Strong bacteria compound preparation as well as preparation method and application thereof

Abstract

The invention discloses a probiotic compound preparation which is composed of probiotics loaded with biomass carbon dots. The probiotic compound preparation provided by the invention utilizes the biomass carbon dots to carry out functional modification on the probiotics, and compared with single use of lactobacillus reuteri, the compound preparation can significantly improve the survival rate of thalli in a harsh digestive tract environment (gastric acid and bile salt), effectively ensures that enough viable bacteria reach intestinal tracts and realize colonization, and improves the survival rate of the thalli in the harsh digestive tract environment (gastric acid and bile salt). The problem that a traditional probiotic preparation is low in efficiency is solved, the synergistic interaction of the carbon dots and the lactobacillus reuteri is achieved, and the L.r-coated CDs show multiple intestinal micro-ecological regulation functions.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and microbiology, specifically relating to a compound preparation of styrax and its preparation method and application. Background Technology

[0002] Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, is a group of chronic inflammatory bowel diseases with complex etiologies, and its global incidence is continuously rising. The pathogenesis of IBD involves multiple factors, including impaired intestinal barrier function, abnormal immune regulation, and gut microbiota dysbiosis. Currently, clinical treatment of IBD mainly relies on aminosalicylic acid preparations, glucocorticoids, and immunosuppressants; however, these drugs often have limited efficacy, significant side effects, and are prone to drug resistance.

[0003] In recent years, probiotic-based microecological therapy has provided new insights into the treatment of IBD. Lactobacillus reuteri (Lactobacillus reuteri) L.r As an essential probiotic, *Lactobacillus reuteri* has been shown to alleviate intestinal inflammation by regulating gut microbiota balance, enhancing intestinal epithelial barrier function, and modulating host immune responses. However, in practical applications, orally administered *Lactobacillus reuteri* must overcome the harsh digestive environment of gastric acid and bile salts before reaching the intestines to colonize and exert its effects. Its survival and colonization rates are often unsatisfactory, severely limiting the full potential of probiotics. Therefore, effectively improving the survival rate, colonization rate, and efficacy of *Lactobacillus reuteri* in the gut is a key technical bottleneck currently facing probiotic therapy.

[0004] Meanwhile, the rise of nanozymes has provided a new approach to solving this problem. Carbon dots (CDs), as a commonly used carbon-based nanomaterial, have attracted widespread attention due to their excellent biocompatibility, low toxicity, ease of surface functionalization, and unique biological properties. Studies have shown that carbon dot materials themselves possess enzyme-like activities, such as superoxide dismutase activity. While some existing technologies involve the physical mixing of nanomaterials with probiotics, these studies primarily focus on utilizing the antibacterial properties of nanomaterials. This strategy is clearly unsuitable for microecological regulation scenarios that require maintaining probiotic vitality. Currently, there is a lack of mature solutions that can comprehensively enhance the resilience, intestinal colonization ability, and anti-inflammatory effects of probiotics through precise compounding of nanomaterials and probiotics without compromising their activity.

[0005] Therefore, developing a novel formulation based on a functionalized complex of carbon dots and Lactobacillus reuteri, aiming to empower probiotics through nanoengineering and synergistically enhance their effect on alleviating intestinal inflammation, has significant scientific and clinical application value for promoting green and efficient treatment of IBD. Summary of the Invention

[0006] Purpose of the Invention: The purpose of this invention is to solve the above-mentioned technical problems and provide a probiotic compound preparation based on carbon dots and functionalized Lactobacillus reuteri, as well as its preparation method. This invention achieves a dual enhancement of anti-inflammatory effects and intestinal microecological regulation efficacy through the synergistic effect of carbon dots and probiotics, providing a novel, highly effective, and biosafe microbial preparation solution for the treatment of inflammatory bowel disease.

[0007] Technical solution: The present invention provides a compound preparation of probiotics (…). L.r @CDs), the compound formulation consists of probiotics loaded with biomass carbon dots derived from the hydrothermal synthesis of licorice powder, and the probiotics are Lactobacillus reuteri.

[0008] This invention provides a method for preparing a Johnson & Johnson probiotic compound formulation, comprising the following steps: 1) Preparation of biomass carbon dots: Dry biomass raw materials are mixed with ultrapure water and subjected to hydrothermal reaction. After the reaction, the mixture is cooled, filtered, and dried to obtain biomass carbon dots. 2) Disperse the biomass carbon dots prepared in step 1) in ultrapure water to obtain a carbon dot aqueous dispersion; 3) Collect the cultured probiotic cells and resuspend them in sterile water to obtain a probiotic solution; 4) Mix the carbon dot aqueous dispersion from step 2) with the probiotic liquid from step 3) to obtain a probiotic liquid containing biomass carbon dots, and incubate under suitable conditions to load the biomass carbon dots onto the surface or inside of the probiotics, thus obtaining a Johnson & Johnson compound preparation.

[0009] Furthermore, the biomass raw material is licorice powder, the ratio of licorice powder to ultrapure water is 1g:(5-20)mL, the hydrothermal reaction temperature is 180-220℃, and the time is 8-16 hours.

[0010] Furthermore, the ratio of licorice powder to ultrapure water is 1g:10mL, and the hydrothermal reaction temperature is 200℃ for 12 hours.

[0011] Furthermore, the concentration of the probiotic solution in step 3) is 1x10⁻⁶. 8 CFU / mL.

[0012] Furthermore, in step 4), the concentration of biomass carbon dots in the probiotic solution containing biomass carbon dots is 8-16 µg / mL.

[0013] Furthermore, the incubation conditions for step 4) are carried out in an oxygen-free or micro-oxygen environment at 35-39°C for 8-16 hours.

[0014] This invention also provides the aforementioned Johnson & Johnson probiotic compound formulation. L.r Application of @CDs in the preparation of drugs for treating intestinal inflammation. Beneficial effects:

[0015] (1) This invention provides a compound preparation of styrax and probiotics. L.r @CDs and their application in the preparation of drugs for treating intestinal inflammation. This invention innovatively utilizes biomass carbon dots to functionalize probiotics. Compared with using Lactobacillus reuteri alone, this compound preparation can significantly improve the survival rate of bacteria in the harsh digestive tract environment (gastric acid, bile salts), effectively ensuring that a sufficient number of live bacteria reach the intestine and achieve colonization, thus solving the problem of low efficiency in traditional probiotic preparations.

[0016] (2) This invention achieves a synergistic effect between carbon dots and Lactobacillus reuteri. It combines the inherent antioxidant activity of carbon dots with the anti-inflammatory properties of Lactobacillus reuteri, compared to using carbon dots alone... L.r In comparison, it can more effectively alleviate pathological damage to colon tissue, promote the restoration of expression of tight junction proteins ZO-1 and Occludin, more effectively alleviate intestinal oxidative stress and inflammatory response, and synergistically promote the expression of tight junction proteins in intestinal epithelium, thereby accelerating the repair of the intestinal physical barrier.

[0017] (3) The present invention L.r @CDs exhibit multiple functions in regulating the gut microbiota. While exerting anti-inflammatory effects, they can promote the proliferation of beneficial bacteria in the gut, inhibit the abundance of pathogenic bacteria, and systematically reshape the healthy gut microbiota structure, providing a novel green treatment strategy that addresses both the symptoms and the root cause of inflammatory bowel disease.

[0018] (4) The present invention L.r The preparation method of @CDs compound formulation is simple and green. The whole process is carried out in an aqueous phase at room temperature. It does not require complicated equipment or harsh reaction conditions, does not damage probiotics, is low in cost, and has good prospects for industrial application. Attached Figure Description

[0019] Figure 1 Prepared as in Example 1 L.r Transmission electron microscopy (A) and laser confocal scanning microscopy (B) images of the @CDs compound formulation.

[0020] Figure 2 For different biomass carbon point concentrations L.r The effect of @CDs on bacterial population growth density.

[0021] Figure 3 For different biomass carbon point concentrations L.r The effect of @CDs on bacterial survival.

[0022] Figure 4 for L.r In vitro and in vitro resistance assessment of @CDs, (A) the effect of different SGF treatment times on the resistance of CDs to SGF. L.r @CDs and L.r (B) The effect of different SIF treatment times on survival rate; L.r @CDs and L.r (C) Effects on survival rate; (D) Results of plate coating of SGF and SIF; (E) Different treatment groups in the ileum (control, DSS, ... L.r @CDs and L.r (group) L.r Quantitative statistics; (E) Different treatment groups in the colon (CK, DSS, L.r @CDs and L.r (group) L.r Quantitative statistics; (F) Different treatment groups (CK, DSS, ...) in fresh feces L.r @CDs and L.r (group) L.r Quantity statistics.

[0023] Figure 5 for L.r The therapeutic efficacy of the @CDs compound formulation on DSS-induced colitis in mice: (A) Optical photographs of the colons of mice in each group. (B) Changes in mouse body weight; (C) Statistics on colon length in mice (n=5).

[0024] Figure 6 For the prepared L.r @CDs' ability to repair intestinal barrier function: (A) H&E stained sections of mouse colon in each group, scale bar 100 μm; (B) Immunofluorescence imaging of ZO-1 protein in mouse colon tissue, scale bar 100 μm; (C) Immunofluorescence imaging of Occludin protein in mouse colon tissue, scale bar 100 μm. Detailed Implementation

[0025] To enhance understanding of the present invention, the present invention will be further described in detail below with reference to embodiments and accompanying drawings. These embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0026] The raw materials used in the examples are as follows: unless otherwise specified, all raw materials are from commercially available industrial products.

[0027] Lactobacillus reuteri was obtained from the China Industrial Microbial Culture Collection Center; licorice was purchased from a Chinese herbal medicine wholesale store; 5-ASA was pentaminosalicylic acid, purchased from Chengdu Kelong Chemical Co., Ltd.; DSS was sodium dextran sulfate, purchased from Meilun Biotechnology Co., Ltd. (Liaoning, China).

[0028] Example 1: Johnson & Johnson compound preparation L.r Preparation of @CDs (concentration 10 µg / mL) 1) Preparation of biomass carbon dots: 1g of dried licorice was pulverized into powder and mixed with 10 mL of ultrapure water. A hydrothermal reaction was carried out at a temperature of 200℃ for 12 hours. After the reaction, the mixture was cooled, filtered, and dried to obtain biomass carbon dots. 2) Disperse 1 mg of the biomass carbon dots prepared in step 1) in 10 mL of ultrapure water to obtain a carbon dot aqueous dispersion; 3) Culture *Lactobacillus reuteri* under sterile, oxygen-free conditions at 37°C. Collect the cultured *Lactobacillus reuteri* and resuspend it in sterile water to obtain a bacterial concentration of 1 x 10⁻⁶. 8 CFU / mL Lactobacillus reuteri bacterial suspension; 4) Take 1 mL of the carbon dot aqueous dispersion from step 2) and mix it with 10 mL of the Lactobacillus reuteri bacterial solution from step 3) to obtain a Lactobacillus reuteri bacterial solution containing biomass carbon dots. Make the concentration of biomass carbon dots in the mixture 10 µg / mL and incubate overnight at 37°C under anaerobic conditions to load the biomass carbon dots on the surface or inside of Lactobacillus reuteri, thus obtaining the Johnson & Johnson compound preparation.

[0029] The Johnson & Johnson probiotic compound preparation prepared in Example 1 was standardized, such as... Figure 1 As shown, from Figure 1 A single Lactobacillus reuteri can be clearly observed by TEM. L.r Carbon dots (CDs) are attached to the surface, and there is a certain tendency for probiotics to actively internalize CDs, exhibiting a "swallowing and spitting" behavior in which the bacteria themselves are involved in the body's metabolic processes. Figure 1 B's CLSM shows that the Nile red channel indicates no fluorescence effect on the surface / inside of the bacterial cells and is unaffected by FITC, indicating that CDs are fully mounted on the cells. L.r The uneven distribution of fluorescence in the Merge morphology also indicates the presence of internalized carbon dots in the bacterial cells.

[0030] Example 2: Preparation of Johnson & Johnson's compound preparation Lr@CDs (preparation of other concentrations) Referring to Example 1, the volume ratio of carbon dot aqueous dispersion to Lactobacillus reuteri bacterial culture in step 4 of Example 1 was modified to obtain mixed solutions with different concentrations of biomass carbon dots in the mixed solution: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25 and 30 µg / mL.

[0031] Example 3: Superbacterial compound preparations with different biomass carbon point concentrations L.r@CDs performance impact (1) Effect on bacterial population growth density All treatment groups were placed under anaerobic conditions at 37°C and incubated with shaking at 150 rpm for 2 hours; 3. Growth density determination: Immediately after incubation, samples were taken from each tube, and the optical density value at a wavelength of 600 nm was measured using a UV spectrophotometer and recorded as OD. 600 Used to assess the immediate effect of carbon dots on bacterial population growth density, OD 600 The trend of value change is as follows Figure 2 As shown, when the concentration of GR-CDs is below 12 µg / mL, the OD of the bacterial culture... 600 The values ​​remained at a high level (approximately 1.5–2.0), showing no significant difference from the control group without added carbon dots. This indicates that within this concentration range, the presence of carbon dots did not significantly interfere with bacterial aggregation. However, when the concentration exceeded 12 µg / mL, the OD value increased. 600 The values ​​began to show a significant dose-dependent decrease, suggesting that high concentrations of carbon dots may have led to a decrease in cell density through adsorption aggregation or cytotoxic effects.

[0032] (2) Effect on bacterial survival rate For viable cell count validation, the incubated bacterial suspension was serially diluted 10-fold. An appropriate dilution was selected, and 100 µL was plated onto MRS agar plates. After anaerobic incubation at 37°C for 48 hours, colony-forming units were counted to calculate the number of viable cells per milliliter of stock solution, accurately assessing the impact of carbon dots on bacterial survival. Viable cell count results are shown below. Figure 3 As shown, the results of plate colony counting are compared with OD. 600 The trends are highly consistent and provide more convincing viability data. Within the carbon point concentration range below 12 µg / mL, the viable bacterial count remains stable at approximately 2.5 × 10⁻⁶. 7 The concentration was high at CFU / mL. Once the concentration exceeded this threshold, the viable count dropped sharply, reaching extremely low levels at 30 µg / mL, providing direct evidence of the strong inhibitory effect of high concentrations of carbon dots on the survival of Lactobacillus reuteri.

[0033] The results of both detection indicators showed that the concentration of added licorice carbon dots had a significant impact on the survival status of *Lactobacillus reuteri*, and a clear concentration-dependent safety window existed. (Combined OD...) 600 Based on viable cell count data, 10 µg / mL is the optimal concentration for co-incubating licorice carbon dots with Lactobacillus reuteri. Below this concentration, the carbon dots have no adverse effect on the growth and survival of probiotics, providing a basis for the formation of a functional complex between the two.

[0034] Example 4 L.r In vitro and ex vivo resistance assessment of CDs Lactobacillus reuteri L.r (Preparation in Step 3 of Example 1), Johnson & Johnson compound preparation L.r @CDs cells (prepared in Example 1) (concentration 1×10⁻⁶) 8 CFU was resuspended in 1 mL of SGF (simulated gastric fluid) and incubated with gentle shaking at 37°C. At specified time points, 200 µL of solution was removed from the culture medium, centrifuged at 5000 rpm for 5 min, and resuspended in 200 µL PBS. Samples were cultured on MRS agar plates at 37°C with appropriate dilution for bacterial counting. SIF (simulated intestinal fluid) resistance was assessed using the same method as for SGF. Figure 4 AC stated that it is used as an oral material. L.r @CDs and L.r During the process of simulating and digesting SGF and SIF L.r @CDs consistently have a higher survival rate than unmodified ones. L.r This demonstrates that the modifying effect of CDs can significantly enhance... L.r Its resistance to adverse conditions.

[0035] In the in vitro experiment, the mice were divided into four groups and treated accordingly as follows: Blank control group (CK group): From day 0 to day 7, the same dose of purified water was administered orally. DSS-induced colitis group (DSS group): From day 0 to day 7, 3% DSS was administered to induce the model, and from day 1 to day 7, 100 μL of PBS solution was administered orally once daily. DSS colitis+ L.r Group( L.r Group 1): From day 0 to day 7, the model was induced by administering 3% DSS, and from day 1 to day 7, 100 μL of Lactobacillus reuteri culture prepared in step 3 of Example 1 was administered orally once a day. DSS colitis+ L.r @CDs Group ( L.r @CDs group (abbreviated as LC in the attached figure): from day 0 to day 7, 3% DSS was administered for model induction, and from day 1 to day 7, 100 μL of the Johnson & Johnson compound preparation prepared in Example 1 of this invention was administered orally once daily.

[0036] Then, ileal contents, colonic contents, and fresh fecal samples were collected from each group of mice for dilution and bacterial plate counting. Figure 4 Data from plate coating of contents taken from different locations in the digestive tract in DF also showed... L.r @CDs are superior L.r The colonization ability further confirms the significant modifying effect of CDs.

[0037] Example 5 L.r The therapeutic efficacy of the CDs compound formulation on DSS-induced colitis in mouse models (1) Establishment of a mouse colitis model: Male mice aged 6-8 weeks were randomly divided into 6 groups of 10-12 mice each. The 6 groups were as follows: Blank control group (CK group): From day 0 to day 7, the same dose of purified water was administered orally. DSS-induced colitis group (DSS group): From day 0 to day 7, 3% DSS was administered to induce the model, and from day 1 to day 7, 100 μL of PBS solution was administered orally once daily. DSS colitis + CDs group (CDs group); from day 0 to day 7, 3% DSS was administered to induce the model, and from day 1 to day 7, 100 μL of biomass carbon dot aqueous dispersion prepared by CDs (step 2 of Example 1) was administered orally once a day. DSS colitis+ L.r Group( L.r Group 1): From day 0 to day 7, the model was induced by administering 3% DSS, and from day 1 to day 7, 100 μL of Lactobacillus reuteri culture prepared in step 3 of Example 1 was administered orally once a day. DSS colitis+ L.r @CDs Group ( L.r @CDs group (abbreviated as LC in the attached figure): from day 0 to day 7, 3% DSS was administered for model induction, and from day 1 to day 7, 100 μL of the Johnson & Johnson compound preparation prepared in Example 1 of this invention was administered orally once a day. DSS colitis + 5-ASA group (5-ASA group): From day 0 to day 7, 3% DSS was administered for model induction, and 5-ASA (20 mg / Kg / d) was administered orally once daily from day 1 to day 7.

[0038] Record the daily body weight of each group. On day 9, euthanize the mice in each group and collect intestinal tissue and contents. Record the daily body weight changes of each group throughout the experiment.

[0039] (1) Evaluation of body weight and colon length: Figure 5 B showed that throughout the experiment, the average body weight of the DSS-induced colitis group (DSS group) was significantly lower than that of the control group and the experimental treatment group. All drug groups experienced varying degrees of weight loss, but the degree of weight loss was less than that of the colitis model group (DSS group). L.r The CDs group experienced the least weight loss, indicating that... L.r@CDs treatment can more effectively alleviate weight loss caused by DSS. The experiment was terminated on day 8, with water and food supplies to the mice stopped. The mice were euthanized on day 9, and their colons were collected. Colon length can serve as a direct indicator of the severity of colitis, such as... Figure 5 In groups A and C, compared to healthy mice, the DSS group showed a significantly shortened colon, colonic redness and swelling, and obvious bloody stool residue in the colon. L.r Compared with the CDs group and the enteritis group, L.r @CDs significantly reduced the production of bloody stools in the colon, indicating that the symptoms of enteritis were relieved and the length of the colon was significantly restored.

[0040] (2) Evaluation of intestinal morphology and structure: Mouse colon and colon tissue were collected, fixed with 4% paraformaldehyde, sectioned, stained with H&E, and the changes in intestinal morphology and structure were observed. Intestinal tissue was fixed with 2.5% glutaraldehyde, rinsed with phosphate rinsing solution, dehydrated, sectioned, stained, and observed under an optical microscope. Figure 6 H&E staining was performed on colon tissues from mice in each group. Histopathological analysis showed that the DSS group mice had severe damage to the intestinal epithelium, severe edema of the submucosa, disappearance of goblet cells, severe destruction of crypt structures, and extensive infiltration of inflammatory cells. The CDs group showed almost no structural recovery, while the 5-ASA group showed a small number of goblet cells and extensive infiltration of inflammatory cells. L.r The group showed some recovery in intestinal functional structure. L.r The @CDs group showed a large number of goblet cells and relatively intact crypt structures, with a reduced area of ​​edema.

[0041] (3) Evaluation of intestinal barrier function: Mouse colon tissue was collected, fixed in 4% paraformaldehyde, incubated overnight at 4°C, then frozen and embedded. After sectioning, it was used for immunofluorescence detection. Intestinal sections were incubated overnight with tight junction protein antibodies ZO-1 and Occludin, washed, and incubated with the corresponding FITC fluorescent secondary antibody for 60 min. After washing, DAPI was added and incubated for 10 min. Images were observed and acquired using a laser confocal microscope. Figure 6 B and C assessed the impact of the materials on intestinal function from the perspective of intestinal barrier proteins. Occludin and ZO-1 are important intestinal tight junction proteins, responsible for maintaining the integrity and permeability of the intestinal mucosal barrier; their integrity characterizes the state of intestinal barrier function. Immunofluorescence staining of Occludin and ZO-1 showed that the immunofluorescence expression levels in the DSS group and the treatment group were significantly lower than those in the control group, indicating that DSS can lead to severe intestinal barrier dysfunction. L.r Immunofluorescence results of CDs and 5-ASA groups L.r @CDs have a more significant effect on restoring the intestinal barrier structure.

[0042] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A probiotic compound preparation, characterized in that, The compound preparation consists of probiotics loaded with biomass carbon dots.

2. The probiotic compound preparation according to claim 1, characterized in that, The biomass carbon dots are derived from the hydrothermal synthesis of licorice powder.

3. The probiotic compound preparation according to claim 1, characterized in that, The probiotic is Lactobacillus reuteri.

4. A method for preparing a probiotic compound preparation according to any one of claims 1-3, characterized in that, Includes the following steps: 1) Preparation of biomass carbon dots: Dry biomass raw materials are mixed with ultrapure water and subjected to hydrothermal reaction. After the reaction, the mixture is cooled, filtered, and dried to obtain biomass carbon dots. 2) Disperse the biomass carbon dots prepared in step 1) in ultrapure water to obtain a carbon dot aqueous dispersion; 3) Collect the cultured probiotic cells and resuspend them in sterile water to obtain a probiotic solution; 4) Mix the carbon dot aqueous dispersion from step 2) with the probiotic liquid from step 3) to obtain a probiotic liquid containing biomass carbon dots, and incubate under suitable conditions to load the biomass carbon dots onto the surface or inside of the probiotics, thus obtaining a Johnson & Johnson compound preparation.

5. The method for preparing the Johnson & Johnson compound preparation according to claim 4, characterized in that, The biomass raw material is licorice powder, and the ratio of licorice powder to ultrapure water is 1g:(5-20)mL. The hydrothermal reaction temperature is 180-220℃ and the time is 8-16 hours.

6. The method for preparing the Johnson & Johnson probiotic compound preparation according to claim 5, characterized in that, The ratio of licorice powder to ultrapure water is 1g:10mL, and the hydrothermal reaction is carried out at a temperature of 200℃ for 12 hours.

7. The method for preparing the Johnson & Johnson probiotic compound preparation according to claim 4, characterized in that, The concentration of the probiotic solution in step 3) is 1x10⁻⁶. 8 CFU / mL.

8. The method for preparing the Johnson & Johnson probiotic compound preparation according to claim 4, characterized in that, In step 4), the concentration of biomass carbon dots in the probiotic solution containing biomass carbon dots is 8-16 µg / mL.

9. The method for preparing the Johnson & Johnson probiotic compound preparation according to claim 4, characterized in that, The incubation conditions for step 4) are carried out in an oxygen-free or micro-oxygen environment at 35-39℃ for 8-16 hours.

10. The use of the probiotic compound preparation according to any one of claims 1-9 in the preparation of a drug for treating intestinal inflammation.

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