Seabuckthorn probiotic with effects of improving intestinal inflammation and relieving anxiety and depression and application thereof

Abstract

The present application belongs to the technical field of microorganism and medicine, and particularly relates to sea-buckthorn probiotics with intestinal inflammation improvement and anxiety and depression relieving effects and application thereof. Research shows that Lactobacillus rhamnosus with preservation number CCTCC NO: M20261363 has relieving effects on intestinal inflammation and accompanying anxiety and depression behaviors, and has a biological mineralization effect on selenium. The oral hydrogel of the Lactobacillus rhamnosus loaded with biological mineralized selenium nanoparticles and prepared by using a covalently coupled chlorogenic acid gel carrier not only has intestinal targeting, but also has disease site responsiveness under intestinal inflammation, and can effectively exert the respective biological activities of Lactobacillus rhamnosus, selenium and chlorogenic acid.

Description

Technical Field

[0001] This invention belongs to the fields of microbial technology and pharmaceutical technology, specifically relating to sea buckthorn probiotics that have both effects on improving enteritis and relieving anxiety and depression, and their applications. Background Technology

[0002] Inflammatory bowel disease (IBD) involves elevated reactive oxygen species (ROS) and microbial imbalance in the gut. Probiotics are one of the promising means to alleviate IBD. Although some probiotics or selenium-enriched probiotics have been found to alleviate enteritis, such as Lactobacillus reuteri provided by CN113943687A which can improve ulcerative colitis, there are few reports of the effects of such probiotics on improving enteritis-associated anxiety and depressive behaviors.

[0003] In addition, probiotics often have difficulty surviving in the harsh environment of the stomach, and taking them through the gastrointestinal tract can result in a significant reduction in the number of live bacteria, and it is also difficult to accurately deliver them to inflamed parts of the intestine. Summary of the Invention

[0004] To address the aforementioned problems, the purpose of this invention is to provide a sea buckthorn probiotic that combines the effects of improving enteritis with alleviating anxiety and depression, and its applications. This invention includes the following technical solutions:

[0005] A sea buckthorn probiotic strain with both enteritis-improving and anxiety / depression-relieving effects has been identified. The probiotic is *Lactobacillus reuteri*, with accession number CCTCC NO: M20261363; accession date: June 17, 2026; depositary institution: China Center for Type Culture Collection; deposit address: Wuhan University, Wuhan, China; and taxonomic name: *Lactobacillus reuteri* JBS01.

[0006] The use of probiotics in the preparation of drugs that have both effects on improving enteritis and relieving anxiety and depression, wherein the probiotic is *Lactobacillus reuteri*, the accession number of which is: CCTCC NO: M20261363; the accession date is: June 17, 2026; the depositary institution is: China Center for Type Culture Collection; the depositary address is: Wuhan University, Wuhan, China; and the taxonomic name of which is: *Lactobacillus reuteri* JBS01.

[0007] A drug that combines the effects of improving enteritis and relieving anxiety and depression, the drug containing *Lactobacillus reuteri*, the accession number of which is: CCTCC NO: M20261363; the accession date is: June 17, 2026; the depositary institution is: China Center for Type Culture Collection; the depositary address is: Wuhan University, Wuhan, China; the taxonomic name of which is: *Lactobacillus reuteri* JBS01.

[0008] Preferably, the drug further contains biomineralized nano-selenium.

[0009] Preferably, the dosage form of the drug is an oral hydrogel.

[0010] More preferably, the oral hydrogel has a gel matrix dispersion phase containing a coupling product of methacrylamide hyaluronic acid and chlorogenic acid, and a continuous phase of vegetable oil, and is prepared by microfluidic and photocuring technology.

[0011] Preferably, the hyaluronic acid used in the methacrylamide hyaluronic acid has a viscosity-average molecular weight of 200 kDa to 400 kDa.

[0012] Preferably, the preparation method of the oral hydrogel formulation includes the following steps:

[0013] Take activated Lactobacillus reuteri with preservation number CCTCC NO: M20261363, add it to a culture medium containing sodium selenite, culture it anaerobically, centrifuge it, and collect the first precipitate containing biomineralized nano-selenium and Lactobacillus reuteri.

[0014] Methacrylamide hyaluronic acid and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride were dissolved in N,N-dimethylformamide. 3-aminophenylboronic acid was added, and the pH was adjusted to 6-7. The reaction was carried out for 36-72 hours. The product was then dialyzed and lyophilized to obtain the first reactant. The first reactant was dissolved and mixed with chlorogenic acid. The pH was adjusted to 9-11, and the reaction was carried out for 6-10 hours. The product was then dialyzed and lyophilized to obtain the second reactant.

[0015] The second reactant, the photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, the first precipitate, and water were mixed to prepare a dispersed phase. Vegetable oil was used as the continuous phase. The dispersed phase and the continuous phase were pumped into the channels of the hydrogel microfluidic control device to promote the formation of dispersed droplets. The dispersed phase was then irradiated under visible light for 10 to 25 seconds to perform photocuring and obtain an oral hydrogel.

[0016] Preferably, the concentration of sodium selenite in the culture medium is 0.1 mM to 0.5 mM.

[0017] Preferably, when preparing the second reactant, the mass ratio of the first reactant to chlorogenic acid is 2 to 5:1.

[0018] Preferably, the content of the second reactant in the dispersed phase is 1wt% to 2wt%.

[0019] Preferably, the content of photoinitiator in the dispersed phase is 0.05 wt% to 0.2 wt%.

[0020] Preferably, the content of *Lactobacillus reuteri* with accession number CCTCC NO: M20261363 in the dispersed phase is 1.0 × 10⁻⁶. 3 ~1.0×10 12 CFU / mL.

[0021] Preferably, the volume ratio of the dispersed phase to the continuous phase is 1:8~15, and the dispersed phase and the continuous phase are pumped into the channels of the hydrogel microfluidic control device at rates of 0.5 μL / min~1.5 μL / min and 5 μL / min~15 μL / min, respectively.

[0022] Beneficial effects:

[0023] The *Lactobacillus reuteri* of this invention has both the effect of improving enteritis and relieving anxiety and depression, and can complete the biomineralization of selenium, which is conducive to the preparation of probiotic preparations containing biomineralized selenium nanoparticles, making full use of the antioxidant effect of selenium and the anti-inflammatory effect of probiotics.

[0024] The probiotic formulation containing biomineralized selenium nanoparticles of this invention utilizes methacrylamide-chlorogenic acid gel carriers constructed with borate ester bonds, which facilitates the endowment of the gel system with inflammation-targeting and ROS-responsive release capabilities. Hyaluronic acid exhibits good biocompatibility, modifiability, and affinity for the positive charge of inflamed tissues. Chlorogenic acid, as a natural polyphenol compound, can not only covalently crosslink biopolymer chains to enhance network structure but also endow them with reactive oxygen species (ROS) scavenging activity. The borate ester bonds formed between phenylboronic acid and polyphenol molecules are ROS-sensitive, and bond breakage or network relaxation can occur in the oxidative stress microenvironment (enteritis microenvironment), thereby triggering the release of active substances. It possesses intestinal targeting and disease site responsiveness in the enteritis environment, effectively leveraging the individual bioactivities of *Lactobacillus reuteri*, selenium, and chlorogenic acid.

[0025] Based on the above advantages, the formulation of the present invention, which combines the effects of improving enteritis and relieving anxiety and depression, is conducive to giving full play to the antioxidant effect of selenium, the anti-inflammatory effect of probiotics, the ROS scavenging activity of chlorogenic acid itself, etc., and achieves the protection of probiotics, oral targeted delivery and disease site response release. Attached Figure Description

[0026] Figure 1 Images of LR and LR@Se samples and their transmission electron microscope (TEM) and scanning electron microscope (SEM) images;

[0027] Figure 2 Annular dark-field scanning transmission electron microscope images of LR@Se cultured with LR and different concentrations of Na2SeO3;

[0028] Figure 3 The results of energy-dispersive X-ray spectroscopy elemental analysis for LR@Se;

[0029] Figure 4 For LR, LR@Se, the following are the (a) DPPH, (b) ·OH, and (c) ·O2. - Free radical scavenging effect and (d) growth curves of LR at different Na2SeO3 concentrations;

[0030] Figure 5 The nuclear magnetic resonance spectra and Fourier transform infrared spectra of hydrogen atoms for different products;

[0031] Figure 6 (a) Encapsulation efficiency of oral hydrogels prepared for HA of different molecular weights, (b) LR survival rate after 2 h of digestion in simulated gastric juice, (c) LR survival rate after 4 h of digestion in simulated intestinal juice, (d) LR cumulative release rate after 4 h of reaction in simulated ROS environment, and (e) LR cumulative release rate after 4 h of reaction in PBS;

[0032] Figure 7 (a) Results of vial tilting test and (b) Results of gel strength test for oral hydrogels with different HM contents;

[0033] Figure 8 (a) Microscopic observation images and (b) results of average particle size and encapsulation efficiency measurements of oral hydrogels with different HM contents;

[0034] Figure 9 The survival rates of free LR, LR@Se, and oral hydrogels with different HM contents were measured in (a) simulated gastric juice for 2 h and (b) simulated intestinal juice for 4 h after digestion.

[0035] Figure 10 (a) Cumulative release curve of oral hydrogel in PBS and H2O2 systems and (b) Gel microstructure image;

[0036] Figure 11 The whole fluorescence distribution of mice with colitis at different time points after gavage administration of free LR, LR@Se and oral hydrogel;

[0037] Figure 12The fluorescence distribution of the intestinal tract of mice with colitis at different time points after gavage administration of free LR, LR@Se and oral hydrogel;

[0038] Figure 13 The intestinal fluorescence distribution and average fluorescence efficiency of mice with colitis at different time points after gavage administration of LR@Se / HPCA and LR@Se / PCA.

[0039] Figure 14 The results of (a) body weight, (b) disease activity index, and (c) colon length measurements for each group of animals are as follows:

[0040] Figure 15 Images of colonic pathological staining in each group of animals;

[0041] Figure 16 The results of colonic anti-inflammatory and pro-inflammatory factor level detection in each group of animals;

[0042] Figure 17 Heat maps of the behavioral trajectories and movement paths of each group of animals in the open field experiment;

[0043] Figure 18 The results of the elevated cross maze experiment for each group of animals;

[0044] Figure 19 The results of the forced swimming and tail suspension experiments for each group of animals are shown. Detailed Implementation

[0045] The present invention and its effects are further illustrated below with reference to specific embodiments and accompanying drawings. It should be understood that these embodiments are merely illustrative of the implementation methods and effects of the present invention and are not intended to limit the scope of the invention. After reading the contents of this invention, those skilled in the art can make various simple modifications or alterations to the invention without creative effort, and these equivalent / identical transformations also fall within the protection scope of this invention.

[0046] The main chemicals and biological reagents used in the following examples were sourced from the following sources: sodium selenite (Shanghai Yuanye Biotechnology Co., Ltd.), hyaluronic acid (HA, Shanghai Maclean Biochemical Technology Co., Ltd.), chlorogenic acid (CA, Shanghai Maclean Biochemical Technology Co., Ltd.), methacrylic anhydride (MA, Shanghai Yuanye Biotechnology Co., Ltd.), 3-aminophenylboronic acid (PBA, Shanghai Aladdin Biochemical Technology Co., Ltd.), lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (Lap, Shanghai Yuanye Biotechnology Co., Ltd.), Cy5.5-se (Shanghai Yuanye Biotechnology Co., Ltd.), sodium dextran sulfate (DSS, Dalian Meilun Biotechnology Co., Ltd.), CFDA-SE (Shanghai Yuanye Biotechnology Co., Ltd.), pepsin (Beijing Bio-Top Technology Co., Ltd.), trypsin (Beijing Bio-Top Technology Co., Ltd.), and porcine bile salts (Beijing Bio-Top Technology Co., Ltd.). HT-29-MTX-E12 cells were purchased from Shanghai Enzyme Research Biotechnology Co., Ltd. and preserved in the Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University. The hydrogel microfluidic control equipment is a JSP A / B series high-precision split-type injection pump (Wuhan Jianmi Intelligent Control Technology Co., Ltd.).

[0047] Example 1: Activation, culture and identification of Lactobacillus reuteri JBS01

[0048] *Lactobacillus reuteri* JBS01 (hereinafter referred to as LR) was isolated from *Hippophae rhamnoides* by the Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University, with accession number CCTCC NO: M20261363; accession date: June 17, 2026; depositary institution: China Center for Type Culture Collection, Wuhan University, Wuhan, China; taxonomic name: *Lactobacillus reuteri* JBS01. After two generations of anaerobic culture in MRS medium at 37 °C, the strain was used for subsequent experiments. The 16S rRNA nucleotide sequence of this strain is shown in SEQ ID NO: 1.

[0049] Example 2: Preparation and Condition Investigation of Lactobacillus reuteri JBS01 Containing Biomineralized Selenium Nanoparticles

[0050] LR containing biomineralized selenium nanoparticles (LR@Se) was prepared as follows: LR strains activated for two generations were anaerobically cultured at 37 °C for 24 h in MRS medium supplemented with different concentrations of Na2SeO3 (0.05, 0.1, 0.5, 1.0 mM) (as an example, in this embodiment, the LR inoculum was 2%), and the brick-red precipitate, namely LR@Se, was collected by centrifugation at 8000 rpm for 10 min.

[0051] Products obtained from different concentrations of Na₂SeO₃ were labeled as Se₀.05, Se₀.1, Se₀.5, and Se₁.0, respectively. The microstructure of the strain was observed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Energy-dispersive X-ray spectroscopy (EDS) was used to analyze the elemental composition and content of LR@Se. OD values ​​at 600 nm were measured at different culture times to assess the effect of biomineralization on LR growth. The ROS scavenging capacity of LR and LR@Se (with the same LR concentration) was tested using the following methods:

[0052] DPPH free radical scavenging capacity assay: LR and LR@Se were resuspended, and 500 μL of bacterial suspension (bacterial concentration 1×10⁻⁶) was taken. 8 Mix 500 μL of DPPH solution (concentration 0.2 mmol / L) dissolved in anhydrous ethanol with deionized water (as a control) or deionized water (as a control), shake well, incubate at 37 °C in the dark for 30 min, then centrifuge at 6000 rpm for 10 min, and measure the absorbance of the supernatant at a wavelength of 517 nm.

[0053] •OH radical scavenging ability was assessed using the Solarbio reagent kit, following the kit instructions.

[0054] ·O2 - Free radical scavenging capacity assay: Resuspend LR and LR@Se, and take 0.1 mL of bacterial suspension (bacterial concentration 1×10⁻⁶). 8 Add 0.1 mL of pyrogallol (0.05 mol / L) and 2.8 mL of Tris-HCl (0.05 mol / L, pH 8.2) solution to the sample (CFU / mL). Mix well and react in the dark for 4 min. Then add 1 mL of HCl (8 mol / L) and measure the absorbance of the sample at 320 nm. The blank group is deionized water instead of the sample.

[0055] The results are as follows:

[0056] (1) To enhance the ROS scavenging activity of LR, *Lactobacillus reuteri* JBS01 was exposed to Na2SeO3, and biomineralized Se nanoparticles were generated through bacterial metabolic reduction processes to obtain LR@Se. To determine the optimal process window for selenium loading, the cell surface color, microstructure, and free radical scavenging ability of cultures under different Na2SeO3 concentrations were compared.

[0057] The results showed that LR bacterial solution and powder were white, with rod-shaped cells and a clear, impurity-free surface; LR@Se bacterial solution and powder became more pink with increasing Na2SeO3 concentration, maintaining a typical rod-shaped morphology, and nanoparticles could be observed inside the cells. Figure 1Furthermore, with increasing selenium loading, the distribution of selenium gradually shifted from predominantly endogenous to increasingly exogenous. Annular dark-field scanning transmission electron microscopy revealed a high degree of overlap between Se elements and the bacterial cell region, indicating that Se nanoparticles were successfully integrated into the probiotic cells, and the Se content increased with increasing Na₂SeO₃ concentration. Figure 2 Correspondingly, after treatment, the relative content of Se increased to 5.71%~17.55% ( ). Figure 3 ).

[0058] (2) The free radical scavenging ability of LR@Se was then tested. The results showed that the DPPH, ·OH, and ·O2 of LR@Se obtained under different Na2SeO3 concentrations were different. - The free radical scavenging ability increased by 1.04~31.98%, 10.29~28.46%, and 3.20~18.20%, respectively. Figure 4 ac). Figure 4 In the figure, ns, *, **, and *** represent no statistical difference compared with LR (p>0.05), p<0.05, p<0.01, and p<0.001, respectively.

[0059] (3) Using LR cultured under the same conditions in MRS medium without Na2SeO3 as a control, the OD value at 600 nm wavelength was measured at different culture times to evaluate the effect of biomineralization on LR growth. Figure 4 As shown in d, the growth of LR was almost unaffected after treatment with low levels of Na2SeO3 (0.05~0.5 mM); however, as the concentration of Na2SeO3 increased, the growth of LR was gradually inhibited, exhibiting obvious growth toxicity.

[0060] Based on the above results, 0.5 mM Na2SeO3 was selected for biomineralization of LR for subsequent studies.

[0061] Example 3: Preparation and efficacy validation of LR@Se oral hydrogel

[0062] This embodiment provides information on the preparation of oral hydrogels loaded with LR@Se and their efficacy validation. Although this embodiment provides a method for preparing methacryloyl hyaluronic acid (HM), HM can also be obtained commercially. In this embodiment, the LR@Se used was prepared using the method of Example 2 (Na2SeO3 concentration 0.5 mM; LR inoculum amount 2%). In all oral hydrogel preparations, the amount of LR added to the dispersed phase was 5 × 10⁻⁶. 9 CFU / mL.

[0063] The method for preparing oral hydrogels loaded with LR@Se is as follows:

[0064] I. Preparation of HM

[0065] Methacrylic acid modification of HA: 1 g of HA (to investigate the effect of HA molecular weight on gel, four viscosity-average molecular weights of HA were investigated, namely 2.5~3.5 kDa, 40~100 kDa, 200~400 kDa, and 1500~2500 kDa) was dissolved in 90 mL of solvent (DMF:deionized water volume ratio = 1:2) and stirred until completely dissolved. Under ice bath conditions, 3 mL of methacrylic anhydride (MA) was slowly added dropwise. After adjusting the pH of the system to 8.0~9.0 with NaOH, the mixture was stirred at low temperature (4 °C for example) in the dark for 24 h. Then, 3 volumes of anhydrous ethanol were added, and the mixture was allowed to stand for 12 h. The resulting crude precipitate was dissolved in a small amount of deionized water, transferred to a dialysis bag, and dialyzed continuously with deionized water for 3 days, with frequent changes of the dialysate. The dialysis aqueous solution was pre-frozen and then freeze-dried under vacuum to obtain HM solid powder, which was sealed, protected from light, and stored at low temperature for later use. The HM powder was analyzed by nuclear magnetic resonance spectroscopy (NMR 1H). 1 The molecular characteristics of HM were characterized by 1H NMR. Taking HM prepared from HA with a molecular weight of 200 kDa to 400 kDa as an example, its... 1 H NMR such as Figure 5 As shown. Figure 5 HA represents hyaluronic acid, HM represents methacrylamide hyaluronic acid, and HP represents methacrylamide hyaluronic acid with a borate ester structure; HPCA is the product of the reaction between HP and chlorogenic acid. HM's... 1 A new characteristic peak appears in the 1H NMR at 5.5-7 ppm, indicating the formation of HM.

[0066] II. Preparation of HPCA

[0067] HM (200 mg) prepared from HA of different molecular weights was dissolved in 10 mL of N,N-dimethylformamide (DMF) aqueous solution (DMF:water volume ratio = 2:3), along with 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM, 148 mg). 3-Aminophenylboronic acid (PBA, 125 mg) was added, the pH was adjusted to 6.5, and the reaction was carried out for 48 h. After dialyzing and lyophilization, HM-PBA (i.e., HP, approximately 100 mg) was obtained. HP was dissolved in PBS (adjusted to a final HP concentration of 1.5 wt%), and chlorogenic acid was dissolved in PBS (adjusted to a final chlorogenic acid concentration of 1.5 wt%). The HP and chlorogenic acid solutions were mixed at a 3:1 volume ratio, the pH was adjusted to 10, and the reaction was carried out for 8 h. After dialyzing and lyophilization, HPCA was obtained. 1 H nuclear magnetic resonance (H nuclear magnetic resonance) 1 The structural characteristics of the product were analyzed by 1H NMR and Fourier transform infrared spectroscopy (FTIR). Taking HPCA obtained from HM prepared with HA of molecular weight 200 kDa ~ 400 kDa as an example, its...1 H NMR such as Figure 5 As shown: A new characteristic peak appears in the 1H NMR spectrum of HPCA at 7.0-8.0 ppm, indicating the formation of HP. The FTIR spectrum is shown below. Figure 5 As shown, by Figure 5 It can be seen at 1609 cm -1 and 1410 cm -1 COO at the location - The asymmetric stretching vibration and the symmetric OCO vibration of the pyranose ring reveal the typical structure of HA. (1720 cm⁻¹) -1 and 1630 cm -1 The stretching vibrations of the ester group C=O and the C=C double bond indicate the formation of HM. 710 cm⁻¹ -1 and 1375cm -1 The out-of-plane bending vibrations of the C–H group and the stretching vibrations of the BO group indicate the introduction of PBA. Compared with HP, HPCA exhibits higher activity at 1500 cm⁻¹. -1 More pronounced aromatic ring skeletal vibration peaks were observed at 1275 and 1125 cm⁻¹. -1 The peaks observed at the [specific location] are attributed to the C–O stretching vibration and B–OC related vibration of the phenolic hydroxyl group, indicating the formation of HPCA. These results collectively confirm the successful synthesis of HM, HP, and HPCA.

[0068] III. Preparation of LR@Se oral hydrogel

[0069] 1. Screening experiment on HA molecular weight and HM content

[0070] Since the physical barrier protection ability of HPCA depends on the contribution of HM (hydrogel moiety), the effects of HM prepared with different molecular weight HA and HM content (content in the dispersed phase) on gel performance were screened in preliminary experiments. Chlorogenic acid and PBA were not added during the preparation of the preliminary hydrogels; the purpose was solely to investigate the effects of HM prepared with different molecular weight HA and HM content on gel performance, thus providing a reference for screening the optimal gel preparation process.

[0071] In the HA molecular weight screening experiment, the hydrogel was prepared according to the following formulation: photoinitiator Lap (0.1 wt%), LR@Se (5.0 × 10⁻⁶) were added. 9 HM (1.0 wt%, dissolved in deionized water) prepared by dispersing HA of different molecular weights (CFU / mL) as the dispersed phase, and soybean oil as the continuous phase.

[0072] In the HM concentration screening experiment, the hydrogel was prepared according to the following formulation: photoinitiator Lap (0.1 wt%), LR@Se (5.0 × 10⁻⁶) were added. 9CFU / mL) was dispersed in different concentrations of HM (1.0wt%, 1.5wt%, 2.0wt%, dissolved in deionized water) as the dispersed phase, and soybean oil as the continuous phase.

[0073] In the two screening experiments, the dispersed phase and the continuous phase (volume ratio of dispersed phase to continuous phase: 1:10) were pumped into the corresponding channels of the microfluidic chip of the hydrogel microfluidic preparation device at 1 μL / min and 10 μL / min, respectively, to promote the formation of dispersed droplets and uniformly sized monodisperse droplets. At the same time, they were irradiated under 405 nm visible light for about 15 s to solidify them into uniform microgels.

[0074] With a fixed HM content of 1.0 wt%, the resulting oral hydrogels were named HA1, HA2, HA3, and HA4 according to their HA molecular weight from low to high (HA viscosity-average molecular weights of 2.5–3.5 kDa, 40–100 kDa, 200–400 kDa, and 1500–2500 kDa, respectively). Using a fixed HA content of 200–400 kDa, the resulting oral hydrogels were named HM10, HM15, and HM20 according to HM contents of 1.0 wt%, 1.5 wt%, and 2.0 wt%, respectively.

[0075] In the following experiments, the encapsulation efficiency was calculated as the ratio of the number of viable LR bacteria loaded in the gel microspheres to the number of viable LR bacteria used in the preparation process. The gel microspheres were vortexed at 37 °C for 1 hour to completely release the bacteria before being diluted and coated to test the number of viable LR bacteria loaded.

[0076] In the following experimental investigation, the in vitro simulated digestion method was as follows: Oral hydrogel samples with an equal number of added live bacteria (1×10⁻⁶) were taken. 9 CFU was resuspended in an equal volume of simulated gastric fluid (SGF, pH=2, containing 0.85% NaCl and 0.3% pepsin). The samples were then incubated with shaking at 37 °C. After 2 h of simulated gastric digestion, 10 μL of each sample was taken, diluted to the desired concentration, and inoculated onto MRS agar plates. The plates were then incubated at 37 °C for 48 h to count LR colonies. After 2 h of gastric digestion, the samples were collected by centrifugation and resuspended in an equal volume of simulated intestinal fluid (SIF, pH=6.8, containing 0.68% KH2PO4, 1% trypsin, and 0.3% bile salts), and incubated with shaking at 37 °C. After 4 h of simulated gastric digestion, 10 μL of each sample was taken, diluted to the appropriate concentration, and inoculated onto MRS solid medium. The plates were then incubated at 37 °C for 48 h to count LR colonies.

[0077] The survival rate of LR in different simulated digestive fluids is calculated as follows: simulated gastric fluid survival rate (%) = (logN2 / logN1) × 100%; simulated intestinal fluid survival rate = (logN3 / logN1) × 100%, where N2 is the number of viable bacteria after 2 hours of simulated gastric fluid treatment, N3 is the number of viable bacteria after 4 hours of simulated intestinal fluid treatment, and N1 is the number of viable bacteria before simulated digestion.

[0078] The ROS-responsive degradation experiment method is as follows: Take an oral hydrogel sample with an equal number of viable bacteria (1×10⁻⁶). 9 CFU was resuspended in PBS or H2O2 aqueous solution and incubated with shaking at 37 °C. After 4 h, the supernatant was collected, diluted to the required concentration, and inoculated onto MRS agar plates. The plates were then incubated at 37 °C for 48 h to count the colonies.

[0079] (1) Screening results of HA molecular weight

[0080] To screen the optimal molecular weight of HA suitable for constructing photocrosslinked oral hydrogels, this study selected four different molecular weights of HA (HA1~HA4) for methacrylation modification, and further prepared oral hydrogels to evaluate the regulatory effect of HA molecular weight on the functionality of microgels. The assays included encapsulation efficiency, simulated gastrointestinal digestion experiments, and ROS-responsive degradation experiments. Figure 6 In the figure, ns, ***, and **** represent no statistical difference between the two groups (p>0.05), p<0.01, and p<0.001, respectively.

[0081] 1) such as Figure 6 As shown in Figure a, the LR encapsulation efficiency first increases and then decreases with increasing HA molecular weight. Gels constructed with medium molecular weight HA3 exhibit superior performance, while excessively low or high molecular weight HAs fail to achieve optimal results. HA3 shows the highest encapsulation efficiency improvement of 78.67% compared to other molecular weight HAs, indicating that HA molecular weight within a suitable range can effectively enhance the microgel's encapsulation capacity for LR@Se. Lower molecular weight HAs, due to their shorter molecular chain length, form a relatively loose three-dimensional network structure during cross-linking, resulting in insufficient microgel structural stability and encapsulation capacity. With increasing HA molecular weight, the longer polymer chains provide more effective cross-linking sites, facilitating the formation of a denser and more uniform HPCA network structure, thereby improving the gel's encapsulation efficiency and protective ability for LR@Se. However, when the HA molecular weight further increases, the high viscosity of the system limits the improvement in encapsulation performance.

[0082] 2) In simulated gastrointestinal digestion experiments ( Figure 6(bc) The protective effects of gels prepared with HA of different molecular weights varied significantly. Low molecular weight HA microgels showed almost no protective effect. Compared to the low molecular weight HA group, the medium and high molecular weight HA groups exhibited higher bacterial survival rates under gastric acid and bile salt environments; HA3 showed a 13% and 18% increase in survival rate after gastrointestinal digestion, respectively, compared to HA4. This indicates that the dense network structure formed by medium molecular weight HA can effectively reduce the damage to probiotics caused by the external digestive environment.

[0083] 3) ROS-responsive degradation experiments showed that the microgel cross-linked network constructed with high molecular weight HA4 was more compact, resulting in reduced degradation ability under ROS stimulation and limiting the release of probiotics; while the HA3 system maintained good ROS-responsive release characteristics while ensuring the gastrointestinal tolerance and survival rate of probiotics. Figure 6 d). HA1 and HA2 also exhibited high release rates even in PBS ( Figure 6 (e) indicates that the increased release was not caused by ROS, which may be related to its loose structure. In summary, the network structure formed by excessively low molecular weight HA is insufficiently protective, while excessively high molecular weight HA may limit the rapid response degradation induced by ROS. Therefore, HA with a molecular weight of 200 kDa to 400 kDa was selected for the construction of the gel delivery system.

[0084] (2) Screening results of HM content

[0085] The gel state of HM10, HM15, and HM20 gels before photocuring was observed (vial tilting experiment). Gel formation was determined when the sample in the vial lost its fluidity. The gel strength was measured using a TA.XT Plus C texture analyzer; and microscopic morphology, embedding rate, and simulated gastrointestinal digestion experiments were performed.

[0086] 1) such as Figure 7 As shown in Figure a, HM10, HM15, and HM20 can all form hydrogels with a certain degree of self-support. Under inclined conditions, the gels did not exhibit significant flow, indicating that methacryloyl hyaluronic acid can form a stable three-dimensional network structure after photocrosslinking. Gel strength is a fundamental characteristic of hydrogels. Figure 7 As shown in b, the gel strength increases significantly with increasing HM content.

[0087] 2) The results of the gel microstructure analysis are as follows: Figure 8 As shown in Figure a, after photocrosslinking, the resulting gel maintained a complete spherical structure with clear particle boundaries and no obvious collapse or breakage, indicating that the HM network formed after photocrosslinking can maintain the integrity of the microgel morphology. Further particle size analysis showed that with the increase of HM concentration, the gel particle size gradually increased, with the average particle size increasing from 116 µm to 177 µm, indicating that HM concentration is an important factor in regulating gel size. Figure 8 b).

[0088] 3) Encapsulation efficiency is a crucial parameter affecting production efficiency during the probiotic encapsulation process. For example... Figure 8 As shown in b, the encapsulation efficiency gradually increases with increasing HM concentration. The encapsulation efficiencies for HM10, HM15, and HM20 are 95.61%, 99.23%, and 99.03%, respectively. This is because as the HM concentration increases, the system viscosity rises and the cross-linked network becomes denser.

[0089] 4) The successful passage of probiotics through the gastrointestinal tract is a key prerequisite for their effectiveness. Using free LR and LR@Se as controls, the survival rate of LR in gels prepared with dispersed phases of different HM concentrations was detected after digestion with simulated gastric and intestinal juices (the amount of LR was equal).

[0090] like Figure 9 As shown in Figure a, the survival rates of free LR and LR@Se in the gastrointestinal environment were low, indicating that although selenium modification can enhance antioxidant capacity, its physical barrier effect against acid and bile salt stress is limited. In the gel system, the survival rate of LR increased significantly with increasing HM concentration. After treatment with simulated gastric juice, the survival rates of HM15 and HM20 groups remained at approximately 90%, while those of free LR and LR@SeNPs were only about 50%. After treatment with simulated intestinal juice, the survival rates of HM10, HM15, and HM20 increased by 30.10%, 43.60%, and 42.96% respectively compared to LR, with HM15 showing the highest survival rate. These results demonstrate that the gel network can reduce the direct contact between acid, bile salts, and digestive enzymes and the bacterial cells, thereby improving the gastrointestinal tolerance of LR.

[0091] Based on the above results, HM15 can maintain a moderate particle size and good morphology while exhibiting high encapsulation efficiency and simulated digestion survival rate, making it the preferred concentration for subsequent studies.

[0092] In the final selected optimal hydrogel preparation process, HA with a molecular weight of 200 kDa to 400 kDa was chosen. The HM concentration in the dispersed phase was preferably 1.5 wt%. Since the molecular weight difference between HM and HPCA is small, the HPCA concentration in the dispersed phase was set to 1.5 wt%, referencing HM. The final optimal process window hydrogel preparation formulation is as follows: Photoinitiator Lap (0.1 wt%), LR@Se (5.0 × 10⁻⁶)... 9HPCA (1.5 wt%, dissolved in deionized water) prepared by dispersing in 200-400 kDa HA at CFU / mL was used as the dispersed phase, and soybean oil was used as the continuous phase. The dispersed phase and continuous phase (volume ratio: 1:10) were pumped into the corresponding channels of the microfluidic chip in the hydrogel microfluidic preparation device at 1 μL / min and 10 μL / min, respectively, to promote the formation of dispersed droplets. The dispersed phase formed uniform monodisperse droplets, and was simultaneously irradiated under 405 nm visible light for approximately 15 s to solidify into a uniform microgel. The HPCA was prepared using the method described in Section II, "Preparation of HPCA," of this embodiment.

[0093] The samples prepared according to this optimal preparation process were used for subsequent experiments on "the ability of oral hydrogel loaded with LR@Se to enhance the ROS-responsive degradation and inflamed intestinal adhesion of LR" and "the alleviating effect of the gel on DSS-induced colitis and anxiety and depression". The sample preparation methods for LR (without Na2SeO3 culture) and LR@Se (with 0.5 mM Na2SeO3 added during culture, and LR inoculum of 2%) used in these studies are described in Examples 1 and 2, respectively.

[0094] 2. Oral hydrogels loaded with LR@Se enhance LR's ROS-responsive degradation and inflamed gut adhesion.

[0095] Oral hydrogels loaded with LR@Se were prepared according to the optimal hydrogel preparation process obtained in the aforementioned study, and ROS-responsive degradation experiments and in vivo retention capacity analysis were conducted.

[0096] The experimental method for analyzing in vivo retention capacity was as follows: A BALA / c mouse model of enteritis was established using male BALA / c mice (6-8 weeks old, weighing 20±2g) induced with sodium dextran sulfate (DSS). The modeling method was as follows: After a 7-day acclimatization period, mice were allowed free access to 3% DSS aqueous solution for 7 days to establish the enteritis model. After modeling, mice were randomly divided into 3 groups (n=3). LR, LR@Se, and oral hydrogels (LR@Se / HPCA) were stained with Cy5.5-Se and then administered via gavage to the animal models once (the LR samples contained 1×10⁻⁶ live LR bacteria). 9 CFU (Cyclic Fusion Imaging Unit) is used to monitor the distribution of samples in the intestine by detecting fluorescence signals using a small animal in vivo imaging system (IVIS Spetrum Nano).

[0097] (1) such as Figure 10 As shown, under PBS conditions, the gel network remained relatively stable, which helped limit premature leakage of probiotics in non-pathological environments; in a ROS-simulated environment (H2O2), the cumulative release rate of LR from the gel was 66.67% higher than that under PBS conditions. Figure 10a). Microscopic observation of the gel structure showed that it remained relatively intact in the PBS environment, but disintegrated after treatment in a ROS-simulated environment. This indicates that the gel system can convert pathological signals in the inflammatory microenvironment into release trigger signals, achieving "response activation at the disease site" (a). Figure 10 b).

[0098] (2) The dynamic distribution of different probiotic preparations in the gastrointestinal tract was tracked using an IVIS in vivo imaging system. IVIS images and in vitro intestinal imaging results showed that the fluorescence signals of free LR and LR@Se decayed rapidly over time, almost emptying out within 48 h; while the oral hydrogel group (LR@Se / HPCA) maintained a high fluorescence signal in the gastrointestinal tract region for 72 h, with the strain retention time increasing by at least 24 h compared to the LR group. Figure 11 , 12 ).

[0099] This result indicates that LR@Se / HPCA gel can not only protect probiotics from the complex gastrointestinal environment and peristaltic clearance, but also enhance their interaction with the intestinal mucosa through its hydrogel network, thereby achieving more durable intestinal adhesion.

[0100] Considering that DSS-induced colitis is often accompanied by distal colonic mucosal barrier disruption and exposure of inflammatory adhesion sites, the targeting ability of HA-containing LR@Se / HPCA to the inflamed area was further investigated compared to HA-free LR@Se / PCA. The HA-free LR@Se / PCA was prepared by adding LR@Se (5.0 × 10⁻⁶ ppm) to the inflammatory area. 9 CFU / mL) was dispersed in a PBA-CA mixture dissolved in deionized water.

[0101] The model was established using the aforementioned method, and then LR@Se / HPCA and LR@Se / PCA were administered by gavage (the live LR bacteria in each sample were 1×10⁻⁶). 9 CFU), fluorescence detection was performed using the aforementioned method. The average fluorescence efficiency was determined using the statistical function of Living Image 4.4 software.

[0102] like Figure 13As shown, LR@Se / HPCA exhibited significantly enhanced fluorescence signals in DSS-induced inflamed colon at 6 h and 72 h after oral administration, while its signal was relatively weak in healthy colon, indicating that the gel has a preferential enrichment capacity dependent on the inflammatory microenvironment. Compared with LR@Se / PCA (without HA), LR@Se / HPCA containing HA showed more significant retention in inflamed colon tissue, suggesting that HA-mediated inflammation targeting is an important basis for its lesion enrichment. In particular, LR@Se / HPCA could still maintain a detectable fluorescence signal in inflamed colon 72 h after administration, while the signal in the control system was significantly weakened, further demonstrating that this microgel can achieve long-term retention in inflamed colon.

[0103] 3. The alleviating effect of the gel on DSS-induced colitis and anxiety-depressive behaviors.

[0104] BALA / c mice (male, 6-8 weeks old, weighing 20±2g) were randomly divided into 5 groups (n=10) after a 1-week acclimatization period: normal control group (NC), model group (DSS), free LR group (LR), LR@Se group, and oral hydrogel group (LR@Se / HPCA). The normal control group was not modeled and administered PBS by gavage for 14 days; the DSS group was modeled and administered PBS by gavage for 7 days; each drug administration group was modeled and administered the corresponding test drug by gavage for 14 days (LR dosage was 1×10⁻⁶). 9 CFU), with each group receiving an equal volume of medication via gavage. The modeling method was as follows: 7 days (8-14 days) after gavage administration of the corresponding drug, the DSS group, LR group, LR@Se group, and LR@Se / HPCA group were switched from free drinking water to free drinking of 3% DSS aqueous solution to induce colitis, while the NC group maintained free drinking water.

[0105] After the start of DSS treatment, the mice's body weight was measured daily, and the severity of diarrhea and bloody stools were monitored. The daily disease activity index (DAI) was calculated according to Table 1.

[0106] Table 1 Disease Activity Index (DAI) Scoring Table

[0107] On day 15, mice were given a 30-minute acclimatization period. During this period, the mice were allowed to adapt to their new environment. After the acclimatization period, behavioral experiments were conducted. The activity of each mouse was recorded for 5–8 minutes. Then, the specific behaviors or activities exhibited by the mice during the experiment were analyzed and quantified using the ANY-maze Animal Behavior Analysis System (Stoelting, USA).

[0108] At the end of the experiment, the mice were euthanized after fasting, and blood, intestinal, and fecal samples were quickly collected for further testing.

[0109] In histopathological analysis, tissue samples were fixed in 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin and itson. They were then examined under a microscope at 40× magnification.

[0110] The levels of typical pro-inflammatory factors (IL-1β, IL-6, TNF-α) and anti-inflammatory factors (IL-10) were detected using an ELISA kit according to the kit instructions.

[0111] (1) such as Figure 14 As shown, DSS treatment significantly induced typical colitis disease features, including weight loss, increased DAI, and colonic shortening, indicating that the colitis model was successfully constructed. Figure 14 LR@Se / HPCA intervention significantly slowed DSS-induced weight loss in mice, reduced DAI, and increased colon length, with significantly better results than LR and LR@Se. Figure 14 ).

[0112] (2) Histopathological examination and inflammatory levels in the colon further confirmed the protective effect of LR@Se / HPCA. For example... Figure 15 As shown, the DSS group showed severe epithelial damage, disordered crypt structure, and extensive inflammatory cell infiltration in the colon; while the LR@Se / HPCA group mice had intact colonic epithelium and regular crypt structure, indicating that LR@Se / HPCA can significantly promote colonic tissue repair.

[0113] The levels of typical anti-inflammatory factors (IL-10) and pro-inflammatory factors (TNF-α, IL-6, IL-1β) in the colon of mice in each group were detected by ELISA. Figure 16 As shown, LR@Se / HPCA intervention increased the levels of anti-inflammatory factors and significantly decreased the levels of pro-inflammatory cytokines in mice. Overall, oral administration of LR@Se / HPCA showed a beneficial effect on colitis by improving colonic tissue damage and alleviating epithelial inflammation.

[0114] (3) The harm of enteritis is not limited to local intestinal damage, but can also induce neuroinflammation, blood-brain barrier dysfunction and emotional and behavioral disorders through the gut-brain axis. Therefore, the effect of LR@Se / HPCA on improving colitis-related anxiety / depression-like behaviors was evaluated.

[0115] The open field test is used to assess the spontaneous activity and anxiety-like behavior of mice. Figure 17Behavioral trajectory heatmaps and movement path results showed that the NC group mice had a wide range of activity in the open area and exhibited a high tendency to explore the central area; the DSS model group mice had a significantly limited range of activity, with reduced lingering and traversing behavior in the central area, suggesting that colitis induced significant anxiety-like behavior. LR@Se / HPCA intervention increased central area activity in mice, indicating that LR@Se / HPCA improved the mice's activity level and exploratory behavior.

[0116] The elevated cross maze was used to assess the moderating effect of LR@Se / HPCA on anxiety-like behaviors. For example... Figure 18 As shown, DSS treatment significantly reduced the number of times mice entered the open arms and the time spent in them, with more time spent in the closed arm region, indicating that colitis induced a typical anxiety-like phenotype. In contrast, LR@Se / HPCA intervention significantly increased mice's exploration behavior in the open arms and improved their spatial activity distribution in the elevated cruciate maze. Compared with free LR and LR@Se, LR@Se / HPCA showed superior effectiveness in restoring open arm exploration ability.

[0117] The effects of LR@Se / HPCA on DSS-induced depressive-like behavior were further evaluated using forced swimming and tail suspension tests. Figure 19 As shown, mice in the DSS group exhibited significantly prolonged immobility in the forced swimming and tail suspension tests, reflecting a marked state of behavioral despair. After LR@Se / HPCA treatment, the immobility time of mice was significantly shortened, while movement or struggling behaviors increased, indicating that LR@Se / HPCA can effectively alleviate colitis-induced depressive-like behaviors. This result is consistent with the findings of the open field and elevated cruciate maze tests, further supporting the comprehensive ameliorative effect of LR@Se / HPCA on colitis-related mood disorders.

[0118] Example 4 Cytotoxicity test of LR and its formulation

[0119] The cytotoxicity of the hydrogel was evaluated using the HT-29-MTX-E12 intestinal cell model. Cells were seeded into multi-well plates (seeding density 1×10⁻⁶). 4Cells / wells were treated with different concentrations of LR, LR@Se (prepared according to Example 2, Na2SeO3 concentration 0.5 mM; LR seeding amount 2%), and LR@Se / HPCA (prepared according to Example 3 "Final Selected Optimal Hydrogel Preparation Process") for 2, 4, 6, and 12 h. Cell viability was analyzed using CCK-8 reagent (Apex BIO, USA). The blank control group was added with HT-29-MTX-E12 cell culture medium and CCK-8 reagent, without adding samples or cells; the negative control group was added with HT-29-MTX-E12 cells, HT-29-MTX-E12 cell culture medium, and CCK-8 reagent, without adding samples. After co-incubation of different samples with cells for 2, 4, 6, and 12 h, the viability was measured by LR viability concentration, 1×10⁻⁶. 12 When the CFU / mL level was below 97%, the cell viability of each sample remained at an excellent level (approximately 97%–104%, with no statistical difference compared to the negative control group), indicating that the probiotics and their preparations have good cell compatibility and safety.

Claims

1. A seabuckthorn probiotic strain, characterized in that, The probiotic is *Lactobacillus reuteri*, with accession number CCTCC NO: M20261363; accession date: June 17, 2026; depositary institution: China Center for Type Culture Collection; depositary address: Wuhan University, Wuhan, China; and taxonomic name: *Lactobacillus reuteri*. Limosilactobacillus reuteri JBS01 .

2. The use of probiotics in the preparation of drugs that have both effects on improving enteritis and relieving anxiety and depression, characterized in that, The probiotic is *Lactobacillus reuteri*, with accession number CCTCC NO: M20261363; accession date: June 17, 2026; depositary institution: China Center for Type Culture Collection; depositary address: Wuhan University, Wuhan, China; and taxonomic name: *Lactobacillus reuteri*. Limosilactobacillus reuteri JBS01 .

3. A drug that combines the effects of improving enteritis and relieving anxiety and depression, characterized in that, The drug contains *Lactobacillus reuteri*, whose accession number is CCTCC NO: M20261363; accession date is June 17, 2026; the depositary institution is the China Center for Type Culture Collection (CCTCC); the depositary address is Wuhan University, Wuhan, China; and the taxonomic name of *Lactobacillus reuteri* is *Lactobacillus reuteri*. Limosilactobacillus reuteri JBS01 .

4. A drug according to claim 3 that has both effects of improving enteritis and relieving anxiety and depression, characterized in that, The drug contains biomineralized nano-selenium.

5. A drug according to claim 4 that has both effects of improving enteritis and relieving anxiety and depression, characterized in that, The drug is in the form of an oral hydrogel.

6. A drug according to claim 5 that has both effects of improving enteritis and relieving anxiety and depression, characterized in that, The oral hydrogel has a gel matrix in which the dispersed phase contains a coupling product of methacrylamide hyaluronic acid and chlorogenic acid, and the continuous phase is plant oil. It is prepared by microfluidics and photocuring technology.

7. A drug according to claim 6 that has both effects of improving enteritis and relieving anxiety and depression, characterized in that, The hyaluronic acid used in the methacrylamide hyaluronic acid has a viscosity-average molecular weight of 200 kDa to 400 kDa.

8. A drug according to claim 6 that has both effects of improving enteritis and relieving anxiety and depression, characterized in that, The preparation method of the drug includes the following steps: Take activated Lactobacillus reuteri with preservation number CCTCC NO: M20261363, add it to a culture medium containing sodium selenite, culture it anaerobically, centrifuge it, and collect the first precipitate containing biomineralized nano-selenium and Lactobacillus reuteri. Methacrylamide hyaluronic acid and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride were dissolved in N,N-dimethylformamide. 3-aminophenylboronic acid was added, and the pH was adjusted to 6-7. The reaction was carried out for 36-72 hours. The product was then dialyzed and lyophilized to obtain the first reactant. The first reactant was dissolved and mixed with chlorogenic acid. The pH was adjusted to 9-11, and the reaction was carried out for 6-10 hours. The product was then dialyzed and lyophilized to obtain the second reactant. The second reactant, the photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, the first precipitate, and water were mixed to prepare a dispersed phase. Vegetable oil was used as the continuous phase. The dispersed phase and the continuous phase were pumped into the channels of the hydrogel microfluidic control device to promote the formation of dispersed droplets. The dispersed phase was then irradiated under visible light for 10 to 25 seconds to perform photocuring and obtain an oral hydrogel.

9. A drug according to claim 8 that has both effects of improving enteritis and relieving anxiety and depression, characterized in that, The concentration of sodium selenite in the culture medium is 0.1 mM to 0.5 mM.

10. A drug according to claim 8 that has both effects of improving enteritis and relieving anxiety and depression, characterized in that, The content of the second reactant in the dispersed phase is 1wt%~2wt%.

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