Screening method and application of acid-producing bacteroides

Abstract

The invention belongs to the technical field of strain screening, and particularly relates to a screening method of acid-producing bacteroides, and the screening method specifically comprises the following steps: taking a fresh sample, mixing and dispersing, and then carrying out gradient centrifugal purification to obtain a single-cell suspension; injecting the single-cell suspension into a micro-fluidic chip, and distributing the cells into a culture room one by one to obtain single cells; inoculating the single cells subjected to micro-control flow treatment into a bioreactor, simulating an intestinal environment for culture, observing the morphology and growth condition of the cells through a microscope, measuring the pH value of a culture solution and the content of short-chain fatty acid, and selecting a strain with the highest acid yield and good growth state; the method comprises the following steps: centrifuging a bacterial strain with higher activity to obtain pure thalli, preparing a bacterial suspension, culturing the bacterial strain by adopting a solid culture medium taking gel polysaccharide as a unique carbon source, and selecting the bacterial strain with higher activity as a target bacterial strain. The defects in the prior art are overcome, and the acid-producing bacteroides capable of degrading and utilizing the gel polysaccharide is screened by adopting the technologies of micro-control flow and simulated intestinal culture.

Description

Technical Field

[0001] The present invention belongs to the technical field of strain screening, and particularly relates to a screening method and application of acidogenic Bacteroides. Background Art

[0002] The human gut is home to one of the densest and most diverse microbial communities known, with the gene content of the gut microbiome being a staggering hundred times higher than that of the host. The colon is the primary home of intestinal bacteria, with bacterial levels estimated at 1012 / ml. In recent years, a growing body of research has demonstrated the inextricable relationship between the gut microbiome and human health.

[0003] Bacteroides is a very important group of intestinal bacteria. It is a Gram-negative, non-spore-forming, anaerobic, rod-shaped bacterium with an outer membrane, a peptidoglycan layer, and a cytoplasmic membrane. The main byproducts of its anaerobic respiration are acetic acid, isovaleric acid, and succinic acid. Members of the genus Bacteroides are the foundation of the colonic flora and play multiple roles in the human intestine: they not only provide nutrition to the host and other microorganisms living near them, but also, as one of the next generation of probiotics, benefit host health through various pathways.

[0004] Members of the genus Bacteroides possess a broad range of polysaccharide degradation capabilities—an ability not inherent in the human body. They can degrade a wide range of polysaccharides, providing nutrients for the host and other intestinal microorganisms. This crucial role in maintaining intestinal microbial homeostasis is crucial, and in the process, they produce numerous metabolites that benefit host health. For example, Bacteroides thetaiotaomicron strain VPI-5482 possesses nearly 100 polysaccharide utilization sites, enabling it to degrade a wide range of polysaccharides.

[0005] Probiotics, a group of bacteria beneficial to human health, are widely loved by the public, and the probiotics market has enormous potential. Data shows that the scale of China's probiotics market has been growing year by year.

[0006] Currently, the majority of probiotics used commercially on a large scale come from foreign companies, and the majority of probiotic strains used in the Chinese probiotic market are still from foreign companies. The gut microbiome of Western cultures may not be suitable for Chinese people, and the intestinal environment of Chinese people may not be suitable for the colonization of these strains, which may affect their efficacy. Furthermore, the current dietary diversity of Chinese people means that intestinal issues in China require domestically produced probiotics tailored to the Chinese population. Therefore, screening for probiotics that are more suitable for the Chinese population's constitution holds great market potential. Summary of the Invention

[0007] The purpose of the present invention is to provide a screening method and application of acid-producing Bacteroides, which overcomes the shortcomings of the existing technology. The technology of micro-controlled flow and simulated intestinal culture is used to screen out acid-producing Bacteroides that can degrade and utilize curdlan, and make a biological agent that helps intestinal digestion.

[0008] In order to solve the above problems, the technical solutions adopted by the present invention are as follows:

[0009] A method for screening acidogenic Bacteroides comprises the following steps:

[0010] S1. Sample initial processing: fresh human fecal samples were collected, mixed and dispersed, and then purified by gradient centrifugation to obtain a single-cell suspension;

[0011] S2, micro-control flow processing: inject the single cell suspension into the microfluidic chip, and use the microvalves and microchannels in the chip to distribute the cells one by one to the culture chamber to obtain single cells;

[0012] S3. Simulated culture: Single cells treated with micro-flow are inoculated into a bioreactor and cultured in a simulated intestinal environment. Cell morphology and growth are observed under a microscope. The pH value and short-chain fatty acid content of the culture medium are measured. The strain with the highest acid production and the best growth status is selected.

[0013] S4. Purification culture: After centrifugation of the strain with higher activity to obtain pure bacteria, resuspend it in PBS to prepare a bacterial suspension, and culture the strain in a solid culture medium with curdlan as the only carbon source. The strain with higher activity is selected as the target strain.

[0014] Furthermore, the gradient centrifugation purification after mixing and dispersion in S1 includes the following specific steps:

[0015] S11. Place the human fecal sample into a sterile sampling tube, add 20 mL of sterile PBS buffer, and vortex for 5 minutes to fully disperse the sample.

[0016] S12. Transfer the dispersed sample to a 50 mL centrifuge tube, centrifuge at 3000 × g at 4°C for 10 minutes, discard the supernatant, and collect the precipitate; add 10 mL of lysis buffer containing 1 mg / mL proteinase K and 10 mg / mL lysozyme to the precipitate, and incubate at 37°C for 30 minutes; then add DNase I and RNase A, and incubate at 37°C for 15 minutes to degrade nucleic acids; filter the treated sample through a 0.22 μm filter membrane to remove unlysed impurities to obtain a crude microbial cell suspension;

[0017] S13. Place the crude cell suspension in a centrifuge tube containing 40% and 60% Percoll gradient solutions, centrifuge at 8000×g at 4° C. for 30 minutes, and collect the high-purity single-cell suspension at the interface between 40% and 60%.

[0018] Furthermore, the micro-control flow processing in S2 includes the following specific steps:

[0019] S21, injecting the single-cell suspension into the microfluidic chip at a flow rate of 1 μL / min through a peristaltic pump, and using the microvalves and microchannels in the chip to distribute the cells one by one to the culture chamber;

[0020] S22. Turn on the pH sensor built into the chip and continuously monitor the pH value. When it is detected that the pH value of a culture chamber drops by more than 0.5 units, the chip automatically activates the microvalve to isolate the culture chamber and collect single cells.

[0021] Furthermore, the simulated intestinal environment described in S3 includes:

[0022] (1) Start the anaerobic incubator, set the temperature to 37°C, and adjust the gas ratio to 85% N, 10% H, and 5% CO;

[0023] (2) Turn on the peristaltic simulation device, set the frequency to 6 times / min, and the amplitude to 5 mm;

[0024] (3) Fresh culture medium was added to the reactor at a flow rate of 0.5 mL / h using a constant flow pump to simulate intestinal nutrient flow.

[0025] Furthermore, the fresh culture medium is a GAM liquid culture medium, which contains L-cysteine hydrochloride, histidine, vitamin K3, hemin, vitamin B12, vancomycin hydrochloride, and kanamycin sulfate.

[0026] The present invention also protects the use of the acidogenic Bacteroides in degrading curdlan.

[0027] The present invention finally protects a probiotic preparation, which contains the above-mentioned acidogenic Bacteroides; the content of acidogenic Bacteroides in the probiotic preparation is 1.875×108 CFU / mL

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The acidogenic Bacteroides screened by the present invention has good curdlan degradation ability. It can degrade curdlan added to various foods that is difficult for the human body to digest and utilize, and produce laminarin oligosaccharides that have been proven to be beneficial to the human body. It not only provides more nutrition for the human body and other intestinal flora, but also is beneficial to human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the culture results of Bacteroides acidophilus on solid culture medium with curdlan as the sole carbon source.

[0031] Figure 2Schematic diagram of the changes in transcription levels of acidogenic Bacteroides genes.

[0032] Figure 3 Schematic diagram of the results of curdlan degradation by Bacteroides acidophilus. DETAILED DESCRIPTION

[0033] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0034] Example 1

[0035] This embodiment discloses a method for screening acidogenic Bacteroides, which specifically comprises the following steps:

[0036] S1. Sample initial processing: fresh human fecal samples were collected, mixed and dispersed, and then purified by gradient centrifugation to obtain a single-cell suspension;

[0037] S2, micro-control flow processing: inject the single cell suspension into the microfluidic chip, and use the microvalves and microchannels in the chip to distribute the cells one by one to the culture chamber to obtain single cells;

[0038] S3. Simulated culture: Single cells treated with micro-flow are inoculated into a bioreactor and cultured in a simulated intestinal environment. Cell morphology and growth are observed under a microscope. The pH value and short-chain fatty acid content of the culture medium are measured. The strain with the highest acid production and the best growth status is selected.

[0039] S4. Purification culture: After centrifugation of the strain with higher activity to obtain pure bacteria, resuspend it in PBS to prepare a bacterial suspension, and culture the strain in a solid culture medium with curdlan as the only carbon source. The strain with higher activity is selected as the target strain.

[0040] The gradient centrifugation purification after mixing and dispersion in S1 includes the following specific steps:

[0041] S11. Place the human fecal sample into a sterile sampling tube, add 20 mL of sterile PBS buffer, and vortex for 5 minutes to fully disperse the sample.

[0042] S12. Transfer the dispersed sample to a 50 mL centrifuge tube, centrifuge at 3000 × g at 4°C for 10 minutes, discard the supernatant, and collect the precipitate; add 10 mL of lysis buffer containing 1 mg / mL proteinase K and 10 mg / mL lysozyme to the precipitate, and incubate at 37°C for 30 minutes; then add DNase I and RNase A, and incubate at 37°C for 15 minutes to degrade nucleic acids; filter the treated sample through a 0.22 μm filter membrane to remove unlysed impurities to obtain a crude microbial cell suspension;

[0043] S13. Place the crude cell suspension in a centrifuge tube containing 40% and 60% Percoll gradient solutions, centrifuge at 8000×g at 4° C. for 30 minutes, and collect the high-purity single-cell suspension at the interface between 40% and 60%.

[0044] The micro-control flow processing in S2 includes the following specific steps:

[0045] S21, injecting the single-cell suspension into the microfluidic chip at a flow rate of 1 μL / min through a peristaltic pump, and using the microvalves and microchannels in the chip to distribute the cells one by one to the culture chamber;

[0046] S22. Turn on the pH sensor built into the chip and continuously monitor the pH value. When it is detected that the pH value of a culture chamber drops by more than 0.5 units, the chip automatically activates the microvalve to isolate the culture chamber and collect single cells.

[0047] The simulated intestinal environment in S3 includes:

[0048] (3) Start the anaerobic incubator, set the temperature to 37°C, and adjust the gas ratio to 85% N, 10% H, and 5% CO;

[0049] (4) Turn on the peristaltic simulator, set the frequency to 6 times / min, and the amplitude to 5 mm;

[0050] (3) Fresh culture medium was added to the reactor at a flow rate of 0.5 mL / h using a constant flow pump to simulate intestinal nutrient flow.

[0051] The fresh culture medium is GAM liquid medium, which contains L-cysteine hydrochloride, histidine, vitamin K3, hemin, vitamin B12, vancomycin hydrochloride, and kanamycin sulfate.

[0052] Example 2

[0053] This embodiment discloses the use of Bacteroides acidophilus in degrading curdlan.

[0054] The screened acidogenic Bacteroides were inoculated on a solid culture medium containing KH2PO4, KOH, NaCl, (NH4)2SO4, MgCl2, CaCl2, FeSO4, L-cysteine hydrochloride, histidine, vitamin K3, hemin, vitamin B12, vancomycin hydrochloride, and kanamycin sulfate as the sole carbon source, and placed in an anaerobic incubator at 37°C for inverted culture. OD600 was continuously monitored. The results are shown in the attached figure. Figure 1 .

[0055] The results showed that the screened acidogenic Bacteroides strain J21 could grow rapidly on solid culture medium with curdlan as the sole carbon source, indicating that it has a certain ability to degrade curdlan.

[0056] Using dbCAN3 prediction and prokaryotic transcriptome sequencing, we initially screened for glycoside hydrolases that may have curdlan degradation activity. Prokaryotic transcriptome sequencing involved sequencing bacteria grown on curdlan-only carbon source plates for days 0 and 1. Fluorescence quantitative PCR was then used to verify changes in the transcriptional levels of these genes. The results are shown in the attached figure. Figure 2 .

[0057] The results showed that the transcription level of this gene in Bacteroides acidophilus continued to increase over time, indicating that this gene may be involved in the degradation of curdlan.

[0058] The function of this gene was further verified by in vitro TCL enzyme activity detection. Figure 3 .

[0059] The results showed that the protein of acidogenic Bacteroides can indeed degrade curdlan into laminarin oligosaccharides, thereby producing metabolites that are beneficial to the human body.

[0060] Based on the application of acidogenic Bacteroides in degrading curdlan, this embodiment further discloses a probiotic preparation containing the acidogenic Bacteroides; the bacterial content of acidogenic Bacteroides in the probiotic preparation is 1.875×108 CFU / mL.

[0061] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

Claims

1. A method for screening acidogenic Bacteroides, characterized in that: The specific steps include: S1. Sample initial processing: fresh human fecal samples were collected, mixed and dispersed, and then purified by gradient centrifugation to obtain a single-cell suspension; S2, micro-control flow processing: inject the single cell suspension into the microfluidic chip, and use the microvalves and microchannels in the chip to distribute the cells one by one to the culture chamber to obtain single cells; S3. Simulated culture: Single cells treated with micro-flow are inoculated into a bioreactor and cultured in a simulated intestinal environment. Cell morphology and growth are observed under a microscope. The pH value and short-chain fatty acid content of the culture medium are measured. The strain with the highest acid production and the best growth status is selected. S4. Purification culture: After centrifugation of the strain with higher activity to obtain pure bacteria, resuspend it in PBS to prepare a bacterial suspension, and culture the strain in a solid culture medium with curdlan as the only carbon source. The strain with higher activity is selected as the target strain.

2. The method for screening acidogenic Bacteroides according to claim 1, wherein: The mixed dispersion and subsequent gradient centrifugation purification described in S1 includes the following specific steps: S11. Place the human fecal sample into a sterile sampling tube, add 20 mL of sterile PBS buffer, and vortex for 5 minutes to fully disperse the sample. S12. Transfer the dispersed sample to a 50 mL centrifuge tube, centrifuge at 3000 × g at 4°C for 10 minutes, discard the supernatant, and collect the precipitate; add 10 mL of lysis buffer containing 1 mg / mL proteinase K and 10 mg / mL lysozyme to the precipitate, and incubate at 37°C for 30 minutes; then add DNase I and RNase A, and incubate at 37°C for 15 minutes to degrade nucleic acids; filter the treated sample through a 0.22 μm filter membrane to remove unlysed impurities to obtain a crude microbial cell suspension; S13. Place the crude cell suspension in a centrifuge tube containing 40% and 60% Percoll gradient solution, centrifuge at 8000×g at 4°C for 30 minutes, and collect the high-purity single-cell suspension at the interface between 40% and 60%.

3. The method for screening acidogenic Bacteroides according to claim 1 or 2, wherein: The micro-control flow processing described in S2 includes the following specific steps: S21, injecting the single-cell suspension into the microfluidic chip at a flow rate of 1 μL / min through a peristaltic pump, and using the microvalves and microchannels in the chip to distribute the cells one by one to the culture chamber; S22. Turn on the pH sensor built into the chip to continuously monitor the pH value. When it is detected that the pH value of a culture chamber drops by more than 0.5 units, the chip automatically activates the microvalve to isolate the culture chamber and collect single cells.

4. The method for screening acidogenic Bacteroides according to claim 3, wherein: The simulated intestinal environment described in S3 includes: (1) Start the anaerobic incubator, set the temperature to 37°C, and adjust the gas ratio to 85% N, 10% H, and 5% CO; (2) Turn on the peristaltic simulation device, set the frequency to 6 times / min, and the amplitude to 5 mm; (3) Fresh culture medium was added to the reactor at a flow rate of 0.5 mL / h using a constant flow pump to simulate intestinal nutrient flow.

5. The method for screening acidogenic Bacteroides according to claim 4, characterized in that: The fresh culture medium is a GAM liquid culture medium, which contains L-cysteine hydrochloride, histidine, vitamin K3, hemin, vitamin B12, vancomycin hydrochloride, and kanamycin sulfate.

6. Use of the acidogenic Bacteroides according to claim 1 in degrading curdlan.

7. A probiotic preparation, characterized in that The probiotic preparation contains the acidogenic Bacteroides according to claim 1; the bacterial content of the acidogenic Bacteroides in the probiotic preparation is 1.875×108 CFU / mL.

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