A strain of Klebsiella pneumoniae and its application in preparing a product for degrading valine

Abstract

The invention discloses a strain of Klebsiella pneumoniae and its application in preparing a degraded valine product, belonging to the technical fields of microbiology, biochemistry and fermentation engineering. The Klebsiella pneumoniae degrading valine of the invention is Klebsiella pneumoniae with a deposit number of CCTCC M 20241526 ( Klebsiella pneumoniae ) Tongji, which can reduce the host's valine level and is highly sensitive to multiple antibiotics, has clinical application prospects.

Description

Technical Field

[0001] The invention relates to the technical fields of microbiology, biochemistry and fermentation engineering, and in particular to a strain of Klebsiella pneumoniae and application thereof in preparing a product for degrading valine. Background Art

[0002] Valine is involved in multiple biochemical pathways that are essential for neuronal health and function. It is a precursor for the synthesis of glycine and serine, two amino acids that play important roles in synaptic transmission and neural signaling. In addition, valine metabolism contributes to the production of acetyl-CoA, an important substrate for energy production and lipid synthesis. Studies have shown that valine metabolism may be affected in patients with Alzheimer's disease. Abnormal valine metabolism may lead to decreased levels of glycine and serine, thereby affecting the normal function of neurons. Recent studies have shown that an imbalance in the intestinal flora is associated with the development of a variety of diseases, such as intestinal diseases, metabolic diseases, and immune diseases. The development of these diseases may affect the way valine is metabolized and utilized, increasing health risks.

[0003] Therefore, it is necessary to provide a strain that can reduce host valine. Summary of the invention

[0004] The present invention aims to provide a strain of Klebsiella pneumoniae and application thereof in preparing a product for degrading valine, wherein the strain can effectively degrade valine.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] In a first aspect of the present invention, a strain of Klebsiella pneumoniae is provided. The deposit number of the Klebsiella pneumoniae is: CCTCC NO: M 20241526.

[0007] In the second aspect of the present invention, provided is the use of the Klebsiella pneumoniae in preparing a product for degrading valine.

[0008] In a third aspect of the present invention, a fermentation agent is provided, wherein the fermentation agent comprises:

[0009] Fermentation of the Klebsiella pneumoniae to obtain a fermentation broth;

[0010] Or the fermentation liquid is spray-dried to obtain a dry powder bacterial agent.

[0011] In a fourth aspect of the present invention, a product for degrading valine is provided, wherein the product for degrading valine comprises the Klebsiella pneumoniae or the fermentation bacterial agent.

[0012] Furthermore, the product for degrading valine also includes pharmaceutically acceptable excipients.

[0013] In a fifth aspect of the present invention, a method for degrading valine using the Klebsiella pneumoniae is provided, the method comprising:

[0014] The Klebsiella pneumoniae or the fermentation bacteria agent is added into the culture medium, or administered intragastricly or orally.

[0015] Furthermore, the culture medium is LB culture medium.

[0016] Furthermore, the culture temperature is 36° C.-38° C., the culture pH range is 6.5-7.5, and the most suitable inoculation amount is 5%-10%.

[0017] As a specific embodiment, the method for degrading valine using Klebsiella pneumoniae specifically includes:

[0018] The Klebsiella pneumoniae was inoculated into a test tube containing LB liquid culture medium and cultured overnight at 35°C in a shaking incubator for activation;

[0019] The bacterial solution after overnight culture was centrifuged, the supernatant was discarded, and the solution was resuspended with sterile water. The solution was centrifuged again, the supernatant was discarded, and the solution was resuspended with sterile water. The OD600 of the resuspended bacterial solution was transferred to a triangular shake flask containing valine as LB culture medium and cultured on a shaker.

[0020] Furthermore, the method further comprises: measuring the degradation rate of valine, specifically comprising:

[0021] A valine standard solution was prepared, and the peak area of ​​valine was measured by gas chromatography-mass spectrometry, and a standard curve of valine was obtained based on the relationship between the peak area and the concentration;

[0022] The sample to be tested is detected by gas chromatography-mass spectrometry to obtain the peak area of ​​the sample to be tested, and the peak area is substituted into the standard curve to obtain the concentration C2 of valine in the sample to be tested;

[0023] The control group samples were detected by gas chromatography-mass spectrometry to obtain the peak area of ​​the control group samples, which was substituted into the standard curve to obtain the concentration C1 of valine in the control group;

[0024] The degradation rate of valine was obtained by the formula (C1-C2) / C1.

[0025] The control group sample is a blank culture medium, which is cultured under the same conditions as the experimental group (sample to be tested).

[0026] Furthermore, the model of the gas chromatograph-mass spectrometer is G1540N, the chromatographic column used is TraceGOLDTG-5MS chromatographic column 30m×0.25 μm, the gas flow rate: high-purity nitrogen: 1.5 mL / min, hydrogen: 30 ml / min, air: 300 ml / min. The injection port and detector temperature are 280°C; column temperature: initial temperature 50°C maintained for 2 min, increased to 230°C at a rate of 40°C per minute, increased to 320°C at a rate of 20°C per minute and maintained for 20 min; injection volume is 1 μL; detector temperature: 325°C.

[0027] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0028] 1. The present invention provides a strain of Klebsiella pneumoniae and its application, which can separate bacteria capable of degrading high-concentration valine from fresh fecal samples of volunteers with low valine content, with a degradation rate of 30% within 24 hours and 51% within 72 hours.

[0029] 2. The present invention provides an intestinal bacterium involved in valine metabolism, which can provide a new approach for regulating human valine metabolism in order to further understand the effect of intestinal flora on valine.

[0030] The Klebsiella pneumoniae of the present invention is deposited on July 9, 2024, and the deposit number is CCTCC NO: M20241526. Klebsiella pneumoniae Tongji ， The name of the depository is China Center for Type Culture Collection, and its address is Wuhan University, Wuhan, Hubei, China, Postal Code: 430072. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0032] Figure 1 The Klebsiella pneumoniae isolated in Example 1 Tongji (KP Tongji) Pictures;

[0033] Figure 2 This is the phylogenetic tree of Klebsiella pneumoniae (named Tongji in the figure);

[0034] Figure 3 For Example 2 KP Tongji Drug sensitivity testing;

[0035] Figure 4 For Example 3 KP Tongji Detection results of degraded valine;

[0036] Figure 5 In Example 4, KP Tongji The results of serum valine detection in mice after oral administration. DETAILED DESCRIPTION

[0037] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0038] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0039] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0040] The following is a detailed description of a strain of Klebsiella pneumoniae and its application in the present application in combination with embodiments and experimental data.

[0041] Example 1: Isolation, purification and identification of Klebsiella pneumoniae

[0042] (1) Isolation of Klebsiella pneumoniae

[0043] Based on the assumption that intestinal bacteria can degrade valine, the inventors mixed 0.5 g of fresh fecal samples from low-valine volunteers with selective culture medium (valine as the sole carbon source). The culture was incubated in the dark at 37°C and 250 rpm on a rotary shaker. The culture medium was changed after three to four days of culture. After five repetitions, the culture suspension was inoculated on blood agar plates to isolate strains, such as Figure 1 (2) Identification of Klebsiella pneumoniae

[0044] The isolated strains were inoculated in LB liquid medium containing 600 mg / L valine, and cultured in a shaking incubator at 37°C and 200 rpm for 24 h. The bacterial solution was mixed with 30% glycerol at a volume ratio of 1:1 and sent to Qingke Biotechnology for sequence sequencing. The 16S rDNA sequence of the strain obtained by sequencing was analyzed by BLAST in the NCBI database, and the strain was systematically analyzed with the model strain using MEGA11 software, and the phylogenetic tree was constructed using the neighbor-joining method, as shown in Figure 2. Figure 2 As shown;

[0045] Combined with physiological and biochemical characteristics, matrix-assisted laser desorption ionization-time of flight mass spectrometry, it was identified as Klebsiella pneumoniae ( Klebsiella pneumoniae ), we named the bacterium Klebsiella pneumoniae Tongji ( KP Tongji). It was deposited in the China Center for Type Culture Collection (deposit address: Wuhan University, Wuhan, China) on July 9, 2024, and its classification was named Klebsiella pneumoniae , with a deposit number of CCTCC M 20241526. The Klebsiella pneumoniae can utilize and degrade valine.

[0046] Embodiment 2, M. paraoxydans Tongji drug sensitivity test

[0047] Using the disc diffusion method, MH medium containing blood was used to KP Tongji was used for drug sensitivity test.

[0048] The results are as follows Figure 3 As shown, KP Tongji was resistant to ampicillin and sensitive to amoxicillin, sulbactam, cefazolin, cefotaxime, cefoxitin, meropenem, amikacin, tobramycin, levofloxacin, minocycline, cefoperazone, piperacillin, cefuroxime, ceftazidime, aztreonam, imipenem, gentamicin, ciprofloxacin, and co-trimoxazole.

[0049] Drug sensitivity test can quickly and effectively detect the sensitivity of pathogens to various antimicrobial drugs and guide the rational use of drugs in clinical practice. M. paraoxydans Tongji has clinical application prospects.

[0050] Embodiment 3, KP Verification of valine degradation by Tongji

[0051] Will KP Tongji was inoculated in LB medium, and 0.5 mg / mL valine was added to the LB medium as KP Tongji group, no additions KP Tongji was used as the control group. The culture flask was placed on a shaker at 37°C and incubated at 220 rpm. Samples were taken once a day. Finally, the concentration of valine was detected by LC-MS / MS. The results are as follows Figure 4 shown.

[0052] Depend on Figure 4 It can be seen that KP Tongji degraded glutamate in a time-dependent manner without adding KP The control group of Tongji did not degrade. KP Tongji's degradation rate reached 30% in 24 hours and 51% in 72 hours.

[0053] Embodiment 4, KP Verification of Tongji's effect on host serum valine levels

[0054] Will KP Tongji was administered intragastrically to mice (10 8 CFU / mL, 3 times a week for 4 weeks) as KP Tongji group. The control group did not receive any treatment. KP Serum valine levels of mice in the Tongji group and the control group. Figure 5 As shown, KP The serum valine level of mice in the Tongji group was lower than that in the control group, and the difference was statistically significant. KP Tongji can reduce the host valine level.

[0055] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0056] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0057] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0058] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A strain of Klebsiella pneumoniae, characterized in that: The classification name of the Klebsiella pneumoniae is Klebsiella pneumoniae, and the deposit number is CCTCC M 20241526.

2. A fermentation agent, characterized in that: The fermentation agent comprises: Fermentation broth obtained by fermenting the Klebsiella pneumoniae according to claim 1; Or the fermentation liquid is spray-dried to obtain a dry powder bacterial agent.

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