Active peptide TK-9 and application of active peptide TK-9 in inhibition of Alistipes indinctus

Abstract

The invention provides an active peptide TK-9 and an application of the active peptide TK-9 in inhibition of Alistipes indinctus. The active peptide TK-9 can be used for inhibiting Alistipes indinctus. The amino acid sequence of the active peptide TK-9 is shown as SEQ ID No.1. The nonapeptide provided by the invention can be chemically synthesized, and also can be directionally prepared from sea cucumber proteolysis products or other animal proteolysis products. The active peptide TK-9 can inhibit the activity of Alistipes indinctus, the minimum inhibitory concentration (MIC) value of the active peptide TK-9 is 7.8 ng / L, and the minimum bactericidal concentration (MBC) value of the active peptide TK-9 is 31.25 ng / L; a flow cytometry and laser confocal analysis also show that the active peptide has the activity of inhibiting Alistipes indinctus. The invention also discloses a preparation method of the active peptide for inhibiting Alistipes indinctus. The compound disclosed by the invention is expected to be used as an inhibitor of Alistipes indinctus in the future, and has an application value in the aspects of prevention and treatment of metabolic diseases and braingut axis diseases, precise micro-ecological intervention and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of food and biomedicine, and in particular to an active peptide TK-9 and its application in inhibiting Alistipes indistinctus. Alistipes indistinctus BACKGROUND

[0002] Alistipes indistinctus is a gram-negative anaerobic bacteria belonging to Bacteroidetes and Rikenellaceae family. It was first isolated and named by Rautio et al. in 2007 from human clinical specimens. It is a member of the Alistipes genus and widely exists in the intestines of healthy people and patients with various diseases as an important component of human intestinal microbiota. In recent years, Alistipes indistinctus has gradually attracted attention due to its role in intestinal flora homeostasis, metabolic regulation, and disease correlation. For example, in metabolic diseases such as obesity, type 2 diabetes, and non-alcoholic fatty liver disease, the abundance of Alistipes indistinctus usually increases, which may be related to metabolic disorders and inflammatory states. In patients with mental diseases such as depression and anxiety, the abundance of Alistipes indistinctus is also often abnormally increased, suggesting that it may participate in neural regulation through the brain-gut axis.

[0003] Currently, no study has found an active peptide that can inhibit the growth of Alistipes indistinctus. SUMMARY

[0004] The present application aims to provide an active peptide TK-9 and its application in inhibiting Alistipes indistinctus. The primary structure amino acid sequence of the active peptide is TDEVYTVPK.

[0005] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: An active peptide TK-9, which has any of the following amino acid sequences: (1) SEQ ID No. 1; (2) an amino acid sequence obtained by substituting, deleting, and / or adding one or more amino acids from SEQ ID No. 1 and having the same function as SEQ ID No. 1; (3) an amino acid sequence having more than 90% homology with SEQ ID No. 1 and having the same function as SEQ ID No. 1.

[0006] ​The antimicrobial peptide TK-9 of this invention has an amino acid sequence as shown in SEQ ID No. 1: TDEVYTVPK. It can also undergo point mutations on the amino acid sequence shown in SEQ ID No. 1, such as substitution, deletion, and / or addition of one or more amino acids, as long as it has the same function as SEQ ID No. 1. Alternatively, it can be an amino acid sequence with more than 90% homology to SEQ ID No. 1 and with the same function as SEQ ID No. 1, such as having 99%, 98%, 97%, 96%, 95%, 94%, 93%, or 92% homology.

[0007] The present invention does not impose any particular limitation on the preparation method of the active peptide TK-9; it can be prepared by chemical synthesis or by targeted preparation from protease hydrolysis products.

[0008] The present invention also provides the application of the above-mentioned active peptides in the preparation of antibacterial and bactericidal agents.

[0009] Furthermore, the antibacterial spectrum of the bacteriostatic agent includes Gram-negative anaerobic bacteria.

[0010] Furthermore, the Gram-negative anaerobic bacterium is *Alistipes indistinctus*.

[0011] The present invention also provides an inhibitor of Alistipes indistinctus, the active ingredient of which includes the above-mentioned active peptide TK-9.

[0012] This invention also provides the application of the described active peptide in the preparation of products that regulate gut microbiota homeostasis, wherein the gut microbiota is Alistipes indistinctus.

[0013] The advantages of this invention are: The bioactive peptide TK-9 of this invention exhibits inhibitory activity against Alistipes indistinctus, with a minimum inhibitory concentration (MIC) of 7.8 ng / µL and a minimum bactericidal concentration (MBC) of 31.25 ng / µL. Flow cytometry and laser confocal microscopy analyses also confirm that this bioactive peptide has inhibitory activity against Alistipes indistinctus. This invention holds promise for future applications as an inhibitor of Alistipes indistinctus in the prevention and treatment of metabolic diseases, gut-brain axis disorders, and precise microecological intervention. Attached Figure Description

[0014] Figure 1The minimum inhibitory concentration (MIC) of TK-9 against Alistipes indistinctus was determined by one-way ANOVA. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0015] Figure 2 The minimum bactericidal concentration (MBC) of TK-9 against Alistipes indistinctus.

[0016] Figure 3 To analyze the antibacterial activity of TK-9 against Alistipes indistinctus using flow cytometry.

[0017] Figure 4 To analyze the antibacterial activity of TK-9 against Alistipes indistinctus using laser confocal microscopy. Detailed Implementation

[0018] To facilitate understanding of the present invention, the technical solutions described below are further illustrated with specific embodiments. These embodiments are intended to illustrate the invention and are not intended to limit the invention in any way.

[0019] The materials used in the following embodiments are as follows: Colombian broth was purchased from Shandong Top Biotechnology Co., Ltd., batch number M0052; Colombian blood platelets were purchased from Guangdong Huankai Microbial Technology Co., Ltd., batch number CP0160; Alistipes indistinctus was purchased from Beina Biotechnology, batch number BNCC353575; fluorescein isothiocyanate was purchased from MedchemExpress, batch number HY-66019; fluorescein succinimide 5(6)-carboxydiacetic acid was purchased from MedchemExpress, batch number HY-D0938; and propidium iodide was purchased from MedchemExpress, batch number HY-D0815.

[0020] The instruments and equipment used are as follows: Anaerobic workbench (Concept 400, Baker, Britain), flow cytometer (Beckman Coulter-CytoFLEX LX), laser confocal microscopy (Leica STELLARIS5, Germany).

[0021] Example 1 Synthesis of active peptide TK-9 Based on the designed amino acid sequence of the active peptide TK-9, TDEVYTVPK was synthesized using a conventional solid-phase method (purity greater than 98%, molecular weight 1051.531 Da, isoelectric point 3.94, normalized hydrophobicity -0.53).

[0022] Example 2: Determination of the antibacterial activity of active peptide TK-9 2.1 Determination of MIC Minimum inhibitory concentration (MIC): The lowest peptide concentration that can inhibit the growth of Alistipes indistinctus is the minimum inhibitory concentration.

[0023] Alistipes indistinctus was inoculated into Colombian broth and anaerobically cultured at 37°C (80% N2 + 10% CO2 + 10% H2) for 24 h. The bacterial suspension was then centrifuged and adjusted to a bacterial concentration of 2 × 10⁸ CFU / mL with PBS buffer (0.01 M, pH = 7.2). 100 µL of the bacterial suspension was added to 100 µL of TK-9 active peptide dissolved in PBS (final concentrations of 0 ng / µL, 7.8 ng / µL, 15 ng / µL, 31.25 ng / µL, 62.5 ng / µL, 12 ng / µL, 250 ng / µL, and 500 ng / µL) in a 96-well plate. After anaerobic culture for another 24 hours, the OD600 nm of each well was measured to determine the minimum inhibitory concentration (MIC).

[0024] 2.2 MBC determination: Minimum bactericidal concentration (MBC): The lowest peptide concentration that will not form colonies on an agar plate after incubation.

[0025] Alistipes indistinctus was inoculated into Colombian broth and anaerobically cultured at 37°C (80% N2 + 10% CO2 + 10% H2) for 24 h. The bacterial suspension was then centrifuged and adjusted to a bacterial concentration of 2 × 10⁻⁶ using PBS buffer (0.01 M, pH 7.2). 8 CFU / mL, 100µL of bacterial culture was added to 100µL of active peptide TK-9 (final concentrations of 0ng / µL, 6.25ng / µL, 12.5ng / µL, 15ng / µL, 31.25ng / µL, and 62.5ng / µL) dissolved in PBS (0.01M, pH=7.2) in a 96-well plate. The mixture was thoroughly mixed, and then 50µL of the mixture was spread onto Columbia blood agar for anaerobic culture. After 24 hours of incubation, the MBC value was determined by visual inspection of bacterial growth.

[0026] The results showed that TK-9 exhibited significant antibacterial activity against Alistipes indistinctus, with a minimum inhibitory concentration (MIC) of 7.8 ng / µL. Figure 1 The minimum bactericidal concentration (MBC) is 31.25 ng / µL. Figure 2 ).

[0027] 2.3 Flow cytometry analysis The *Alistipes indistinctus* bacterial solution was centrifuged at 12000 r / min for 1 min (3H24R1, Changsha Xiangzhi Centrifuge Instrument Co., Ltd., China), washed three times with sterile PBS (0.01M, pH=7.2), and resuspended. The bacteria and active peptides were mixed in an equal volume ratio of 1:1 (total volume >200 μL), with a final bacterial concentration of 2 × 10⁻⁶. 8 CFU / mL, with final concentrations of active peptides of 2×MBC and 4×MBC, followed by incubation at 37°C for 2 hours. Dual fluorescence staining was performed using propidium iodide and carboxyfluorescein diacetate succinimide (1:1, v / v), followed by incubation at 4°C for 15 minutes. Fluorescence intensity was quantified by flow cytometry (Beckman Coulter CytoFLEX, USA), and data were analyzed using FlowJo (v10.8.1, USA). PBS served as a negative control.

[0028] In the control group, the fluorescence was located on the left side and was weaker, with P2 covering only 3.55% of the area, indicating that the Alistipes indistinctus membrane was not damaged. Figure 3 In the TK-9 (2×MBC, MBC=31.25 ng / µL) treatment group, fluorescence shifted to the right, and the fluorescence intensity increased significantly, occupying 18.7% of the P2 region, indicating damage to Alistipes indistinctus and altered membrane permeability. In the high-dose TK-9 (4×MBC, MBC=31.25 ng / µL) treatment group, the fluorescence intensity increased significantly, occupying 32.1% of the P2 region. This suggests that with increasing TK-9 concentration, more PI and cFDA penetrate the membrane and bind to DNA, leading to increased Alistipes indistinctus cell death.

[0029] 2.4 Laser confocal Laser confocal microscopy (LSM) was used to observe the antibacterial activity of the active peptide against *Alistipes indistinctus*. The green fluorescent probe fluorescein isothiocyanate (FITC) was labeled onto the N-terminal site of the peptide. 1 mL of logarithmic growth phase *Alistipes indistinctus* (2 × 10⁻⁶) was used.8 The FITC-labeled active peptides (concentrations of 1×MBC, 2×MBC, and 4×MBC) were incubated in the dark with 1 mL of FITC-labeled active peptides (CFU / mL). Images were taken using a structured light illumination microscope (22 Hz, 90 nm) and a laser confocal microscope, and the fluorescence intensity was analyzed.

[0030] Laser confocal results are as follows Figure 4 As shown, the antibacterial ability of TK-9 against Alistipes indistinctus is such that the higher the concentration of TK-9, the more bacteria are stained, indicating that the higher the concentration of TK-9, the stronger its antibacterial ability against Alistipes indistinctus.

[0031] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. An active peptide TK-9, characterized in that: The active peptide TK-9 has any of the following amino acid sequences: (1) SEQ ID No.1; (2) An amino acid sequence of SEQ ID No. 1 that has undergone substitution, deletion and / or addition of one or more amino acids and has the same function as SEQ ID No. 1; (3) An amino acid sequence that has more than 90% homology with SEQ ID No.1 and has the same function as SEQ ID No.

1.

2. The application of the active peptide TK-9 as described in claim 1 in the preparation of antibacterial and bactericidal agents.

3. The application according to claim 2, characterized in that: The antibacterial spectrum of the bacteriostatic agent includes Gram-negative anaerobic bacteria.

4. The application according to claim 3, characterized in that: The Gram-negative anaerobic bacteria are Alistipes indistinctus .

5. A kind Alistipes indistinctus The inhibitor is characterized by: Its active ingredient includes the active peptide TK-9 as described in claim 1.

6. The application of the active peptide TK-9 as described in claim 1 in the preparation of products that regulate intestinal flora homeostasis.

7. The application according to claim 6, characterized in that: The gut microbiota is Alistipes indistinctus .

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