Streptococcus thermophilus CT-01 and application thereof in preparation of immunomodulatory preparation

Abstract

The invention relates to a streptococcus thermophilus CT-01 and application of the streptococcus thermophilus CT-01 in preparation of an immunomodulatory preparation, the classification name of the streptococcus thermophilus CT-01 is Streptococcus thermophilus, the preservation number of the streptococcus thermophilus CT-01 is GDMCC No: 64473, and the preservation date of the streptococcus thermophilus CT-01 is March 29, 2024. The bacterium has an excellent immune regulation effect which is specifically shown as follows: comprehensive regulation is performed on the behavior of secreting inflammatory factors by macrophages, excessive activation of TNF-alpha, IL-1beta and IL-6 can be avoided, the level of IL-10 can be improved, and the bacterium has a potential anti-inflammatory effect while regulating 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 microorganisms and relates to thermophilic streptococcus CT-01 and application thereof in preparing an immunomodulatory preparation. Background Art

[0002] Immunomodulation requires appropriate immune stimulation. Immunosuppression and excessive immune stimulation both have negative effects on the body. Streptococcus thermophilus can regulate immunity in the following ways: Activate the immune system: Streptococcus thermophilus can activate the body's immune system and improve the body's defense against pathogens by promoting the proliferation and differentiation of immune cells. This activation helps to enhance the body's disease resistance, thereby playing an auxiliary role in resisting infection and disease.

[0003] Improve intestinal microenvironment: Thermophilic Streptococcus can regulate the balance of intestinal flora, inhibit the growth and reproduction of harmful bacteria, and thus improve intestinal health. A good intestinal environment is the basis for the normal functioning of the immune system, so thermophilic Streptococcus indirectly promotes the improvement of immunity by improving the intestinal microenvironment.

[0004] Promote wound healing: Thermophilic Streptococcus can also help wound healing and promote the growth and repair of new granulation tissue, which to a certain extent also reflects its positive effect on the immune system. Because the wound healing process requires the participation of the immune system to remove damaged tissue and promote the growth of new tissue.

[0005] After macrophages are stimulated and activated, they produce cytokines such as TNF-α, IL-6, and IL-10, indicating their immune activation effect. However, excessive production of TNF-α may bring some negative effects, including but not limited to: Excessive TNF-α may inhibit the body's normal immune response ability, making patients more susceptible to bacterial and viral infections, which may lead to severe infection symptoms. Excessive TNF-α may induce the body to produce autoantibodies against TNF-α receptors, thereby triggering abnormal activation of the immune system, which may lead to symptoms such as fatigue and joint swelling. For some individuals, excessive TNF-α may trigger allergic reactions such as urticaria, itching, and shortness of breath. In extreme cases, this may lead to anaphylactic shock.

[0006] Therefore, it would be very meaningful to develop a probiotic product that can comprehensively regulate immune factors, does not over-activate promoting factors, and can increase the level of anti-inflammatory factors, that is, it has potential anti-inflammatory effects while regulating immunity. Summary of the invention

[0007] In view of the deficiencies in the prior art, the object of the present invention is to provide a thermophilic Streptococcus CT-01 and its use in the preparation of immunomodulatory preparations.

[0008] 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-01, wherein the thermophilic Streptococcus CT-01 is classified as Streptococcus thermophilus , the deposit number is GDMCC No: 64473, and the deposit date is March 29, 2024.

[0009] The present invention isolates and preserves a new strain of thermophilic Streptococcus CT-01 with immunomodulatory efficacy 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 efficacy, which is specifically manifested in: comprehensive regulation of the behavior of macrophages secreting inflammatory factors, which can not only avoid the overactivation of TNF-α, IL-1β, and IL-6, but also increase the level of IL-10, and has potential anti-inflammatory effects while regulating immunity. It is promising to use the strain to prepare products with immunomodulatory efficacy or products with inflammatory factor regulation efficacy.

[0010] In a second aspect, the present invention provides the use of the thermophilic Streptococcus CT-01 or its postbiotic preparation described in the first aspect in the preparation of a product with immunomodulatory or auxiliary immunomodulatory efficacy.

[0011] Preferably, the number of Streptococcus thermophilus CT-01 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), 1×10 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.

[0012] Preferably, the product includes food, health care products or medicines.

[0013] In the above products, Streptococcus thermophilus CT-01 can exist in both live bacteria state and inactivated state (postbiotics).

[0014] In a third aspect, the present invention provides the use of the thermophilic Streptococcus CT-01 or its postbiotic preparation described in the first aspect in the preparation of a product having the efficacy of regulating inflammatory factors or assisting in the regulation of inflammatory factors.

[0015] Preferably, the number of Streptococcus thermophilus CT-01 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), 1×10 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.

[0016] In the above products, Streptococcus thermophilus CT-01 can exist in both live bacteria state and inactivated state (postbiotics).

[0017] In a fourth aspect, the present invention provides a probiotic preparation, wherein the strain in the probiotic preparation comprises the thermophilic Streptococcus CT-01 described in the first aspect in a living and / or inactivated state.

[0018] Preferably, the strains in the probiotic preparation further include Lactobacillus gasseri CKCC1913 in a live and / or inactivated state, and the classification name of the Lactobacillus gasseri CKCC 1913 is Lactobacillus gasseri Lactobacillus gasseri The deposit number is CGMCC No. 23175, and the deposit date is August 23, 2021.

[0019] The present invention also creatively discovered that the above-mentioned Streptococcus thermophilus CT-01 strain can be used in combination with Lactobacillus gasseri CKCC 1913 strain to regulate immune and inflammatory factors, and also has excellent effects. Compared with a single bacterial agent, the composite bacterial agent has a more significant immune activation and anti-inflammatory effect, indicating that the CT-01 strain and the CKCC 1913 strain have potential synergistic effects in the above-mentioned effects.

[0020] Preferably, the ratio of the number of Streptococcus thermophilus CT-01 to that of Lactobacillus gasseri CKCC 1913 is (2-10):1, for example 2:1, 3:1, 4:1, 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.

[0021] Based on the potential interaction between the two strains in activating immunity and simultaneously fighting inflammation, the effect will be better when the two meet the above-mentioned specific ratio relationship.

[0022] Preferably, the total bacterial count in the probiotic preparation is not less than 1×10 4 / mL or 1×10 4 Pieces / g, for example 1×10 4 pieces / g (pieces / mL), 1×10 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.

[0023] Preferably, the dosage form of the probiotic preparation includes a solution or a powder.

[0024] The dosage form of the probiotic preparation involved in the present invention is not limited, including the most commonly used solution, powder, or further prepared capsules, tablets or granules.

[0025] The probiotic preparation also contains bacteria protective agents and / or nutritional components.

[0026] In a fifth aspect, the present invention provides use of the probiotic preparation described in the fourth aspect in the preparation of a product having immunomodulatory or auxiliary immunomodulatory efficacy.

[0027] Preferably, the product includes food, health care products or medicines.

[0028] In a sixth aspect, the present invention provides the use of the probiotic preparation described in the fourth aspect in the preparation of a product having the function of regulating or assisting in regulating inflammatory factors.

[0029] Compared with the prior art, the present invention has the following beneficial effects: The present invention isolates and preserves a new strain of thermophilic Streptococcus CT-01 with immunomodulatory efficacy 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 efficacy, which is specifically manifested in: comprehensive regulation of the behavior of macrophages secreting inflammatory factors, which can not only avoid the overactivation of TNF-α, IL-1β, and IL-6, but also increase the level of IL-10, that is, while activating immunity, it has a potential anti-inflammatory effect. It is promising to use the strain to prepare products with immunomodulatory efficacy or products with inflammatory factor regulation efficacy.

[0030] The classification of the CT-01 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: 64473, and the address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0031] The CKCC 1913 strain involved in the present invention is classified as Lactobacillus gasseri Lactobacillus gasseri The depository is the General Microbiology Center of China Microbiological Culture Collection Administration. The deposit date is August 23, 2021. The deposit number is CGMCC No. 23175. The address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a microscopic image of the Streptococcus thermophilus CT-01 strain; Figure 2 This is the appearance of the colony of Streptococcus thermophilus CT-01 strain; Figure 3 This is a statistical chart showing the effects of different experimental groups on the secretion of TNF-α by macrophages; Figure 4 This is a statistical chart showing the effects of different experimental groups on the secretion of IL-1β by macrophages; Figure 5 This is a statistical chart showing the effects of different experimental groups on the secretion of IL-6 by macrophages; Figure 6 This is a statistical chart showing the effects of different experimental groups on the secretion of IL-10 by macrophages. DETAILED DESCRIPTION

[0033] 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.

[0034] Test materials and instruments: The taxonomic designation of the CT-01 strain involved is Streptococcus thermophilus , deposited on March 29, 2024, with a deposit number of GDMCC No: 64473; the classification name of the CKCC 1913 strain involved is Lactobacillus gasseri Lactobacillus gasseri The deposit date is August 23, 2021, and the deposit number is CGMCC No.23175; the other strains involved are all commercial strains available on the market.

[0035] 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.

[0036] 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

[0037] Isolation and screening of thermophilic Streptococcus CT-01: 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-01. Example 2

[0038] Identification of Streptococcus thermophilus CT-01: (1) Observe the selected CT-01 strain under a microscope. Figure 1 As shown, the strain is spherical in appearance, arranged in short chains, and is Gram-positive.

[0039] (2) The appearance of the CT-01 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.

[0040] (3) 16S rDNA molecular biological identification: The screened CT-01 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.

[0041] SEQ ID No: 1: Example 3

[0042] Cellular immune regulation test: (1) Experimental groups: The experiment was divided into CONTROL group (not treated with probiotic solution), CT-01 group (intervention with CT-01 bacterial solution), CKCC1913 group (CKCC 1913 bacterial solution), CT-01 + CKCC 1913 group 1 (combined intervention of the two bacteria, with a bacterial number ratio of 2:1), CT-01 + CKCC 1913 group 2 (combined intervention of the two bacteria, with a bacterial number ratio of 10:1), commercially available LR group (intervention with Bacillus rhamnosus ATCC7469), commercially available BB group (intervention with Bifidobacterium animalis subsp. lactis ATCC700541), commercially available ST group (intervention with Streptococcus thermophilus ATCC19258), and commercially available ST + commercially available LG group (combined intervention with commercially available Streptococcus thermophilus ATCC19258 and commercially available Lactobacillus gasseri ATCC19992, with a bacterial number ratio of 2:1).

[0043] (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.

[0044] (3) Cellular intervention methods: Collect Raw264.7 mouse macrophages in normal growth state to prepare cell suspension, so that it reaches 50,000 cells per milliliter. Take 1mL of the cell solution and place it on a 24-well experimental plate, and then put the plate into a constant temperature incubator at 37°C and 5% carbon dioxide environment for 2 hours to allow the cells to adhere to the wall. After incubation for 2 hours, each strain intervention group was added with RPMI1640 culture medium with different bacterial suspensions prepared in step (2) (the final total bacterial concentration of each group in the culture system 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.

[0045] (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.

[0046] The RT-qPCR method was used to detect the cytokine levels in the supernatant of each group. First, RNA was extracted from each group and reverse transcribed. Then, the levels of four immune factors, TNF-α, IL-1β, IL-6, and IL-10, were detected by RT-qPCR. The statistical results are shown in Figure 3-Figure 6 shown.

[0047] Depend on Figure 3-Figure 6 The results show that the thermophilic Streptococcus CT-01 involved in the present invention has excellent immunomodulatory effect and can perform immunomodulation by activating macrophages to secrete inflammatory factors. Compared with other thermophilic Streptococci or rhamnosus and animal Bifidobacterium lactis subspecies that also have immunomodulatory potential, this strain can avoid the overactivation of proinflammatory factors TNF-α, IL-1β, and IL-6, and can also increase the level of anti-inflammatory factor IL-10, indicating that thermophilic Streptococcus CT-01 has more obvious advantages in activating immunity while potentially fighting inflammation.

[0048] In addition, the immunomodulatory effect of the above-mentioned aspects is further improved by compounding thermophilic Streptococcus CT-01 with Lactobacillus gasseri CKCC 1913, indicating that the CT-01 strain and the CKCC 1913 strain have potential synergistic effects on the above-mentioned effects. Compared with other commercially available thermophilic Streptococcus and commercially available Lactobacillus gasseri compounding methods, the compounding method involved in the present invention has obvious advantages.

[0049] 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.

[0050] 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.

[0051] 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-01, characterized in that The thermophilic Streptococcus CT-01 is classified as Streptococcus thermophilus , the deposit number is GDMCC No: 64473, and the deposit date is March 29, 2024.

2. Use of the thermophilic Streptococcus CT-01 or its postbiotic preparation according to claim 1 in the preparation of a product having immunomodulatory or auxiliary immunomodulatory effects; The bacterial count of the thermophilic Streptococcus CT-01 in the product is not less than 1×10 4 / mL or 1×10 4 pcs / g; The products include food, health products or medicines.

3. Use of the thermophilic Streptococcus CT-01 or its postbiotic preparation according to claim 1 in the preparation of a product having the function of regulating inflammatory factors or assisting in regulating inflammatory factors.

4. A probiotic preparation, characterized in that The strains in the probiotic preparation include the thermophilic Streptococcus CT-01 according to claim 1 in a living and / or inactivated state.

5. The probiotic preparation according to claim 4, characterized in that The strains in the probiotic preparation also include Lactobacillus gasseri CKCC 1913 in a live and / or inactivated state, and the classification name of Lactobacillus gasseri CKCC 1913 is Lactobacillus gasseri Lactobacillus gasseri The deposit number is CGMCC No. 23175, and the deposit date is August 23, 2021.

6. The probiotic preparation according to claim 5, characterized in that The ratio of the bacterial counts of the thermophilic Streptococcus CT-01 to the Lactobacillus gasseri CKCC 1913 is (2-10):

1.

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

8. The probiotic preparation according to claim 4, characterized in that The dosage form of the probiotic preparation includes solution or powder; The probiotic preparation also contains bacteria protective agents and / or nutritional components.

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

10. Use of the probiotic preparation according to any one of claims 4 to 8 in preparing a product having the function of regulating or assisting in regulating inflammatory factors.

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