Lactobacillus rhamnosus YDJ-05 as well as targeted delivery composition and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to lactobacillus rhamnosus YDJ-05 as well as a targeted delivery composition and application of the lactobacillus rhamnosus YDJ-05. The invention provides a lactobacillus rhamnosus YDJ-05. The lactobacillus rhamnosus strain is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms on April 11, 2025, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) NO.34174. The lactobacillus rhamnosus YDJ-05 has the advantages that the lactobacillus rhamnosus YDJ-05 can be used for preparing the lactobacillus rhamnosus YDJ-05, and the lactobacillus rhamnosus YDJ-05 can be used for preparing the lactobacillus rhamnosus YDJ-05; meanwhile, the invention provides a target delivery composition and application of the rhamnosus cheese bacillus YDJ-05, the YDJ-05 strain is a newly separated and preserved strain, plays an anti-inflammatory role by regulating and controlling a specific immune signal channel and is clear in mechanism, and moreover, the double-layer polysaccharide hydrogel delivery system adopted by the invention has the advantages that the anti-inflammatory effect is achieved, and the application range is wide. The survival rate of thalli in a gastric acid environment can be remarkably improved, rapid and accurate release in an intestinal environment is realized, and the utilization efficiency of probiotics is greatly improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology, and particularly relates to a Lactobacillus rhamnosus YDJ-05, a targeted delivery composition thereof and application. BACKGROUND

[0002] Probiotics, as a kind of beneficial active microorganisms, play an important role in maintaining intestinal health, enhancing immune barrier and regulating immune function. Lactobacillus rhamnosus is one of the most widely studied probiotics. However, there are two key technical bottlenecks in the practical application of traditional oral probiotic preparations. First, live bacteria are easily inactivated when passing through the gastric acid environment, resulting in a significant reduction in the number of live bacteria reaching the intestine and affecting the exertion of their efficacy. Second, conventional dosage forms (such as ordinary freeze-dried powder or capsules) are difficult to achieve effective delivery and release of active bacteria to specific intestinal environments or local pathological sites.

[0003] In the prior art, although some embedding techniques have been used to improve the gastric acid tolerance of probiotics, the protection effect and precision of intestinal targeted release still need to be improved. In addition, it is still a challenge to apply probiotics in a localized and high-activity form, and there is a lack of effective dosage form support. In terms of functional application, although some probiotic strains have been reported to have anti-inflammatory potential, the specific mechanism of action, especially the regulation of key immune signaling pathways (such as the STING / TBK1 / RELA pathway), is still unclear and needs further research and more targeted strain development.

[0004] Therefore, there is an urgent need in the art for a new probiotic strain with a clear efficacy mechanism and a high-efficiency targeted delivery system that can effectively overcome the above technical bottlenecks. SUMMARY

[0005] The main purpose of the present application is to provide a Lactobacillus rhamnosus YDJ-05, a targeted delivery composition thereof and application, to overcome the problems in the prior art.

[0006] To achieve the aforementioned purposes, the technical solutions adopted by the present application comprise: According to a first aspect of an embodiment of the present application, a Lactobacillus rhamnosus YDJ-05 is provided, which was deposited with the China General Microbiological Culture Collection Center on April 11, 2025, and has a deposit number of CGMCC NO. 34174.

[0007] According to a second aspect of an embodiment of the present application, a probiotic composition is provided, comprising the above-mentioned Lactobacillus rhamnosus YDJ-05 and a pharmaceutically or dietetically acceptable carrier.

[0008] Further, the carrier is an intestinal-targeted delivery system, which is a double-layer polysaccharide hydrogel; wherein the double-layer polysaccharide hydrogel comprises an inner gel mesh composed of carboxymethyl cellulose, and an outer pH-responsive shell composed of oxidized sodium alginate cross-linked carboxymethyl chitosan.

[0009] Further, the double-layer polysaccharide hydrogel is configured to protect the Lactobacillus rhamnosus YDJ-05 in simulated gastric fluid with a pH of 2.0, with a survival rate of not less than 85.8%; and to release not less than 90% of the live bacteria within 6 hours in simulated intestinal fluid with a pH of 7.0.

[0010] Further, the dosage form of the probiotic composition is a fibrous dressing loaded with active probiotics; wherein the Lactobacillus rhamnosus YDJ-05 is encapsulated in agarose microcapsules and uniformly embedded in a hydrophobic fiber matrix by electrospinning technology, and the live bacteria load in the fibrous dressing is not less than 10 -6 CFU / cm 2 .

[0011] Further, the fibrous dressing has an inhibitory effect on Escherichia coli and Staphylococcus aureus.

[0012] According to a third aspect of the embodiments of the present application, a method for preparing any of the above-mentioned probiotic compositions is provided, comprising the following steps: mixing the Lactobacillus rhamnosus YDJ-05 bacteria after activation culture with carrier materials; When the carrier is an intestinal-targeted delivery system, the double-layer polysaccharide hydrogel is used to embed the bacteria by using the emulsification-internal gelation combined ion cross-linking method; When the dosage form of the composition is a fibrous dressing loaded with active probiotics, agarose microcapsules containing bacteria are spun into a fiber matrix by electrospinning technology.

[0013] According to a fourth aspect of the embodiments of the present application, the use of the above-mentioned Lactobacillus rhamnosus YDJ-05 or the above-mentioned probiotic composition in the preparation of a drug, health product or food for preventing and / or treating depression and inflammatory bowel disease is provided.

[0014] Further, the inflammatory bowel disease is colitis.

[0015] According to a fifth aspect of the embodiments of the present application, the use of the above-mentioned Lactobacillus rhamnosus YDJ-05 or any of the above-mentioned probiotic compositions in the preparation of a health product or food for regulating depression, intestinal flora, enhancing intestinal barrier function, or promoting defecation is provided.

[0016] Compared with the prior art, the present application has the following advantages: The present application provides a lactosaccharo-rhamnosus YDJ-05, its targeted delivery composition and application, the YDJ-05 strain is a newly isolated and preserved strain, which plays an anti-inflammatory role by regulating specific immune signal pathways, and the mechanism is clear.The double-layer polysaccharide hydrogel delivery system used in the present application can significantly improve the survival rate of bacterial bodies in gastric acid environment, and realize rapid and accurate release in intestinal environment, greatly improving the utilization efficiency of probiotics.The developed fiber dressing dosage form provides a new paradigm for the local application of probiotics on skin mucosa and wound surface, and exhibits sustained antibacterial activity.The combination of the targeted delivery system and the specific efficacy strain produces a synergistically enhanced anti-inflammatory and intestinal protection effect, and the technical advantage is obvious. DETAILED DESCRIPTION

[0017] In view of the deficiencies in the prior art, the present inventors have obtained the technical solution of the present application through long-term research and a large number of practices. The technical solution, its implementation process and principles will be further explained as follows.

[0018] The present application will be further described in detail below in conjunction with specific embodiments, and the examples given are only for illustrating the present application, rather than limiting the scope of the present application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not constitute any limitation on the present application in any way.

[0019] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0020] The present application provides a lactosaccharo-rhamnosus YDJ-05, which was preserved in the China General Microbiological Culture Collection Center on April 11, 2025, and the preservation number is CGMCC NO. 34174.

[0021] The embodiment of the present application provides a probiotic composition, which comprises the above-mentioned Lactobacillus rhamnosus YDJ-05 and a pharmaceutically or dietetically acceptable carrier. The carrier is an intestinal-targeted delivery system, which is a double-layer polysaccharide hydrogel; wherein the double-layer polysaccharide hydrogel comprises an inner gel grid composed of carboxymethyl cellulose, and an outer pH-responsive shell composed of oxidized sodium alginate cross-linked carboxymethyl chitosan. The double-layer polysaccharide hydrogel is configured to protect the Lactobacillus rhamnosus YDJ-05 in simulated gastric juice with a pH of 2.0, and the survival rate is not less than 85.8 %; and in simulated intestinal fluid with a pH of 7.0, not less than 90 % of the viable bacteria are accumulated and released within 6 hours. The dosage form of the probiotic composition is a fiber dressing loaded with active probiotics; wherein the Lactobacillus rhamnosus YDJ-05 is encapsulated in agarose microcapsules and uniformly embedded in a hydrophobic fiber matrix by an electrospinning technology, and the viable bacteria load in the fiber dressing is not less than 10 -6 CFU / cm 2 The fiber dressing has an inhibitory effect on Escherichia coli and Staphylococcus aureus.

[0022] The present application provides a method for preparing any of the above-mentioned probiotic compositions, comprising the following steps: mixing the Lactobacillus rhamnosus YDJ-05 bacteria after activation culture with carrier materials; when the carrier is an intestinal-targeted delivery system, the double-layer polysaccharide hydrogel is used to embed the bacteria by adopting an emulsification-internal gelation combined ion cross-linking method; when the dosage form of the composition is a fiber dressing loaded with active probiotics, the agarose microcapsules containing the bacteria are spun into a fiber matrix by an electrospinning technology.

[0023] The present application provides the use of the above-mentioned Lactobacillus rhamnosus YDJ-05 or the above-mentioned probiotic composition in the preparation of a drug, health product or food for preventing and / or treating depression and inflammatory bowel disease. The inflammatory bowel disease is colitis.

[0024] The present application provides the use of the above-mentioned Lactobacillus rhamnosus YDJ-05 or any of the above-mentioned probiotic compositions in the preparation of a health product or food for regulating depression, intestinal flora, enhancing intestinal barrier function, or promoting defecation.

[0025] In order to better understand the technical scheme of the present application, the following will be discussed in detail in combination with specific embodiments.

[0026] Embodiment 1: Fermentation culture of Lactobacillus rhamnosus YDJ-05 and preparation of double-layer polysaccharide hydrogel composition.

[0027] 1. Strain culture: Lactobacillus rhamnosus YDJ-05 with the preservation number of CGMCC NO. 34174 was inoculated into MRS liquid medium and incubated at 37°C for 20 hours under anaerobic condition to obtain a bacterial solution with a viable cell concentration of 5.0 x 10 -9 CFU / mL. The MRS medium: 20 g / L glucose, 10 g / L tryptone, 10 g / L beef extract, 5 g / L yeast extract, 5 g / L sodium acetate, 2 g / L potassium phosphate dibasic, 2 g / L triammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1 mL / L Tween-80.

[0028] 2. Strain identification: identified by 16S rRNA gene sequencing, the sequence has a homology of more than 99.9% with the known Lacticaseibacillus rhamnosus standard sequence. The strain has typical morphological and physiological and biochemical characteristics of Lactobacillus rhamnosus.

[0029] Example 2: Preparation and performance evaluation of double-layer polysaccharide hydrogel (DPH) composition.

[0030] 1. Preparation of DPH composition: YDJ-05 strain was cultured as in Example 1, and the bacterial cells were collected by centrifugation.

[0031] Inner layer preparation: the bacterial cells were mixed with a 2% (w / v) carboxymethyl cellulose (CMC) solution, and a bacterial cell-containing microcapsule embryo was prepared by emulsification.

[0032] Outer layer coating: the microcapsule embryo was suspended in a solution containing 1.5% (w / v) oxidized sodium alginate (DAA) and 1% (w / v) carboxymethyl chitosan (CMCS), and a pH-responsive shell was formed by ionic crosslinking to obtain DPH-embedded probiotic microspheres.

[0033] 2. Gastric acid protection performance test: DPH microspheres and naked YDJ-05 bacteria were placed in simulated gastric fluid with a pH of 2.0, and shaken at 37°C for 2 hours. The results are shown in Table 1 below: Table 1:

[0034] DPH embedding significantly improved the survival rate of bacterial cells in a gastric acid environment (p<0.01).

[0035] 3. Intestinal targeting release test: the samples treated with gastric juice were transferred to simulated intestinal fluid with a pH of 7.0. The DPH-embedded group released 92.5% of the active probiotic bacteria within 6 hours, showing good intestinal targeting release characteristics.

[0036] Example 3: Preparation of fiber dressing loaded with YDJ-05.

[0037] 1. Dressing preparation: The YDJ-05 bacterial cells obtained in Example 1 were mixed with agarose solution to prepare agarose microcapsules. Subsequently, the microcapsules were blended with a hydrophobic solution of polylactic acid (PLA) and prepared into a fiber dressing using microchip electrospinning technology.

[0038] 2. Viable bacterial load determination: Scanning electron microscopy (SEM) observation showed that the probiotics were evenly distributed in the fibrous matrix. The viable bacterial load of the dressing, determined by plate counting, reached 3.5 × 10⁻⁶. -6 CFU / cm 2 .

[0039] 3. Antibacterial activity evaluation: The inhibition zone method was used. The dressing sample was applied to a dressing coated with Staphylococcus aureus (Staphylococcus aureus). S. aureus ) or E. coli ( E. coli The dressing was incubated on agar plates at 37°C for 24 hours. The results showed that the dressing produced significant inhibition zones (12.3 mm and 10.1 mm in diameter, respectively) against both common wound pathogens, indicating that it has sustained antibacterial activity.

[0040] Example 4: The therapeutic effect of YDJ-05 on DSS-induced colitis in mice.

[0041] 1. Model Establishment and Grouping: An acute colitis model was established in C57BL / 6 mice by inducing sodium dextran sulfate (DSS). Mice were randomly divided into 4 groups (n=8): normal control group, DSS model group, YDJ-05 common probiotic powder intervention group, and YDJ-05 DPH composition intervention group.

[0042] 2. Intervention regimen: The intervention group was given 1.0×10⁹ CFU of YDJ-05 (bacterial powder or DPH combination) by gavage daily for 7 days.

[0043] 3. Evaluation of Effects: Disease Activity Index: Compared with the model group, both YDJ-05 intervention groups significantly reduced the disease activity index. Among them, the DPH combination group showed the most significant improvement (p<0.01).

[0044] Colon length: The colon length of mice in the model group was significantly shortened. After intervention with YDJ-05 DPH composition, the colon length was close to that of the normal group and significantly longer than that of the model group and the ordinary bacterial powder group (p<0.05).

[0045] Inflammatory factor levels: ELISA kits were used to detect the levels of inflammatory factors in colon tissue homogenate. The results showed that, compared with the model group, the YDJ-05 DPH composition could significantly up-regulate the expression of anti-inflammatory factor IL-10 (increased by about 2.1 times), and significantly reduce the levels of pro-inflammatory factors TNF-α and IL-6 (reduced by 58.3% and 49.7%, respectively). Western Blotting analysis further confirmed that YDJ-05 activated the STING / TBK1 / RELA signaling pathway in the colon tissue.

[0046] Intestinal flora analysis: 16S rRNA sequencing was performed on the feces of mice. The results showed that YDJ-05 intervention reshaped the intestinal flora structure, increased the relative abundance of beneficial bacteria such as Lactobacillus, and reduced the abundance of conditionally pathogenic bacteria associated with inflammation.

[0047] Example 5: Regulation of intestinal barrier function by YDJ-05 In the animal experiment of Example 4, the colon tissue of the mice was also taken for pathological examination. Immunohistochemical staining showed that the expression of tight junction proteins (such as ZO-1 and Occludin) in the intestinal mucosa of mice in the YDJ-05 DPH composition intervention group was significantly higher than that in the model group and the ordinary bacteria powder group, indicating that YDJ-05 helps to enhance the physical barrier function of the intestinal epithelium. The data in the above examples fully show that the Rhamnosus casei YDJ-05 and the targeted delivery composition provided by the present application have significant and excellent effects in improving the gastric acid tolerance of the bacterial body, achieving precise release in the intestine, relieving intestinal inflammation, regulating the flora, and enhancing the barrier function.

[0048] Example 6: Anti-depression effect of YDJ-05 on depression model mice.

[0049] 1. Purpose of the experiment: The present experimental example aims to verify that the Rhamnosus casei YDJ-05 of claim 1 and the probiotic composition comprising a double-layer polysaccharide hydrogel of claim 3 have a significant improvement effect on the depression-like behavior of mice induced by chronic unpredictable mild stress (CUMS), thereby providing experimental data support for the "preparation of drugs, health products or food for preventing and / or treating depression" in claims 8 and 10.

[0050] 2. Experimental materials: Experimental strain: Rhamnosus casei YDJ-05 (CGMCC NO. 34174) of claim 1.

[0051] Experimental preparation: YDJ-05 ordinary bacteria powder group: the activated YDJ-05 bacteria were collected by centrifugation, mixed with a freeze-drying protective agent, and then vacuum freeze-dried to prepare ordinary bacteria powder.

[0052] YDJ-05 hydrogel group: according to the method of claim 3 and 4, a YDJ-05 probiotic composition embedded in a double-layer polysaccharide hydrogel (carboxymethyl cellulose inner layer, cross-linked carboxymethyl chitosan outer layer) was prepared. It was verified by simulated gastrointestinal fluid that the survival rate in gastric juice (pH 2.0) was 87.3%, and the live bacteria release rate in intestinal juice (pH 7.0) within 6 hours reached 92.1%.

[0053] Control strain: Lactobacillus rhamnosus GG (LGG) purchased commercially.

[0054] Positive control drug: fluoxetine hydrochloride (a commonly used antidepressant in clinical practice).

[0055] Experimental animals: healthy male C57BL / 6J mice, SPF level, 8 weeks old, randomly divided into groups.

[0056] 3. Experimental method: Depression model construction: the classic chronic unpredictable mild stress (CUMS) model was used for 5 weeks. Stressors included water and food deprivation, day-night reversal, cage tilting, wet bedding, restraint stress, etc., one of which was randomly applied daily.

[0057] Experimental grouping and drug administration: Blank control group: no CUMS stress was applied, and an equal volume of normal saline was administered intragastrically daily.

[0058] Model control group: CUMS stress was applied, and an equal volume of normal saline was administered intragastrically daily.

[0059] Positive control group: CUMS stress was applied, and fluoxetine hydrochloride (10 mg / kg) was administered intragastrically daily.

[0060] LGG control group: CUMS stress was applied, and LGG ordinary bacteria powder (about 1×10 -9 CFU per mouse) was administered intragastrically daily.

[0061] YDJ-05 ordinary bacteria powder group: CUMS stress was applied, and YDJ-05 ordinary bacteria powder (about 1×10 -9 CFU per mouse) was administered intragastrically daily.

[0062] YDJ-05 hydrogel group: CUMS stress was applied, and an equal dose of YDJ-05 hydrogel preparation was administered intragastrically daily.

[0063] Behavioral evaluation (5th week): Sucrose preference test: to evaluate the anhedonia of mice, one of the core symptoms of depression. Record the consumption of 1% sucrose solution and pure water within 4 hours, and calculate the percentage of sucrose preference.

[0064] Forced swimming test: to evaluate the despair behavior of mice. Put the mice into a water tank that cannot escape, and record the immobility time within 6 minutes, and the last 4 minutes. The shorter the immobility time, the stronger the antidepressant-like behavior.

[0065] Tail suspension test: the principle is the same as the forced swimming test, and the immobility time of the mouse within 6 minutes is recorded.

[0066] Biochemical index detection: after the behavioral test, the hippocampus tissue of the mouse is taken.

[0067] ELISA method for detecting inflammatory factors: to detect the content of pro-inflammatory cytokines TNF-α and IL-1β.

[0068] ELISA method for detecting neurotransmitters: to detect the levels of brain-derived neurotrophic factor (BDNF) and 5-hydroxytryptamine (5-HT).

[0069] 4. Experimental results: Table 1: Effect of Rhamnosus Lactobacillus YDJ-05 on the behavior of CUMS depression model mice

[0070] Note: compared with the model control group, p<0.01; p<0.05, *p<0.01.

[0071] Table 2: Effect of Rhamnosus Lactobacillus YDJ-05 on the biochemical indicators of hippocampus of CUMS depression model mice:

[0072] Note: compared with the model control group, p<0.01; p<0.05, *p<0.01.

[0073] 5. Experimental conclusion: This example fully proves by strict animal models and multiple evaluation indexes that: 1). Rhamnosus Lactobacillus YDJ-05 of the application itself has a significant antidepressant effect, which is better than the ordinary commercial strain LGG in behavior (improving anhedonia and despair behavior) and biochemical level (reducing neural inflammation, improving BDNF and 5-HT levels), and the effect is comparable to that of the positive drug fluoxetine.

[0074] 2) The probiotic composition comprising double-layer polysaccharide hydrogel of the present application has better antidepressant effect than YDJ-05 ordinary bacteria powder. This strongly proves that the intestinal targeted delivery system of the present application not only improves the survival rate of bacteria in the gastrointestinal tract, but also significantly enhances its final in vivo biological efficacy, embodying the synergistic advantages and advancement of the product invention.

[0075] The above merely describes some embodiments of the present application, and it should be noted that, for those skilled in the art, other modifications and improvements can be made without departing from the inventive concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A strain of Lacticaseibacillus rhamnosus YDJ-05, characterized in that: The Lactobacillus rhamnosus strain was deposited at the China General Microbiological Culture Collection Center on April 11, 2025, with accession number CGMCC NO.34174.

2. A probiotic composition, characterized in that: It comprises Lactobacillus rhamnosus YDJ-05 as described in claim 1 and a pharmaceutically or food-grade acceptable carrier.

3. The probiotic composition according to claim 2, characterized in that: The carrier is an intestinal targeted delivery system, which is a bilayer polysaccharide hydrogel; wherein the bilayer polysaccharide hydrogel comprises an inner gel mesh composed of carboxymethyl cellulose and an outer pH-responsive shell composed of sodium alginate crosslinked with carboxymethyl chitosan.

4. The probiotic composition according to claim 3, characterized in that: The bilayer polysaccharide hydrogel is configured to protect *Lactobacillus rhamnosus* YDJ-05 in simulated gastric fluid at pH 2.0 with a survival rate of not less than 85.8%; and to cumulatively release not less than 90% of live bacteria in simulated intestinal fluid at pH 7.0 within 6 hours.

5. The probiotic composition according to claim 2, characterized in that, The probiotic composition is formulated as a fiber dressing loaded with active probiotics; wherein, *Lactobacillus rhamnosus* YDJ-05 is encapsulated in agarose microcapsules and uniformly embedded in a hydrophobic fiber matrix using electrospinning technology, and the viable bacterial load in the fiber dressing is not less than 10. -6 CFU / cm 2 .

6. The probiotic composition according to claim 5, characterized in that, The fiber dressing has an inhibitory effect on Escherichia coli and Staphylococcus aureus.

7. A method for preparing the probiotic composition according to any one of claims 2-6, characterized in that, Includes the following steps: The activated Lactobacillus rhamnosus YDJ-05 cells were mixed with the carrier material; When preparing the probiotic composition as described in claim 3 or 4, the bacterial cells are encapsulated in a bilayer polysaccharide hydrogel constructed by an emulsification-internal gelation combined with ionic crosslinking method. When preparing the probiotic composition as described in claim 5 or 6, electrospinning technology is used to spin agarose microcapsules containing bacteria into a fiber matrix.

8. The use of the Lactobacillus rhamnosus YDJ-05 of claim 1 or the probiotic composition of any one of claims 2 to 6 in the preparation of a medicament, health product or food for the prevention and / or treatment of depression and inflammatory bowel disease.

9. The application according to claim 8, characterized in that, The inflammatory bowel disease mentioned is colitis.

10. The use of the Lactobacillus rhamnosus YDJ-05 of claim 1 or the probiotic composition of any one of claims 2-6 in the preparation of health products or foods for regulating depression, gut microbiota, enhancing intestinal barrier function, or promoting bowel movements.

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