 8 As can be seen, after spraying, the Lactobacillus reuteri liquid microcapsules contain smaller droplets, and the bacterial cells still aggregate. This indicates that pressure makes the liquid microcapsule system more uniformly dispersed, and the coating is not damaged. At the same time, the Lactobacillus reuteri liquid microcapsule system sample treated with spraying is whiter and more uniformly mixed.

[0103] Depend on Figure 9 It can be seen that the total number of colonies of *Lactobacillus reuteri* liquid microcapsules before and after the spraying treatment is around 10. 9 The difference was not significant, indicating that the spraying had little effect on bacterial survival.

[0104] 2) Experimental conditions: The *Lactobacillus reuteri* liquid microcapsules provided in this embodiment were diluted 100-fold and sprayed using a sprayer at different gauge pressures. The total bacterial count and encapsulation rate of the diluted liquid microcapsule system after spraying were then measured and calculated. The gauge pressures of the sprayer were 2, 3, 4, 5, and 6 Bar (1 Bar = 100 kPa = 0.1 MPa). The results are as follows: Figure 10 As shown.

[0105] from Figure 10It can be seen that as the pressure increases, the total number of colonies first increases and then decreases. The total number of colonies is highest at a pressure of 3 bar, but the total number of colonies is around 10 at pressures of 2-6 bar. 9 This indicates that the Lactobacillus reuteri liquid microcapsules have good pressure resistance. The encapsulation rate first increases and then decreases with increasing pressure, reaching its peak at 98% at 3 bar. Encapsulation rates remain above 90%, even exceeding 96%, at pressures of 2-6 bar, indicating good encapsulation performance. This demonstrates that the aforementioned liquid microcapsules can protect the microbial cells from damage caused by pressure and other factors during post-coating processes, thereby increasing the survival rate of the cells in the processing of health products, food, pharmaceuticals, or feed.

[0106] Therefore, the *Lactobacillus reuteri* liquid microcapsules provided in this invention are a milky white liquid with good dispersibility. Under a 40x microscope, they appear as uniformly dense spherical particles, while under a 100x microscope, the bacterial cells aggregate. They exhibit excellent heat resistance and sustained-release properties, improving the tolerance of *Lactobacillus reuteri* to adverse factors such as digestive enzymes, and possessing high survival and encapsulation rates. Furthermore, the post-encapsulation spraying of *Lactobacillus reuteri* avoids the influence of external factors such as pressure during processing, ensuring a sufficient number of live bacteria enter the intestines to exert their probiotic effects. This expands the application range of *Lactobacillus reuteri* and lays a theoretical foundation for its use in food processing.

[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in the present invention.

Claims

1. A liquid microcapsule system, characterized in that, The liquid microcapsule system is a stable emulsion formed by uniform dispersion of liquid microcapsules. The liquid microcapsule system is composed of 1 part microbial sludge, 2 parts enzymatically hydrolyzed soybean protein, 3-7 parts concentrated phospholipids, 10 parts soybean oil, 2-10 parts maltodextrin, 1 part glycerol, and the remainder is water, based on 40 parts by weight. The liquid microcapsule system is mainly prepared by the following methods: Preparation of oil phase substance: Concentrated phospholipids and soybean oil are uniformly mixed at 40℃~60℃ to obtain oil phase substance; Preparation of aqueous phase substance: Maltodextrin, glycerol and a portion of water are mixed evenly to obtain an aqueous phase substance; To prepare the capsule system: First, the microbial sludge, enzymatically hydrolyzed soybean protein, and remaining water are mixed under vacuum for 15-45 minutes to form a mixed bacterial solution; then, the oil phase and aqueous phase are added to the mixed bacterial solution, and emulsified at room temperature under vacuum for 60-120 minutes to form a stable emulsion, thus obtaining the uniformly dispersed liquid microcapsule system. The liquid microcapsule system is applied using a post-coating process in the processing of health products, food, probiotic agents, or probiotic feed.

2. The liquid microcapsule system according to claim 1, characterized in that, The microbial sludge is one or more of the following: Bacillus spp. sludge, yeast sludge, Clostridium butyricum sludge, and Bifidobacterium spp. sludge.

3. The liquid microcapsule system according to claim 1, characterized in that, The microbial sludge is lactic acid bacteria sludge.

4. A method for preparing the liquid microcapsule system according to any one of claims 1-3, comprising the steps of: Preparation of oil phase substance: Concentrated phospholipids and soybean oil are uniformly mixed at 40℃~60℃ to obtain oil phase substance; Preparation of aqueous phase substance: Maltodextrin, glycerol and a portion of water are mixed evenly to obtain an aqueous phase substance; To prepare the capsule system: First, the microbial sludge, enzymatically hydrolyzed soybean protein, and remaining water are mixed under vacuum for 15-45 minutes to form a mixed bacterial solution; then, the oil phase and aqueous phase are added to the mixed bacterial solution, and emulsified at room temperature under vacuum for 60-120 minutes to form a stable emulsion, thus obtaining the liquid microcapsule system.

5. The application of the liquid microcapsule system according to any one of claims 1-3 in the processing of products containing microorganisms, wherein, The microbial products mentioned are health products, food, probiotic preparations, or probiotic feed.

6. The application according to claim 5, characterized in that, The liquid microcapsule system is applied to the processing of health products, food, probiotic agents or probiotic feed using a post-coating process, and the spraying pressure of the post-coating process is less than or equal to the gauge pressure of the spraying machine of 0.7 MPa.

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