A type of acid-producing Klebsiella oxytoca KN-L and its application

Abstract

The application belongs to the technical field of biology, and particularly relates to a Klebsiella oxytoca KN-L and application thereof. The Klebsiella oxytoca KN-L is preserved in the China Center for Type Culture Collection, which is located in Wuhan University, Wuhan, China, and has a postal code of 430072. The preservation date is April 12, 2024, the preservation number is CCTCC NO: M2024683, and the taxonomic name is Klebsiella oxytoca. The Klebsiella oxytoca KN-L provided in the application can significantly increase the metabolic production of DOPC when MAOS is added during in-vitro fermentation. It is proved through experiments that the DOPC produced by the Klebsiella oxytoca KN-L can relieve nerve injury in-vivo and in-vitro. The biosynthesis method for producing DOPC through fermentation and metabolism of the Klebsiella oxytoca KN-L can significantly reduce the production cost of DOPC.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to an acid-producing Klebsiella oxytoca KN-L and its applications. Background Technology

[0002] The blood-brain barrier (BBB) ​​is the physical and biochemical barrier between the brain and the bloodstream. It plays a crucial physiological role in preventing external toxins and pathogens from entering brain tissue from the bloodstream, maintaining the stability of the brain's internal environment. Furthermore, it selectively regulates the entry and exit of specific molecules through active transport and passive diffusion mechanisms, ensuring the brain receives sufficient nutrients and oxygen. When the BBB is damaged or disrupted, harmful substances such as bacteria, viruses, and toxins can easily enter the brain, increasing the risk of neuronal damage and inflammatory responses, and even triggering brain diseases or neurological dysfunction.

[0003] Mannuronic acid oligosaccharides (MAOS) are sodium salts of β-D-mannuronic acid oligosaccharides with varying degrees of polymerization, prepared from alginate through specific enzymatic degradation, fractionation, and gel chromatography. Studies have reported that sodium mannitol capsules (GV-971) prepared from MAOS can remodel the gut microbiota, inhibit neuroinflammation, and thus improve cognitive function in Alzheimer's disease (AD) patients.

[0004] Dioleoyl lecithin (DOPC) is a common phospholipid compound and a key component of cell membranes, particularly in liposome formation. It forms a stable lipid bilayer, significantly influencing cell membrane fluidity and permeability. In the gut, DOPC plays various regulatory roles, including promoting cell membrane stability, participating in lipid metabolism, and cell signaling. In cardiovascular diseases, DOPC participates in the regulation of lipid metabolism and vascular endothelial function. Abnormal DOPC metabolism or distribution may be associated with the development of diseases such as atherosclerosis. Furthermore, in neurological diseases, DOPC is important for the stability and function of nerve cell membranes, and its changes may be associated with neurodegenerative diseases. Currently, DOPC is mainly extracted and purified from egg yolks and soybeans using physical methods. It can also be synthesized chemically as a phospholipid, but both physical and chemical methods are complex and have limited yields.

[0005] Due to the high production cost of DOPC in existing technologies, there is an urgent need to find a method and strain for high-yield, low-cost production of DOPC through biosynthesis. Summary of the Invention

[0006] This invention aims to provide an acid-producing Klebsiella oxytoca KN-L strain and its application method for producing DOPC. The addition of MAOS during in vitro fermentation significantly increases DOPC production, and experiments have demonstrated that the DOPC produced by this strain can alleviate nerve damage both in vivo and in vitro. The biosynthetic method of producing DOPC using the fermentation metabolism of this strain can significantly reduce the production cost of DOPC.

[0007] The specific technical solution adopted is as follows: an acid-producing Klebsiella oxytoca KN-L strain, which is deposited at the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China; postcode 430072, deposited on April 12, 2024, with accession number: CCTCC NO: M2024683, and taxonomically named Klebsiella oxytoca.

[0008] Furthermore, the 16S rDNA sequence of this strain is shown in SEQ ID NO.1.

[0009] Furthermore, the strain is used to prepare dioleoyl lecithin.

[0010] Furthermore, the method for preparing dioleoyl lecithin using the aforementioned strain is as follows:

[0011] Step 1: Inoculate the suspension of Klebsiella oxytoca KN-L bacteria into LB medium;

[0012] Step 2: Add mannouronic acid oligosaccharides to the culture medium;

[0013] Step 3: Incubate the strain at 200 rpm in a shaker at 37°C for 12 hours;

[0014] Step 4: The OD value of the bacterial suspension was found to be 1.0;

[0015] Step 5: Centrifuge at 8000 rpm for 5 min to collect the supernatant.

[0016] Furthermore, the strain is used to prepare formulations for the production of dioleoyl lecithin, wherein the number of Klebsiella oxytoca KN-L in the formulation is ≥1×10⁻⁶. 7 CFU / g or 1×10 7 CFU / mL.

[0017] And the use of the strain in the preparation of drugs for protecting the intestinal barrier.

[0018] And the use of the strain in the preparation of drugs for protecting the blood-brain barrier.

[0019] And the use of the strain in the preparation of a drug for inhibiting neuroinflammation.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] (1) The acid-producing Klebsiella oxytoca KN-L provided by this invention can metabolize dioleoyl lecithin (DOPC) under the induction of MAOS. The biosynthetic method of producing DOPC by fermentation metabolism using this strain is lower in cost than traditional physical and chemical methods, and more environmentally friendly and efficient than traditional chemical synthesis methods.

[0022] (2) Experiments have shown that DOPC produced by Klebsiella oxytoca KN-L can protect the intestinal barrier function and significantly protect the integrity of the blood-brain barrier when used in mice.

[0023] (3) Experiments have shown that DOPC produced by Klebsiella oxytoca KN-L can significantly inhibit neuroinflammation in vitro. Attached Figure Description

[0024] Figure 1 Identification of strains of Klebsiella acidogenic bacteria

[0025] Figure 2 MAOS induces Klebsiella oxytoca to metabolize KN-L and produce DOPC.

[0026] Figure 3 The protective effect of DOPC produced by Klebsiella oxytoca KN-L on the intestinal barrier in mice.

[0027] Figure 4 The protective effect of DOPC produced by Klebsiella oxytoca KN-L on the blood-brain barrier in mice.

[0028] Figure 5 The inhibitory effect of DOPC produced by Klebsiella oxytoca KN-L on neuroinflammation. Detailed Implementation

[0029] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. However, the content of the present invention is not limited to the following embodiments.

[0030] Sources of experimental materials and composition of some components of this invention

[0031] (1) Source of MAOS: Mannuronic acid oligosaccharide was purchased from Qingdao Bozhi Huili Biotechnology Co., Ltd. Its molecular weight is 6.44kDa, M / G≈12.56, and purity is 80%.

[0032] (2) BHI medium: 200.0 g / L bovine brain, 250.0 g / L bovine heart extract, 10.0 g / L peptone, 2.0 g / L glucose, 5.0 g / L NaCl, 20.0 g / L agar.

[0033] (3) LB medium: 10.0 g / L tryptone, 5.0 g / L yeast extract, 10.0 g / L sodium chloride.

[0034] (4) MacConkey agar: peptone 20.0 g / L, lactose 10.0 g / L, ox bile salt 5.0 g / L, sodium chloride 5.0 g / L, neutral red 0.075 g / L, agar 12.0 g / L.

[0035] (5) Identification of 16S rRNA by PCR of isolated bacteria: Purified single colonies were picked for 16S rRNA PCR amplification. The primer sequences for bacterial 16S rRNA were: upstream primer: 5'-AGAGTTTGATCCTGGCTCAG-3'; downstream primer: 5'-GGTTACCTTGTTACGACTT-3'. The expected amplified product length was 1466 bp. The PCR reaction system consisted of 50 μL: 25 μL TaqMaster Mix, 2 μL each of upstream and downstream primers (10 μmol / L), and 21 μL ddH2O. PCR reaction conditions: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 90 s, for a total of 30 cycles; 72℃ extension for 10 min. After the PCR reaction, 10 μL was collected. The amplified products were detected by 1.0% agarose gel electrophoresis, and the PCR products with matched band lengths were sequenced. The obtained sequences were compared using the BLAST program in NCBI to determine the strain information.

[0036] Klebsiella oxytoca KN-L, a strain of which is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China 430072, on April 12, 2024, with accession number CCTCC NO: M2024683, and taxonomically named Klebsiella oxytoca.

[0037] Example 1

[0038] Identification of strains of Klebsiella acidogenic bacteria

[0039] Approximately 1 g of fecal sample was collected from MAOS-fed mice. The fecal sample was then inoculated into BHI broth supplemented with 10% MAOS and incubated at 37°C for 24 h to increase Klebsiella abundance. Subsequently, the enriched bacterial suspension was inoculated into BHI plates and incubated at 37°C for 48 h. After observing growth, single colonies were selected using a toothpick and inoculated into BHI medium for further culture.

[0040] like Figure 1 As shown, the isolated strain appears pink, round, smooth, and with a metallic sheen on MacConkey agar. Gram staining revealed that the isolated Klebsiella pneumoniae is a Gram-negative bacterium. The strain appears rod-shaped under a microscope and produces acid and gas after incubation.

[0041] Klebsiella oxytoca KN-L was obtained from the feces of MAOS-fed C57BL mice. A specific isolation method is as follows:

[0042] Step 1: Obtaining mouse feces: Collect mouse feces after feeding donor mice with mannuronic acid oligosaccharide for three weeks, wherein the dosage of mannuronic acid oligosaccharide is 100 mg / kg / d;

[0043] Step 2: Dilution of feces: Dilute the collected mouse feces by adding 1g of feces to 10mL of PBS;

[0044] Step 3: Expanding the culture of fecal microbiota: The fecal samples were then inoculated into BHI broth with 10% MAOS added and cultured at 37°C for 24 hours to increase the abundance of Klebsiella spp.

[0045] Step 4: Inoculation: Inoculate the enriched bacterial suspension onto BHI plates and incubate at 37°C for 48 hours.

[0046] Step 5: Single colony culture: Select a single colony with a toothpick and inoculate it into BHI culture medium for further culture.

[0047] Step 6: Gram staining of the strain.

[0048] Step 7: Detection of the biochemical characteristics of the strain.

[0049] Furthermore, in step 6, strains that are Gram-negative are selected.

[0050] Moreover, the Klebsiella pneumoniae isolated in step 7 is a smooth, round, milky-white colony that can ferment glucose to produce acid and gas; it appears pink and has a metallic sheen on MacConkey agar.

[0051] Example 2

[0052] MAOS induces DOPC production from Klebsiella oxytoca KN-L metabolism.

[0053] Klebsiella acidogenic bacteria were cultured in LB medium, with two groups: a CON group and a MAOS (20 μg / mL) group, each with six replicates. Klebsiella acidogenic bacteria were inoculated into LB medium at a ratio of 1% and cultured at 37°C for 12 h at 200 rpm. The supernatant was then collected by centrifugation at 8000 rpm for 5 min. The effects of MAOS on Klebsiella metabolites were investigated using non-targeted metabolomics (LC-MS). Metabolites were identified by M / Z (mass-to-charge ratio, the ratio of the mass of a charged ion to its charge) based on the time-of-flight of each metabolite ion in positive and negative ion scanning modes, and abundance was calculated based on the peak area.

[0054] PLS-DA analysis can effectively differentiate samples between groups. The results of PLS-DA analysis of the metabolome in positive ion mode are as follows: Figure 2 As shown in Figure A, the horizontal axis represents differences between groups, and the vertical axis represents differences within groups. After MAOS treatment, the spatial distribution of the KN-L strain metabolome was clearly separated. The results indicate that MAOS can detect changes in the metabolite composition of Klebsiella pneumoniae.

[0055] After standardizing the relative abundance of all DAMs, hierarchical clustering was performed, and it was found that after MAOS treatment, the production of DOPC by Klebsiella acidogenic KN-L metabolism was significantly increased. Figure 2 Extractive ion chromatography (XIC) of DOPC in B showed a retention time (RT) of 8.95 min. After MAOS treatment, the peak value and peak area of ​​DOPC increased, indicating an increase in the concentration of DOPC.

[0056] A specific method for preparing DOPC using the fermentation metabolism of Klebsiella oxytoca KN-L is as follows:

[0057] Step 1: Inoculate 20 μL of KN-L bacterial suspension into 100 mL of LB medium;

[0058] Step 2: Add mannouronic acid oligosaccharides to the culture medium;

[0059] Step 3: Incubate the strain at 200 rpm in a shaker at 37°C for 12 hours;

[0060] Step 4: The OD value of the bacterial suspension was found to be approximately 1.0;

[0061] Step 5: Centrifuge at 8000 rpm for 5 min and collect the supernatant;

[0062] Step 6: Detect the DOPC content in the supernatant using LC-MS technology.

[0063] The MAOS used in step 2 has a concentration of 20 μg / mL.

[0064] Example 3

[0065] The protective effect of DOPC produced by Klebsiella oxytoca KN-L on the intestinal barrier in mice.

[0066] Twenty-four 7-week-old male C57BL / 6J mice were randomly divided into three groups: a control group, a DSS group, and a DOPC group, with eight mice in each group. The DOPC group received DOPC solution (20 mg / mL) in their drinking water for 21 consecutive days. Simultaneously, the control and DSS groups received PBS in their drinking water for 21 days. Subsequently, mice in the DSS and DOPC groups received 2% DSS in their drinking water for 7 consecutive days. Mice were housed in a standardized laboratory at 25±2℃, 50±5% relative humidity, and a 12h light-12h dark cycle. After acclimatization and normal feeding for 3 days, the experiment lasted for 28 days. After sacrifice, approximately 1 cm of colonic tissue was taken from each mouse and placed in a 1.5 mL EP tube. The tissue was fixed with 4% formaldehyde fixative at room temperature for 24 h, then embedded in paraffin, sectioned to a thickness of 6 μm, dewaxed, and rehydrated. After antigen retrieval, sections were blocked with 5% BSA for 60 min, then incubated with primary antibody overnight at 4℃. Subsequently, sections were incubated with the appropriate fluorescent secondary antibody at room temperature for 2 hours. Finally, the intestinal tight junction protein was stained with DAPI for 15 minutes and observed using a fluorescence microscope (Nikon Eclipse C1) to determine its localization and expression level.

[0067] Transmission electron microscopy (TEM) observations of the intestinal barrier structure showed that the colonic tissue of DSS-treated mice was damaged, with shortened microvilli and widened intercellular spaces. Figure 3 A). However, compared to the DSS group, administration of DOPC resulted in the restoration of regularly arranged microvilli and a reduction in the gaps between tight junctions between cells (A). Figure 3 A). Immunofluorescence staining showed that Claudin-1 and ZO-1 expression was disordered and reduced in DSS-treated mouse colon tissue. Figure 3 B). Conversely, the expression of Claudin-1 and ZO-1 in the intestines of mice supplemented with DOPC increased. This indicates that DOPC can protect the intestinal barrier function in mice.

[0068] Example 4

[0069] The protective effect of DOPC produced by Klebsiella oxytoca KN-L on the blood-brain barrier in mice.

[0070] Twenty-four 7-week-old male C57BL / 6J mice were randomly divided into three groups: a control group, a DSS group, and a DOPC group, with eight mice in each group. The DOPC group received DOPC solution (20 mg / mL) in their drinking water for 21 consecutive days. Simultaneously, the control and DSS groups received PBS in their drinking water for 21 days. Subsequently, mice in the DSS and DOPC groups received 2% DSS in their drinking water for 7 consecutive days. Mice were housed in a standardized laboratory at 25±2℃, 50±5% relative humidity, and a 12h light-12h dark cycle. After acclimatization and normal feeding for 3 days, the experiment lasted for 28 days. After sacrifice, approximately 1 cm of colonic tissue was taken from each mouse and placed in a 1.5 mL EP tube. The tissue was fixed with 4% formaldehyde fixative at room temperature for 24 h, then embedded in paraffin, sectioned to a thickness of 6 μm, dewaxed, and rehydrated. After antigen retrieval, sections were blocked with 5% BSA for 60 min, then incubated with primary antibody overnight at 4℃. Subsequently, sections were incubated with the appropriate fluorescent secondary antibody at room temperature for 2 hours. Finally, the intestinal tight junction protein was stained with DAPI for 15 minutes and observed using a fluorescence microscope (Nikon Eclipse C1) to determine its localization and expression level.

[0071] We found that DSS treatment led to diffuse damage to vascular tight junctions and endothelial cells in brain tissue. Conversely, administration of DOPC improved tight junctions, resulting in clear, dense bands, and reduced endothelial cell damage. Figure 4 A). Claudin-5 is mainly located on the cell membrane of brain capillary endothelial cells and plays a crucial role in the integrity of the blood-brain barrier. Immunofluorescence analysis showed that, compared with the control group, DSS led to a decrease in the expression of Claudin-5 and ZO-1 around brain capillaries, while DOPC supplementation effectively restored the levels of these tight junction proteins in the blood-brain barrier. Figure 4 B).

[0072] Example 5

[0073] The inhibitory effect of DOPC produced by Klebsiella oxytoca KN-L on neuroinflammation.

[0074] Lipopolysaccharide (LPS) was used to stimulate BV2 microglia to simulate an inflammatory environment in the brain. BV2 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS, Gibco) and penicillin-streptomycin (100 μg / mL, Gibco) at 37°C and 5% CO2. Then, they were cultured at 10... 5 cells / cm 2BV2 cells were seeded into 6-well plates at a density of [insert density here] and incubated overnight at 37°C. Subsequently, the cells were treated with 20 μg / mL DOPC at 37°C for 24 h. The cells were washed three times with PBS, then stimulated with LPS (20 μg / mL) for 3 h. Finally, the BV2 cells were collected for further analysis.

[0075] like Figure 5 The results showed that LPS treatment significantly increased the levels of pro-inflammatory cytokines IL-6, IL-8, and TNF-α, while decreasing the levels of anti-inflammatory cytokines IL-4 and IL-10. Figure 5 (AE). Notably, the addition of DOPC effectively attenuated the inflammatory response of BV2 microglia, leading to a decrease in pro-inflammatory cytokines (IL-6, IL-8, TNF-α) and an increase in anti-inflammatory cytokines (IL-4, IL-10).

[0076] In summary, the beneficial effects of the present invention are as follows:

[0077] (1) The acid-producing Klebsiella oxytoca KN-L provided by this invention can metabolize dioleoyl lecithin (DOPC) under the induction of MAOS. The biosynthetic method of producing DOPC by fermentation metabolism using this strain is lower in cost than traditional physical and chemical methods, and more environmentally friendly and efficient than traditional chemical synthesis methods.

[0078] (2) Experiments have shown that DOPC produced by Klebsiella oxytoca KN-L can protect the intestinal barrier function and significantly protect the integrity of the blood-brain barrier when used in mice.

[0079] (3) Experiments have shown that DOPC produced by Klebsiella oxytoca KN-L can significantly inhibit neuroinflammation in vitro.

Claims

1. A Klebsiella oxytoca KN-L strain, which is deposited with the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on April 12, 2024, and has the accession number CCTCC NO: M2024683, and is taxonomically named as Klebsiella oxytoca.

2. The Klebsiella oxytoca KN-L according to claim 1, characterized by: The 16S rDNA sequence of the strain is shown in SEQ ID NO.

1.

3. The use of Klebsiella oxytoca KN-L according to claim 1, characterized in that: A method for preparing dioleoyl lecithin.

4. The use of Klebsiella oxytoca KN-L according to claim 3, characterized in that, The method for preparing dioleoyl lecithin is as follows: Step 1: inoculate the Klebsiella oxytoca KN-L strain suspension into LB medium; Step 2: add mannuronic acid oligosaccharide to the medium; Step 3: cultivate the strain in a 37℃ shaking bed at 200 rpm for 12 hours; Step 4: detect the OD value of the bacterial suspension as 1.0; Step 5: centrifuge at 8000 rpm for 5 minutes to collect the supernatant.

5. The use of Klebsiella oxytoca KN-L according to claim 1, characterized in that: For the production of a preparation of dioleoyl lecithin, the number of Klebsiella oxytoca KN-L in the preparation is ≥ 1 x 10 7 CFU / g or 1 x 10 7 CFU / mL.

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