Double-layer microcapsules containing lactobacillus plantarum, and preparation method and application thereof

Abstract

The application discloses a double-layer microcapsule containing lactobacillus plantarum and a preparation method and application thereof, and application of lactobacillus plantarum EASY 0352 in preparation of anxiolytic and depressive products. 2+ The metal-phenol network coating is formed on the surface of the bacteria by coordination self-assembly, the single-cell coating microcapsule can significantly enhance the adhesion of lactobacillus plantarum EASY 0352 to intestinal epithelial cells, and is beneficial to the colonization of lactobacillus plantarum EASY 0352 in the intestinal tract; on the basis of the single-cell coating microcapsule, the application further coats transglutaminase (TG) and whey protein concentrate (WPC) composite wall materials to form a double-layer microcapsule; and simulation of gastric and intestinal fluid digestion experiment results show that the double-layer microcapsule prepared by the application can significantly improve the survival rate of lactobacillus plantarum EASY 0352 in simulated gastric and intestinal fluid, and effectively protects the probiotics to pass through the gastrointestinal tract smoothly.

Description

Technical Field

[0001] This invention relates to the field of Lactobacillus plantarum technology, and more particularly to a double-layered microcapsule containing Lactobacillus plantarum, its preparation method, and its application. Background Technology

[0002] Anxiety disorders are a common mental illness characterized by excessive worry, anxiety, fear, and other emotional symptoms, often accompanied by autonomic dysfunction. Epidemiological surveys show that the global lifetime prevalence of anxiety disorders is approximately 16%, and is increasing year by year, placing a heavy burden on patients' quality of life and public health systems.

[0003] Currently, the treatment of anxiety disorders is still mainly medication-based, primarily including selective serotonin reuptake inhibitors (SSRIs) and benzodiazepines. However, these drugs have drawbacks such as slow onset of action, numerous adverse reactions (e.g., drowsiness, cognitive impairment, drug dependence), and high relapse rates after discontinuation. Therefore, developing safer alternative or adjunctive treatment strategies with fewer side effects is of significant clinical importance.

[0004] In recent years, with researchers’ in-depth study of the relationship between human microbiota and mental illnesses such as anxiety and depression, intervention or treatment methods based on gut microbiota have emerged, such as probiotic supplementation and fecal microbiota transplantation, showing promising application prospects. Studies have shown that probiotics (including Lactobacillus plantarum) can exert anti-anxiety effects by regulating gut microbiota structure, affecting neurotransmitter (such as γ-aminobutyric acid, GABA) levels, regulating the function of the hypothalamus-pituitary-adrenal (HPA) axis, and reducing neuroinflammation.

[0005] However, probiotics face severe challenges from gastric acid, bile salts, and digestive enzymes during oral delivery, resulting in a significantly reduced survival rate of live bacteria reaching the intestines, thus affecting their efficacy. Meanwhile, the ability of probiotics to adhere to intestinal epithelial cells is a key factor in their colonization and function in the intestines. Therefore, developing encapsulation technologies that can improve the gastrointestinal fluid tolerance and intestinal adhesion of probiotics is of great significance for enhancing the efficacy of probiotic formulations. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides the following technical solution: In the first aspect, we provide the application of Lactiplantibacillus plantarum EASY 0352 in the preparation of products to relieve anxiety and depression. The Lactiplantibacillus plantarum EASY0352 was deposited at the China General Microbiological Culture Collection Center on July 7, 2025, with the accession number CGMCC No. 35111.

[0007] Secondly, a method for preparing bilayer microcapsules is provided, comprising the following steps: S1: Mix the activated *Lactobacillus plantarum* EASY 0352 suspension with Zn 2+ The solution and EGCG solution were mixed, the pH was adjusted to 7.0, and then washed and freeze-dried under vacuum to obtain single-cell coated microcapsules; S2: Add single-cell coated microcapsules to the composite wall material solution, mix well, and then freeze-dry under vacuum to obtain double-layer microcapsules.

[0008] As an improvement to the above technical solution, the composite wall material includes transglutaminase (TG) and whey protein concentrate (WPC).

[0009] As an improvement to the above technical solution, the mass ratio of TG to WPC in the composite solution is (1-5):(9-5).

[0010] As an improvement to the above technical solution, the mass ratio of TG to WPC in the composite solution is 2:8.

[0011] Thirdly, the plant lactobacillus bilayer microcapsules prepared according to the preparation method described above.

[0012] Fourthly, this invention provides an application of *Lactobacillus plantarum* double-layer microcapsules in probiotic preparations; Fifthly, this invention provides the application of *Lactobacillus plantarum* EASY 0352 in probiotic preparations.

[0013] The beneficial effects of this invention are: Intervention with *Lactobacillus plantarum* EASY 0352 significantly improved anxiety-like behaviors in CRS model mice: central activity time increased by 49.10 s in the open field test (P<0.05); number of entries into the open arm and time spent in the open arm increased by 87.77% and 62.31% respectively in the elevated cross maze test (P<0.05); activity time in the light box increased by 32.45% in the light box test (P<0.05); and the number of buried beads decreased by 31.89% in the marble burial test (P<0.05). CRS modeling resulted in a 25.73% and 17.69% decrease in GABA levels in the prefrontal cortex and hippocampus of mice, respectively (P<0.05). After intervention with *Lactobacillus plantarum* EASY 0352, colonic GABA levels increased by 18.96% (P<0.05), and prefrontal cortex and hippocampal GABA levels recovered to 37.50 ng / g and 47.60 ng / g, respectively (P<0.05). After intervention with Lactobacillus plantarum EASY 0352, the serum CORT level in CRS model mice decreased by 14.05% (P<0.05), and the levels of IL-1β, IL-6 and TNF-α in hippocampus tissue decreased by 8.25%, 8.28% and 12.99%, respectively (P<0.05). After intervention with Lactobacillus plantarum EASY 0352, the relative abundance of Lactobacillus in the gut microbiota of CRS model mice significantly increased, while the relative abundance of anxiety-related bacteria Desulfovibrio and Alternaria significantly decreased (P<0.05). This invention uses EGCG and Zn 2+ A metal-phenol network coating was formed by coordination self-assembly on the bacterial cell surface. The HT-29 cell adhesion experiment results showed that the single-cell coated microcapsules prepared in this invention can significantly enhance the adhesion ability of Lactobacillus plantarum EASY0352 to intestinal epithelial cells, which is beneficial to its colonization in the intestine. Based on single-cell coated microcapsules, this invention further encapsulates transglutaminase (TG) and whey protein concentrate (WPC) composite wall material to form double-layer microcapsules. Simulated gastrointestinal digestion experiments show that the double-layer microcapsules prepared by this invention can significantly improve the survival rate of Lactobacillus plantarum EASY 0352 in simulated gastrointestinal fluid and effectively protect probiotics to pass smoothly through the gastrointestinal tract. Attached Figure Description

[0014] Figure 1 shows the effect of the plant lactobacillus EASY 0352 of the present invention on the body weight of CRS mice; Figure 2 The effect of *Lactobacillus plantarum* EASY 0352 of this invention on open field behavior in CRS mice was investigated, where (A) represents the activity trajectory; (B) represents the total distance traveled; and (C) represents the central activity time. Figure 3 The present invention describes the effect of *Lactobacillus plantarum* EASY 0352 on the behavior of CRS mice in an elevated cruciate maze, wherein (A) represents the activity trajectory; (B) represents the number of times the mouse entered the open arm; and (C) represents the time the mouse spent in the open arm. Figure 4 The effect of Lactobacillus plantarum EASY 0352 on the behavior of CRS mice in a light-dark box experiment, where (A) is the number of shuttles and (B) is the time spent in the light box. Figure 5 This invention relates to the effect of *Lactobacillus plantarum* EASY 0352 on the number of embedded beads in CRS mice. Figure 6The present invention describes the effect of *Lactobacillus plantarum* EASY 0352 on GABA levels in mice, wherein (A) is the serum GABA level in mice; (B) is the colonic GABA level; (C) is the prefrontal cortex GABA level; and (D) is the hippocampal GABA level. Figure 7 The present invention describes the effect of *Lactobacillus plantarum* EASY 0352 on serum HPA-related hormone levels in CRS mice, wherein (A) represents CRH level and (B) represents CORT level. Figure 8 The present invention relates to the effect of *Lactobacillus plantarum* EASY 0352 on the levels of pro-inflammatory factors in the hippocampus of CRS mice; wherein, (A) IL-1β level in hippocampus; (B) IL-6 level in hippocampus; (C) TNF-α level in hippocampus; Figure 9 This invention illustrates the effect of *Lactobacillus plantarum* EASY 0352 on the hippocampus of CRS mice, with blue arrows indicating areas of significant damage. Figure 10 The effect of *Lactobacillus plantarum* EASY 0352 on the α-diversity of gut microbiota in CRS mice was investigated in this invention, wherein (A) Simpson index; (B) Shannon index; Figure 11 This invention relates to the effect of *Lactobacillus plantarum* EASY 0352 on the β-diversity of gut microbiota in CRS mice. Figure 12 The present invention describes the effect of *Lactobacillus plantarum* EASY 0352 on the intestinal flora of CRS mice at the genus level, where (A) is the relative abundance at the genus level, (B) is the relative abundance of the genus *Lactobacillus*, (C) is the relative abundance of the genus *Desulfovibrio*, and (D) is the relative abundance of the genus *Alternaria*. Figure 13 This is a CLSM image of the *Lactobacillus plantarum* EASY 0352 single-cell coated microcapsules of the present invention; Figure 14 The cell adhesion rate of the *Lactobacillus plantarum* EASY 0352 single-cell coated microcapsules of this invention; Figure 15 The survival status of the *Lactobacillus plantarum* EASY 0352 double-layer microcapsules of this invention was simulated during gastrointestinal digestion. Figure 16 This is a SEM image of the *Lactobacillus plantarum* EASY 0352 double-layer microcapsules of the present invention; Figure 17 The particle size and zeta potential of the *Lactobacillus plantarum* EASY 0352 bilayer microcapsules of the present invention are shown in the figures, wherein (A) is the particle size and (B) is the zeta potential. Figure 18 The FT-IR spectrum of the *Lactobacillus plantarum* EASY 0352 bilayer microcapsules of the present invention is shown, wherein (A) is a glycoprotein; and (B) is a single- or double-layer microcapsule. Figure 19 This is a TEM image of the *Lactobacillus plantarum* EASY 0352 double-layer microcapsule of the present invention. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0016] In this invention, Lactiplantibacillus plantarum EASY0352 was deposited on July 7, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35111, and its 16S rDNA sequence is shown in SEQ ID NO:1. Example 1 1. Materials and Methods 1.1 Activation and culture of Lactobacillus plantarum After being activated twice on MRS medium, *Lactobacillus plantarum* EASY 0352 was inoculated into GABA fermentation medium (glucose yeast extract peptone medium) and cultured at 37°C for 48 h. 1.2 Experimental Methods 1.2.1 Experimental animal design Male Balb / c mice aged 6 to 8 weeks were housed in a controlled environment with a temperature of 22 ± 2 ℃ and a relative humidity of 45 ± 5%, and exposed to a 12 h light / dark cycle. All mice were acclimatized for 1 week before the experiment, during which they had free access to food and water. After the adaptation period, the mice were divided into three groups: NC group, MC group, and LP group, with 10 mice in each group. The normal control group (NC group) did not undergo CRS modeling and received 200 μL of PBS solution by gavage daily; the model control group (MC group) underwent CRS modeling and received 200 μL of PBS solution by gavage daily; the probiotic group (LP group) underwent CRS modeling and received 200 μL of PBS solution containing 10 μg / mL of PBS solution by gavage daily. 8 CFU of Lactobacillus plantarum EASY 0352 in PBS suspension; The CRS modeling method is as follows: Place the mouse in a restraint tube so that it can breathe normally in the tube but cannot move freely. At the same time, the device will not cause external injury to the mouse. Restrain the mouse for 3 hours each time after daily administration, and continue the modeling for 21 days. After 21 days of modeling, behavioral tests were started. Each mouse underwent one behavioral test daily, and the open field test, elevated cross maze test, light and dark box test, and marble burial test were completed in sequence. All tests were completed in 4 days. After all behavioral tests were completed, the mice were fasted for 12 hours, then anesthetized by intraperitoneal injection of 1.25% tribromoethanol (20 μL / g), and blood was drawn from the orbital cavity. Finally, the mice were euthanized and dissected for tissue collection.

[0017] 1.2.2 Weight Changes Before each day's gavage, the mice in each group were weighed and their weights recorded.

[0018] 1.2.3 Behavioral Testing 1.2.3.1 Open space activities The specific steps of the open field test are as follows: The experiment was conducted in an open field box, which measures 40 cm × 40 cm × 40 cm and is divided into 16 small squares. The central area consists of four squares in the center. The area around the box and the bottom are black. The test was conducted in a quiet environment. Mice were placed in one corner of the open field, and the test time was recorded for 6 minutes, consisting of a 1-minute adaptation period and a 5-minute testing period. After the test, the mice were removed, and the open field box was wiped with a 70% ethanol solution. 1.2.3.2 Elevated Cross Maze Activity The elevated cross maze apparatus consisted of two open arms and two closed arms positioned opposite each other in a cross shape. Each arm was 30 cm long, the track width was 5 cm, the maze was 30 cm above the ground, and the height of the closed arm sidewalls was 15 cm. The experiment was conducted in a quiet environment. At the start of the experiment, mice were placed into the maze from the center towards the closed arm, and their activity was recorded for 5 minutes. One mouse was tested at a time using the same apparatus. After the test, the mice were removed, and the maze was cleaned by wiping it with a 70% ethanol solution.

[0019] 1.2.3.3 Open and Closed Box Activities The specific steps are as follows: The dark box occupies 2 / 3 of the light box and the light box occupies 1 / 3 of the light box. There is a small square hole between the light and dark boxes that allows the mouse to pass through freely. The light box is illuminated by an incandescent bulb. At the beginning of the experiment, the mouse's head is facing the dark box, and its activity is recorded for 5 minutes. After the experiment, the device is wiped with a 70% ethanol solution. 1.2.3.4 Marble Burial Experiment Marble burial experiment: A 5 cm thick layer of corn cob bedding was laid flat in a cage measuring 40 cm × 25 cm × 20 cm. Twenty glass marbles with a diameter of 2.5 cm were arranged in a "4×5" pattern. Mice were placed alone in the cage for 20–30 minutes to acclimatize, and the number of glass marbles buried within 10 minutes was recorded. The burial criterion was that a glass marble was considered buried if 2 / 3 or more of its volume was covered by the corn cob bedding.

[0020] 1.2.4 Measurement of GABA levels in serum, colon, and different brain regions GABA levels in the serum, colon, prefrontal cortex, and hippocampus of mice in each group were determined using an enzyme-linked immunosorbent assay (ELISA) kit, according to the manufacturer's instructions.

[0021] 1.2.5 Measurement of serum HPA axis related indicators The levels of corticotropin-releasing hormone (CRH) and corticosterone (CORT) in the serum of mice in each group were determined using an enzyme-linked immunosorbent assay kit according to the manufacturer's instructions.

[0022] 1.2.6 Measurement of inflammatory factor levels in hippocampal tissue The levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in the hippocampus of mice in each group were determined using an enzyme-linked immunosorbent assay kit according to the manufacturer's instructions.

[0023] 1.2.7 HE staining of hippocampal tissue Mouse brain tissue was fixed in 4% paraformaldehyde by weight-to-volume ratio, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). The morphological features and pathological damage of the hippocampus were observed under a 25x optical microscope.

[0024] 1.2.8 Gut microbiota 16S rRNA sequencing Mouse colon contents were collected, flash-frozen in liquid nitrogen, and preserved. Genomic DNA was extracted from the samples, a 16S rDNA library was constructed, and sequencing analysis was performed.

[0025] 1.3 Experimental Results 1.3.1 Effects of Lactobacillus plantarum EASY 0352 on body weight in CRS mice Anxiety disorders can lead to abnormal weight changes; to investigate the effects of CRS and *Lactobacillus plantarum* EASY 0352 gavage intervention on mouse weight, this application recorded the weight changes of mice in each group during the experiment, and the results are as follows: Figure 1As shown, at week 0, the body weight of mice in the NC, MC, and LP groups ranged from 18.79 to 21.12 g, with no significant difference (P>0.05). From week 0 to week 3, the body weight of mice in the NC group showed a continuous increasing trend, the body weight of mice in the MC group showed a continuous decreasing trend, and the body weight of mice in the LP group showed a trend of first decreasing and then increasing. At week 3, the body weight of mice in the MC group decreased to 18.71 ± 0.56 g, which was significantly lower than that of the NC group by 3.72 g (P<0.05), while the body weight of mice in the LP group was 20.53 ± 0.72 g, which was significantly higher than that of the MC group by 9.73% (P<0.05).

[0026] 1.3.2 Effects of Lactobacillus plantarum EASY 0352 on anxiety-like behavior in CRS mice 1.3.2.1 Open space activities The open field test utilizes the fear and exploratory nature of rodents in new open environments to reflect their anxiety state. Quantitative statistical analysis of the activity trajectories, total distance traveled, and central activity time of mice in the NC, MC, and LP groups within the open field area is shown below. Figure 2 ; Figure 2 (A) indicates that the open field activity trajectories of the NC group mice were distributed in both the central and peripheral areas, while the MC group's trajectories were mainly concentrated in the peripheral area. After intervention with *Lactobacillus plantarum* EASY 0352, the activity trajectories of the LP group mice were redistributed throughout the entire open field area. Figure 2 As shown in (B) and (C), the total distance traveled and the central activity time of mice in the NC group were 12.45 ± 3.30 m and 158.10 ± 41.63 s, respectively. Compared with the NC group, the decrease rates of total distance traveled and central activity time in the MC group were 44.38% and 70.38%, respectively (P<0.05). Compared with the MC group, the central activity time of mice in the LP group increased significantly by 49.10 s (P<0.05) and the total distance traveled increased by 4.41 m, but there was no statistical difference (P>0.05).

[0027] 1.3.2.2 Elevated Cross Maze Activity The elevated cross maze experiment utilizes the conflict between rodents' innate fear of open arms and their exploratory nature to assess their anxiety levels. Figure 3 The activity trajectories of mice in the NC, MC, and LP groups in the elevated cross maze are presented, along with the quantitative analysis results of anxiety-like behavior-related indicators for each group, such as... Figure 3 As shown in (A), the activity trajectories of the NC group mice in the elevated cross maze were distributed in both the open and closed arms, while the activity trajectories of the MC group mice were smaller and mainly concentrated in the closed arms, exhibiting typical anxiety-like behavior. The activity trajectory distribution of the LP intervention group mice was similar to that of the NC group. Figure 3(B) and (C) showed quantitative statistical analysis of the number of arm-opening entries and the duration of arm-opening residence. The results indicated that the number of arm-opening entries and the duration of arm-opening residence in the MC group were significantly lower than those in the NC group (P<0.05), decreasing to 6.83 ± 2.32 times and 73.07 ± 20.41 s, respectively. After intervention with *Lactobacillus plantarum* EASY 0352, compared with the MC group, the number of arm-opening entries and the duration of arm-opening residence in the LP group were significantly increased (P<0.05), with increases of 87.77% and 62.31%, respectively.

[0028] 1.3.2.3 Open and Closed Box Activities The light-dark box experiment utilizes the innate aversion to bright environments and the instinctive conflict between rodents' spontaneous exploration of novel environments to assess their anxiety levels. Quantitative statistical results of the number of times mice traversed the light-dark box apparatus and the duration of activity in the bright box for each group are shown below. Figure 4 The number of light-dark box shuttles and the duration of activity in the light box were 10.83 ± 2.79 times and 243.70 ± 14.83 s, respectively, in the NC group, significantly higher than those in the MC group (P<0.05). Compared with the MC group, the duration of activity in the light box in the LP group was significantly increased by 32.45% (P<0.05), to 195.50 ± 40.35 s. The number of light-dark box shuttles increased by 1.83 times, but the difference was not statistically significant (P>0.05).

[0029] 1.3.2.4 Marble Burial Experiment The marble burial test assesses anxiety-like behaviors in rodents by observing their repetitive digging and burial behaviors under anxiety or stress. As shown in Figure 5, the average number of marbles buried in the NC group was 5.00 ± 2.68, while the MC group showed a significant increase of 130.00% (P<0.05), with 11.50 ± 2.81 marbles buried. After intervention with *Lactobacillus plantarum* EASY 0352, the number of marbles buried in the LP group was 7.83 ± 1.47, a significant decrease of 31.89% (P<0.05) compared to the MC group, with no statistically significant difference between the LP and NC groups (P>0.05).

[0030] 1.3.3 Effects of *Lactobacillus plantarum* EASY 0352 on GABA levels in multiple sites of CRS mice To investigate whether intervention with the high-GABA-producing strain *Lactobacillus plantarum* EASY 0352 could successfully affect intestinal GABA levels, and whether the GABA produced by it could enter the bloodstream via the intestinal epithelium and thus affect brain GABA levels, the study quantitatively measured GABA levels in serum, colon, prefrontal cortex, and hippocampus of mice in the NC, MC, and LP groups. Specific results are detailed below. Figure 6The results showed that, compared with the NC group, the GABA levels in the prefrontal cortex and hippocampus of mice in the MC group decreased significantly by 25.73% and 17.69%, respectively (P<0.05), while there was no significant change in serum and colonic GABA levels (P>0.05). The GABA level in the colon of the MC group was 69.05 ± 5.12 ng / g, while the GABA level in the colon of the LP group was significantly increased by 18.96% compared with the MC group (P<0.05), reaching 82.14 ± 5.33 ng / g. The levels of GABA in the prefrontal cortex and hippocampus of mice in the LP group were significantly higher than those in the MC group by 37.50 ng / g and 47.60 ng / g, respectively (P<0.05). The GABA level in the prefrontal cortex increased to 213.00 ± 12.22 ng, which was not significantly different from that in the NC group (P>0.05). In addition, no significant difference in serum GABA levels was observed between the LP group and the MC group (P>0.05).

[0031] 1.3.4 Effect of Lactiplantibacillus plantarum EASY 0352 on the HPA axis in CRS mice TC "3.2.4 Effect of Lactiplantibacillus plantarum EASY 0352 on the HPA axis in CRS mice"\l 3 \*MERGEFORMAT The HPA axis is a core component of the neuroendocrine system, playing a crucial role in the initiation and regulation of the body's stress response. CRH and CORT are key regulatory factors of the HPA axis. Therefore, this application measured the serum CRH and CORT levels in mice from the NC, MC, and LP groups. The results are shown below. Figure 7 The results showed that the serum CRH and CORT levels in the MC group mice were significantly higher than those in the NC group by 24.12% and 32.12% (P<0.05), respectively, at 2.17 ± 0.21 ng / mL and 52.52 ± 4.60 ng / mL. Compared with the MC group, the serum CORT level in the LP group mice was significantly lower by 14.05% (P<0.05), and the serum CRH level decreased by 15.58%, but the difference was not statistically significant (P>0.05).

[0032] 1.3.5 Effect of Lactiplantibacillus plantarum EASY 0352 on inflammatory factors in hippocampal tissue of CRS mice TC "3.2.5 Effect of Lactiplantibacillus plantarum EASY 0352 on inflammatory factors in hippocampal tissue of CRS mice"\l 3 \* MERGEFORMAT IL-1β, IL-6, and TNF-α are important pro-inflammatory cytokines in neuroinflammation, participating in the occurrence and persistence of neuroinflammation. Figure 8 The results of measuring the levels of IL-1β, IL-6, and TNF-α in the hippocampus of mice in the NC, MC, and LP groups are presented. As shown in the figure, compared with the NC group, the levels of IL-1β, IL-6, and TNF-α in the hippocampus of mice in the MC group were significantly increased (P<0.05), by 0.86 pg / mL, 0.97 pg / mL, and 0.58 pg / mL, respectively. The levels of the above inflammatory factors in the LP group were significantly decreased compared with those in the MC group, by 8.25%, 8.28%, and 12.99%, respectively (P<0.05).

[0033] 1.3.6 Effect of Lactiplantibacillus plantarum EASY 0352 on hippocampal tissue in CRS mice TC "3.2.6 Effect of Lactiplantibacillus plantarum EASY 0352 on hippocampal tissue in CRS mice"\l3 \* MERGEFORMAT Neuronal damage was assessed by observing the HE staining results of the dentate gyrus (DG) region of the hippocampus in each group of mice. The results are as follows: Figure 9 As shown, the results indicated that neurons in the DG region of NC group mice were morphologically intact, tightly arranged, and uniformly stained. Compared with the NC group, neurons in the DG region of MC group mice showed obvious morphological abnormalities, including cell body shrinkage, intensified cytoplasmic staining, and loose arrangement of some neurons. After intervention with *Lactobacillus plantarum* EASY 0352, the morphology of neurons in the DG region of LP group was somewhat restored compared with that of MC group, the degree of cell shrinkage was reduced, and the staining tended to be normal.

[0034] 1.3.7 Effect of Lactiplantibacillus plantarum EASY 0352 on gut microbiota in CRS mice TC "3.2.7 Effect of Lactiplantibacillus plantarum EASY 0352 on gut microbiota in CRS mice"\l 3 \*MERGEFORMAT Based on the 16S rDNA gene sequencing results of gut microbiota Figures 10 to 12 The effects of *Lactobacillus plantarum* EASY0352 intervention on the gut microbiota of CRS mice were comprehensively evaluated. Figure 10(A) The α diversity of gut microbiota in the three groups of mice was assessed based on the Simpson and Shannon indices. As shown in the figure, no significant differences were observed in the Simpson and Shannon indices of gut microbiota in the three groups of mice (P>0.05). Figure 11 The results of the gut microbiota β diversity analysis showed that the gut microbiota composition of the LP group mice was more similar to that of the NC group, while the MC group was much different from the NC group. Figure 12 (A) shows the composition of the top twenty bacterial communities in terms of relative abundance at the genus level for groups NC, MC, and LP. Figure 12 (B) Quantitative statistical analysis was performed on the relative abundance of the probiotic genus Lactobacillus. The results showed that, at the genus level, compared with the NC group, the relative abundance of Lactobacillus in the gut microbiota of mice in the MC group was significantly decreased (P<0.05), while the relative abundance of Lactobacillus in the LP group recovered to a level that was not significantly different from that in the NC group (P>0.05). Figure 12 (C) and (D) quantitative statistical analysis was performed on the relative abundance of anxiety-related bacteria genus *Desulfovibrio* and *Alistipes*. The results showed that, compared with the NC group, the relative abundance of *Desulfovibrio* and *Alistipes* in the gut microbiota of mice in the MC group was significantly increased (P<0.05), while the relative abundance of *Lactobacillus* was significantly increased in the LP group, and the relative abundance of *Desulfovibrio* and *Alistipes* was significantly decreased (P>0.05). There was no significant difference in the relative abundance of *Alistipes* between the LP group and the NC group (P>0.05).

[0035] Example 2 2.1 Preparation of *Lactobacillus plantarum* EASY 0352 double-layer microcapsules 2.1.1 Preparation of single-cell coated microcapsules One mL of activated Lactobacillus plantarum EASY 0352 bacterial suspension was washed three times with phosphate buffered saline (PBS) to remove residual MRS medium. The suspension was then resuspended in 1 mL of sterile PBS. 80 μL of zinc chloride (48 mmol / L) solution and 40 μL of EGCG (48 mmol / L) solution were added sequentially while shaking to mix. After the mixture was thoroughly shaken and the pH adjusted to 7.0, it was washed twice with PBS to remove unbound ZnCl2 and EGCG. Finally, the mixture was freeze-dried under vacuum to obtain single-cell coated microcapsules of Lactobacillus plantarum EASY 0352. 2.1.2 CLSM Observation Take 40 μL of EGCG (48 mmol / L) solution, add 20 μL of 0.01% Rhodamine B solution, mix well and set aside. Separately, take 1 mL of activated Lactobacillus plantarum EASY 0352 bacterial suspension, wash 3 times with PBS to remove residual MRS medium, resuspend in 1 mL of sterile PBS, add 80 μL of zinc chloride (48 mmol / L) and 60 μL of pre-prepared EGCG-Rhodamine B mixture to the bacterial suspension, shake to mix, adjust pH to 7.0, and incubate at 4℃ in the dark for 2 h. After the reaction is complete, take an appropriate amount of sample and drop it onto a glass slide, cover with nail polish and incubate overnight. Use a laser confocal microscope to observe the micromorphology of fluorescently labeled single-cell coated microcapsules and acquire images. 2.1.3 HT-29 cell adhesion experiment Using the HT-29 human colon adenocarcinoma cell line as an in vitro cell model, HT-29 cells frozen in liquid nitrogen were revived and cultured in a 37 ℃, 5% CO2 incubator. After passage and expansion to the required number, they were seeded into 6-well plates. Adhesion experiments were performed when cell confluence reached 80%. 1 mL of *Lactobacillus plantarum* EASY 0352 bacterial suspension and single-cell coated probiotic microcapsule bacterial suspension were washed three times with sterile PBS buffer. The bacterial pellet was collected and added to the corresponding wells of the plate, shaken well, and incubated for 2 h. The supernatant was discarded, and the cells were washed three times with sterile PBS buffer to remove unadhesive cells. The cells were digested and collected in sterile centrifuge tubes, serially diluted, and plated onto MRS solid medium plates. The plates were incubated at 37 ℃ for 48 h, and the colony count was recorded. The adhesion rate was calculated using the formula: 2.1.4 Preparation of bilayer microcapsules 20 mL of TG solutions with mass-volume fractions of 1%, 2%, 3%, 4%, and 5% were prepared respectively, and stirred in a 90 ℃ constant temperature water bath for 2 h until completely dissolved. After the solutions cooled to room temperature, whey protein concentrate (WPC) was added to each solution to achieve final mass fractions of 9%, 8%, 7%, 6%, and 5%, respectively. The solutions were thoroughly mixed to obtain composite solutions with TG to WPC mass ratios of 1:9, 2:8, 3:7, 4:6, and 5:5, respectively. The prepared *Lactobacillus plantarum* EASY 0352 single-cell coated microcapsules were added to each composite solution, thoroughly mixed, and then freeze-dried under vacuum to obtain *Lactobacillus plantarum* EASY 0352 double-layer microcapsule lyophilized powder. 2.1.5 Gastrointestinal tolerance test Methods for preparing in vitro simulated gastric and intestinal fluids; specific preparation methods are as follows: Preparation of artificial gastric juice: Take 16.4 mL of dilute hydrochloric acid, add 800 mL of water and 10 g of pepsin, shake well, dilute with water to 1000 mL, adjust the pH to 2.5, filter with a 0.22 μm filter membrane to remove bacteria, and the artificial gastric juice is obtained. Store at 4 ℃ for later use. Preparation of artificial intestinal fluid: Dissolve 6.8 g of potassium dihydrogen phosphate in 500 mL of water, and adjust the pH to 6.8 with 0.1 mol / L sodium hydroxide solution; separately dissolve 10 g of pancreatic enzyme in an appropriate amount of water. Mix the two solutions, dilute with water to 1000 mL, add porcine bile salts to make the bile salt mass-volume fraction 0.3%, and filter through a 0.22 μm filter membrane for sterilization to obtain the artificial intestinal fluid. Store at 4 ℃ for later use. Accurately weigh out *Lactobacillus plantarum* EASY 0352 naked bacteria (LP), single-cell coated microcapsules (SL), and lyophilized powders of each group of double-layer microcapsules (DL-1:9, DL-2:8, DL-3:7, DL-4:6, DL-5:5), and adjust the viable count to 10. 9 CFU was resuspended in 1 mL of sterile PBS and inoculated into 9 mL of simulated gastric fluid. The mixture was treated at 37 °C for 3 h, and samples were taken at 0 h and 3 h. Viable bacterial counts were determined using the plate count method to evaluate the strain's tolerance in simulated gastric fluid. Subsequently, 1 mL of the above mixture was added to 9 mL of simulated intestinal fluid, and the mixture was treated at 37 °C for another 4 h. Viable bacterial counts were again determined using the dilution plate count method.

[0036] 2.1.6 SEM Observation A small amount of naked Lactobacillus plantarum EASY 0352, single-cell coated microcapsules, and lyophilized powders of various groups of double-layer microcapsules were attached to the observation stage, sputtered with gold, and their morphology was observed and photographed using SEM at different magnifications.

[0037] 2.1.7 Particle size and zeta potential determination An appropriate amount of naked Lactobacillus plantarum EASY 0352 bacteria, single-cell coated microcapsules, and lyophilized powders of each group of bilayer microcapsules were reconstituted in PBS buffer and dispersed evenly. The microcapsule solutions of each group were added to particle size cuvettes and potential cuvettes. The particle size and zeta potential of the probiotic bilayer microcapsules were determined using a Malvern nanoparticle size and zeta potential analyzer.

[0038] 2.1.8 FTIR Spectroscopy Measurement Appropriate amounts of TG powder, WPC powder, powder prepared by dissolving and mixing TG and WPC and then freeze-drying under vacuum (TG-WPC), lyophilized powder of *Lactobacillus plantarum* EASY 0352 single-cell coated microcapsules (SL-MPN), and lyophilized powders of various groups of double-layer microcapsules (DL-1:9, DL-2:8, DL-3:7, DL-4:6, DL-5:5) were subjected to FT-IR spectral scanning, and the spectral data were obtained at 500~4000 cm-1.

[0039] 2.1.9 TEM Observation An appropriate amount of *Lactobacillus plantarum* EASY 0352 naked bacteria, single-cell coated microcapsules, and double-layer microcapsule bacterial powder were diluted with ultrapure water and dropped onto a carbon-coated copper grid without staining. After adsorption, the morphology was observed using a transmission electron microscope and photographed for preservation.

[0040] 2.2 Experimental Results 2.2.1 CLSM observation of single-cell coated microcapsules Rhodamine B was used to fluoresce in single-cell coated microcapsules encapsulating *Lactobacillus plantarum* EASY 0352, and the red fluorescence signal was observed using CLSM to evaluate the encapsulation effect of the single-cell-coated probiotic microcapsules. Figure 13 As shown, clear short rod-shaped bacterial cells can be observed under bright field. Under fluorescence excitation, the corresponding positions exhibit short rod-shaped red fluorescence. The merged image shows that the fluorescence signal basically overlaps with the bacterial cell position under bright field.

[0041] 2.2.2 Adhesion of Single-Cell Coated Microcapsules Metallo-phenol networks possess adhesive properties, which can enhance the adhesion ability of probiotics, thereby improving their colonization effect in the intestine. The effect of the metallo-phenol network formed by EGCG-Zn2+ on the adhesion of bacterial strains was evaluated using an HT-29 cell adhesion assay. The statistical results of the adhesion rates of *Lactobacillus plantarum* EASY 0352 naked bacteria and probiotic microcapsules encapsulated in single-cell coatings to HT-29 cells are as follows: Figure 14 As shown, the adhesion rates of HT-29 cells in the LP group and the SL group were 5.78 ± 0.25% and 11.87 ± 0.73%, respectively, with the cell adhesion rate in the SL group being significantly higher than that in the LP group (P<0.05).

[0042] 2.2.3 Tolerance test of bilayer microcapsules in simulated gastrointestinal fluid After entering the gastrointestinal tract, probiotics face severe challenges from the digestive environment. Based on single-cell coated microcapsules, different mass concentrations of TG and WPC were embedded in the outer layer, designated as DL-1:9, DL-2:8, DL-3:7, DL-4:6, and DL-5:5 groups, respectively. The optimal ratio was screened through simulated gastrointestinal fluid digestion experiments. The survival of *Lactobacillus plantarum* EASY 0352 naked bacteria, single-cell coated microcapsules, and bilayer microcapsules with different polysaccharide-protein ratios in simulated gastrointestinal fluid was quantitatively statistically analyzed as follows: Figure 15 As shown, the results indicated that after 3 h of treatment in simulated gastric fluid, the viable bacterial count in the LP group was 7.01 ± 0.06 Log CFU, while the viable bacterial count in the SL group was significantly higher (P < 0.05) at 7.47 ± 0.01 Log CFU, an increase of 6.56% compared to the LP group. The viable bacterial counts in all five double-layer embedded groups were significantly higher than those in the LP group (P < 0.05). As the ratio of outer TG to WPC concentration changed from 1:9 to 5:5, the viable bacterial count showed a trend of first increasing and then decreasing. The DL-2:8 group had the highest viable bacterial count, reaching 8.40 ± 0.05 Log CFU, an increase of 19.82% and 11.07% compared to the LP and SL groups, respectively (P < 0.05). After further treatment in simulated intestinal fluid for 4 h, the viable bacterial counts in all groups further decreased, with the DL-2:8 group having the highest viable bacterial count at 6.51 ± 0.03 Log CFU, and the SL group at 6.97 ± 0.07 Log CFU. The CFU count was 7.07% higher than that of the LP group (P<0.05). The viable bacterial count in all five double-layer embedded groups was higher than that in the LP group. The viable bacterial count in the double-layer group was still the highest in the DL-2:8 group, with a viable bacterial count of 8.25±0.05 Log CFU, which was significantly higher than that in the LP group and the SL group by 21.09% and 15.52% respectively (P<0.05).

[0043] 2.2.4 SEM observation of bilayer microcapsules The surface morphology, smoothness, and pore characteristics of naked *Lactobacillus plantarum* EASY 0352, single-cell coated microcapsules, and the microcapsule system were observed using SEM to clarify the encapsulation state of this strain. The results are as follows: Figure 16 As shown, the results indicate that the LP group of bacteria had smooth surfaces, regular shapes, short rod-shaped structures, and dense distribution, while the SL group of bacteria had significantly rough surfaces with a layer of coating visible on the periphery. After double-layer embedding, it was almost impossible to observe any exposed bacteria in the field of view. In terms of the smoothness and density of the wall material surface, the DL-2:8 group of embedding system had the smoothest surface and no obvious pores.

[0044] 2.2.5 Determination of Particle Size and Zeta Potential of Bilayer Microcapsules The influence of the encapsulation process on the physicochemical properties of microcapsules was analyzed by examining the particle size and zeta potential of the naked bacteria and the various groups of *Lactobacillus plantarum* EASY 0352 encapsulation systems. The results are shown in […]. Figure 17 , Figure 17 (A) The results showed that the particle size of *Lactobacillus plantarum* EASY 0352 naked bacteria (LP group) was 699.8 ± 18.9 nm. After EGCG and Zn... 2+ After the metal-phenol network formed coordinated self-assembly on the bacterial cell surface, the particle size of the single-cell coated microcapsules (SL group) significantly increased to 830.0 ± 17.0 nm (P<0.05). Upon further coating with an outer layer, the particle size of the bilayer microcapsules continued to increase, showing an increasing trend with the ratio of TG to WPC, significantly rising from 933.8 ± 8.9 nm to 1900.0 ± 41.4 nm (P<0.05). Figure 17 As shown in (B), the surface of the naked bacteria is negatively charged, with a Zeta potential of -18.25 ± 0.39 mV. After being processed with EGCG-Zn... 2+ After inner layer embedding, Zn 2+ Neutralizing some of the negative charge significantly increased the potential of the single-cell coated microcapsules to -16.59 ± 0.13 mV (P<0.05). After further coating with an outer layer, the potential change showed a dependence on the ratio of TG to WPC, exhibiting a trend of first decreasing and then increasing. At a ratio of 2:8, the Zeta potential was the peak value, which was -20.62 ± 1.24 mV.

[0045] 2.2.6 FTIR Spectroscopy Determination of Bilayer Microcapsules The interaction between TG and WPC in the outer wall material of *Lactobacillus plantarum* EASY 0352 bilayer microcapsules was analyzed by FTIR spectroscopy. Specific results are shown in […]. Figure 18 , Figure 18 (A) shows that in the region near the amide A band, the peak positions of TG and WPI are 3404.7 cm⁻¹, respectively. -1 With 3307.6 cm -1 When the two are combined, the wavenumber redshifts to 3304.4 cm⁻¹. -1 The peak position is lower than that of pure protein, indicating that hydrogen bonds have formed between the sugar and the protein. Figure 18 As shown in (B), single-cell coated microcapsules at 3304.8 cm⁻¹ -1 An absorption peak appears at 3300 cm⁻¹. After coating with the outer layer, the bilayer microcapsule exhibits an absorption peak at 3300 cm⁻¹. -1 The absorption peak near the surface increases from 3304.05 cm⁻¹ with increasing outer TG to WPC mass concentration ratio. -1 Gradually shifted blue to 3400.2 cm. -1 When the mass concentration ratio of outer TG to WPC is between 1:9 and 3:7, the wavenumber stabilizes at approximately 1659 cm⁻¹. -1 However, at ratios of 4:6 and 5:5, the wavenumber drops sharply to approximately 1655 cm⁻¹. -1.

[0046] 2.2.7 TEM observation of bilayer microcapsules TEM images of probiotic double-layer microcapsules are shown below. Figure 19 Short rod-shaped bacteria were visible in all three groups. In the LP group, the bacteria were naked and had no outer coating. In the SL group, a thin protective layer was adsorbed on the outside of the bacteria. In the DL group, a wall material was wrapped around the bacteria on the basis of the SL group, which had the largest thickness and formed a dense encapsulation structure for the probiotics.

[0047] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it.

Claims

1. The application of *Lactobacillus plantarum* EASY 0352 in the preparation of products for relieving anxiety and depression, characterized in that... The Lactiplantibacillus plantarum EASY0352 was deposited on July 7, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35111.

2. A method for preparing bilayer microcapsules, characterized in that, Includes the following steps: S1: The activated *Lactobacillus plantarum* EASY 0352 suspension of claim 1 is mixed with Zn 2+ The solution and EGCG solution were mixed, the pH was adjusted to 7.0, and then washed and freeze-dried under vacuum to obtain single-cell coated microcapsules; S2: Add single-cell coated microcapsules to the composite wall material solution, mix well, and then freeze-dry under vacuum to obtain double-layer microcapsules.

3. The method for preparing bilayer microcapsules according to claim 2, characterized in that, The composite wall material includes transglutaminase (TG) and whey protein concentrate (WPC).

4. The method for preparing bilayer microcapsules according to claim 2, characterized in that, The mass ratio of TG to WPC in the composite wall material solution is (1-5):(9-5).

5. The method for preparing bilayer microcapsules according to claim 4, characterized in that, The mass ratio of TG to WPC in the composite wall material solution is 2:

8.

6. The *Lactobacillus plantarum* bilayer microcapsules prepared by the preparation method according to any one of claims 2-5.

7. The use of a double-layer microcapsule of *Lactobacillus plantarum* as described in claim 6 or *Lactobacillus plantarum* EASY 0352 as described in claim 1 in a probiotic preparation.

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