Klebsiella oxytoca KN-L and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an acid-producing Klebsiella oxytoca KN-L. The acid-producing Klebsiella oxytoca KN-L. is preserved in the China Center for Type Culture Collection, the address is Wuhan University, Wuhan, China, and the preservation number is CGMCC NO. The strain is preserved in China Center for Type Culture Collection (CCTCC) on April 12, 2024, the postal code is 430072, the preservation number is CCTCC NO: M2024683, and the strain is named as Klebsiella oxytoca in taxonomy. When the Klebsiella oxytoca KN-L provided by the invention is subjected to in-vitro fermentation, MAOS is added, so that the metabolic production of DOPC can be obviously increased, and experiments prove 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 by fermentation metabolism of the strain can significantly reduce the production cost of DOPC.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to Klebsiella oxytocaKN-L and application thereof. Background Art

[0002] The blood-brain barrier is a physical and biochemical barrier between the brain and the blood. It has extremely important physiological functions. It can prevent external toxins and pathogens from entering the brain tissue from the blood and maintain the stability of the internal environment of the brain. It can also selectively regulate the entry and exit of specific molecules through active transport and passive diffusion mechanisms to ensure that the brain receives sufficient nutrition and oxygen. When the blood-brain barrier is damaged or destroyed, harmful substances such as bacteria, viruses and toxins can easily enter the brain, increasing the risk of neuronal damage and inflammatory response, and even causing brain diseases or nervous system dysfunction.

[0003] Mannosyl oligosaccharides (MAOS) are sodium salts of β-D-mannuronic acid oligosaccharides with different polymerization degrees, which are prepared from alginate by specific enzyme degradation, classification and gel chromatography separation. Studies have reported that mannosyl oligosaccharides (GV-971) prepared from MAOS can reshape intestinal flora, inhibit neuroinflammation, and thus improve cognitive function in patients with Alzheimer's disease (AD).

[0004] Dioleoylphosphatidylcholine (DOPC) is a common phospholipid compound and a key component of cell membranes. In particular, in the formation of liposomes, it can form a stable lipid bilayer and has an important influence on the fluidity and permeability of cell membranes. In the intestine, DOPC can play a variety of regulatory roles, including promoting the stability of cell membranes, participating in lipid metabolism and cell signal transduction. In cardiovascular diseases, DOPC is involved in the regulation of blood lipid metabolism and endothelial function. Abnormal DOPC metabolism or distribution may be related to the occurrence and development of diseases such as atherosclerosis. In addition, in nervous system diseases, DOPC is of great significance to the stability and function of nerve cell membranes, and its changes may be related to neurodegenerative diseases. DOPC is currently mainly extracted and purified from egg yolks and soybeans by physical means, and can also be synthesized by chemical phospholipids. The physical and chemical processes are complex and the yield is limited.

[0005] Since the production cost of DOPC in the prior art is high, it is urgent to find a method and strain for producing DOPC by biosynthesis with high yield and low cost. Summary of the invention

[0006] The present invention aims to provide a strain of Klebsiella oxytoca KN-L and an application method thereof for producing DOPC. The strain can significantly increase the metabolic production of DOPC by adding MAOS during in vitro fermentation, and experiments have shown that the DOPC produced by the strain can alleviate nerve damage both in vivo and in vitro. The biosynthetic method of producing DOPC by fermentation metabolism using the strain can significantly reduce the production cost of DOPC.

[0007] The specific technical solution adopted is: an acid-producing Klebsiella oxytoca KN-L, which is deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China; postal code 430072, the preservation date is April 12, 2024, the preservation number is: CCTCC NO: M2024683, and the taxonomic name is Klebsiella oxytoca.

[0008] Moreover, the 16SrDNA sequence of the strain is shown as SEQ ID NO.1.

[0009] And, the strain is used for preparing dioleoyl phosphatidylcholine.

[0010] And, the method for preparing dioleoyl lecithin using the strain is:

[0011] Step 1: inoculate a suspension of Klebsiella oxytoca KN-L into LB medium;

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

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

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

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

[0016] And, the strain is used to prepare a preparation for producing dioleoyl lecithin, and the number of Klebsiella oxytoca KN-L in the preparation is ≥1×10 7 CFU / g or 1×10 7 CFU / mL.

[0017] And, use of the strain in preparing a drug for protecting the intestinal barrier.

[0018] And, use of the strain in preparing a drug for protecting the blood-brain barrier.

[0019] And, use of the strain in preparing drugs for inhibiting neuroinflammation.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The Klebsiella oxytoca KN-L provided by the present invention can metabolize and produce dioleoylphosphatidylcholine (DOPC) under the induction of MAOS. The biosynthesis method of producing DOPC by fermentation metabolism using the strain is lower in cost than traditional physical and chemical methods, and is 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 intervened in mice.

[0023] (3) Experiments have shown that DOPC produced by Klebsiella oxytoca KN-L can significantly inhibit neuroinflammation in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 :Identification of strains of Klebsiella oxytoca

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

[0026] Figure 3 :Protective effect of DOPC produced by Klebsiella oxytoca KN-L on the intestinal barrier of mice

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

[0028] Figure 5 :Inhibitory effect of DOPC produced by Klebsiella oxytoca KN-L on neuroinflammation DETAILED DESCRIPTION

[0029] The present invention is described in detail below with reference to the accompanying drawings and embodiments, but the present invention is not limited to the following embodiments.

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

[0031] (1) Source of MAOS: Mannosyl oligosaccharide was purchased from Qingdao Bozhihuili Biotechnology Co., Ltd. Its molecular weight is 6.44 kDa, M / G≈12.56, and its 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, and 20.0 g / L agar.

[0033] (3) LB medium: 10.0 g / L tryptone, 5.0 g / L yeast powder, and 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) 16S rRNA PCR identification of isolated bacteria: single colonies were picked after purification for 16S rRNA PCR amplification. The primer sequences of bacterial 16S rRNA were: upstream primer: 5'-AGAGTTTGATCCTGGCTCAG-3'; downstream primer: 5'-GGTTACCTTGTTACGACTT-3'. The expected amplified product length was 1466 bp. PCR reaction system 50 μL: TaqMaster Mix 25 μL, upstream and downstream primers (10 μmol / L) 2 μL each, ddH2O 21 μL. PCR reaction conditions: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 90 s, 30 cycles in total; 72℃ extension for 10 min. After the PCR reaction was completed, 10 μL was taken. The amplified products were detected by 1.0% agarose gel electrophoresis, and the PCR products with matching band lengths were sequenced. The sequences obtained by sequencing were compared using the BLAST program in NCBI to determine the strain information.

[0036] Klebsiella oxytoca KN-L, the strain is deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China; postal code 430072, the deposit date is April 12, 2024, the deposit number is: CCTCC NO: M2024683, and the taxonomic name is Klebsiella oxytoca.

[0037] Example 1

[0038] Identification of Klebsiella oxytoca

[0039] Fecal samples of approximately 1 g were collected from mice fed with MAOS. The fecal samples were then inoculated into BHI broth supplemented with 10% MAOS and cultured at 37°C for 24 h to enhance the abundance of Klebsiella. Subsequently, the enriched bacterial suspension was streaked onto BHI plates and incubated at 37°C for 48 h. After observing the growth, individual colonies were selected with a toothpick and inoculated into BHI broth for further culture.

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

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

[0042] Step 1: Obtaining mouse feces: The feces of donor mice were collected after feeding mannuronic acid oligosaccharides for three weeks, wherein the dosage of mannuronic acid oligosaccharides was 100 mg / kg / d;

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

[0044] Step 3: Expansion of fecal flora: The fecal samples were then inoculated into BHI broth supplemented with 10% MAOS and cultured at 37°C for 24 h to increase the abundance of Klebsiella spp.

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

[0046] Step 5: Single colony culture: Use a toothpick to select a single colony and inoculate it in BHI culture medium for further culture.

[0047] Step 6: Gram staining of strains.

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

[0049] Furthermore, in step 6, a strain that is Gram-negative is selected.

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

[0051] Example 2

[0052] MAOS induces Klebsiella oxytoca KN-L to metabolize and produce DOPC

[0053] LB medium was used to culture acid-producing Klebsiella, with CON group and MAOS (20 μg / mL) group, each group had six replicates, and the acid-producing Klebsiella was inoculated into LB medium at a ratio of 1%, and cultured at 200 rpm and 37°C for 12 hours. Then the supernatant was collected by centrifugation at 8000 rpm for 5 minutes. The effect of MAOS on the metabolites of Klebsiella was studied using non-targeted metabolomics (LC-MS) technology. According to the flight time of each metabolite ion in the positive and negative ion scanning mode of the mass spectrometer, the metabolites were qualitatively identified by M / Z (mass-to-charge ratio, the ratio of the mass of the charged ion to the charge carried), and the abundance was calculated based on the mass spectrometry peak area.

[0054] PLS-DA analysis can distinguish samples between groups very well. The PLS-DA analysis results of the metabolome in positive ion mode are as follows Figure 2 As shown in A, the horizontal axis represents the difference between groups, and the vertical axis represents the difference within groups. After treatment with MAOS, the spatial distribution of the metabolome of the KN-L strain was significantly separated. The results showed that MAOS can change the metabolite composition of Klebsiella.

[0055] The relative abundances of all DAMs were standardized and hierarchical clustering was performed, and it was found that after MAOS treatment, the production of DOPC by Klebsiella oxytoca KN-L metabolism increased significantly. Figure 2 The extracted ion chromatogram (XIC) of DOPC in B showed that the retention time (RT) of DOPC was 8.95 min. After MAOS treatment, the peak value and peak area of ​​DOPC became larger, indicating that the concentration of DOPC increased.

[0056] A specific preparation method of DOPC produced by 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 mannuronic acid oligosaccharides to the culture medium;

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

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

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

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

[0063] The content of MAOS used in step 2 is 20 μg / mL.

[0064] Example 3

[0065] Protective effect of DOPC produced by Klebsiella oxytoca KN-L on the intestinal barrier of mice

[0066] Seven-week-old C57BL / 6J male mice (a total of 24 mice) were randomly divided into three groups, namely the control group, the DSS group, and the DOPC group, with 8 mice in each group. The DOPC group was given DOPC solution (20 mg / mL) in drinking water for 21 consecutive days. At the same time, the control and DSS groups were given PBS drinking water for 21 days. Thereafter, the mice in the DSS and DOPC groups drank 2% DSS for 7 consecutive days. The mice were housed in a standardized laboratory at 25±2℃, relative humidity of 50±5%, 12h light and 12h dark. The 28-day experiment began after 3 days of adaptation to normal feeding. After the mice were killed, about 1 cm of colon tissue was taken from each mouse in a 1.5mL EP tube, fixed with 4% formaldehyde fixative at room temperature for 24h, and then the tissue was embedded in paraffin, sliced ​​at 6μm thickness, dewaxed, and rehydrated. After antigen retrieval, the sections were blocked with 5% BSA for 60min and then incubated with primary antibodies at 4℃ overnight. The sections were then incubated with corresponding fluorescent secondary antibodies for 2 hours at room temperature. Finally, the cells were stained with DAPI for 15 min, and the localization and expression of intestinal tight junction proteins were observed using a fluorescence microscope (Nikon Eclipse C1).

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

[0068] Example 4

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

[0070] Seven-week-old C57BL / 6J male mice (a total of 24 mice) were randomly divided into three groups, namely the control group, the DSS group, and the DOPC group, with 8 mice in each group. The DOPC group was given DOPC solution (20 mg / mL) in drinking water for 21 consecutive days. At the same time, the control and DSS groups were given PBS drinking water for 21 days. Thereafter, the mice in the DSS and DOPC groups drank 2% DSS for 7 consecutive days. The mice were housed in a standardized laboratory at 25±2℃, relative humidity of 50±5%, 12h light and 12h dark. The 28-day experiment began after 3 days of adaptation to normal feeding. After the mice were killed, about 1 cm of colon tissue was taken from each mouse in a 1.5mL EP tube, fixed with 4% formaldehyde fixative at room temperature for 24h, and then the tissue was embedded in paraffin, sliced ​​at 6μm thickness, dewaxed, and rehydrated. After antigen retrieval, the sections were blocked with 5% BSA for 60min and then incubated with primary antibodies at 4℃ overnight. The sections were then incubated with corresponding fluorescent secondary antibodies for 2 hours at room temperature. Finally, the cells were stained with DAPI for 15 min, and the localization and expression of intestinal tight junction proteins were observed using a fluorescence microscope (Nikon Eclipse C1).

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

[0072] Example 5

[0073] Inhibitory effect of DOPC produced by Klebsiella oxytoca KN-L on neuroinflammation

[0074] Lipopolysaccharide (LPS) was used to stimulate BV2 microglia to simulate the 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. 5 cells / cm 2BV2 cells were seeded into 6-well plates at a density of 1.5 and incubated overnight at 37°C. Subsequently, the cells were treated with 20 μg / mL DOPC for 24 h at 37°C. The cells were washed three times with PBS, stimulated with LPS (20 μg / mL) for 3 h, and finally BV2 cells were collected for further analysis.

[0075] like Figure 5 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:

[0077] (1) The Klebsiella oxytoca KN-L provided by the present invention can metabolize and produce dioleoylphosphatidylcholine (DOPC) under the induction of MAOS. The biosynthesis method of producing DOPC by fermentation metabolism using the strain is lower in cost than traditional physical and chemical methods, and is 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 intervened 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 in China Center for Type Culture Collection, address: Wuhan University, Wuhan, China; postal code 430072, deposit date: April 12, 2024, deposit number: CCTCC NO: M2024683, taxonomic name: Klebsiella oxytoca.

2. The Klebsiella oxytoca KN-L according to claim 1, characterized in that: The 16SrDNA 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: Used to prepare dioleoyl lecithin.

4. The use of Klebsiella oxytoca KN-L according to claim 3, characterized in that: The method for preparing dioleoyl lecithin is: Step 1: inoculate a suspension of Klebsiella oxytoca KN-L into LB medium; Step 2: Add mannuronic acid oligosaccharides to the culture medium; Step 3: Incubate the strain in a shaker at 37°C at 200 rpm for 12 h; Step 4: Detect the OD value of the bacterial suspension to be 1.0; Step 5: Centrifuge at 8000 rpm for 5 min and collect the supernatant.

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

6. Use of Klebsiella oxytoca KN-L according to claim 1 in the preparation of a medicament for protecting the intestinal barrier.

7. Use of Klebsiella oxytoca KN-L according to claim 1 in the preparation of a medicament for protecting the blood-brain barrier.

8. Use of Klebsiella oxytoca KN-L according to claim 1 in the preparation of a medicament for inhibiting neuroinflammation.

Images