Anti-infection probiotic agent containing CT-02 strain and preparation method and application thereof

Abstract

This invention belongs to the field of microbial technology and relates to an anti-infective probiotic agent containing the CT-02 strain, its preparation method, and its application. The strains in the agent include *Streptococcus thermophilus* CT-02 strain (GDMCC No: 64474) and *Lactococcus lactis* subsp. lactis 220007 strain (CGMCC No. 16583). The probiotic agent prepared by this invention can regulate the intestinal barrier and alleviate intestinal infectious inflammation. Furthermore, the *Streptococcus thermophilus* CT-02 strain and the *Lactococcus lactis* subsp. lactis 220007 strain can synergistically enhance the above-mentioned effects.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to an anti-infective probiotic agent containing CT-02 strain, its preparation method, and its application. Background Technology

[0002] The interaction between the gut microbiota and host health is a current research hotspot in the life sciences. Probiotics play a key role by regulating gut immune homeostasis, maintaining epithelial barrier function, and influencing stem cell activity. While *Streptococcus thermophilus*, a widely used probiotic strain, has been shown to have potential in improving lactose metabolism and inhibiting pathogen colonization, its multidimensional regulatory mechanisms on the gut microenvironment, particularly its comprehensive effects on immune response, gut dryness maintenance, and barrier function, remain unclear.

[0003] The expression levels of intestinal stem cell-related genes (such as key molecules in the WNT pathway, Lgr5, and olfm4) directly determine the self-renewal and differentiation capacity of stem cells, and abnormalities may lead to impaired intestinal epithelial regeneration or increased risk of cancer. Changes in the expression of intestinal barrier function genes (such as the antimicrobial peptide Lyz, the mucin muc2, and the tight junction protein occludin) can reflect the integrity of the epithelial barrier.

[0004] Probiotics, as biological agents that regulate the balance of the human gut microbiota, are used to improve gut health and enhance immunity due to their high safety and few side effects. Selecting specific types of probiotics and developing probiotic preparations that strengthen the intestinal barrier and alleviate intestinal infectious inflammation is of great significance. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of this invention is to provide an anti-infective probiotic agent containing CT-02 strain, its preparation method and application.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention relates to an anti-infective probiotic agent containing strain CT-02, wherein the strains in the agent include Streptococcus thermophilus CT-02 strain with accession number GDMCC No:64474 and Lactococcus lactis subsp.lactis220007 strain with accession number CGMCC No.16583.

[0008] The probiotic preparation obtained by this invention can regulate the intestinal barrier and alleviate intestinal infectious inflammation. Furthermore, the *Streptococcus thermophilus* CT-02 strain and *Lactococcus lactis* subsp. *lactococcus* 220007 strain exhibit synergistic effects in achieving the aforementioned efficacy. Specifically, when using the same bacterial quantity, the combination of the two strains significantly enhances the above-mentioned efficacy compared to using either the CT-02 strain or the 220007 strain alone. Since both the CT-02 and 220007 strains are probiotics, they are highly safe and unlikely to induce resistance when used in the preparation of related products.

[0009] Preferably, the ratio of the number of *Streptococcus thermophilus* CT-02 strain and *Lactococcus lactis* subsp. lactis 220007 strain is (1-10):(1-10). Specific values ​​in (1-10) can be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. Other specific values ​​within the above range can also be selected, and will not be elaborated here.

[0010] Preferably, the probiotic agent has a bacterial count of not less than 1×10⁻⁶. 6 cells / mL or 1×10 6 pcs / g, for example 1×10 6 cells / mL, 2×10 6 cells / mL, 3×10 6 cells / mL, 4×10 6 cells / mL, 5×10 6 cells / mL, 6×10 6 cells / mL, 7×10 6 cells / mL, 8×10 6 cells / mL, 9×10 6 cells / mL, 1×10 7 cells / mL, 5×10 7 cells / mL, 1×10 8 cells / mL, 1×10 9 cells / mL, 1×10 10 cells / mL, or 1×10 6 Units / g, 2×10 6 pcs / g, 3×10 6 pcs / g, 4×10 6 pcs / g, 5×10 6 pcs / g, 6×10 6 pcs / g, 7×10 6 pcs / g, 8×10 6 pcs / g, 9×10 6Units / g, 1×10 7 pcs / g, 5×10 7 Units / g, 1×10 8 Units / g, 1×10 9 Units / g, 1×10 10 The values ​​for each item / g, etc., can be selected from other specific point values ​​within the above range, which will not be elaborated here.

[0011] Preferably, the dosage form of the probiotic agent includes solution, lyophilized powder, capsule, tablet or granule.

[0012] Preferably, the probiotic agent further includes acceptable excipients.

[0013] Preferably, the acceptable excipients include protective agents.

[0014] In a second aspect, the present invention provides a method for preparing an anti-infective probiotic agent containing strain CT-02 according to the first aspect, the preparation method comprising: activating the strains separately and then fermenting them to obtain a fermentation broth, mixing the fermentation broth with a protectant, freeze-drying, and mixing the freeze-dried bacterial powder to obtain the final product.

[0015] Thirdly, the present invention provides the use of the anti-infective probiotic agent containing the CT-02 strain according to the first aspect in the preparation of a medicine for preventing or alleviating intestinal infectious inflammation.

[0016] Fourthly, the present invention provides the use of the anti-infective probiotic agent containing the CT-02 strain according to the first aspect in the preparation of a medicine that enhances the intestinal barrier.

[0017] Fifthly, the present invention provides the use of the anti-infective probiotic agent containing the CT-02 strain according to the first aspect in the preparation of a medicament for preventing or alleviating pulmonary infectious inflammation.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The probiotic preparation obtained by this invention can regulate the intestinal barrier and alleviate intestinal infectious inflammation. Furthermore, the *Streptococcus thermophilus* CT-02 strain and *Lactococcus lactis* subsp. *lactococcus* 220007 strain exhibit synergistic effects in achieving the aforementioned efficacy. Specifically, when using the same bacterial quantity, the combination of the two strains significantly enhances the above-mentioned efficacy compared to using either the CT-02 strain or the 220007 strain alone. Since both the CT-02 and 220007 strains are probiotics, they are highly safe and unlikely to induce resistance when used in the preparation of related products. Attached Figure Description

[0020] Figure 1This is a graph showing the relative expression levels of genes related to gut stemness. Graph A represents the Lgr5 gene, Graph B represents the wnt gene, and Graph C represents the olfm gene.

[0021] Figure 2 This is a graph showing the relative expression levels of genes related to the intestinal barrier. Graph A represents the Lyz gene, Graph B represents the ZO-1 gene, Graph C represents the occludin gene, Graph D represents the muc2 gene, and Graph E represents the KI67 gene.

[0022] Figure 3 This is a graph showing the relative expression levels of ileal cytokines, where A represents IL-10, B represents IL-17a, C represents TNF-α, and D represents IL-6.

[0023] Figure 4 This is a graph showing the relative expression levels of the ileal barrier protein ZO-1.

[0024] Figure 5 These are the results of short-chain fatty acid analysis of intestinal metabolites. Figure A shows the butyric acid content, Figure B shows the isovaleric acid content, and Figure C shows the isobutyric acid content.

[0025] Figure 6 This is a graph showing the relative expression levels of lung cytokines, where graph A represents IL-10 and graph B represents IL-17a. Detailed Implementation

[0026] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0027] The Streptococcus thermophilus strain CT-02 mentioned below is classified as Streptococcus thermophilus, with accession number GDMCC No: 64474, accession date March 29, 2024, and deposited at Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0028] The strain Lactococcus lactis subsp. lactis220007 mentioned below is classified as Lactococcus lactis subsp. lactis, with accession number CGMCC No.16583, accession date October 15, 2018, and deposited at the China General Microbiological Culture Collection Center, located at No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0029] Example 1

[0030] This embodiment provides an anti-infective bacterial suspension, which includes Streptococcus thermophilus CT-02 strain and Lactococcus lactis subsp. lactis 220007 strain with a live bacteria ratio of 10:1.

[0031] The preparation method is as follows: the strains were inoculated into MRS liquid medium and activated by culturing at 37°C for 24 h. The activation was repeated twice to obtain the activated solution. The activated solution was inoculated into MRS liquid medium at an inoculation rate of 4% (v / v) and cultured at 37°C for 30 h to obtain the bacterial suspension. The bacterial suspension was centrifuged at 4°C and 5000 rpm for 10 min, filtered, and the bacterial cells were obtained. The bacterial cells were resuspended in PBS solution. The suspension of Streptococcus thermophilus CT-02 and the suspension of Lactococcus lactis subsp. lactis 220007 were mixed to obtain the final product.

[0032] Example 2

[0033] This embodiment provides an anti-infective bacterial suspension, which includes Streptococcus thermophilus CT-02 strain and Lactococcus lactis subsp. lactis 220007 strain with a live bacteria ratio of 1:1.

[0034] The preparation method is the same as in Example 1.

[0035] Example 3

[0036] This embodiment provides an anti-infective bacterial suspension, which includes Streptococcus thermophilus CT-02 strain and Lactococcus lactis subsp. lactis 220007 strain with a live bacteria ratio of 1:10.

[0037] The preparation method is the same as in Example 1.

[0038] Comparative Example 1

[0039] This comparative example provides an anti-infective bacterial suspension, which includes Streptococcus thermophilus CT-02 strain.

[0040] The preparation method is the same as in Example 1.

[0041] Comparative Example 2

[0042] This comparative example provides an anti-infective bacterial suspension, which includes Lactococcus lactis subsp. lactis strain 220007.

[0043] The preparation method is the same as in Example 1.

[0044] Comparative Example 3

[0045] This comparative example provides an anti-infective bacterial suspension, which differs from Example 1 only in that the viable bacteria such as Streptococcus thermophilus CT-02 strain are replaced with Streptococcus thermophilus ATCC19258 strain.

[0046] The preparation method is the same as in Example 1.

[0047] Comparative Example 4

[0048] This comparative example provides an anti-infective bacterial suspension, which differs from Example 1 only in that the viable count of Lactococcus lactis subsp. lactis 220007 strain is replaced with Lactococcus lactis subsp. lactis ATCC19435 strain.

[0049] The preparation method is the same as in Example 1.

[0050] Test Example 1

[0051] Test method:

[0052] Primary small intestinal cells were extracted from 6-week-old male C57 mice. After being mixed 1:1 with culture medium and matrix gel, 50 μL of the mixture was seeded into 24-well plates, with 100-200 small intestinal organoids per well. Once solidified, 500 mL of culture medium was added, and the cells were incubated at 37 ℃ in a 5% CO2 incubator. On the second day of culture, the culture medium was removed and replaced with 750 mL of fresh medium. Cells were passaged on the fifth day, and intervention could be initiated after the third passage.

[0053] Organoids in good growth condition were digested and passaged. After being mixed 1:1 with culture medium and matrix gel, 50 μL of the mixture was inoculated into 24-well plates, with 100-200 small intestinal organoids per well. Once solidified, 750 mL of culture medium was added, and the plates were incubated at 37 ℃ in a 5% CO2 incubator until the organoids began to bud (day 3). The culture medium was then removed, and 500 mL of fresh culture medium and 50 μL of bacterial suspension (total viable count 1×10⁻⁶) were added. 6 CFU), after intervention until day 5, total RNA was isolated and cDNA first strand was synthesized according to the reverse transcription kit instructions. qPCR was performed using β-actin and GADPH as internal reference genes to measure the relative expression levels of intestinal stemness-related genes and intestinal barrier-related genes, analyzing the probiotic strain's ability to regulate the intestinal barrier. The relative expression levels of intestinal stemness-related genes are shown below. Figure 1 As shown, the relative expression levels of intestinal barrier-related genes are as follows: Figure 2 As shown in the figure. The blank group was given an equal volume of culture medium, and the LPS group was given 50 μg of LPS.

[0054] Depend on Figure 1 and Figure 2Data shows that the bacterial agent of this invention can increase the expression levels of intestinal stem genes and intestinal barrier-related genes. Furthermore, the Streptococcus thermophilus CT-02 strain and Lactococcus lactis subsp. lactis 220007 strain have a certain synergistic effect in increasing the expression levels of intestinal barrier-related genes, indicating that the bacterial agent can be applied to products that enhance the intestinal barrier.

[0055] Test Example 2

[0056] Test methods: Six-week-old C57BL / 6 mice were selected for in vivo experiments and randomly divided into groups (blank control group, model group, positive drug group, Example 1-3 groups, and Comparative Example 1-4 groups), with 10 mice in each group. After 7 days of acclimatization feeding, the mice were weighed and their feces were collected. From day 8 to day 28, each mouse in the experimental group was administered 200 μL of bacterial suspension (1×10⁻⁶) by gavage daily. 9 The model group, positive drug group, and blank control group were administered the bacterial suspension (CFU / mL) by gavage with an equal volume of physiological saline. From day 26 to day 28, each mouse in the positive drug group was injected intraperitoneally with 3 mg / kg TAK-242 daily. On day 29, except for the blank control group, the mice in the other groups were injected intraperitoneally with 8 mg / kg LPS to induce infectious inflammation. The mice's feces were collected one day before modeling (day 28) and after modeling (day 29). The mice were sacrificed 6 hours after modeling and ileal samples were collected.

[0057] Mouse ileum tissue was extracted using an RNA extraction kit. The purified RNA was then subjected to real-time quantitative polymerase chain reaction (RT-qPCR). The expression levels of cytokines (IL-10, IL-17a, TNF-α, IL-6) and the barrier protein (ZO-1) were calculated using β-actin as an internal reference gene. The results are as follows: Figure 3-4 As shown.

[0058] Depend on Figure 3 It is known that the compound bacterial agent and the CT-02 strain of the present invention can both promote the secretion of multiple inflammation-related cytokines such as IL-10, IL-17a, TNF-α and IL-6 in ileal tissue, and exert anti-infective inflammatory effects.

[0059] Depend on Figure 4 It is known that the compound bacterial agent and CT-02 strain involved in this invention can increase the content of tight junction protein ZO-1, and there is a certain synergistic effect between the strains, which indicates that the bacterial agent and CT-02 have the potential to improve the intestinal barrier.

[0060] The feces collected from the animal experiments were analyzed for short-chain fatty acids, which are intestinal metabolites. The results are as follows: Figure 5 As shown.

[0061] from Figure 5The results showed that the bacterial agent of the present invention can regulate the content of fatty acids in the intestine. Both the compound bacterial agent and the intervention of CT-02 strain can significantly promote the secretion of short-chain fatty acids, and there is a certain synergistic effect between the strains.

[0062] In summary, the probiotic agent and strain CT-02 described in this invention can improve intestinal inflammation and intestinal barrier function by upregulating the expression of key inflammatory cytokines and tight junction proteins. The results of intestinal stemness and tight junction protein-related indicators such as Wnt in the in vitro experiment of Test Example 1 also confirm the effects of the probiotic agent and CT-02 in alleviating intestinal inflammation and promoting the repair and regeneration of intestinal epithelial cells.

[0063] Mouse lung tissue was extracted using an RNA extraction kit. The purified RNA was then subjected to real-time quantitative polymerase chain reaction (RT-qPCR). The expression levels of cytokines (IL-10 and IL-17a) were calculated using β-actin as an internal reference gene. The results are as follows: Figure 6 As shown.

[0064] The bacterial agent and CT-02 of this invention can increase the relative expression level of the anti-inflammatory factor IL-10 and decrease the relative expression level of the pro-inflammatory factor IL-17a, indicating that while improving intestinal inflammation and the intestinal barrier, the bacterial agent can further improve the pulmonary inflammation status, and there is a certain synergistic effect among the bacterial agents. This also shows that the bacterial agent of this invention has the potential to improve pulmonary inflammatory infections.

[0065] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. An anti-infective probiotic agent containing strain CT-02, characterized in that, The bacterial strains in the bacterial agent consist of Streptococcus thermophilus CT-02 strain with accession number GDMCC No:64474 and Lactococcus lactis subsp. lactis 220007 strain with accession number CGMCC No.16583. The ratio of the number of *Streptococcus thermophilus* strain CT-02 to the number of *Lactococcus lactis* subsp. lactis 220007 strain is (1-10):(1-10).

2. The anti-infective probiotic agent containing CT-02 strain according to claim 1, characterized in that, The number of bacteria in the probiotic agent is not less than 1 x 10 6 / mL or 1 x 10 6 / g.

3. The anti-infective probiotic agent containing CT-02 strain according to claim 1, characterized in that, The dosage forms of the probiotic agent include solutions, lyophilized powders, capsules, tablets, or granules.

4. The anti-infective probiotic agent containing CT-02 strain according to claim 1, characterized in that, The probiotic agent also includes acceptable excipients.

5. The anti-infective probiotic agent containing CT-02 strain according to claim 4, characterized in that, The acceptable excipients include protective agents.

6. The method for preparing the anti-infective probiotic agent containing strain CT-02 according to any one of claims 1-5, characterized in that, The preparation method includes: activating the bacterial strains separately and then fermenting them to obtain a fermentation broth; mixing the fermentation broth with a protective agent; freeze-drying the mixture; and mixing the freeze-dried bacterial powder to obtain the final product. The strains mentioned are Streptococcus thermophilus CT-02 strain with accession number GDMCC No:64474 and Lactococcus lactis subsp. lactis 220007 strain with accession number CGMCC No.16583.

7. The use of the anti-infective probiotic agent containing the CT-02 strain according to any one of claims 1-5 in the preparation of a medicine for enhancing the intestinal barrier.

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