Streptococcus thermophilus CT-02 with immunoregulation effect and application thereof

Abstract

The invention relates to streptococcus thermophilus CT-02 with an immune regulation effect and application of the streptococcus thermophilus CT-02, the classification name of the streptococcus thermophilus CT-02 with the immune regulation effect is Streptococcus thermophilus, the preservation number of the streptococcus thermophilus CT-02 is GDMCC No: 64474, and the preservation date of the streptococcus thermophilus CT-02 is March 29, 2024. The strain has an excellent immune regulation effect which is specifically shown as follows: by moderately activating macrophages, not only can the excessive activation of TNF-alpha, IL-1beta, IL-6 and IL-12 be avoided, but also the activation level of IL-10 can be improved, that is, the levels of various inflammatory factors secreted by the macrophages are comprehensively regulated, and the strain has a potential anti-inflammatory effect while activating immunity. The strain has a prospect for preparing a product with an immune regulation effect or a product with an inflammatory factor regulation effect.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotics and relates to Streptococcus thermophilus CT-02 with immunomodulatory effect and application thereof. Background Art

[0002] Immunomodulation is the key to maintaining the stability of the body's internal environment. If the immune regulation function is abnormal, a strong immune attack will be generated on the body's own components, causing cell damage and loss of function, and autoimmune diseases will occur. If there is no appropriate response to external pathogenic microorganisms (too low a response can cause serious infection, and too strong a response can cause allergic reactions), it can also cause harmful effects on the body.

[0003] Macrophages originate from blood monocytes and can non-specifically phagocytize bacteria, foreign bodies, etc., participate in natural anti-infection and anti-tumor processes, and are an important part of the natural immune system. The specific role of macrophages in the immune process is as follows: 1) In the initial stage of immunity, macrophages can act as antigen-presenting cells to absorb and process antigens and present them to helper T cells to initiate specific immune responses; 2) In the immune response stage, activated macrophages chemotaxis to the vicinity of the lesion, more effectively phagocytize bacteria and kill target cells; at the same time, they secrete a variety of active substances, such as lysozyme, complement, cytokines, etc., to exert corresponding biological functions.

[0004] At present, there are two main methods used in clinical practice to regulate immunity: one is immunomodulatory drugs, which enhance or inhibit the body's immune function by affecting the body's immune response and immunopathological reaction; the other is probiotic products, which can be used as microbial preparations to regulate the body's immunity. Strains have strain specificity for regulating immunity, and different strains have differences in their ability to regulate immunity. Moreover, there are relatively few probiotic strategies that can effectively regulate immunity. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide a Streptococcus thermophilus CT-02 with immunomodulatory efficacy and its application.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a thermophilic Streptococcus CT-02 having an immunomodulatory effect, wherein the thermophilic Streptococcus CT-02 having an immunomodulatory effect is classified as Streptococcus thermophilus , the deposit number is GDMCC No: 64474, and the deposit date is March 29, 2024.

[0007] The present invention separates and preserves a new strain of thermophilic streptococcus CT-02 with immunomodulatory effect from a traditional fermented yogurt sample. The strain is sequenced and analyzed, and its 16S rDNA sequence is shown in SEQ ID No: 1. The sequence obtained by sequencing is compared with the nucleic acid sequence in GeneBank, and the result shows that the strain is thermophilic streptococcus. The bacteria has excellent immunomodulatory effect, which is specifically manifested as: moderate activation of macrophages, which can avoid excessive activation of TNF-α, IL-1β, IL-6, and IL-12, and increase the activation level of IL-10, that is, comprehensive regulation of the levels of various inflammatory factors secreted by macrophages, while activating immunity, has potential anti-inflammatory effects. It is promising to use the strain to prepare products with immunomodulatory effect or products with inflammatory factor regulation effect.

[0008] In a second aspect, the present invention provides the use of the thermophilic Streptococcus CT-02 or the inactivated bacteria thereof described in the first aspect in the preparation of a product having immunomodulatory or auxiliary immunomodulatory efficacy.

[0009] Preferably, the number of thermophilic Streptococcus CT-02 or its inactivated bacteria in the product is not less than 1×10 4 / mL or 1×10 4 Pieces / g, for example 1×10 4 pieces / g (pieces / mL), 5×10 4 pieces / g (pieces / mL), 1×10 5 pieces / g (pieces / mL), 2.5×10 5 pieces / g (pieces / mL), 1×10 6 pieces / g (pieces / mL), 5×10 6 pieces / g (pieces / mL), 1×10 7 pieces / g (pieces / mL), 5×10 7 pieces / g (pieces / mL), 1×10 8 pieces / g (pieces / mL), 3×10 8 pieces / g (pieces / mL), 5×10 8 pieces / g (pieces / mL), 1×10 9 Pieces / g (pieces / mL), etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0010] Preferably, the product includes food, health care products, medicines, cosmetics or daily chemical products.

[0011] In the above products, Streptococcus thermophilus CT-02 can exist in either a live bacterial state or an inactivated state.

[0012] In a third aspect, the present invention provides a use of the thermophilic Streptococcus CT-02 or an inactivated bacterium thereof described in the first aspect in the preparation of a preparation having anti-inflammatory or auxiliary anti-inflammatory effects.

[0013] Preferably, the number of Streptococcus thermophilus CT-02 in the product is not less than 1×10 4 / mL or 1×10 4 Pieces / g, for example 1×10 4 pieces / g (pieces / mL), 5×10 4 pieces / g (pieces / mL), 1×10 5 pieces / g (pieces / mL), 2.5×10 5 pieces / g (pieces / mL), 1×10 6 pieces / g (pieces / mL), 5×10 6 pieces / g (pieces / mL), 1×10 7 pieces / g (pieces / mL), 5×10 7 pieces / g (pieces / mL), 1×10 8 pieces / g (pieces / mL), 3×10 8 pieces / g (pieces / mL), 5×10 8 pieces / g (pieces / mL), 1×10 9 Pieces / g (pieces / mL), etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0014] In the above products, Streptococcus thermophilus CT-02 can exist in either a live bacterial state or an inactivated state.

[0015] In a fourth aspect, the present invention provides a microbial preparation, wherein the strain in the microbial preparation includes the thermophilic Streptococcus CT-02 or an inactivated strain thereof as described in the first aspect.

[0016] Preferably, the strain in the microbial preparation also includes animal Bifidobacterium lactis subspecies C-2 or its inactivated bacteria, and the classification of the animal Bifidobacterium lactis subspecies C-2 is named Bifidobacterium animalis subsp. lactis , the deposit number is GDMCC No: 65455, and the deposit date is November 8, 2024.

[0017] The present invention also creatively discovered that the above-mentioned thermophilic Streptococcus CT-02 strain can be used in combination with the animal Bifidobacterium lactis subspecies C-2 strain for regulating immunity and inflammatory factors, and also has excellent effects. Under the premise of consistent bacterial intervention amounts, compared with single strain intervention, the composite strain intervention method has a more significant effect of activating immunity while having potential anti-inflammatory effects, indicating that the CT-02 strain and the C-2 strain have potential synergistic effects in the above-mentioned effects.

[0018] Preferably, the ratio of the number of thermophilic Streptococcus CT-02 to that of animal Bifidobacterium lactis subspecies C-2 is (5-10):1, for example 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, etc. Other specific point values ​​within this numerical range can be selected and will not be described one by one here.

[0019] Based on the potential interaction between the two strains in regulating immune and inflammatory factors, the effect is better when the two meet the above-mentioned specific ratio relationship.

[0020] Preferably, the total bacterial count in the microbial preparation is not less than 1×10 4 / mL or 1×10 4 Pieces / g, for example 1×10 4 pieces / g (pieces / mL), 5×10 4 pieces / g (pieces / mL), 1×10 5 pieces / g (pieces / mL), 2.5×10 5 pieces / g (pieces / mL), 1×10 6 pieces / g (pieces / mL), 5×10 6 pieces / g (pieces / mL), 1×10 7 pieces / g (pieces / mL), 5×10 7 pieces / g (pieces / mL), 1×10 8 pieces / g (pieces / mL), 3×10 8 pieces / g (pieces / mL), 5×10 8 pieces / g (pieces / mL), 1×10 9 Pieces / g (pieces / mL), etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0021] The dosage form of the microbial preparation involved in the present invention is not limited, including the most commonly used solutions, powders, or further prepared capsules, tablets or granules.

[0022] Preferably, the microbial preparation also contains bacteria protective agents and / or nutritional components.

[0023] In a fifth aspect, the present invention provides use of the microbial preparation described in the fourth aspect in the preparation of a product having immunomodulatory or auxiliary immunomodulatory efficacy.

[0024] Preferably, the product includes food, health care products, medicines, cosmetics or daily chemical products.

[0025] In a sixth aspect, the present invention provides use of the microbial preparation described in the fourth aspect in the preparation of a preparation having anti-inflammatory or auxiliary anti-inflammatory effects.

[0026] Compared with the prior art, the present invention has the following beneficial effects: The present invention separates and preserves a new strain of thermophilic streptococcus CT-02 with immunomodulatory effect from a traditional fermented yogurt sample. The strain is sequenced and analyzed, and its 16S rDNA sequence is shown in SEQ ID No: 1. The sequence obtained by sequencing is compared with the nucleic acid sequence in GeneBank, and the result shows that the strain is thermophilic streptococcus. The bacteria has excellent immunomodulatory effect, which is specifically manifested as: moderate activation of macrophages, which can avoid excessive activation of TNF-α, IL-1β, IL-6, and IL-12, and increase the activation level of IL-10, that is, comprehensive regulation of the levels of various inflammatory factors secreted by macrophages, while activating immunity, has potential anti-inflammatory effects. It is promising to use the strain to prepare products with immunomodulatory effect or products with inflammatory factor regulation effect.

[0027] The classification of the CT-02 strain involved in the present invention is named Streptococcus thermophilus The preservation unit is Guangdong Provincial Microbiological Culture Collection Center, the preservation time is March 29, 2024, the preservation number is GDMCC No: 64474, and the address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0028] The classification of the C-2 strain involved in the present invention is named Bifidobacterium animalis subsp. lactis The preservation unit is Guangdong Provincial Microbiological Culture Collection Center, the preservation time is November 8, 2024, the preservation number is GDMCC No: 65455, and the address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a microscopic image of the Streptococcus thermophilus CT-02 strain; Figure 2 This is the appearance of the colony of Streptococcus thermophilus CT-02 strain; Figure 3 This is a statistical result diagram of the effects of different experimental groups on the secretion of TNF-α by macrophages; Figure 4 This is a statistical result diagram of the effects of different experimental groups on the secretion of IL-1β by macrophages; Figure 5 This is a statistical result diagram of the effects of different experimental groups on the secretion of IL-6 by macrophages; Figure 6 This is a statistical result diagram of the effects of different experimental groups on the secretion of IL-12 by macrophages; Figure 7 This is a statistical graph showing the effects of different experimental groups on the secretion of IL-10 by macrophages. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0031] Test materials and instruments: The taxonomic designation of the CT-02 strain involved is Streptococcus thermophilus , deposited on March 29, 2024, with a deposit number of GDMCC No: 64474; the classification of the C-2 strain involved is named Bifidobacterium animalis subsp. lactis , deposited on November 8, 2024, with a deposit number of GDMCC No: 65455. The other strains involved are all commercial strains.

[0032] Mouse mononuclear macrophage RAW 264.7 was provided by the laboratory of West China School of Public Health, Sichuan University. They were cultured in RPMI 1640 complete medium containing 88% RPMI 1640 medium, 10% heat-inactivated fetal bovine serum (FBS), and 2% penicillin-streptomycin solution at 37°C in a cell culture incubator environment with 5% CO. Cell monolayers were passaged when they were 80% confluent.

[0033] M17 medium (Beijing Luqiao Technology Co., Ltd.), RPMI 1640 medium (Thermo Fisher Scientific Instrument Co., Ltd.), PBS pH 7.2 buffer (Guangzhou Saiku Biotechnology Co., Ltd.), LPS working solution (L4130, Sigma), total cell RNA isolation kit (Nanjing Novozyme Biotechnology Co., Ltd.), iScript TM cDNA synthesis kit (1708890, Bio-Rad, USA), SsoFast™ EvaGreen® supermix (1725200, Bio-Rad, USA), ELISA kit (Thermo Fisher Scientific). Example 1

[0034] Isolation and screening of thermophilic Streptococcus CT-02: A certain amount of traditional fermented yogurt sample was placed in physiological saline with a mass concentration of 0.85% for ten-fold dilution. 100 μL of sample was taken from the three dilution gradients of -4, -5, and -6 and spread on M17 agar medium. The sample was placed in an incubator at 37°C and inverted for 48 h. Colonies with obvious morphological differences were selected on the plate of the -5 dilution gradient and streaked to obtain pure single colonies. A strain with good growth status was isolated and named CT-02. Example 2

[0035] Identification of Streptococcus thermophilus CT-02: (1) Observe the selected CT-02 strain under a microscope. Figure 1 As shown, the strain has a spherical shape, is arranged in short chains, and is Gram-positive.

[0036] (2) The appearance of the CT-02 colonies on the plate is as follows: Figure 2 As shown, the colonies are round, with a smooth surface, smooth edges, milky white and opaque.

[0037] (3) 16S rDNA molecular biological identification: The screened CT-02 strain was inoculated into a centrifuge tube containing 20 mL of M17 liquid culture medium at a ratio of 2%, and then centrifuged at 37°C for 18 hours, centrifuged at 5000rpm for 10 minutes, and the supernatant was removed to collect the bacteria. The genome of the strain was extracted, and bacterial universal primers were added for PCR amplification, and the amplified product was sequenced and identified. The strain was sequenced and analyzed, and its 16S rDNA sequence was shown in SEQ ID No: 1. The sequence obtained by sequencing was compared with the nucleic acid sequence in GeneBank, and the results showed that the strain was Streptococcus thermophilus.

[0038] SEQ ID No: 1: Example 3

[0039] Cellular immune regulation test: (1) Experimental groups: The experiment was divided into a blank control group (not treated with probiotic solution), CT-02 group 1 (intervention with low-concentration CT-02 bacterial solution), CT-02 group 2 (intervention with high-concentration CT-02 bacterial solution), C-2 group (C-2 bacterial solution), CT-02 + C-2 group 1 (combined intervention with the two bacteria, with a bacterial count ratio of 5:1), CT-02 + C-2 group 2 (combined intervention with the two bacteria, with a bacterial count ratio of 10:1), commercially available ST group 1 (intervention with low-concentration bacterial solution of Streptococcus thermophilus ATCC14485), commercially available ST group 2 (intervention with high-concentration bacterial solution of Streptococcus thermophilus ATCC14485), and commercially available ST + commercially available BB group (commercially available Streptococcus thermophilus ATCC14485 and commercially available Bifidobacterium animalis CICC24932 were combined for intervention, with a bacterial count ratio of 5:1).

[0040] (2) Preparation of test strain suspension: Each test strain was subcultured once every 48 hours on a sterile workbench, and a single strain was cultured on a new M17 medium plate for a total of 3 subcultures. An appropriate amount of colonies cultured on the surface of the M17 medium were collected, placed in 4 mL of sterile saline, and fully mixed using a vortex mixer. According to the standard curve between the number of units of bacteria and absorbance, the bacterial density in the suspension was gradually adjusted, and then centrifuged at 4°C and 12000g for 5 minutes, the supernatant was discarded, and the sediment was dissolved with 4 mL of sterile saline, and the centrifugation steps under the same conditions were repeated. After discarding the supernatant, RPMI1640 culture medium was added to resuspend and dilute the bacteria to obtain a bacterial suspension of the test strain.

[0041] (3) Cellular intervention methods: Raw264.7 mouse macrophages in normal growth state were collected to prepare cell suspension, and the concentration was accurately calculated to reach 50,000 cells per milliliter. 1 mL of the cell solution was extracted and placed on a 24-well experimental plate, which was then placed in a constant temperature incubator at 37°C and 5% carbon dioxide for 2 h to allow the cells to adhere to the wall. After incubation for 2 h, each strain intervention group continued to add RPMI1640 culture medium with different bacterial suspensions prepared in step (2) (except for CT-02 group 2 and commercially available ST group 2, the final total bacterial concentration in the culture system was 2.5×10 5 CFU / mL, the final total bacterial concentration in the culture system of the other groups was 5×10 4 CFU / mL), and the blank control group was added with an equal amount of RPMI1640 medium. Five parallel groups were set up for each group, and the final results were averaged.

[0042] (4) Index detection methods and results: After 24 hours of incubation, the supernatant in the culture solution was removed and then centrifuged at 12,000 rpm for 5 minutes at 4° C. Afterwards, the extracted supernatant was transferred to a 1.5 ml centrifuge tube and placed at 4° C. for subsequent use.

[0043] The RT-qPCR method was used to detect the mRNA expression level of cytokines in the supernatant of each group. First, RNA was extracted from each group. All steps were carried out according to the instructions of the cell total RNA extraction kit of Chengdu Fuji Biotechnology Co., Ltd. Then, RNA was reverse transcribed, and the cDNA obtained by reverse transcription was frozen in a -80℃ refrigerator for standby use. Then, qPCR of cDNA was performed. Finally, the relative expression of the target gene mRNA was measured using β-actin as the internal reference gene and 2 －△△Ct The mRNA expression levels of five immune factors, TNF-α, IL-1β, IL-6, IL-10, and IL-12, were calculated by the method. The results are as follows Figure 3-Figure 7 shown.

[0044] Depend on Figure 3-Figure 7 It can be seen from the data results that compared with other thermophilic Streptococcus strains, the thermophilic Streptococcus CT-02 strain involved in the present invention has a better comprehensive immunomodulatory effect, whether under low concentration conditions or high concentration conditions, and can activate macrophages to secrete inflammatory factors to exert an immune activation effect, while avoiding the overactivation of pro-inflammatory factors TNF-α, IL-1β, IL-6, and IL-12 and increasing the level of anti-inflammatory factor IL-10, that is, it exerts anti-inflammatory effects while exerting immune activation effects.

[0045] In addition, compared with the use of a single CT-02 strain or C-2 strain, the combined use of thermophilic Streptococcus CT-02 and animal Bifidobacterium lactis subspecies C-2 further improved the comprehensive immunomodulatory efficacy in the above aspects, indicating that the CT-02 strain and the C-2 strain have potential synergistic effects in the above effects.

[0046] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0047] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0048] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A thermophilic Streptococcus CT-02 having immunomodulatory effect, characterized in that: The classification of the thermophilic Streptococcus CT-02 with immunomodulatory efficacy is named Streptococcus thermophilus , the deposit number is GDMCC No:64474, and the deposit date is March 29, 2024.

2. Use of the thermophilic Streptococcus CT-02 or its inactivated bacteria according to claim 1 in the preparation of products with immunomodulatory or auxiliary immunomodulatory effects; The number of thermophilic Streptococcus CT-02 or its inactivated bacteria in the product is not less than 1×10 4 / mL or 1×10 4 pcs / g; The products include food, health products, medicines, cosmetics or daily chemical products.

3. Use of the thermophilic Streptococcus CT-02 or its inactivated bacteria according to claim 1 in the preparation of a preparation having anti-inflammatory or auxiliary anti-inflammatory effects.

4. A microbial preparation, characterized in that: The strain in the microbial preparation includes the thermophilic Streptococcus CT-02 or an inactivated strain thereof according to claim 1.

5. The microbial preparation according to claim 4, characterized in that The strains in the microbial preparation also include animal Bifidobacterium lactis subspecies C-2 or its inactivated bacteria, and the classification of the animal Bifidobacterium lactis subspecies C-2 is named Bifidobacterium animalis subsp. lactis , the deposit number is GDMCC No: 65455, and the deposit date is November 8, 2024.

6. The microbial preparation according to claim 5, characterized in that The ratio of the number of thermophilic Streptococcus CT-02 to the number of animal Bifidobacterium lactis subspecies C-2 is (5-10):

1.

7. The microbial preparation according to claim 4, characterized in that The total bacterial count in the microbial preparation is not less than 1×10 4 / mL or 1×10 4 Pieces / g.

8. The microbial preparation according to claim 4, characterized in that The dosage form of the microbial preparation includes solution or powder; The microbial preparation also contains a bacterial protective agent and / or a nutritional component.

9. Use of the microbial preparation according to any one of claims 4 to 8 in the preparation of a product having immunomodulatory or auxiliary immunomodulatory efficacy; The products include food, health products, medicines, cosmetics or daily chemical products.

10. Use of the microbial preparation according to any one of claims 4 to 8 in the preparation of a preparation having anti-inflammatory or auxiliary anti-inflammatory effects.

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