Streptococcus infantis and application

Abstract

The invention discloses infant streptococcus which is preserved in China General Microbiological Culture Collection Center (CGMCC) on April 11, 2024, and the preservation number is CGMCC NO.30306. The strain has the function of reducing saliva nitrate and can relieve hypertension symptoms. The invention further discloses application of the streptococcus infantis to preparation of products with the saliva nitrate reduction function, products with the blood pressure reduction function, products with the hypertension symptom relieving function and other drugs.

Description

Technical Field

[0001] The present application belongs to the field of microbial technology, and specifically relates to a Streptococcus infantis. Background Art

[0002] Hypertension is one of the leading causes of morbidity and mortality worldwide, and is also a major risk factor for cardiovascular diseases such as heart disease and stroke, and chronic kidney disease. As one of the complex microecological systems of the human body, the oral cavity plays an important role in cardiovascular disease. When digesting different foods, oral flora produces metabolites such as short-chain fatty acids (SCFAs), hydrogen sulfide (H2S), nitric oxide (NO), trimethylamine oxide (TAMO), and lipids, which affect oral and cardiovascular health. Among them, NO is an important vasodilator in the human body, responsible for arterial relaxation and dilation. Reduced NO production or increased metabolism leading to reduced NO bioactivity is considered to be the potential pathogenesis of essential hypertension and is related to aging, kidney, cardiovascular and metabolic disorders.

[0003] When NO3 - When supplemented, oral microorganisms reduce nitrate to nitrite, via NO3 - -NO2 - -NO pathway into nitrite and NO. Oral bacteria reducing salivary nitrate helps activate the nitrate-nitrite-NO pathway in intestinal saliva, increasing systemic NO levels, thereby improving diseases such as hypertension and diabetes. The ability of oral bacteria to reduce nitrate substances varies from individual to individual, so isolating and screening oral strains with nitrate reduction ability under aerobic conditions will help clinical research on the effects of oral flora treatment on oral and systemic health. Summary of the invention

[0004] In order to solve the above-mentioned technical problems, the first object of the present application is to provide a Streptococcus infantis PKU608 strain; it was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on April 11, 2024, with a deposit number of CGMCC NO.30306. The 16S rDNA sequence of Streptococcus infantis is shown in SEQ ID NO.1, which can reduce salivary nitrate and relieve symptoms of hypertension.

[0005] The second object of the present application is to provide the application of the above-mentioned infantile streptococcus, including:

[0006] Application in the preparation of products with the function of reducing salivary nitrate.

[0007] or,

[0008] Application in the preparation of products with the function of lowering blood pressure.

[0009] or,

[0010] Application in the preparation of products having the function of relieving hypertension symptoms.

[0011] In some embodiments, the hypertensive symptom is a symptom of aortic inflammation.

[0012] or,

[0013] Application in the preparation of medicines.

[0014] In some embodiments, the above-mentioned drug is a drug for treating oral diseases, which are caused by insufficient nitrate reduction function of saliva.

[0015] Alternatively, in other embodiments, the drug is a drug for relieving high blood pressure. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Oral bacteria initial screening process;

[0017] Figure 2 Mass spectrum of Streptococcus infantis PKU608;

[0018] Figure 3 Qualitative test results of nitrate reduction ability of Streptococcus infantis PKU608 strain;

[0019] Figure 4 Streptococcus infantis PKU608 strain morphology;

[0020] Figure 5 Quantitative test results of nitrate reduction ability of Streptococcus infantis PKU608 strain;

[0021] Figure 6 Schematic diagram of the treatment process of Streptococcus infantis PKU608 strain on the hypertensive mouse model;

[0022] Figure 7 Efficacy of Streptococcus infantis PKU608 strain in the treatment of hypertension. DETAILED DESCRIPTION

[0023] The present application is further described below with reference to embodiments, but is not limited thereto.

[0024] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. The present invention has signed an informed consent and project commitment letter with the sample provider in accordance with the 2023 version of the "Regulations on the Administration of Human Genetic Resources". The operation of the present invention is strictly carried out in accordance with the standard operating procedures of the biological sample bank.

[0025] Embodiment 1:

[0026] This embodiment provides a strain of infant Streptococcus. It also discloses a screening method and identification process for infant Streptococcus strains, and quantitatively detects the nitrate reduction ability of the screened infant Streptococcus strains. A hypertensive mouse model is established, and oral transplantation of the screened infant Streptococcus strains is performed on the hypertensive mice to verify the efficacy of the infant Streptococcus strain in treating hypertension.

[0027] The culture medium and reagents involved in this example are:

[0028] Nitrate liquid medium: Add 10g peptone and 0.5524g NaNO3 to every 1L water.

[0029] BHI (brain heart infusion broth) liquid culture medium: add 10 g tryptone, 2.5 g disodium hydrogen phosphate, 17.5 g beef heart infusion powder, 5.0 g sodium chloride and 2.0 g glucose to every 1 L of water.

[0030] BHI agar medium: Add 1.5% agar to BHI liquid medium.

[0031] GriessA reagent: 1 g of p-aminobenzenesulfonic acid, 100 mL of 25% dilute hydrochloric acid.

[0032] Griess B reagent: 0.2 g of α-naphthylamine, 100 mL of 25% dilute hydrochloric acid, heat and stir the solution.

[0033] 1. Strain Screening

[0034] 1.1. Oral bacteria screening

[0035] Oral bacteria screening process Figure 1 As shown in the figure, it includes four steps: saliva sample collection, culture medium cultivation, time-of-flight mass spectrometry detection and bacterial species identification.

[0036] Saliva sample collection: Unstimulated saliva was collected by drooling through a sterile tube in a quiet room in the morning 1 hour after eating or drinking.

[0037] Culture medium: dilute the saliva in a gradient manner and take 10 -2 , 10 -3 , 10 -4100 μL of each of the three dilution gradients was evenly spread on the sterilized nitrate liquid culture medium prepared in advance and cultured aerobically at 37°C for 48 h.

[0038] Time-of-flight mass spectrometry detection and identification of bacterial species: After a single colony grew on the culture medium, a time-of-flight mass spectrometer (MALDI-TOF mass spectrometer) was used to quickly identify the colony. The mass spectrum obtained by the mass spectrometer was compared with the known protein sequence database, and a strain of infant Streptococcus was initially screened and labeled as Streptococcus infantis PKU608.

[0039] 1.2. Rescreening of nitrate-reducing strains

[0040] (1) Streak and activate the infantile Streptococcus obtained in 1 on BHI agar medium, then pick a single colony and place it in BHI liquid medium, culture it aerobically at 37°C and 180 rpm for 12 h to obtain a fresh bacterial liquid, centrifuge it at 4500 rpm for 10 min, discard the supernatant, wash it twice with sterilized 1× PBS to obtain the infantile Streptococcus bacteria, and set it aside.

[0041] (2) Use 2 mL of nitrate liquid culture medium to resuspend the cells of infant Streptococcus infantis and culture them aerobically at 37°C and 180 rpm for 8 h. To test the nitrate reduction ability of infant Streptococcus infantis, take an appropriate amount of culture medium and add 1 drop of GriessA reagent and 1 drop of GriessB reagent respectively. The qualitative test results of the nitrate reduction ability of infant Streptococcus infantis are as follows: Figure 3 As shown, the culture solution of the strain turned into rose red, indicating that it has the ability to reduce nitrate.

[0042] 2. Identification of Streptococcus infantis PKU608

[0043] 2.1. Colony morphology identification

[0044] The Streptococcus infantis PKU608 strain was inoculated on BHI agar medium and cultured aerobically at 37°C for 24 hours. The morphology of the Streptococcus infantis PKU608 strain is shown in the following figure. Figure 4 As shown, the single colony is milky white and shiny, with a smooth and moist surface and neat edges.

[0045] 2.2 Molecular Biological Identification

[0046] (1) Genomic DNA extraction

[0047] A single colony of Streptococcus infantis PKU608 was picked and inoculated into BHI liquid culture medium and cultured at 37°C for 24 h. 1 mL of bacterial solution was taken and operated according to the Tiangen Bacterial Genomic DNA Extraction Kit to obtain the genome of Streptococcus infantis PKU608.

[0048] (2) 16S rDNA gene amplification

[0049] PCR amplification uses total DNA as template and the amplification primers are as follows:

[0050] The forward primer is 27F: 5'-AGAGTTTGATCCTGGCTCA-3'

[0051] Reverse primer is 1492R: 5'-GGTTACCTTGTTACGACTT-3'

[0052] The PCR reaction was carried out in a 20 μL reaction system, and the reaction system composition was as follows: dNTP (mix) 10 μL, DNA template 1 μL, forward primer 27F 0.5 μL, reverse primer 1492R 0.5 μL, and double distilled water was added to the total volume of the reaction system to 20 μL.

[0053] PCR reaction procedure: pre-denaturation at 95°C for 3 min; denaturation at 94°C for 25 sec, annealing at 58°C for 45 sec, extension at 72°C for 20 sec, for a total of 30 cycles; repair and extension at 72°C for 5 min, termination of the reaction at 4°C to obtain the PCR product.

[0054] (3) PCR product detection

[0055] The PCR product was sequenced to obtain the 16S rDNA sequence of the Streptococcus infantis PKU608 strain as shown in SEQ ID NO. 1. The sequence was homologously compared in the NCBI database to confirm that the Streptococcus infantis PKU608 strain was Streptococcus infantis.

[0056] (4) Protein mapping

[0057] Use a sterile toothpick to pick up a single colony of the Streptococcus infantis PKU608 strain and transfer it to the mass spectrometry sample target plate, spread it evenly to form a biofilm. Use a pipette to take 1 μL of component I (50% acetonitrile and 70% formic acid) and cover it on the above biofilm, and dry it at room temperature. Use a pipette to take 1 μL of component II (2.5% trichloroacetic acid, 50.% acetonitrile, saturated α-cyano-4-hydroxycinnamic acid) and cover it on the above mass spectrometry sample target plate, and dry it at room temperature. Place the mass spectrometry sample target plate in a time-of-flight mass spectrometry system for detection. The protein spectrum identification results are shown in the figure below. Figure 2 As shown, the strain was identified as Streptococcus infantum.

[0058] After the above detection and identification, the obtained Streptococcus infantis PKU608 strain was named Streptococcus infantis PKU608 strain. On April 11, 2024, the strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number CGMCC NO.30306.

[0059] 3. Quantitative detection of nitrate reduction ability of Streptococcus infantis PKU608

[0060] 3.1. Streak and activate the Streptococcus infantis PKU608 strain on BHI agar medium, then pick a single colony and place it in BHI liquid medium, culture it aerobically at 37°C and 180 rpm for 12 h to obtain fresh bacterial liquid, centrifuge it at 4500 rpm for 10 min, discard the supernatant, wash it twice with sterilized 1× PBS to obtain Streptococcus infantis PKU608 bacteria for later use.

[0061] 3.2. Determination of nitrate reduction capacity

[0062] Resuspend the cells in nitrate liquid medium, OD 600 0.6, 37℃, 180rpm aerobic culture for 4h, centrifuge at 4500rpm for 10min, take the supernatant to detect NO3 - -N, NO2 - -N and NO content. The culture medium without bacteria inoculation was used as blank control, and 3 replicates were set for each group. Figure 5 As shown, after 4 hours of cultivation, NO3 - -N concentration decreased from the initial 75μg / mL to 44.5μg / mL, NO3 - -N reduction rate was 40.4%. At the same time, NO2 --N and NO contents increased to 30.9 μg / mL and 2 mM, respectively, while the control group had no NO2 - -N and NO are produced.

[0063] 4. Efficacy of Streptococcus infantis PKU608 in treating hypertension

[0064] 4.1 Construction of Hypertensive Mouse Model

[0065] Schematic diagram of the treatment of hypertensive mouse model with Streptococcus infantis PKU608 strain Figure 6 As shown. Using 8-week-old C57BL / 6J male mice, a hypertensive mouse model was induced by gavage of L-arginine methyl ester (L-NAME, 100 mg / kg / day). The mice were gavaged once a day, and the systolic blood pressure of the mice was tested by the tail cuff method every week. The systolic blood pressure of the mice was higher than 130 mmHg for 3 consecutive weeks after the 6th week of the hypertensive mouse model construction, indicating that the hypertensive mouse model was successfully established after the 9th week of gavage, and oral transplantation of Streptococcus infantis PKU608 strain can be started.

[0066] 4.3. Oral transplantation of Streptococcus infantis PKU608

[0067] The Streptococcus infantis PKU608 strain was transferred to BHI liquid medium and cultured at 37°C, 180 rpm for 12 h. The bacterial cells were obtained by centrifugation, washed three times with sterilized 1× PBS, and resuspended with 2% sodium carboxymethyl cellulose to an appropriate concentration (5×10 9 CFU / mL) to obtain the bacterial suspension resuspended in sodium carboxymethyl cellulose.

[0068] 0.2 mL of sodium carboxymethylcellulose resuspended bacterial solution was delivered to the mouse's mouth each time by oral feeding, and the frequency of bacterial administration was once every two days.

[0069] 4.3. Experiment on the efficacy of Streptococcus infantis PKU608 in treating hypertension

[0070] This example sets two experimental groups and two control groups. Figure 6 As shown:

[0071] Control group CK: normal mice;

[0072] Control group HP: 0.2 mL of 2% sodium carboxymethyl cellulose was delivered into the oral cavity of hypertensive mice by oral feeding, and the frequency of administration was once every two days.

[0073] Experimental group 1 (HP+S. infantis PKU608): 0.2 mL of sodium carboxymethyl cellulose resuspended bacterial solution was delivered to the oral cavity of hypertensive mice by oral feeding, and the frequency of bacterial administration was once every two days.

[0074] Experimental group 2 (HP+Nitrate+S.infantis PKU608): 0.2 mL of sodium carboxymethylcellulose resuspended bacterial solution was delivered to the oral cavity of hypertensive mice by oral feeding, the frequency of bacteria administration was once every two days, and 1 mM sodium nitrate was contained in the drinking water.

[0075] The experimental results are as follows Figure 7 As shown. As can be seen from the figure, after oral transplantation of Streptococcus infantis PKU608 strain, the systolic blood pressure of mice decreased. After 8 weeks, the systolic blood pressure of hypertensive mice in experimental group 1 decreased by 16.6 mmHg. In contrast, the systolic blood pressure of hypertensive mice in experimental group 2 was not significantly different from that of normal mice (control group CK) after 5 weeks, and remained stable for 3 consecutive weeks, with no significant difference from normal mice. The relative expression levels of proinflammatory factors (IL-6 and TNF-α) in the aorta were determined by qRT-PCR. The results showed that oral transplantation of Streptococcus infantis PKU608 strain can alleviate the symptoms of aortic inflammation caused by hypertension.

[0076]

Claims

1. A Streptococcus infantis, characterized in that: The infant Streptococcus is the infant Streptococcus PKU608 (Streptococcus infantis PKU608) strain; the infant Streptococcus was deposited in the General Microbiology Center of the China Culture Collection Administration on April 11, 2024, with the deposit number CGMCC NO.30306.

2. The Streptococcus infantis according to claim 1, characterized in that: The 16S rDNA sequence of the Streptococcus infantum is shown in SEQ ID NO.

1.

3. The Streptococcus infantis according to claim 1, characterized in that: The S. infantum bacteria can reduce salivary nitrate.

4. The Streptococcus infantis according to claim 1, characterized in that: The infantile streptococcus can relieve the symptoms of hypertension.

5. Use of the infantis streptococcus as claimed in claim 1 in preparing a product having the function of reducing salivary nitrate.

6. Use of the infantis streptococcus as claimed in claim 1 in the preparation of a product having the function of lowering blood pressure.

7. Use of the infantis streptococcus as claimed in claim 1 in the preparation of a product having the function of relieving hypertension symptoms.

8. A use according to claim 7, characterized in that: The hypertension symptoms are symptoms of aortic inflammation.

9. Use of the infantis streptococcus as claimed in claim 1 in the preparation of medicines.

10. A use as claimed in claim 9, wherein the drug is a drug for treating oral diseases caused by insufficient nitrate reduction function of saliva; or, the drug is a drug for relieving hypertension.

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