Lactobacillus plantarum microcapsule, preparation method and application thereof

Abstract

The application discloses a plant Lactobacillus rhamnosus microcapsule and a preparation method and application thereof, and the plant Lactobacillus rhamnosus is embedded by adopting a low-temperature spray drying process with lily starch, citrus pectin and whey protein as wall materials, and mainly includes the following steps: preparation of a bacterial suspension, preparation of a wall material solution, preparation of a microcapsule solution and low-temperature spray drying for preparing the microcapsule. The preparation technology of the stable encapsulated plant Lactobacillus rhamnosus microcapsule provided by the application has the advantages that the viable bacterial count in the microcapsule is high after spray drying, the microcapsule has strong gastrointestinal fluid resistance, is stable in storage, and can effectively solve the problem that the bacterial activity of the plant Lactobacillus rhamnosus and products thereof is reduced or even inactivated in the production and processing, storage and transportation and in-vivo digestion process; the prepared microcapsule can be used as a probiotic bacterial agent and applied to foods such as cheese, yogurt, ice cream, milk, chocolate, lactic acid bacterial beverages and baked products, and helps to promote the healthy development of the probiotic industry, and has obvious economic and social benefits.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a microcapsule of *Lactobacillus plantarum*, its preparation method, and its application. Background Technology

[0002] Lactobacillus plantarum is a type of lactic acid bacteria distributed in the intestinal mucosa. It can be used to ferment various foods, regulate the balance of intestinal flora, lower cholesterol levels, and enhance immune function. However, when Lactobacillus plantarum passes through the human digestive tract, its probiotic effects are often limited because it cannot tolerate the challenges posed by environmental factors such as gastric acid, digestive enzymes, and bile acids.

[0003] Microencapsulation is considered an effective method for protecting probiotics. Low-temperature spray drying technology produces microcapsules with small particle size, uniform texture, and maximum preservation of bacterial activity. By adjusting the inlet air temperature and feed flow rate in low-temperature spray drying, a solution containing *Lactobacillus plantarum* and wall materials can be rapidly converted into microcapsule powder, thus achieving rapid encapsulation and fixation of *Lactobacillus plantarum*. Furthermore, suitable microcapsule wall materials can effectively isolate the potential threats of extreme external environments to probiotics, thereby enhancing their tolerance in the gastrointestinal tract and extending shelf life. For example, polysaccharides (such as starch, pectin, arabinose, and maltodextrin) have good thickening, gelling, water-retention, and stabilizing properties, forming dense capsule walls that effectively isolate adverse external factors. However, simply using polysaccharides to encapsulate probiotics is insufficient to achieve ideal bacterial viability retention. Proteins (such as whey protein, gelatin, and soy protein isolate) have good nutritional value and bioactivity; when combined with polysaccharides through simple stirring, they can form stable colloidal structures, thereby enhancing the functionality of the microcapsules.

[0004] Therefore, it is necessary to develop a new composite wall material and optimize spray drying parameters to prepare plant lactobacillus microcapsules with high viable bacterial counts, thereby reducing the reduction of probiotic activity during the production, thermal processing, and digestion of the microcapsules. Summary of the Invention

[0005] This invention aims to provide a microcapsule of *Lactobacillus plantarum*, its preparation method, and its application. A composite wall material is prepared by using lily starch, citrus pectin, and whey protein. After encapsulating *Lactobacillus plantarum* NCUTUUAS4 through spray drying, the number of viable bacteria in the microcapsule is significantly increased. After a 4-hour continuous in vitro gastrointestinal digestion simulation experiment, the number of viable bacteria is higher than 8.82 log CFU / g.

[0006] In a first aspect, the present invention provides a plant lactobacillus microcapsule, the plant lactobacillus microcapsule comprising plant lactobacillus and a composite wall material;

[0007] The plant lactobacillus mentioned is Lactiplantibacillus plantarum NCUTUAS4, with the Latin name Lactiplantibacillus plantarum NCUTUAS4 and the accession number CCTCC NO: M 2024858.

[0008] The composite wall material includes lily starch, citrus pectin, and whey protein.

[0009] Optionally, the diameter of the *Lactobacillus plantarum* microcapsules is 3-5 μm.

[0010] Optionally, the viable count of *Lactobacillus plantarum* NCUTUUAS4 in the *Lactobacillus plantarum* microcapsules is (1-5) × 10⁻⁶. 10 CFU / g.

[0011] Secondly, the present invention provides a method for preparing *Lactobacillus plantarum* microcapsules, comprising the following steps:

[0012] Different mass ratios of lily starch, citrus pectin, and whey protein powder were mixed with deionized water and magnetically stirred to prepare a wall material solution. After sterilization, a sterile wall material solution was prepared.

[0013] A suspension of *Lactobacillus plantarum* was mixed with a sterile wall material solution to obtain a microcapsule solution; the volume ratio of the *Lactobacillus plantarum* suspension to the sterile wall material solution was 1:(50-120).

[0014] The microcapsule solution was spray-dried at low temperature to obtain *Lactobacillus plantarum* microcapsules.

[0015] Optionally, the wall material solution includes a one-element wall material solution, a two-element wall material solution, and a three-element composite wall material solution.

[0016] Optionally, in the mono- or binary wall material solution, the mass ratio of lily starch to citrus pectin is 1:(0-1.25), the mass ratio of lily starch to whey protein is 1:(0-2.5), and the mass ratio of whey protein to citrus pectin is 1:(0-1.25).

[0017] Optionally, in the ternary composite wall material solution, the mass ratio of lily starch, citrus pectin and whey protein is (1-3):(1-3):(1-3).

[0018] Optionally, the parameters for the low-temperature spray drying are: the inlet air temperature for spray drying is 100-140℃, the feed flow rate is 150-750mL / h, and the solid content in the wall material solution is 3%-15%.

[0019] Thirdly, the present invention provides the application of *Lactobacillus plantarum* microcapsules in probiotic preparations.

[0020] Fourthly, this invention provides an application of *Lactobacillus plantarum* microcapsules in the thermal processing of food.

[0021] The beneficial effects of this invention include:

[0022] (1) The preparation method of plant lactobacillus microcapsules provided by the present invention uses advanced low-temperature spray drying technology to encapsulate plant lactobacillus NCUTUUAS4, which can retain the bacterial activity to the greatest extent.

[0023] (2) In the plant lactobacillus microcapsules provided by the present invention, natural food raw materials such as lily starch, citrus pectin and whey protein are preferred as microcapsule wall materials and sustained-release carriers. Lily starch is a type of resistant starch with good targeting effect, stronger acid resistance and anti-enzymatic hydrolysis characteristics, and can also be used as a nutrient matrix to promote the proliferation of probiotics such as lactobacillus and bifidobacteria. Citrus pectin is extracted from citrus peel and has very ideal protein stability and viscosity performance. While providing stability, it can also provide a good taste. Moreover, the carboxyl groups on citrus pectin can interact electrostatically with the amino groups of proteins on the interface and form a barrier layer with a stearin-like structure. By combining citrus pectin with whey protein isolate, a stable microcapsule wall material structure can be obtained.

[0024] (3) The plant lactobacillus microcapsules provided by the present invention achieve stable encapsulation of plant lactobacillus. The resulting plant lactobacillus microcapsules with lily starch-citrus pectin-whey protein composite encapsulation have stable morphology, small particle size and uniform texture. A composite protective agent for the industrialization of probiotic preparation spray drying is developed to effectively protect the activity and improve the stability of probiotics, while taking into account the nutritional, safety and scientific nature of the microcapsules and reducing the cost of industrial production. It can also be used as a probiotic preparation in foods such as cheese, yogurt, ice cream, milk, chocolate, lactic acid bacteria beverages, and baked goods.

[0025] (4) The lily starch-citrus pectin-whey protein composite encapsulated plant lactobacillus microcapsules provided by the present invention can resist adverse environments and maintain cell activity in gastric juice, while having certain enteric coagulation and anti-digestive enzyme capabilities, and can better exert probiotic effects in the intestine.

[0026] (5) The lily starch-citrus pectin-whey protein composite encapsulated plant lactobacillus microcapsules provided by the present invention can be stored stably at 4°C and maintain a long shelf life.

[0027] The biological preservation information involved in this invention

[0028] Lactiplantibacillus plantarum NCTUAS4 was deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on April 30, 2024, with accession number CCTCC NO: M 2024858. Attached Figure Description

[0029] Figure 1 The effect of different mass ratios of lily starch and citrus pectin on the viable bacteria count of microcapsules;

[0030] Figure 2 The effect of different mass ratios of lily starch and whey protein on the viable bacterial count of microcapsules;

[0031] Figure 3 The effect of different mass ratios of whey protein and citrus pectin on the viable bacterial count of microcapsules;

[0032] Figure 4 The effect of different mass ratios of lily starch, citrus pectin and whey protein on the viable bacterial count of microcapsules;

[0033] Figure 5 The effect of different inlet air temperatures on the number of viable bacteria in microcapsules;

[0034] Figure 6 The effect of different feed flow rates on the number of viable bacteria in microcapsules;

[0035] Figure 7 The effect of different solid contents on the number of viable bacteria in microcapsules;

[0036] Figure 8 To investigate the interaction of different factors on the number of viable bacteria encapsulated in *Lactobacillus plantarum* microcapsules;

[0037] Figure 9 The change in viable bacterial count of the microcapsules prepared in Example 2 during simulated continuous digestion in gastrointestinal fluid;

[0038] Figure 10 Scanning electron microscope (SEM) images of the *Lactobacillus plantarum* microcapsules prepared in Comparative Examples 4, 9, 16 and 2. Detailed Implementation

[0039] The present invention will be further described in conjunction with the accompanying drawings and through the following embodiments.

[0040] In a first aspect, embodiments of the present invention provide a plant lactobacillus microcapsule, the plant lactobacillus microcapsule comprising plant lactobacillus and a composite wall material;

[0041] The plant lactobacillus mentioned is Lactiplantibacillus plantarum NCUTUAS4, with the Latin name Lactiplantibacillus plantarum NCUTUAS4 and the accession number CCTCC NO: M 2024858.

[0042] The composite wall material includes lily starch, citrus pectin, and whey protein.

[0043] In some embodiments, the diameter of the *Lactobacillus plantarum* microcapsules is 3-5 μm.

[0044] In some embodiments, the viable count of *Lactobacillus plantarum* NCUTUUAS4 in the *Lactobacillus plantarum* microcapsules is (1-5) × 10⁻⁶. 10 CFU / g.

[0045] Secondly, embodiments of the present invention provide a method for preparing *Lactobacillus plantarum* microcapsules, comprising the following steps:

[0046] Different mass ratios of lily starch, citrus pectin, whey protein powder and deionized water were mixed and magnetically stirred to prepare a wall material solution, which was then sterilized to prepare a sterile wall material solution.

[0047] A suspension of *Lactobacillus plantarum* was mixed with a sterile wall material solution to obtain a microcapsule solution; the volume ratio of the *Lactobacillus plantarum* suspension to the sterile wall material solution was 1:(50-120).

[0048] The microcapsule solution was spray-dried at low temperature to obtain *Lactobacillus plantarum* microcapsules.

[0049] In some embodiments, the wall material solution includes a one-element wall material solution, a two-element wall material solution, and a three-element composite wall material solution.

[0050] In some embodiments, the mass ratio of lily starch to citrus pectin in the mono- and binary wall material solutions is 1:(0-1.25), the mass ratio of lily starch to whey protein is 1:(0-2.5), and the mass ratio of whey protein to citrus pectin is 1:(0-1.25).

[0051] In some embodiments, the mass ratio of lily starch, citrus pectin and whey protein in the ternary composite wall material solution is (1-3):(1-3):(1-3).

[0052] In some embodiments, the parameters for the low-temperature spray drying are: the inlet air temperature for spray drying is 100-140°C, the feed flow rate is 150-750 mL / h, and the solid content in the wall material solution is 3%-15%.

[0053] Thirdly, embodiments of the present invention provide the application of *Lactobacillus plantarum* microcapsules in probiotic preparations.

[0054] Fourthly, embodiments of the present invention provide an application of *Lactobacillus plantarum* microcapsules in the thermal processing of food.

[0055] Unless otherwise specified, all raw materials used in the embodiments of this invention are commercially available. The lily starch is pure Longya lily powder, purchased from Wanzai Kangxiang Agricultural Technology Development Co., Ltd.; the citrus pectin is purchased from Shanghai Yuanye Biotechnology Co., Ltd.; the whey protein powder is purchased from Shandong Qilu Technology Co., Ltd.; the bile salts are purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; and the pepsin and trypsin are purchased from Solarbio Technologies Co., Ltd.

[0056] Example 1

[0057] Example 1 of this invention provides a method for preparing *Lactobacillus plantarum* microcapsules, comprising the following steps:

[0058] S0. Streaking Lactobacillus plantarum NCUTUUAS4 into MRS solid medium for activation, taking a loop of bacteria into MRS broth culture medium, and incubating at 37°C for 24 hours.

[0059] The cultured bacterial culture was re-inoculated into MRS broth culture medium at an inoculation amount of 2%, and incubated at 37°C for 24 hours.

[0060] After the culture was completed, the bacterial culture was centrifuged at 4℃ and 5000 rpm for 10 min. The supernatant was removed, the precipitate was collected, and the precipitate was resuspended in sterile deionized water to obtain a *Lactobacillus plantarum* NCUTUUAS4 suspension. The number of viable bacteria in the suspension was 10. 10 -10 12 CFU / mL.

[0061] S1. Take 20g of lily starch, 20g of citrus pectin and 20g of whey protein powder, disperse them in 940g of deionized water to prepare a ternary composite wall material solution with a solid content of 6%, and obtain a lily starch-citrus pectin-whey protein solution with a mass ratio of 1:1:1.

[0062] After magnetic stirring for 1 hour, the mixture was refrigerated at 4°C for 12 hours. After refrigeration, it was taken out and sterilized at 115°C for 20 minutes to obtain a sterile ternary composite wall material solution.

[0063] S2. Mix the plant lactobacillus NCUTUUAS4 suspension with a sterile ternary composite wall material solution at a volume ratio of 1:100 and stir magnetically for 1 hour to obtain a microcapsule solution.

[0064] S3. The microcapsule solution is spray-dried at low temperature to obtain plant lactobacillus microcapsules; the parameters of low temperature spray drying are: inlet air temperature of 130℃ and feed flow rate of 450mL / h.

[0065] Example 2

[0066] Example 2 of this invention provides a method for preparing plant lactobacillus microcapsules. Based on the results of single-factor process optimization, the method differs from Example 1 in that the parameters for spray drying in step S3 are: inlet air temperature of 120°C and feed flow rate of 450 mL / h, while other conditions and steps remain the same, thus obtaining plant lactobacillus microcapsules.

[0067] Example 3

[0068] Example 3 of this invention provides a method for preparing plant lactobacillus microcapsules. According to the response surface optimization results, the method differs from Example 1 in that the solid content in the composite ternary wall material solution in step S1 is 5.25%, the inlet air temperature of spray drying in step S3 is 121℃, and the feed flow rate is 435mL / h; other conditions and steps are kept the same, and plant lactobacillus capsules are obtained.

[0069] Comparative Examples 1-23

[0070] Comparative Examples 1-23 of this invention provide a method for preparing plant lactobacillus microcapsules using mono-, binary, and ternary composite wall materials. The difference from Example 1 is that plant lactobacillus microcapsules are prepared using lily starch, citrus pectin, and whey protein, and the mass ratio of the wall materials in the solution is changed to obtain plant lactobacillus microcapsules as shown in Table 1.

[0071] Table 1. Differences between Comparative Examples 1-23 and Example 1

[0072]

[0073]

[0074] Note: The amount of lily starch, citrus pectin, and whey protein added is g / 1kg of wall material solution.

[0075] Comparative Examples 24-37

[0076] Comparative Examples 24-37 of this invention provide a method for preparing microcapsules of *Lactobacillus plantarum*. The difference from Example 2 is that the parameters in low-temperature spray drying are optimized by changing a single factor, as shown in Table 2.

[0077] Table 2. Differences between Comparative Examples 24-37 and Example 2

[0078]

[0079] Comparative Examples 38-54

[0080] Comparative Examples 38-54 of this invention provide a method for preparing microcapsules of *Lactobacillus plantarum*. The difference from Example 3 is that the method uses Response Surface Methodology (RSM) as the analytical tool. A three-factor, three-level response surface experiment is designed with inlet air temperature of 110-130℃, feed flow rate of 300-600mL / h, and solid content of 3-9% as independent variables to optimize the parameters in low-temperature spray drying, as shown in Table 3.

[0081] Table 3. Differences between Comparative Examples 38-54 and Example 3

[0082]

[0083] Performance verification

[0084] (1) Detection of viable bacteria in microcapsules

[0085] Weigh 0.1 g of spray-dried microcapsules and add 900 μL of sterile physiological saline. Vortex for 5 min to allow the microcapsules to fully disintegrate. Take 100 μL of the microcapsule disintegration solution and perform serial dilutions. Spread the solution onto MRS solid medium and determine the viable cell count using the dilution plate count method. Three parallel control treatments were performed. The plates were incubated at 37°C for 2 days before counting.

[0086] The test results of Comparative Examples 1-6 are as follows Figure 1 As shown; the test results of Comparative Example 7-11 are as follows. Figure 2 As shown; the test results of Comparative Examples 12-17 are as follows. Figure 3 As shown;

[0087] The results of Example 1 and Comparative Examples 18-23 are as follows Figure 4 As shown; the results for comparative examples 24-28 are as follows. Figure 5 As shown; the results for comparative examples 29-33 are as follows. Figure 6 As shown; the results of Example 2 and Comparative Examples 34-37 are as follows. Figure 7 As shown; the results of Example 3 and Comparative Examples 38-54 are as follows. Figure 8 As shown.

[0088] See Figure 1 Comparative Example 1 showed the lowest number of viable bacteria, with a significant difference compared to Comparative Examples 2-6. Compared to adding only lily starch, the binary composite wall material prepared by adding citrus pectin and lily starch significantly increased the number of viable bacteria in the microcapsules. When the amount of lily starch added to each 1 kg of wall material solution was 10 g and the amount of citrus pectin added was 7.5 g (i.e., lily starch:citrus pectin = 1:0.75), Comparative Example 4 showed the highest number of viable bacteria in the microcapsules, at 3.02 × 10⁻⁶. 9 CFU / g.

[0089] See Figure 2 Compared with Comparative Examples 7-11, Comparative Example 1 had the lowest viable bacterial count. Compared with the addition of lily starch alone, the binary composite wall material prepared by adding whey protein and lily starch significantly increased the viable bacterial count of the microcapsules. When the amount of lily starch added to each 1 kg of wall material solution was 10 g and the amount of whey protein added was 15 g, i.e., the ratio of lily starch to whey protein was 1:1.5, Comparative Example 9 had the highest viable bacterial count of microcapsules, at 2.88 × 10⁻⁶. 9 CFU / g.

[0090] See Figure 3 When the amount of citrus pectin added was 0, the microcapsules prepared with whey protein as the single wall material had the lowest viable bacteria count. When whey protein was combined with citrus pectin, the viable bacteria count increased. Furthermore, when the amount of whey protein added was 10g and the amount of citrus pectin added was 10g per 1kg of wall material solution (i.e., whey protein:citrus pectin = 1:1), the microcapsules in Comparative Example 16 achieved the highest viable bacteria count of 2.34 × 10⁻⁶. 9 CFU / g.

[0091] See Figure 4 When the amount of lily starch, citrus pectin, and whey protein added to each 1 kg of composite wall material solution is 20 g, that is, when the ratio of lily starch:citrus pectin:whey protein is 1:1:1, the microcapsule *Lactobacillus plantarum* viable bacteria count in Example 2 is the highest, at 9.37 × 10⁻⁶. 9 CFU / g.

[0092] See Figures 1-4 The viable bacteria count in the *Lactobacillus plantarum* microcapsules prepared from the ternary composite wall material provided by this invention is significantly higher than that in the viable bacteria count in the *Lactobacillus plantarum* microcapsules prepared from the mono- and binary composite wall materials.

[0093] See Figure 5 When the inlet air temperature was set to 120℃, the number of viable bacteria in the microcapsules reached its peak, at 1.11 × 10⁻⁶. 10 CFU / g.

[0094] See Figure 6 When the feed flow rate is 450 mL / h, the viable count is 1.17 × 10⁻⁶. 10 The CFU / g value was significantly different from other feed flow rates (P < 0.05).

[0095] See Figure 7 When the solid content is 6%, the highest viable bacteria count is 2.26 × 10⁻⁶. 10 The CFU / g count of microcapsules was significantly different from that of other microcapsules containing solids (P < 0.05).

[0096] See Figures 5-7After single-factor experiments, the spray drying parameters determined by this invention are: inlet air temperature 120℃, feed flow rate 450mL / h, and solid content 6%, and subsequent response surface experiments were carried out based on these parameters.

[0097] (2) Validation of the response surface model after single-factor optimization

[0098] Based on the results of single-factor optimization obtained from the detection of viable bacteria in microcapsules in (1), a response surface model was established;

[0099] Response surface methodology was conducted using three factors as variables: inlet air temperature of 110℃, 120℃, and 130℃; feed flow rate of 300mL / h, 450mL / h, and 600mL / h; and solid content of 3%, 6%, and 9%. This yielded microcapsules of *Lactobacillus plantarum* with different process parameters, and the viable cell count in the microcapsules was then determined.

[0100] The results are as follows Figure 8 As shown in Table 4, the optimal reaction conditions calculated using Design-expert 13 software were: inlet air temperature set at 120.49℃, feed flow rate at 437.4 mL / min, and solid content controlled at 5.26%. Based on actual operation, the conditions were adjusted as follows: inlet air temperature adjusted to 121℃, feed flow rate slightly adjusted to 435 mL / min, and solid content maintained at 5.25%.

[0101] Table 4 Results of Response Surface Experiment

[0102]

[0103] Since the initial viable counts of *Lactobacillus plantarum* were different in Examples 2 and 3, directly comparing the final encapsulated viable counts was unreliable. Therefore, a new set of experiments was conducted using the conditions from Examples 2 and 3 to verify the reliability of the response surface methodology optimization effect. Under the optimal response surface methodology conditions (Example 3), the viable count of the microcapsules prepared was 4.6 × 10⁻⁶. 10 The number of viable bacteria in the microcapsules prepared under the optimal single-factor conditions (Example 2) was 3.0 × 10⁻⁶ CFU / g. 10 The CFU / g count under the optimal process conditions was 53.33% higher than that under the single-factor optimization conditions, indicating that the response surface methodology results are reliable.

[0104] (3) In vitro simulated digestion experiment

[0105] D1. Preparation of simulated gastric digestive juice (SGF): Adjust the pH of a solution containing 0.2% sodium chloride to 2.0 with 0.08M hydrochloric acid, sterilize at 121℃ for 20 min, add 0.3% pepsin, dissolve thoroughly and set aside.

[0106] D2. Preparation of simulated digestive intestinal fluid (SIF): Prepare a PBS solution with pH=6.4, sterilize at 121℃ for 20 min, add 1% bile salts and 1% trypsin, dissolve thoroughly and set aside.

[0107] D3. Add the 0.1g sample of *Lactobacillus plantarum* microcapsules prepared in Example 2 to a test tube containing 9.9mL of simulated gastric juice (preheated to 37°C);

[0108] Incubate at 37℃ in a water bath with shaking at 100 rpm for 2 hours. Take samples at 0.5h, 1h, 1.5h and 2h, dilute and spread on MRS solid medium, and count after incubation at 37℃ for 2 days.

[0109] D4. After 2 hours of simulated gastric digestion, the pH was adjusted to 7.0 with NaOH solution, centrifuged at 8000 rpm for 10 minutes, the precipitate (about 0.1 g) was collected, and 9.9 mL of simulated intestinal fluid was quickly added.

[0110] Digest at 37℃ for 2 hours, and take samples at 0.5h, 1h, 1.5h and 2h respectively. Dilute and spread on MRS solid medium, and count after incubation at 37℃ for 2 days.

[0111] The dynamic changes in the number of viable bacteria in the *Lactobacillus plantarum* capsules prepared in Example 2 during simulated in vitro digestion in the gastrointestinal tract were determined using the above method. Free *Lactobacillus plantarum* was used as a control, and records were taken every 0.5 hours for a total of 4 hours. The results are as follows: Figure 9 As shown.

[0112] See Figure 9 Compared with free *Lactobacillus plantarum*, the *Lactobacillus plantarum* microcapsules prepared in Example 2 showed better gastric juice tolerance, with the number of viable bacteria decreasing by only 1.50 log CFU / g, and still maintaining an activity of over 8.00 log CFU / g after 4 hours of continuous gastrointestinal digestion.

[0113] (4) Phenomorphological characterization of Lactobacillus plantarum microcapsules

[0114] The microstructure of the microcapsules of Comparative Example 4, Comparative Example 9, Comparative Example 16 and Example 2 was observed using field emission scanning electron microscopy.

[0115] The conductive tape is flatly pasted onto a dedicated stage. Microcapsule powder is taken and evenly adhered to the conductive tape. Sample powder that fails to adhere to the tape is blown away and then gold sputtering is performed.

[0116] After processing, high-resolution images were captured using a field emission scanning electron microscope at a voltage of 5.0 kV for observation and photography. The magnifications were 10000× and 20000×. The results are as follows: Figure 10 As shown.

[0117] See Figure 10 The binary composite microcapsule samples (Comparative Example 4, Comparative Example 9, and Comparative Example 16) exhibited spherical shapes of varying sizes and severe wrinkling and depressions, which may be due to the rapid evaporation of moisture caused by high temperatures during the drying process. This is a typical characteristic of products obtained by spray drying.

[0118] Compared with microcapsules encapsulated in binary composite wall materials, the *Lactobacillus plantarum* microcapsules encapsulated in ternary composite wall materials prepared in Example 2 of this invention are spherical, smooth in appearance, and without obvious cracks, with shallower and fewer wrinkles and depressions. This indicates that the ternary composite wall material can improve the surface structure of the microcapsules and reduce the impact of physical damage on the appearance of the capsules. Furthermore, no free probiotics were found on the surface of the microcapsules, indicating that the selected ternary composite wall material can stably encapsulate *Lactobacillus plantarum* NCUTUUAS4 inside the microcapsules.

[0119] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A microcapsule of *Lactobacillus plantarum*, characterized in that, The plant lactobacillus microcapsules include plant lactobacillus and composite wall material; The *Lactobacillus plantarum* mentioned is *Lactobacillus plantarum* NCUTUUAS4, whose Latin name is... Lactiplantibacillus plantarum NCUTUAS4, accession number CCTCC NO: M 2024858; The composite wall material is composed of lily starch, citrus pectin, and whey protein; wherein the mass ratio of lily starch, citrus pectin, and whey protein is 1:1:

1.

2. The *Lactobacillus plantarum* microcapsules according to claim 1, characterized in that, The diameter of the *Lactobacillus plantarum* microcapsules is 3-5 μm.

3. The *Lactobacillus plantarum* microcapsules according to claim 1, characterized in that, The viable count of *Lactobacillus plantarum* NCUTUUAS4 in the *Lactobacillus plantarum* microcapsules is (1-5) × 10⁻⁶. 10 CFU / g.

4. A method for preparing *Lactobacillus plantarum* microcapsules as described in claim 1, characterized in that, Includes the following steps: Lily starch, citrus pectin, whey protein powder and deionized water were mixed and magnetically stirred to prepare a ternary composite wall material solution. After sterilization, a sterile ternary composite wall material solution was prepared. A suspension of *Bacillus plantarum* was mixed with a sterile ternary composite wall material solution to obtain a microcapsule solution; the volume ratio of the *Bacillus plantarum* suspension to the sterile ternary composite wall material solution was 1:(50-120). The microcapsule solution was spray-dried at low temperature to obtain *Lactobacillus plantarum* microcapsules.

5. The preparation method according to claim 4, characterized in that, In the ternary composite wall material solution, the mass ratio of lily starch, citrus pectin, and whey protein is 1:1:

1.

6. The preparation method according to claim 4, characterized in that, The parameters for the low-temperature spray drying are as follows: the inlet air temperature for spray drying is 100-140 ℃, the feed flow rate is 150-750 mL / h, and the solid content in the ternary composite wall material solution is 3%-15%.

7. The application of the *Lactobacillus plantarum* microcapsules as described in claim 1 in probiotic preparations.

8. The application of the *Lactobacillus plantarum* microcapsules as described in claim 1 in the heat processing of food.

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