Lactobacillus plantarum double-layer microcapsule, preparation method and application thereof

Abstract

The present application relates to the field of probiotic microcapsules, in particular to a bifidobacterium longum double-layer microcapsule, a preparation method and application thereof. The present application is a bifidobacterium longum double-layer microcapsule prepared by extrusion and electrostatic deposition with pectin as a core layer and chitosan and its derivatives as an outer layer wall material. The method can achieve multi-layer encapsulation under mild conditions, and the preparation conditions are simple and mild, and easy to realize large-scale production. The obtained bifidobacterium longum double-layer microcapsule can not only effectively improve its tolerance stability, but also realize targeted delivery and release to colon cells, thereby realizing better probiotic function and providing a new idea for the development of probiotic functional products. At the same time, the bifidobacterium longum double-layer microcapsule of the present application applied in different types of fruit juice reduces the acidification of the fruit juice, reduces the influence of the matrix on the bifidobacterium longum, improves the activity of the bifidobacterium longum, significantly improves the transparency of the fruit juice, and does not affect the original sensory characteristics of the fruit juice.

Description

Technical Field

[0001] This invention relates to the field of probiotic microcapsules, specifically to a double-layer microcapsule of *Lactobacillus plantarum*, its preparation method, and its application. Background Technology

[0002] Probiotics possess a variety of physiological activities and can promote the host's gut health. The sustainability of their health-promoting effects depends on their ability to colonize the host's gut. However, due to the dynamic and diverse environment of the host's gastrointestinal tract, composed of microbial metabolism and physical and chemical factors involved by the host itself (such as oxygen, pH, and pepsin), probiotics, including *Lactobacillus plantarum*, struggle to survive and colonize successfully in high numbers. Therefore, probiotics are constantly challenged by environmental factors during application and food processing, such as various digestive enzymes in the gastrointestinal tract, acid stress, and heat stress. Environmental stress may lead to decreased bacterial cell viability, limiting their application.

[0003] Lactobacillus plantarum is a key probiotic found in the human colon and is widely used in the food and pharmaceutical industries. As a probiotic, Lactobacillus plantarum has great potential to improve the host's nutritional status and health, and plays an important role in regulating the balance of the human gut microbiota and maintaining human health. This has led to increasing attention being paid to the development of Lactobacillus plantarum-based foods.

[0004] Microencapsulation technology is an important means of protecting probiotics. This technology can protect *Lactobacillus plantarum* from external stress during processing and storage, and from protease degradation and pH fluctuations when it reaches its intestinal site of action, thereby improving its acid resistance. Therefore, encapsulating *Lactobacillus plantarum* to ensure its smooth arrival in the intestines and its ability to adhere and colonize the colon with a high viable count is of positive significance for the application of probiotics in functional foods.

[0005] In existing technologies, microencapsulation primarily focuses on the protection and stability of probiotics, neglecting the colonic adhesion that truly affects their efficacy in the human body. Furthermore, common microencapsulation preparation methods, such as spray drying and electrostatic bonding, present significant production challenges, further limiting their application. Additionally, probiotics are more commonly used in dairy products and fermented foods, with limited application in fruit and vegetable juices. Probiotic-based fruit juices and other food products have not been widely explored. Given the low pH environment of fruit and vegetable juices, a sufficiently stable system is essential to protect the strains from the effects of low pH. Therefore, developing alternative food matrices is necessary.

[0006] In summary, the preparation of a bilayer microcapsule of *Lactobacillus plantarum* is of great significance in order to solve the above problems. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a double-layer microcapsule of *Lactobacillus plantarum* to address the shortcomings of the prior art.

[0008] Another technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned *Lactobacillus plantarum* bilayer microcapsules.

[0009] The final technical problem to be solved by this invention is to provide the application of the above-mentioned *Lactobacillus plantarum* bilayer microcapsules.

[0010] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0011] A method for preparing *Lactobacillus plantarum* bilayer microcapsules includes the following steps:

[0012] (1) Cultivate Lactobacillus plantarum, centrifuge to collect the precipitate and resuspend it to obtain Lactobacillus plantarum suspension;

[0013] (2) The plant lactobacillus suspension prepared in step (1) is mixed evenly with pectin solution and cross-linked to obtain a core layer solution;

[0014] (3) The core layer solution obtained in step (2) is added to calcium chloride solution for solidification. After filtration and washing, it is immersed in chitosan compound solution to generate electrostatic deposition to form an outer protective shell. After washing, plant lactobacillus bilayer microcapsules are prepared.

[0015] The plant lactobacillus (Latin name Lactobacillus plantarum, abbreviated as L.plantarum) mentioned herein includes, but is not limited to, plant lactobacillus T1 and JEB. Any strain under the classification of plant lactobacillus in the prior art can be used to prepare plant lactobacillus bilayer microcapsules using the preparation method of this invention.

[0016] Specifically, the *Lactobacillus plantarum* T1 strain, classified as *Lactobacillus plantarum*, with strain number T1, was deposited at the China Center for Type Culture Collection (CCTCC) on August 31, 2023, with accession number CCTCC NO: M20231553, located at Wuhan University, Wuhan, China; and the *Lactobacillus plantarum* JEB strain, classified as *Lactobacillus plantarum*, with strain number JEB, was deposited at the China Center for Type Culture Collection (CCTCC) on January 5, 2024, with accession number CCTCC NO: M 2024023, located at Wuhan University, Wuhan, China.

[0017] In step (1), the cultivation of *Lactobacillus plantarum* specifically involves activating and culturing *Lactobacillus plantarum* in sterile broth medium 2 to 3 times.

[0018] In step (1), the *Lactobacillus plantarum* suspension has a concentration of 10. 8 ~10 10 CFU / mL.

[0019] In step (2), the pectin is apple pectin or citrus pectin, and its degree of esterification is 6-65%.

[0020] Preferably, the pectin is apple pectin with a degree of esterification of 30%.

[0021] In step (2), the concentration of the pectin solution is 1-4% (g / 100mL), i.e., 10-40g / L, and the solvent is water; the volume ratio of the plant lactobacillus suspension to the pectin solution is 1:(8-10).

[0022] Preferably, the concentration of the pectin solution is 2% (g / 100mL), i.e., 20g / L, and the solvent is water; the volume ratio of the *Lactobacillus plantarum* suspension to the pectin solution is 1:9.

[0023] Specifically, the process of achieving uniform mixing involves gently stirring at 37°C for 5 minutes.

[0024] In step (3), the calcium chloride solution has a concentration of 1-2% (g / 100mL), i.e., 10-20g / L, and the solvent is water; the curing time is 20-30min.

[0025] Preferably, the calcium chloride solution has a concentration of 2% (g / 100mL), i.e., 20g / L, and the solvent is water.

[0026] In step (3), the addition is done drop by drop, with an addition volume of 10-50 μL / drop, a drop flow rate of 200-800 μL / min, and a drop height of 10-15 cm from the calcium chloride solution surface.

[0027] Preferably, the droplet infusion is performed at a volume of 10 μL / drop, a flow rate of 200 μL / min, and a height of 10 cm above the calcium chloride solution surface.

[0028] In step (3), the chitosan compound includes any one of chitosan, chitosan hydrochloride, and carboxymethyl chitosan; the chitosan compound solution has a concentration of 0.5-2% (g / 100mL), i.e. 5-20g / L, and the solvent is water.

[0029] Preferably, the chitosan compound is carboxymethyl chitosan; the chitosan compound solution has a concentration of 1% (g / 100mL), i.e., 10g / L, and the solvent is water.

[0030] In step (3), the volume ratio of the core layer solution, calcium chloride solution and chitosan compound solution is 1:10-20:10-20.

[0031] Preferably, the volume ratio of the core layer solution, calcium chloride solution and chitosan compound solution is 1:20:10.

[0032] In step (3), the immersion conditions are: rotation speed 70-100 rpm, time 1-1.5 h.

[0033] The plant lactobacillus bilayer microcapsules prepared by the above method are also within the scope of protection of this invention.

[0034] The application of the above-mentioned Lactobacillus plantarum double-layer microcapsules in improving the colonic adhesion of Lactobacillus plantarum is also within the scope of protection of this invention.

[0035] The application of the above-mentioned *Lactobacillus plantarum* double-layer microcapsules in the preparation of fruit juice beverages is also within the scope of protection of this invention.

[0036] The fruit juice beverages mentioned herein include any one or a combination of grape juice, apple juice, and carrot juice.

[0037] Beneficial effects:

[0038] Compared with the prior art, the present invention has the following advantages:

[0039] (1) Existing technologies focus on the protection and stability of microbial strains through microencapsulation. This technology utilizes a two-layer material for reinforcement and optimizes the wall material for encapsulating Lactobacillus plantarum, thus constructing an efficient and stable encapsulation system.

[0040] (2) The double-layer microcapsules of Lactobacillus plantarum prepared in this invention significantly improve the encapsulation rate of Lactobacillus plantarum and the survival rate of Lactobacillus plantarum after treatment with simulated gastrointestinal fluid.

[0041] (3) The double-layer microcapsules of Lactobacillus plantarum prepared by the present invention have colon-targeted release capability. In the gastrointestinal tract, Lactobacillus plantarum is almost unaffected by gastric digestion and can reach the intestine smoothly and exhibit good colon cell adhesion. That is, while effectively protecting the strain, it further improves the adhesion of Lactobacillus plantarum to colon cells, which is conducive to the targeted release of Lactobacillus plantarum and its colonization in large quantities in the colon, thus achieving better probiotic function and providing a new idea for the development of probiotic functional products.

[0042] (5) The *Lactobacillus plantarum* double-layer microcapsules of the present invention, when applied to different types of fruit juices, reduce acidification, reduce the influence of the matrix on *Lactobacillus plantarum*, improve the activity of *Lactobacillus plantarum*, and significantly improve the transparency of the fruit juice without affecting its original sensory characteristics. Specifically, the three fruit juices with added *Lactobacillus plantarum* microcapsules showed good turbidity during storage, and the solutions were relatively clear and stable. The pectin-carboxymethyl chitosan microcapsules maintained a good spherical shape and a smooth, intact surface after 21 days of storage. The viable count during the storage period was still greater than 10. 8 CFU / mL. Under simulated gastrointestinal fluid conditions, the viable counts of free *Lactobacillus plantarum* T1 and JEB decreased significantly over time, while encapsulation of *Lactobacillus plantarum* significantly improved bacterial activity in the fruit juice.

[0043] (6) The plant lactobacillus double-layer microcapsules of the present invention can achieve multi-layer encapsulation under mild conditions, and the preparation conditions are simple and mild, making it easy to achieve large-scale production. Attached Figure Description

[0044] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0045] Figure 1 Images of different plant lactobacillus bilayer microcapsules. AH represents microcapsules Pec2, Pec2-cs, Pec2-chc, Pec2-cmcs, Pec3, Pec3-c, Pec3-chc, and Pec3-cmcs placed in aqueous solution, respectively; ah represents the corresponding microcapsules placed in air.

[0046] Figure 2 Scanning electron microscopy images of bilayer microcapsules of different plant lactobacilli. A: Pec2; B: Pec2-cs; C: Pec2-chc; D: Pec2-cmcs; E: Pec3; F: Pec3-cs; G: Pec3-chc; H: Pec3-cmcs.

[0047] Figure 3 Fourier transform infrared (FTIR) images of bilayer microcapsules of different plant lactobacilli. In the images, A: Pec2 is the matrix (Pec2, Pec2-cs, Pec2-chc, Pec2-cmcs); B: Pec3 is the matrix (Pec3, Pec3-c, Pec3-chc, Pec3-cmcs).

[0048] Figure 4Release characteristics of different *Lactobacillus plantarum* bilayer microcapsules under different pH and time conditions. Wherein, A: number of viable *Lactobacillus plantarum* released using Pec2 as the matrix; B: number of viable *Lactobacillus plantarum* released using Pec3 as the matrix; C: release rate of *Lactobacillus plantarum* using Pec2 as the matrix; D: release rate of *Lactobacillus plantarum* using Pec3 as the matrix.

[0049] Figure 5 The swelling characteristics of different plant-derived Lactobacillus bilayer microcapsules in simulated gastric (SGF, abbreviated G) and intestinal (SIF, abbreviated I) fluids were studied. In this study, A: Pec2 was used as the matrix; B: Pec3 was used as the matrix.

[0050] Figure 6 The following diagrams simulate the gastrointestinal digestion of different *Lactobacillus plantarum* bilayer microcapsules: A: Digestion diagram of different *Lactobacillus plantarum* bilayer microcapsules with Pec2 matrix in the stomach; B: Digestion diagram of different *Lactobacillus plantarum* bilayer microcapsules with Pec3 matrix in the stomach; C: Digestion diagram of different *Lactobacillus plantarum* bilayer microcapsules with Pec2 matrix in the intestine; D: Digestion diagram of different *Lactobacillus plantarum* bilayer microcapsules with Pec3 matrix in the intestine.

[0051] Figure 7 This diagram shows the adhesion rates of different plant-derived Lactobacillus bilayer microcapsules after digestion in the stomach and intestines. In the diagram, A represents the stomach, and B represents the intestines.

[0052] Figure 8 The storage stability of free and different double-layered encapsulated *Lactobacillus plantarum* at 4°C was evaluated. A: using Pec2 as the substrate; B: using Pec3 as the substrate.

[0053] Figure 9 The turbidity change of juices containing *Lactobacillus plantarum* or double-layered microcapsules was observed after storage at 4°C for 21 days. Here, *carrot*, *grape*, and *apple* represent sterile carrot juice, grape juice, and apple juice, respectively; *carrot+L. plantarum*, *gra+L. plantarum*, and *app+L. plantarum* represent carrot juice, grape juice, and apple juice containing free *Lactobacillus plantarum*, respectively; and *carrot+microcapsule*, *gra+microcapsule*, and *app+microcapsule* represent carrot juice, grape juice, and apple juice containing microcapsules encapsulating *Lactobacillus plantarum*, respectively.

[0054] Figure 10 Morphological changes of pectin-carboxymethyl chitosan beads encapsulating Lactobacillus plantarum in different fruit juices over 21 days.

[0055] Figure 11This figure shows the viable counts of different *Lactobacillus plantarum* bacteria encapsulated in grape juice during different storage periods.

[0056] Figure 12 This figure shows the viable counts of different *Lactobacillus plantarum* bacteria encapsulated in apple juice during different storage periods.

[0057] Figure 13 This figure shows the viable counts of different *Lactobacillus plantarum* bacteria encapsulated in carrot juice during different storage periods.

[0058] Figure 14 A graph showing the number of viable Lactobacillus plantarum bacteria resistant to gastric digestion in grape juice with different storage days, encapsulated using pectin-carboxymethyl chitosan beads.

[0059] Figure 15 A graph showing the number of viable Lactobacillus plantarum bacteria resistant to gastric digestion in apple juice encapsulated with pectin-carboxymethyl chitosan beads after different storage days.

[0060] Figure 16 A graph showing the number of viable Lactobacillus plantarum bacteria in carrot juice with different storage days, encapsulated with pectin-carboxymethyl chitosan beads, tolerating gastric digestion.

[0061] Figure 17 A graph showing the number of viable enterodigestible bacteria of different *Lactobacillus plantarum* strains encapsulated in grape juice with different storage days using pectin-carboxymethyl chitosan beads.

[0062] Figure 18 A graph showing the number of viable enterodigestible bacteria of different *Lactobacillus plantarum* strains encapsulated in apple juice with pectin-carboxymethyl chitosan beads after different storage days.

[0063] Figure 19 A graph showing the number of viable enterodigestible bacteria of different *Lactobacillus plantarum* strains encapsulated in carrot juice with pectin-carboxymethyl chitosan beads after different storage days.

[0064] Figure 20 Sensory evaluations were performed on different Lactobacillus plantarum juice products after 21 days of storage. Here, gra, app, and car represent grape juice, apple juice, and carrot juice without bacteria, respectively; Lgra, Lapp, and Lcar represent grape juice, apple juice, and carrot juice containing free bacteria, respectively; Egra, Eapp, and Ecar represent grape juice, apple juice, and carrot juice containing Lactobacillus plantarum encapsulated with pectin-carboxymethyl chitosan beads, respectively; color represents color; transparency represents transparency; flavor represents flavor; odor represents odor; and texture represents texture. Detailed Implementation

[0065] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0066] In the following embodiments, the *Lactobacillus plantarum* T1, classified as *Lactobacillus plantarum*, strain number T1, was deposited at the China Center for Type Culture Collection (CCTCC) on August 31, 2023, with accession number CCTCC NO: M20231553, and the deposit address is Wuhan University, Wuhan, China; the *Lactobacillus plantarum* JEB, classified as *Lactobacillus plantarum*, strain number JEB, was deposited at the China Center for Type Culture Collection (CCTCC) on January 5, 2024, with accession number CCTCC NO: M 2024023, and the deposit address is Wuhan University, Wuhan, China.

[0067] The pectin described in the following examples is food-grade citrus peel pectin with an esterification degree of 60±5%, 30±3%, and 6±3%, and food-grade apple peel pectin with an esterification degree of 30±3%, both purchased from Yantai Andre Pectin Co., Ltd. (galacturonic acid ≥65.0%, Shandong, China).

[0068] In the following examples, the MRS liquid culture medium has the following formulation: 5.0 g yeast extract, 10.0 g peptone, 5.0 g anhydrous sodium acetate, 2.0 g K2HPO4, 0.589 g MgSO4·7H2O, 10.0 g beef extract, 2.0 g triammonium citrate, 1.0 mL Tween-80, 0.25 g MnSO4·4H2O, 1000 mL distilled water, and pH adjusted to 6.8.

[0069] The calcium chloride solution described in the following examples was prepared as follows: 2.0 g and 4.0 g of anhydrous calcium chloride were weighed into 200 mL of pure water, sterilized at 121 °C for 20 min, and cooled to room temperature for later use.

[0070] The pectin solution was prepared as follows in the following examples: 1.0g, 2.0g, and 3.0g of four different food-grade pectins were weighed into 100mL of sterile pure water in a clean bench, and stirred at 40°C for 30min under a magnetic stirrer until completely dissolved. The solution was then cooled to room temperature for later use.

[0071] The following examples illustrate the preparation of the simulated gastric fluid (SGF): Weigh 2.8 g NaCl, 514.4 mg KCl, 2.1 g NaHCO3, 225.0 mg KH2PO4, 20.3 mg MgCl2·6H2O, and 78.6 mg (NH4)2CO3, dissolve them in 1,000 mL distilled water, and adjust the pH to 2.5 with 0.1 M HC.

[0072] The following examples illustrate the preparation of the simulated intestine fluid (SIF): Weigh 2.2g NaCl, 507.0mg KCl, 7.1g NaHCO3, 108.9mg KH2PO4, and 67.1mg MgCl2·6H2O, dissolve them in 1,000mL distilled water, and adjust the pH to 7.5.

[0073] The chitosan (cs), chitosan hydrochloride (chc), and carboxymethyl chitosan (cmcs) (all with a degree of deacetylation ≥ 90.0%) mentioned in the following examples were all purchased from Shanghai Maclean Co., Ltd.

[0074] The 1% (g / 100mL) chitosan (CS) solution was prepared as follows in the examples below: 1g of chitosan was weighed into 100mL of acetic acid solution and dissolved completely in a magnetic stirrer at 800rpm to obtain a 1% (g / 100mL) chitosan (CS) solution.

[0075] The 1% (g / 100mL) chitosan hydrochloride (CHC) solution and the 1% (g / 100mL) carboxymethyl chitosan (CMCS) solution described in the following examples were prepared as follows: Weigh 1g of chitosan hydrochloride and 1g of carboxymethyl chitosan respectively, and dissolve them completely in 100mL of sterile distilled water using a magnetic stirrer at 800rpm to obtain the 1% (g / 100mL) chitosan hydrochloride (CHC) solution and the 1% (g / 100mL) carboxymethyl chitosan (CMCS) solution.

[0076] The grape juice and apple juice mentioned in the following examples are 100% pure fruit juice purchased from supermarkets, and the carrot juice is 100% pure fruit juice purchased from Yidianyuan flagship store.

[0077] In this invention, Pec1 and pec1, Pec2 and pec2, Pec3 and pec3, and Pec3 and pec3 have the same meaning.

[0078] Example 1: Preparation of *Lactobacillus plantarum* bilayer microcapsules

[0079] (1) Add *Lactobacillus plantarum* T1 and JEB, preserved at -80℃, to MRS medium and incubate at 37℃ for 18 h, activating twice. Then, centrifuge 1 mL of the bacterial solution at 5,000 rpm and 4℃ for 5 min, collect the precipitate, wash with 1 mL of sterile physiological saline for later use, and finally dilute the precipitate with 1 mL of physiological saline to approximately 10. 8 Two different suspensions of *Lactobacillus plantarum* were obtained by dispersing CFU / mL.

[0080] (2) Apple pectin (Pec2) and citrus pectin (Pec3) with a pectin esterification degree of 30% were used as pectin materials. The bacterial suspension was added to different pectin solutions with a concentration of 2% (g / 100mL) (mixed according to the volume ratio of Lactobacillus plantarum suspension to pectin solution of 1:9), and gently stirred at 37℃ for 5min to make a mixed core layer solution.

[0081] (3) Using a 0.4 mm syringe with a capacity of 1 mL, drop 1 mL of core layer solution (about 10 μL / drop) into 20 mL of 2% (g / 100 mL) calcium chloride solution at a fixed height of about 10 cm above the liquid surface. Solidify for 30 min to form microcapsule beads. Filter to obtain the sample, and wash the microcapsules three times with sterile water to remove excess calcium ions. Finally, filter to recover the microbeads (i.e., Lactobacillus plantarum monolayer microcapsules).

[0082] (4) Add 10 mL of 1% (g / 100 mL) chitosan and 1% [unclear] to a clean conical flask containing microbeads.

[0083] A 1% (g / 100mL) chitosan derivative and a 1% (g / 100mL) carboxymethyl chitosan solution were used to completely submerge all microbeads. The microbeads were then stirred at 80 rpm with the three chitosan-related solutions and allowed to contact at room temperature for 1 hour to allow electrostatic deposition, forming an outer protective shell. Finally, the *Lactobacillus plantarum* microspheres encapsulated by chitosan and its derivatives were recovered, washed three times with sterile water to remove excess uncrosslinked solution, and finally filtered to obtain different *Lactobacillus plantarum* bilayer microcapsules, named Pec2-cs, Pec2-chc, Pec2-cmcs, Pec3-cs, Pec3-chc, and Pec3-cmcs, respectively.

[0084] Example 2: Morphological observation, diameter and porosity determination of Bacillus plantarum double-layer microcapsules

[0085] The morphology, diameter, and porosity of the different *Lactobacillus plantarum* bilayer microcapsules prepared in Example 1 were observed and measured. The measurement methods are as follows:

[0086] (1) Morphological observation: The morphology of the Lactobacillus plantarum double-layer microcapsules (wet state) and freeze-dried obtained by different pectin preparations was photographed using a digital camera.

[0087] (2) Diameter determination: For each type of Lactobacillus plantarum double-layer microcapsule (wet state), 50 samples were randomly selected and the average size (wet state) was calculated using a calibrated digital vernier caliper and micrometer (resolution 0.01 mm).

[0088] (3) Porosity determination: Porosity was assessed by measuring the bulk density (ρb) and true density (ρt) of each type of *Lactobacillus plantarum* bilayer microcapsule. Porosity formula: Porosity (%) = (1 - ρb / ρt) × 100%

[0089] Figure 1 The appearance of freshly prepared *Lactobacillus plantarum* bilayer microcapsules is shown (AH: placed in water; ah: dried state). Freshly prepared Pec2 pectin-type bilayer microcapsules are translucent white spheres, while *Lactobacillus plantarum* monolayer microcapsules not encapsulated with chitosan or its derivatives are smaller and have a considerably rougher surface. Pec3 pectin-type bilayer microcapsules are translucent pale yellow spheres. These results indicate that chitosan and its derivatives, as the shell layer, have no significant impact on the appearance of the microcapsules. However, it can be seen that the size of the bilayer-encapsulated *Lactobacillus plantarum* microcapsules is larger than that of the monolayer-encapsulated ones, indicating that the chitosan layer can successfully form a good cross-link with the pectin core layer, forming a protective outer layer that leads to the increased size. Referring to Table 1, it can be seen that the size of the bilayer microcapsules is significantly larger than that of the monolayer. For bilayer microcapsules with Pec2 as the core layer, the size increased by 0.51–0.55 mm, while the size of bilayer microcapsules with Pec3 as the core layer increased by 0.30–0.40 mm. Furthermore, the porosity of the bilayer microcapsules ranged from 31.51% to 39.92%, all lower than that of the monolayer microcapsules, indicating that the bilayer microcapsules improved the integrity of the microbeads. Among the bilayer microcapsules, the beads with carboxymethyl chitosan as the outer layer and apple pectin as the main matrix had the lowest porosity (P<0.05), suggesting that the gelation effect between carboxymethyl chitosan and apple pectin was the strongest.

[0090] Table 1. Average size of bilayer microcapsules of different plant-derived Lactobacillus species.

[0091] Material Average size (mm) Pec2 <![CDATA[1.30±0.08 a ]]> Pec2-cs <![CDATA[1.81±0.02 c ]]> Pec2-chc <![CDATA[1.85±0.12 c ]]> Pec2-cmcs <![CDATA[1.84±0.11 c ]]> Pec3 <![CDATA[1.41±0.08 a ]]> Pec3-cs <![CDATA[1.71±0.20 b ]]> Pec-chc <![CDATA[1.81±0.12 c ]]> Pec-cmcs <![CDATA[1.77±0.14 bc ]]>

[0092] Example 3: Determination of Encapsulation Efficiency (EE) of Bacillus plantarum double-layer microcapsules

[0093] Take 1 mL of the prepared *Lactobacillus plantarum* bilayer microcapsules and completely dissolve them in 1×PBS (pH 7.4). Gently stir at 37℃ and 400 rpm for 15–20 min to allow complete disintegration and release of the microcapsules. Then, centrifuge the mixture at 4℃ and 4,600 rpm for 10 min, resuspend in 1 mL of physiological saline, and then dilute 10-fold. Viable bacteria are counted on MRS agar using the plate count method. For the control group, 100 μL of free *Lactobacillus plantarum* was serially diluted with physiological saline and then plated for count. The encapsulation efficiency (EE) of the *Lactobacillus plantarum* bilayer microcapsules is calculated as follows: EE (%) = (E / E0) × 100%.

[0094] In the formula: E is the total number of viable bacteria embedded in the microcapsule, and E0 is the total number of viable bacteria in the bacterial suspension used to prepare the microcapsule.

[0095] The encapsulation efficiency results are shown in Table 2. As can be seen from the table, the encapsulation efficiency of the bilayer microcapsules for *Lactobacillus plantarum* ranged from 83.12% to 89.47%, with no statistically significant difference between groups (P>0.05). Although there was no statistically significant difference between bilayer and monolayer *Lactobacillus plantarum* microcapsules (P>0.05), the overall encapsulation efficiency of the bilayer microcapsules was high, indicating excellent encapsulation effect. The addition of chitosan and its derivatives as the shell layer did not disrupt the original cross-linked structure of pectin and calcium chloride, maintaining a relatively good encapsulation efficiency.

[0096] Table 2 Encapsulation efficiency (EE) of different bilayer microcapsules for Lactobacillus plantarum T1 and JEB

[0097] Material T1 EE (%) JEB EE (%) Pec2 <![CDATA[82.94±5.85 a ]]> <![CDATA[83.78±4.87 a ]]> Pec2-cs <![CDATA[83.12±4.09 a ]]> <![CDATA[84.62±3.25 a ]]> Pec2-chc <![CDATA[85.47±5.95 a ]]> <![CDATA[85.43±4.81 a ]]> Pec2-cmcs <![CDATA[86.17±1.69 a ]]> <![CDATA[84.16±2.34 a ]]> Pec3 <![CDATA[85.11±2.13 a ]]> <![CDATA[87.96±1.57 a ]]> Pec3-cs <![CDATA[86.90±7.57 a ]]> <![CDATA[84.62±0.95 a ]]> Pec-chc <![CDATA[88.36±3.69 a ]]> <![CDATA[89.90±3.36 a <!-- 7 -->]]> Pec-cmcs <![CDATA[88.53±1.98 a ]]> <![CDATA[89.47±2.30 a ]]>

[0098] Note: Different lowercase letters (a, b, c, d) indicate significant differences between different types of *Lactobacillus plantarum* bilayer microcapsule samples (P<0.05).

[0099] Example 4: SEM analysis of *Lactobacillus plantarum* double-layer microcapsules

[0100] Taking Lactobacillus plantarum T1 as an example, SEM analysis was performed on the double-layer microcapsules of Lactobacillus plantarum.

[0101] To better characterize the structure of *Lactobacillus plantarum* bilayer microcapsules, different types of *Lactobacillus plantarum* bilayer microcapsules were frozen at -20℃ for 6 hours and then freeze-dried for 48 hours to obtain dried microcapsules. The morphology, surface, and internal structure of the microcapsules were observed using scanning electron microscopy. *Lactobacillus plantarum* monolayer microcapsules were used as a control.

[0102] from Figure 2Scanning electron microscopy images show that the surface of the *Lactobacillus plantarum* monolayer microcapsules has many pores, and the material surface is incomplete and somewhat wrinkled. Figure 2 A, Figure 2 E) This may be due to the fact that during the freeze-drying process, the microcapsules are first affected by the formation of ice crystals in the residual water at low temperatures, and then the sublimation of the ice crystals under vacuum drying makes the microcapsules more prone to pore formation. In contrast, the surface of the *Lactobacillus plantarum* bilayer microcapsules is intact and without pores. This indicates that although the two-layer encapsulation did not statistically increase the encapsulation rate of *Lactobacillus plantarum*, the addition of the outer chitosan and its derivatives made the surface material of the *Lactobacillus plantarum* monolayer microcapsules more intact and the structural network more compact. Although the shapes of microcapsules prepared with different chitosan materials differed somewhat, they were generally intact, without small cracks, and had a uniform surface microstructure. Furthermore, the addition of the outer material minimized freeze-thaw losses during the freeze-drying process, resulting in a more compact surface. These results indicate that chitosan and its derivatives, as outer layer materials, can effectively combine with pectin, and the bilayer microcapsules can effectively protect the core *Lactobacillus plantarum*.

[0103] Example 5: Fourier Transform Infrared (FT-IR) Analysis of Bacillus plantarum Double-Layer Microcapsules

[0104] Taking Lactobacillus plantarum T1 as an example, Fourier transform infrared (FT-IR) analysis was performed on the double-layer microcapsules of Lactobacillus plantarum.

[0105] To further investigate the changes in pectin molecular groups, the infrared spectral structure of different *Lactobacillus plantarum* bilayer microcapsules was characterized using a Fourier transform infrared spectrophotometer at wavenumbers of 4000–4000 cm⁻¹. -1 The resolution is 4cm. -1 64 scans were performed. A 1 mg microcapsule sample was pressed into a 1 mm thin film using 100 mg of dry KBr powder for determination.

[0106] The results are as follows Figure 3 As shown, Fourier transform infrared spectroscopy reveals that pectin and pectin-chitosan and its derivative microcapsules are present at approximately 3420 cm⁻¹. -1 There is a broadband band at 2941 cm⁻¹, which is due to the stretching vibrations of OH and NH (hydroxyl and amino groups). -1 The position corresponds to CH extension (CH3 group). The binding of chitosan and its derivatives can be seen, enhancing the pectin's adhesion at 1750 cm⁻¹. -1 and 1630cm -1 The nearby characteristic bands correspond to the asymmetric stretching of esterified C=O and non-esterified C=O. This indicates that the binding of chitosan and pectin is through the electrostatic interaction of carboxyl and amino groups. 1420cm -1The symmetrical stretching vibration corresponding to the (COO) position shows that carboxymethyl chitosan exhibits a higher stretching vibration value than chitosan and chitosan hydrochloride. This indicates that carboxymethyl chitosan can dissociate -COOH in addition to -OH and -NH2, which is more conducive to reinforcing the calcium ion structure of pectin. Compared to pectin, the microcapsules formed by combining pectin and chitosan have approximately 1640 cm³ greater density. -1 The CO extension (amide I) at the location is approximately 1620 cm. -1 The signal is stronger. Besides the C=O extension of pectin, there may also be the NH bending (amide II) of chitosan, 1340cm. -1 CN extension (amide III). 1105cm -1 and 1019cm -1 The bands at that location are caused by the stretching of CC and CO associated with the sugar structure of chitosan.

[0107] Example 6: Determination of the release rate of *Lactobacillus plantarum* bilayer microcapsules

[0108] Taking Lactobacillus plantarum T1 as an example, the release rate of Lactobacillus plantarum double-layer microcapsules was determined.

[0109] The release behavior of different *Lactobacillus plantarum* bilayer microcapsules was evaluated using in vitro experiments in three different pH solutions.

[0110] Method 1: Take 1.0g of encapsulated *Lactobacillus plantarum* double-layer microcapsules and 9.0mL of simulated gastric juice (pH 10).

[0111] Mix with 2.0 (a buffer solution free of pepsin and pepsin), and continuously shake at 37°C and 180 rpm for 16 hours. Every 2 hours, take 100 μL to determine the release of *Lactobacillus plantarum* bilayer microcapsules, and determine the viable cell count using the plate dilution method. After each measurement, add 100 μL of fresh buffer to maintain the total volume.

[0112] The second method: Take 1.0g of encapsulated *Lactobacillus plantarum* double-layer microcapsules and 9.0mL of simulated intestinal fluid (pH 10).

[0113] Mix 6.8 (containing no secretin or bile salts) and stir continuously at 180 rpm for 16 hours. Take 100 μL every 2 hours to measure release, and determine viable cell count using the plate dilution method. After each measurement, add 100 μL of fresh buffer to maintain the total volume.

[0114] The third method: 1.0 g of encapsulated *Lactobacillus plantarum* bilayer microcapsules were mixed with 9.0 mL of 1×PBS (pH 7.4) and stirred continuously at 180 rpm for 16 h. 100 μL of the release mixture was collected every 2 h to measure the release, and the viable cell count was determined by plate dilution. After each measurement, 100 μL of fresh buffer was added to maintain the total volume.

[0115] The cumulative release rate of *Lactobacillus plantarum* after each time interval was calculated using the following formula: Release rate (%) = LogN1 / LogN0 × 100%. Where N1 is the number of live *Lactobacillus plantarum* cells released at that time; N0 is the number of live *Lactobacillus plantarum* cells encapsulated in the pectin microspheres.

[0116] The results are as follows Figure 4 As shown, at pH 1.8, the release rate of *Lactobacillus plantarum* microcapsules encapsulated in both pectin core matrix layers with chitosan and its derivatives was relatively slow. This may be due to the electrostatic effects of chitosan and its derivatives depositing onto the pectin matrix, resulting in a denser microsphere membrane and thus reducing the release rate. Compared to the release in an acidic environment, the release rate and cell count were higher in the neutral culture medium. The double-layer encapsulated microbeads exhibited higher stability in the gastric stage, maintaining a low release count even in the early pH 6.8 phase, which is more conducive to the delivery of *Lactobacillus plantarum* to the intestine. Regarding the binding of Pec2 and Pec3 with CS, CHC, and CMCs, the protective effect of CS, CHC, and CMCs on Pec2 was more pronounced compared to the single-layer encapsulation. Overall, the protection of the double-layer microcapsules improved the release performance in the gastrointestinal tract.

[0117] Furthermore, the release characteristics of *Lactobacillus plantarum* bilayer microcapsules were fitted using the Korsmeyer-Pepas mathematical model. The results are shown in Table 3. By adding shells containing CS, CHC, and CMCs, the diffusion index n increased, and all values ​​were greater than 0.45. Here, n represents the diffusion mechanism of the released substance. When the index n is between 0.45 and 1, the release mechanism is not Fick-type, which differs from the release characteristics of *Lactobacillus plantarum* encapsulated solely by a pectin core layer. This indicates that the addition of the shell layer alters the release characteristics of *Lactobacillus plantarum* from the microcapsules. For the zero-order targeted delivery system, the index n is between 0.45 and 1, meaning that the release rate of *Lactobacillus plantarum* is independent of time. Based on the results of this experiment, it is likely more of a pH-dependent release. This may be because, in addition to the cross-linking formed by the electrostatic interaction between pectin and CS, CHC, and CMCs, hydrogen bonds may also exist between molecules in the complex of pectin calcium, CHC, and CMCs. Furthermore, the influence of pH on the polysaccharide polymer leads to the formation of a diffusion barrier in the biopolymer chains, increasing the release coefficient n.

[0118] Table 3 shows the parameters obtained by fitting the data of *Lactobacillus plantarum* release from the bilayer microcapsules based on the Korsmeyer-Pepas mathematical model.

[0119] parameter Pec2-cs Pec2-chc Pec2-cmcs Pec3-cs Pec3-chc Pec3-cmcs kKP 2.724 4.193 4.488 3.236 2.375 2.708 n 0.496 0.502 0.503 0.468 0.513 0.514 <![CDATA[R 2 ]]> 0.9676 0.9545 0.9406 0.9631 0.9623 0.9715

[0120] Example 7: Determination of swelling properties of *Lactobacillus plantarum* bilayer microcapsules

[0121] Taking Lactobacillus plantarum T1 as an example, the swelling properties of Lactobacillus plantarum double-layer microcapsules were determined.

[0122] The water absorption capacity of *Lactobacillus plantarum* bilayer microcapsules was assessed by evaluating the changes in swelling levels in SGF (pH 1.8) and SIF (pH 6.8) solutions of different types. 50 mg of dried microcapsules were placed in vials containing 50 mL of different solutions. At predetermined 50-minute intervals, the microcapsules were carefully removed, excess liquid was thoroughly cleaned from their surface, and then they were weighed again. The swelling rate at each time interval was calculated using the formula: Swelling rate (g / g) = (S1 – S0) / S0, where S1 and S0 are the weights of the sample that swelled with water under different media and drying conditions, respectively.

[0123] Figure 5 As can be seen, in acidic solutions, the swelling ratio of both Pec2 and Pec3 bilayer microcapsules is lower than that of neutral microbeads, indicating that bilayer microcapsules are more stable in acidic solutions. Furthermore, regardless of the type of pectin used for the core layer, microcapsules with carboxymethyl chitosan as the shell layer exhibit the largest swelling ratio. For Pec2, the water absorption capacity of monolayer pectin is worse than that of bilayer microcapsules under acidic conditions. Under neutral conditions, the network of bilayer microcapsules is more compact, resulting in less volume expansion than monolayer microcapsules, thus leading to lower water absorption capacity and overall greater stability. In conclusion, pectin microbeads prepared from chitosan and its derivatives possess excellent pH sensitivity and can be used in pH-responsive delivery systems.

[0124] Example 8: Tolerance of *Lactobacillus plantarum* bilayer microcapsules under simulated gastric and intestinal fluid conditions

[0125] The method for determining the tolerance of *Lactobacillus plantarum* double-layer microcapsules to simulated gastric and intestinal fluids is as follows:

[0126] (1) Tolerance under simulated gastric juice

[0127] One 1.0 g of two different types of *Lactobacillus plantarum* bilayer microcapsules, one 1.0 g of two different types of *Lactobacillus plantarum* monolayer microcapsules, and one 1.0 mL of unencapsulated *Lactobacillus plantarum* were mixed with 9.0 mL of SGF and incubated at 37°C and 180 rpm for 3 h. After gastric tolerance (0, 1.5, 3 h), the microcapsules were removed and centrifuged at 5,000 rpm for 4 min. After resuspending in PBS, the unencapsulated *Lactobacillus plantarum* could be directly serially diluted and plated for counting. The encapsulated bilayer and monolayer microcapsules were gently stirred at 37°C for 15–20 min to ensure complete release of *Lactobacillus plantarum*. After serial dilution, viable cell counts were performed using the plate count method.

[0128] (2) Tolerance under simulated intestinal fluid

[0129] One 1.0 g of two different types of *Lactobacillus plantarum* bilayer microcapsules, one 1.0 g of two different types of *Lactobacillus plantarum* monolayer microcapsules, and one 1.0 mL of unencapsulated *Lactobacillus plantarum* were each mixed with 9.0 mL of SIF and incubated at 37°C and 180 rpm for 4 h. After intestinal tolerance (0, 2, and 4 h), the microcapsules were removed and centrifuged at 5,000 rpm for 4 min. After resuspending in PBS, the unencapsulated *Lactobacillus plantarum* could be directly serially diluted and plated for counting. The encapsulated microcapsules required gentle stirring at 37°C for 15–20 min to ensure complete release of *Lactobacillus plantarum*. After serial dilution, viable cell counts were performed using the plate count method.

[0130] The survival rates of *Lactobacillus plantarum* encapsulated in single and double layers were compared with those of free bacteria. Figure 6 The results showed that in the experiment simulating gastric digestion ( Figure 6 In experiments involving A / B), exposure to SGF for 180 min significantly reduced the viability of free bacteria (P<0.05). Both single-layer and double-layer encapsulated beads showed significant protective effects against *Lactobacillus plantarum*, with double-layer encapsulated beads exhibiting a more pronounced protective effect. Almost all encapsulated strains showed tolerance to the adverse conditions of low pH and pepsin in gastric juice. In simulated intestinal digestion experiments (… Figure 6 (C / D) After exposure to SIF for 240 min, the tolerance of free bacteria was better than that in gastric juice, indicating that *Lactobacillus plantarum* can exist well in the intestinal environment. It can be seen that the double-layered microbeads further protected the probiotics, resulting in a stable viable count greater than 10⁻⁶. 9 CFU / mL.

[0131] In the simulated gastric fluid phase, compared to the viable bacterial count of free bacteria, JEB was protected by Pec2 monolayer and corresponding bilayer chitosan, chitosan hydrochloride, and carboxymethyl chitosan. Figure 6A), the levels increased by 9.46, 9.70, 9.77, and 9.72 (Log CFU / mL), respectively, while T1 increased by 9.18, 9.31, 9.48, and 9.49 (Log CFU / mL), respectively. Similarly, compared to free bacteria, JEB under the protection of Pec3 monolayer and corresponding bilayer ( Figure 6 B) increased by 9.31, 9.47, 9.63, and 9.69 (Log CFU / mL), respectively, and T1 increased by 8.98, 9.12, 9.23, and 9.44 (Log CFU / mL), respectively. It can be seen that the CMCS coating provides better protection for probiotic cells than the CHC and CS coatings under simulated gastrointestinal conditions. Secondly, the interaction between Pec2 and the outer layer material is more conducive to the survival of the strain in gastric acid, but there is no significant difference in the overall protective effect of the two pectin matrices on bacterial tolerance to gastric acid due to their binding with the shell material.

[0132] In the simulated intestinal fluid stage, compared with the final survival rate of 86.20% for JEB free bacteria, the survival rate under double-layer protection was higher. Pec2 binding to the double layer ( Figure 6 C) resulted in JEB viable bacterial counts increasing by 9.67, 9.66, and 9.76 (LogCFU / mL), respectively. Pec3 binding to the bilayer ( Figure 6 D) increased the viable bacterial count of JEB by 9.53, 9.62, and 9.74 (Log CFU / mL) compared to monolayer embedding, all of which were statistically significant (P<0.05). Compared to monolayer, both Pec2-cmcs and Pec3-cmcs groups showed significant protection. This indicates that the combination of carboxymethyl chitosan and pectin is more beneficial in improving the tolerance of *Lactobacillus plantarum* in intestinal fluid, with viable bacterial counts exceeding 10⁻⁶. 9 CFU / mL.

[0133] In summary, the protection of the double-layered microcapsules is more conducive to the intestinal-targeted delivery of Lactobacillus plantarum, and the protective effect of carboxymethyl chitosan as the shell material is more significant.

[0134] Example 9: Adhesion ability of *Lactobacillus plantarum* bilayer microcapsules in simulated gastric and intestinal fluids

[0135] The adhesion rate of the strains was assessed using monolayer Caco-2 cells (purchased from Xinrun Biotechnology). Two different types of *Lactobacillus plantarum* bilayer microcapsules and their corresponding free bacteria were resuspended in penicillin-streptomycin-free DMEM (purchased from Gibico), and the viable cell count (A0) was determined before adding the microcapsules to the wells. After incubation at 37°C for 2 h, the cells were washed three times with sterile PBS buffer to remove unadhered cells. 0.5 mL of trypsin was added, and the mixture was incubated at 37°C for 7 min. Finally, 0.5 mL of DMEM was added to terminate the reaction. The mixture was transferred to sterile centrifuge tubes, and plate counts were performed using a dilution gradient (A1). The adhesion rate was calculated as follows: Adhesion rate (%) = A1 / A0 × 100%, where A1 and A0 represent the viable *Lactobacillus plantarum* counts before and after adhesion.

[0136] The results are as follows Figure 7 As shown in A / B, it can be seen that microbeads with carboxymethyl chitosan as the shell can effectively improve the adhesion of Lactobacillus plantarum T1 and JEB to colon cells under the two pectin core layers (Pec2 and Pec3), whether in the stomach or the intestine.

[0137] Example 10: Determination of storage stability of *Lactobacillus plantarum* double-layer microcapsules

[0138] The storage stability of free and microencapsulated *Lactobacillus plantarum* was determined periodically at 4°C (1, 3, 5, 7, 14, 28 days), as well as the storage stability of unencapsulated and encapsulated *Lactobacillus plantarum*. 50 mg of each type of *Lactobacillus plantarum* monolayer microcapsule was placed in 4.5 mL of 50 mM PBS (pH 7.4), respectively, while free bacteria were stored in 10 mM PBS (pH 7.4). Microcapsules were removed at time intervals, and viable cell counts were determined using the plate dilution method.

[0139] Figure 8 The storage stability of *Lactobacillus plantarum* bilayer microcapsules during 28 days of storage at 4°C was described. Compared to single-layer microcapsules, the maximum viable count of two *Lactobacillus plantarum* strains was greater than 10⁻⁶ after 28 days of storage. 8 CFU / mL, in a Pec2-based system ( Figure 8 A) The double-layer microcapsule with CMCs and CHC as the outer coating provides the best protection, with a viable count of *Lactobacillus plantarum* JEB greater than 10. 9 CFU / mL. However, in systems using Pec3 as the matrix ( Figure 8 B) All three chitosan-related outer layers significantly increased the viable count of *Lactobacillus plantarum* JEB, all exceeding 10. 9CFU / mL. This indicates that the Pec3-based double-layer encapsulation system is more stable. The Pec2-cmcs and Pec3-cmcs groups showed similar protective results against both bacteria, suggesting that the carboxymethyl chitosan coating has better binding stability with pectin than the other two coating materials. Combined with the above results on gastrointestinal fluid tolerance, pectin beads with carboxymethyl chitosan as the outer layer have good protective effects and better storage stability.

[0140] In summary, carboxymethyl chitosan is an outer layer material with good adhesive properties, which can further improve the adhesion and colonization of *Lactobacillus plantarum*. Among the adhesive effects, the Pec2 pectin-carboxymethyl chitosan (Pec2-cmcs) group showed a significant improvement in the adhesion of *Lactobacillus plantarum*. Therefore, apple pectin and carboxymethyl chitosan bilayer microcapsules were selected for subsequent application experiments.

[0141] Example 11: Application of *Lactobacillus plantarum* double-layer microcapsules in fruit juice beverages

[0142] Pec2 pectin-carboxymethyl chitosan bilayer microcapsules, which have the ability to improve the adhesion of probiotics and have lower porosity, were added to three juices with different pH values. Their viability under in vitro gastrointestinal conditions and stored in different juices was evaluated. The specific process is as follows.

[0143] 11.1 Preparation of different live bacteria beverages

[0144] 10 mL of grape juice, apple juice, and carrot juice were separately dispensed into 15 mL sterile centrifuge tubes. 1.2 g (approximately 100 μL of free bacteria) of the prepared fresh *Lactobacillus plantarum* bilayer microcapsules (Pec2 pectin-carboxymethyl chitosan bilayer microcapsules) were weighed into each of the three different fruit juices to form the experimental group. A control group was also set up; *Lactobacillus plantarum* was collected, resuspended in physiological saline, and then 100 μL of free bacteria was added to each of the three different fruit juices to obtain the control group. The blank group consisted of an equal volume of different fruit juices. All samples were stored at 4 °C.

[0145] 11.2 pH determination

[0146] Taking Lactobacillus plantarum T1 as an example, the pH of the juice containing Lactobacillus plantarum double-layer microcapsules was determined.

[0147] Different fruit juices were stored at 4℃ for 21 days, and pH values ​​were measured periodically (days 1, 3, 7, 14, and 21) using a pH meter to monitor pH changes during storage. Before measurement, the juices were removed from 4℃ and allowed to reach room temperature for 2 hours. The results are shown in Table 4. Among the three juices, grape juice had the highest acidity, followed by apple juice, and then carrot juice. The pH value of grape juice without probiotics remained relatively constant during storage at 4℃, with a final pH value of 3.03 ± 0.01 recorded at the end of storage. The pH value of grape juice containing free probiotics decreased significantly compared to day 1 (P < 0.05), reaching a final pH value of 3.01 ± 0.01 on day 21. However, compared to grape juice without probiotics, there was no significant decrease, indicating that the physiological activity of free *Lactobacillus plantarum* was restricted in the acidic environment of grape juice, showing an inactive state. Grape juice with added *Lactobacillus plantarum* double-layer microcapsules showed relatively stable pH during 21 days of storage, with a decrease only on the last day (P<0.05). This indicates that the microcapsule encapsulation reduced the influence of the substrate on *Lactobacillus plantarum*, protecting its activity. For apple juice, the pH of the blank control group remained relatively stable during storage, with no significant change. However, in apple juice with added *Lactobacillus plantarum*, a clear pH change was observed. On day 14, the pH of the juice decreased to 3.72 (P<0.05), indicating that *Lactobacillus plantarum* was still in an adaptation phase to the substrate environment in the early stages. The pH decrease with increasing storage time may be due to *Lactobacillus plantarum* beginning to utilize the carbon and nitrogen sources in the substrate for survival, reproduction, and acid production. For apple juice with added *Lactobacillus plantarum* double-layer microcapsules, the pH also decreased on day 14, but the decrease was much smaller than that in the free bacteria juice group, again indicating the high stability of the double-layer microcapsules in apple juice. For carrot juice with a pH close to neutral, the pH of the juice solution with added free probiotics decreased significantly during 21 days of storage, eventually dropping to 4.72 ± 0.01. This may be because the environment of carrot juice is more suitable for the growth of *Lactobacillus plantarum*, and lactic acid is released from the free probiotics during storage. The final pH value of carrot juice containing encapsulated *Lactobacillus plantarum* was significantly higher than that of carrot juice containing free probiotics (P < 0.05), indicating that encapsulated *Lactobacillus plantarum* also has good stability under conditions close to neutral pH.

[0148] In summary, during the initial storage phase of fruit juice, regardless of whether free or encapsulated *Lactobacillus plantarum* was added, the pH value of the fruit juice did not change significantly compared to the initial value, and the *Lactobacillus plantarum* remained in a low-activity state. After adapting to the fruit juice substrate conditions, the pH values ​​of all groups decreased to varying degrees with increasing storage time, with the free *Lactobacillus plantarum* group showing the largest decrease. The encapsulated probiotics exhibited greater stability during fruit juice storage. For the three types of fruit juice, carrot juice showed the most significant pH decrease throughout storage, possibly because its initial higher pH value facilitated the release of bacteria and their rapid adaptation to substrate conditions.

[0149] Table 4. pH changes of different added grape juice, apple juice, and carrot juice after 21 days of storage.

[0150]

[0151] 11.3 Turbidity Measurement

[0152] Taking Lactobacillus plantarum T1 as an example, the turbidity of fruit juice containing Lactobacillus plantarum double-layer microcapsules was determined.

[0153] Turbidity of fruit juice is one of the important evaluation indicators in the stability assessment of fruit juice beverages. Different prepared fruit juice solutions were stored at 4℃ for 21 days, and periodically removed (1, 3, 7, 14, and 21 days). 200 μL of each fruit juice was transferred to a new 96-well ELISA plate, and the OD value was measured using a spectrophotometer. 600 The absorbance was measured at a specific value, and the experiment was repeated three times. Turbidity was evaluated based on the absorbance values. The results are as follows: Figure 9As shown, the three different fruit juice solutions exhibited varying degrees of turbidity. The overall turbidity, from highest to lowest, was carrot juice, grape juice, and apple juice. The untreated carrot juice showed little change in turbidity during 21 days of storage, with a slight decrease on day 7. This may be because carrot juice itself has relatively high turbidity and is not a homogeneous solution, leading to some sedimentation during storage. For the carrot juice with added *Lactobacillus plantarum*, the turbidity initially increased and then decreased. This indicates that the bacteria utilized the nutrients in the substrate for growth and metabolism, causing an increase in turbidity. Subsequently, acid production created an environment unfavorable for bacterial growth, leading to bacterial lysis and death as storage time increased, resulting in a decrease in turbidity. For the carrot juice with added double-layer microcapsules, the turbidity results were similar to the untreated group, showing a slight decreasing trend and ultimately lower turbidity than the untreated group. This may be because pectin-carboxymethyl chitosan, as an encapsulation material, has a certain adsorption capacity, reducing and stabilizing the solution turbidity. In grape juice, the untreated group showed no significant change in turbidity over 21 days, remaining relatively stable. The turbidity of grape juice containing free *Lactobacillus plantarum* increased on day 14, which was related to the decrease in pH, indicating that the strain was growing and metabolizing in the juice. Apple juice with unencapsulated *Lactobacillus plantarum* showed the same trend, further demonstrating that free *Lactobacillus plantarum* grows less well in acidic juices than in slightly neutral carrot juice. Increased turbidity in the juice leads to decreased stability during storage. Encapsulating *Lactobacillus plantarum* effectively solves this problem. Among the three juices, the juice with added *Lactobacillus plantarum* double-layer microcapsules showed good turbidity during 21 days of storage, resulting in a clearer and more stable solution.

[0154] 11.4 Morphological analysis of beads during storage

[0155] Taking Lactobacillus plantarum T1 as an example, the morphology of beads during storage was analyzed.

[0156] To better illustrate the storage stability of *Lactobacillus plantarum* in fruit juice, beverages with different live bacterial strains were stored at 4°C for 21 days after preparation, and beads were periodically removed (1, 3, 7, 14, 21 days) for morphological analysis. Figure 10As can be visually observed, the pectin-carboxymethyl chitosan beads maintained their spherical shape well during 21 days of storage, and the surface of the coated beads remained smooth with good gloss. Upon addition of grape juice, the color of the pectin-carboxymethyl chitosan-encapsulated *Lactobacillus plantarum* microcapsules changed from translucent white to translucent red. This is due to the exchange of substances within the grape juice, while the size of the pectin beads did not change significantly. On the other hand, in apple juice and carrot juice, the appearance of the carboxymethyl chitosan and pectin beads remained largely intact, and their color also changed to some extent, although the change was not as pronounced as in grape juice, possibly due to the different pH levels.

[0157] In conclusion, pectin-carboxymethyl chitosan bilayer microcapsules in different fruit juices exhibit good storage stability and a pleasant appearance, which is beneficial for the development of novel Lactobacillus plantarum fruit juices.

[0158] 11.5 Determination of viable bacterial count during storage:

[0159] The method for determining the viable bacterial count is the same as above. The results are as follows: Figures 11-13 As shown. Figures 11 to 13 The survival of free and encapsulated *Lactobacillus plantarum* in grape juice, apple juice, and carrot juice during 21 days of storage was described. The growth trend of free *Lactobacillus plantarum* in grape juice was initially a slow increase followed by a decrease. The viable count of *Lactobacillus plantarum* JEB showed a decreasing trend in the first three days, followed by an increase on the seventh day. *Lactobacillus plantarum* T1 exhibited the same growth trend in grape juice, and its adaptation to grape juice was relatively better than that of *Lactobacillus plantarum* JEB. At the end of storage, the viable count of *Lactobacillus plantarum* JEB decreased to below 10 compared to the first day. 6 The cell count was significantly reduced to CFU / mL. Encapsulation within carboxymethyl chitosan-pectin beads significantly improved cell viability. In apple and carrot juice, *Lactobacillus plantarum* encapsulated in double-layered microcapsules showed a significantly higher viable count than free *Lactobacillus plantarum* after 21 days (P<0.05).

[0160] Overall, the strains showed varying degrees of adaptation to the three types of fruit juice, growing best in carrot juice, followed by apple juice, and lastly grape juice. This indicates that free strains grow poorly in highly acidic fruit juices and cannot adapt well to the substrate environment. Encapsulation of *Lactobacillus plantarum* effectively addresses this issue and improves the survival rate of probiotics.

[0161] 11.6 Survival rate determination under simulated gastric juice conditions during juice storage

[0162] This embodiment investigated the tolerance of free and encapsulated *Lactobacillus plantarum* to simulated gastric juice conditions at 4°C for 7, 14, and 21 days under conditions similar to those described above, when stored in grape juice, apple juice, and carrot juice. The results of this study are as follows: Figures 14-16 As shown in the figure, under simulated gastric juice conditions, the number of free *Lactobacillus plantarum* stored in various fruit juices at 4°C significantly decreased over time in all three fruit juices, with the number of viable bacteria falling below 10 on the last day. 6 CFU / mL. Encapsulation with *Lactobacillus plantarum* significantly improved bacterial viability; for grape juice, the tolerance survival rate of T1 increased from 3.22% to 89.05% (P<0.05). The tolerance gastric fluid viable counts of both T1 and JEB were greater than 10 after 21 days. 8 CFU / mL. In apple juice, the survival rate of JEB increased from 0.02% to 50.2%, and T1 increased from 15.09% to 99.05% (P<0.05). In carrot juice, the overall number of viable bacteria resistant to gastric juice was higher than that in the previous two juices, because the pH was more favorable for bacterial survival and it contained more nutrients. Moreover, the viable bacterial count of *Lactobacillus plantarum* encapsulated on day 21 was higher after 3 hours of resistant gastric juice than that on day 14, indicating that the encapsulated *Lactobacillus plantarum* had good resistance. After culturing at 37°C for 3 hours in simulated gastric juice digestion, the bacterial culture was resistant and still had good growth viability.

[0163] In summary, *Lactobacillus plantarum* maintained high activity in fruit juices at three different pH levels under the protection of pectin-carboxymethyl chitosan. The double-layer encapsulation of *Lactobacillus plantarum* improves its storage stability in fruit juice and enhances its ability to pass through the gastrointestinal tract.

[0164] 11.7 Survival rate determination under simulated intestinal fluid conditions during fruit juice storage

[0165] Further simulated intestinal fluid tolerance experiments were conducted on free and encapsulated *Lactobacillus plantarum* in the three fruit juices, using the same simulation method as above. The results are as follows: Figures 17-19As shown, the overall decrease in the survival rate of free bacteria during the intestinal model was less than that during the gastric simulated tolerance period, while the survival rate of encapsulated *Lactobacillus plantarum* was lower during the intestinal simulated tolerance period than in the gastric model. However, it still had a significant protective effect overall because the outer carboxymethyl chitosan formed a membrane on the pectin outer layer, allowing it to maintain capsule stability under intestinal matrix conditions. In the simulated intestinal fluid tolerance of grape juice stored for 21 days, the survival rate of encapsulated *Lactobacillus plantarum* was similar to that of day 7, while the survival rate of free bacteria decreased significantly. Comparing the intestinal fluid tolerance survival rates of bacteria in apple juice at different storage times, the encapsulated group showed higher stability compared to free bacteria. In carrot juice, since previous studies have shown that free bacteria have good growth in carrot juice, it can be seen that at day 7, free and encapsulated *Lactobacillus plantarum* had similar tolerance. However, over time, the number of free bacteria decreased, resulting in a lower survival rate in the tolerant intestinal fluid compared to the encapsulated *Lactobacillus plantarum* protection group.

[0166] In conclusion, the pectin-carboxymethyl chitosan encapsulation technology effectively protects Lactobacillus plantarum during storage, making it more stable under adverse conditions, including gastrointestinal conditions, which is beneficial for the development and application of novel Lactobacillus plantarum products.

[0167] 11.8 Sensory Evaluation

[0168] Taking Lactobacillus plantarum T1 as an example, sensory evaluation of Lactobacillus plantarum double-layer microcapsules was conducted.

[0169] Sensory evaluations were conducted on three types of fruit juice: samples containing free and encapsulated *Lactobacillus plantarum*, and a control sample without *Lactobacillus plantarum*, stored at 4°C for 21 days. All samples were placed at room temperature for 6 hours prior to sensory testing to ensure temperature did not affect sensory scores, and were uniformly packaged in plastic cups to minimize sensory variations caused by packaging. These samples were evaluated by a panel of ten trained assessors with experience in food sensory analysis. The sensory panel used reference standards to evaluate the different groups of fruit juice products based on relevant indicators such as color, clarity, flavor, aroma, texture, and overall acceptability. Each panel member evaluated the sample through a rating system.

[0170] The results are as follows Figure 20As shown in the figure. Scores for color, flavor, clarity, odor, and texture indicated that *Lactobacillus plantarum* encapsulation significantly improved all sensory properties (P<0.05). Furthermore, pectin-carboxymethyl chitosan beads were visible in the apple juice samples, providing the panel members with improved appearance and taste. Additionally, apple juice containing encapsulated *Lactobacillus plantarum* showed lower acidity than apple juice containing free bacteria, likely due to the release of fermentation metabolites from free bacteria, while encapsulation limited acidification. Meanwhile, the three fruit juices containing free probiotics also exhibited higher turbidity. Overall, among the three fruit juices, *Lactobacillus plantarum* encapsulation significantly improved juice clarity without affecting the original sensory properties, resulting in better stability and enhanced probiotic function.

[0171] This invention provides a method and approach for preparing and applying *Lactobacillus plantarum* bilayer microcapsules. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for preparing *Lactobacillus plantarum* bilayer microcapsules, characterized in that, Includes the following steps: (1) Cultivate *Lactobacillus plantarum*, centrifuge to collect the precipitate and resuspend it to obtain *Lactobacillus plantarum* bacterial suspension; (2) Mix the plant lactobacillus suspension prepared in step (1) with the pectin solution to obtain the core layer solution; (3) The core layer solution obtained in step (2) is added to calcium chloride solution for solidification. After filtration and washing, it is immersed in chitosan compound solution to prepare plant lactobacillus bilayer microcapsules. Wherein, the chitosan compound is carboxymethyl chitosan; the chitosan compound solution has a concentration of 5~20 g / L and is in water as the solvent; the volume ratio of the core layer solution, calcium chloride solution and chitosan compound solution is 1:10~20:10~20. In step (2), the pectin is apple pectin or citrus pectin; the degree of esterification of the pectin is 6-65%. In step (2), the pectin solution has a concentration of 10-40 g / L and is in water as the solvent; the volume ratio of the plant lactobacillus suspension to the pectin solution is 1:8-10. In step (3), the calcium chloride solution has a concentration of 10-20 g / L and the solvent is water; the curing time is 20-30 min.

2. The preparation method according to claim 1, characterized in that, In step (3), the immersion conditions are: rotation speed 70~100 rpm, time 1~1.5 h.

3. The *Lactobacillus plantarum* bilayer microcapsules prepared by the preparation method according to any one of claims 1 or 2.

4. The application of the *Lactobacillus plantarum* double-layer microcapsules according to claim 3 in the preparation of fruit juice beverages.

5. The application according to claim 4, characterized in that, The fruit juice beverages mentioned include any one or a combination of grape juice, apple juice, and carrot juice.

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