Method for enriching and screening bifidobacterium intestinal bacteria

Abstract

The invention relates to a method for enriching and screening bifidobacterium intestinal bacteria, and aims to efficiently separate bifidobacteria from complex intestinal flora. The method comprises the steps of basic anaerobic culture medium preparation, enrichment culture, enrichment culture, intestinal bacterium nucleic acid extraction, high-throughput sequencing and analysis, strain separation and culture, intestinal bacterium molecular identification and the like. Through a unique culture medium formula and culture conditions, efficient enrichment and screening of bifidobacteria can be realized under common laboratory conditions without professional anaerobic equipment. The method overcomes the problems of harsh growth conditions of bifidobacteria, low selective culture medium efficiency and the like in the prior art, and provides technical support for deep research and application of bifidobacteria.

Description

Technical Field

[0001] The present invention relates to the enrichment and isolation of a specific type of microorganism, and particularly to the isolation of strains with research and application value from human intestinal bacteria. Combined with a specially designed in vitro culture device and operation method, it is suitable for ordinary laboratories to carry out work under conventional experimental conditions. Background Art

[0002] A huge and diverse microbial community inhabits the human intestine. There are close interactions between them and the host, jointly maintaining the balance of the intestinal microecology and playing a crucial role in the host's nutrient metabolism, immune regulation, disease defense, etc. In recent years, with the rapid development of high-throughput sequencing technology and bioinformatics analysis, breakthroughs have been made in the research of the gut microbiome. More and more studies have shown that gut microbiota dysbiosis is closely related to the occurrence and development of various diseases, such as obesity, diabetes, inflammatory bowel disease, colorectal cancer, etc. The genus Bifidobacterium is an important member of the gut microbiota, belonging to the phylum Actinobacteria, family Bifidobacteriaceae. It is a group of Gram-positive, anaerobic, sporeless bacilli with Y or V-shaped branches. Bifidobacteria are widely present in the intestines, mouths, and vaginas of humans and animals, and are particularly abundant in the intestines of breastfed infants. As a representative strain of probiotics, Bifidobacterium has various physiological functions. For example, it can regulate the balance of the gut microbiota: Bifidobacterium can maintain the balance of the gut microbiota and prevent and treat intestinal diseases such as diarrhea and constipation by competitively inhibiting the growth of pathogenic bacteria and producing antibacterial substances; enhance immune function: Bifidobacterium can stimulate the intestinal immune system, promote the proliferation and differentiation of immune cells, and enhance the body's immune function; improve metabolic function: Bifidobacterium can participate in the metabolism of carbohydrates, proteins, and fats, reduce cholesterol levels, improve insulin sensitivity, and prevent and treat metabolic diseases such as obesity and diabetes; have an anti-tumor effect: Bifidobacterium can play an anti-tumor role by regulating the immune response and inducing apoptosis of tumor cells. Given the important physiological functions of Bifidobacterium, it is of great significance to develop efficient and convenient methods for enriching and screening Bifidobacterium. However, the current enrichment and screening of Bifidobacterium still face the following challenges: (1) Complexity of the gut microbiota: The gut microbiota is complex, consisting of hundreds of bacteria. Bifidobacterium is only a small part of it, and its abundance is affected by various factors such as age, diet, and environment; (2) Harsh growth conditions of Bifidobacterium: Bifidobacterium is a strict anaerobe and has high requirements for the growth environment, requiring special equipment such as an anaerobic incubator, with cumbersome operations and high costs; (3) Lack of efficient selective media: Currently commonly used selective media for Bifidobacterium, such as MRS medium, TPY medium, etc., although they can promote the growth of Bifidobacterium, may also promote the growth of other bacteria, resulting in low enrichment efficiency of Bifidobacterium. To solve the above problems, the present invention provides a new method for enriching and screening Bifidobacterium. This method combines a unique medium formula and culture conditions, and can efficiently and conveniently enrich and screen Bifidobacterium from complex gut microbiota, providing technical support for the in-depth study of Bifidobacterium and the development of new products. Summary of the Invention

[0003] In view of the above technical problems, the object of the present invention is to enrich and isolate the genus Bifidobacterium by using a unique device, process and culture medium formula in the case of the lack of professional instrument and equipment such as anaerobic workstations in ordinary laboratories, specifically including the following steps: (1) Preparation of basic anaerobic culture medium; (2) Preparation of enrichment culture; (3) Enrichment culture; (4) Extraction of intestinal bacteria nucleic acid; (5) High-throughput sequencing and analysis; (6) Isolation and culture of strains; (7) Molecular identification of intestinal bacteria; Preferably, the step (1) of preparing the basic anaerobic culture medium includes the following steps: ① Prepare the basic medium components according to the growth rules of most intestinal bacteria and adjust the pH value; these culture media generally contain necessary sugars, proteins, vitamins, and some short-chain fatty acids; ② Add resazurin at a concentration of one-thousandth as an oxygen indicator, fill with nitrogen, and sterilize by autoclaving or filter sterilization. Although the oxygen indicator is not necessary for the culture of all intestinal bacteria, it provides an important basis for the operation of culturing various intestinal bacteria. When the oxygen indicator shows red, the culture medium conditions cannot meet the growth of strict anaerobes, but can still meet the growth of facultative anaerobes and microaerophiles; ③ Prepare a cysteine solution with a concentration of more than 1% separately, fill with nitrogen, and sterilize by autoclaving or filtration, and use it before inoculation. Cysteine is a commonly used water-soluble deoxidizer, and cysteine can be directly added, or cysteine hydrochloride can be added, and the effects are equivalent; Preferably, the step (2) of preparing the special enrichment culture is carried out according to the following steps: ① Take out the preserved Pichia fermentans strain 15B1 (CGMCC No. 19317 of the China Center for Microbial Culture Collection) from the -80°C ultra-low temperature refrigerator and inoculate it into PDB medium and shake the bacteria at 32°C for 24 h; ② Centrifuge the obtained 15B1 bacterial liquid at 10000 rpm for 1 min and take the supernatant for standby; Preferably, the step (3) of enrichment culture includes the following steps: ① Add 1 / 2 pore volume of the basic liquid medium in step (1) to a 5 ml vial in sequence, and then add 1 / 20 pore volume of the fermentation supernatant in the above step (2); ② Add 1 / 10 volume of silicone oil, oil-soluble antioxidant and liquid paraffin respectively, and let them naturally stratify to block oxygen. The oil-soluble antioxidant can be vitamin E and unsaturated fatty acids; ③ Add a cysteine solution with a concentration of 1% and a volume of 1 / 10. Seal the container with a lid and place it in the refrigerator or at room temperature for more than 12 hours until the red color of the oxygen indicator fades. ④ Take 1 g of fresh feces from multiple people and add it to 10 ml of a basic medium solution containing 1% cysteine deoxidizer. Shake and mix well, and let it stand for 3 - 5 minutes. ⑤ Add the supernatant of the mixed bacterial liquid with a volume of 1 / 20 to the above ampoule bottle, and culture it in an incubator at 37°C. After the bacteria grow, directly dispense it into 1 - ml tubes, add glycerol with a final concentration of 15%, and store it frozen. Preferably, the intestinal bacteria nucleic acid extraction in step (4) includes the following steps: ① Take 1 ml of the above intestinal bacteria liquid sample, centrifuge it at 4000 g for 5 minutes and retain the precipitate. Add 95% zirconia grinding beads. Add 500 μl of CTAB DNA extraction solution to the centrifuge tube with grinding beads, and grind it on a grinder for 5 minutes. ② Place it in a water bath at 65°C and incubate for 15 minutes, then centrifuge at 12000 g for 1 minute. Take 400 μl of the supernatant into the adsorption column, and add 200 μl of absolute ethanol to the adsorption column. Mix well. After centrifuging at 12000 g for 1 minute, replace the collection tube. ③ Add 500 μl of 70% ethanol, centrifuge at 12000 g for 1 minute, replace the collection tube, and repeat this step once. Replace the collection tube again, place the washed adsorption column AC, drop 50 μl of elution buffer EB, centrifuge at 12000 g for 1 minute in a centrifuge, collect the DNA after elution, and perform electrophoresis and imaging scanning on the extracted DNA sample. Preferably, the high - throughput sequencing and analysis in step (5) includes the following steps: ① Design primers targeting the 16s rRNA v3 - v4 region for high - throughput sequencing, and estimate the proportion of each genus of bacteria through sequence alignment. ② Analyze the proportion of bacteria belonging to the genus Bifidobacterium in the sample. If it is less than 5%, take out the frozen tube and repeat the cultivation in step (3) and the nucleic acid extraction process in step (4). ③ If the proportion of bacteria belonging to the genus Bifidobacterium is greater than 5%, continue to step (6) for strain isolation. Preferably, the strain isolation and culture in step (6) includes the following steps: ① Prepare a solid medium plate containing the components of the above steps (1) and (2) in advance, and a ready - to - use anaerobic bag for coating the bacterial liquid (a ready - to - use anaerobic bag for anaerobic plate separation and culture, patent number: ZL201920214535.7). Prepare a ready - to - use anaerobic bag for coating the bacterial liquid in advance. ② Take 50 μl of the cryopreserved intestinal bacteria sample, dilute it 10 times, and spread it on the above solid medium plate, then culture it at 37 °C for 1 - 2 days; ③ Pick the clones and perform enrichment culture as in step (3) above. After the bacteria grow, use them for subsequent nucleic acid extraction; Preferably, the intestinal bacteria molecular identification in step (7) includes the following steps: ① Extract the nucleic acid of the single colony culture one by one according to the nucleic acid extraction method in step (4) above; ② Perform PCR experiments according to the following formula: The reaction volume is 20 μl, 2 μl of 10X Taq Buffer, 1.6 μl of 2.5 mM dNTP mixture, 1 u of Taq, 0.8 μl of each 10 μM primer. The upstream primer is BifLP: AACGCCATGCTGACCGAT, and the downstream primer BifRP is: AAGGCAGGTTGACGTCCAG. The template volume is 1 μl, and the rest is made up with pure water; The PCR reaction program is: 95 °C for 300 s; 94 °C for 15 s, 56 °C for 45 s, 72 °C for 45 s, for a total of 35 - 40 cycles; 72 °C for 300 s; ③ After the product is detected by electrophoresis, if there is a characteristic band of 200 bp, it is a positive colony. The bacteria to which this positive band belongs is the Bifidobacterium genus bacteria. Compared with the prior art, the present invention has the following beneficial effects: 1) It can culture Bifidobacterium bacteria under ordinary aerobic experimental conditions, breaking through the harsh growth conditions at low cost; 2) It can find samples suitable for screening Bifidobacterium bacteria through enrichment and sequencing, overcoming the sample complexity; 3) It has found a food additive formula that can induce the large growth of Bifidobacterium bacteria, reducing the screening workload. Description of the Drawings

[0004] Figure 1 . Positive band for Bifidobacterium bacteria screening. M is the marker of DL2000. Detailed Embodiments

[0005] Example 1. Preparation of the composition of YCFA anaerobic medium Preparation steps: (1) Weigh and add reagents according to the formula of YCFA liquid medium (1 L), and at the same time prepare a mother liquor of resazurin with a concentration of one-thousandth; (2) Add 1 ml of the resazurin mother liquor (1000 times) to 1 L of the liquid medium, so that the final concentration of resazurin in the medium is 1 ppm; (3) Fill the anaerobic bottle with liquid medium, pass high-purity nitrogen for 1 min, immediately cover the cap, and reserve it after autoclaving; (4) Prepare a 1% cysteine mother liquor of deoxidizer, pass high-purity nitrogen for 1 min, immediately cover the cap, and reserve it after autoclaving; Example 2. Preparation of Bifidobacterium enriched culture ① Take out the preserved Pichia fermentans strain 15B1 (CGMCC No. 19317 of China Center for Microbial Culture Collection) from the -80 °C ultra-low temperature refrigerator, inoculate it into PDB medium, and shake the bacteria at 32 °C for 24 h; ② Centrifuge the obtained 15B1 bacterial liquid at 10000 rpm for 1 min, and take the supernatant for standby; Example 3. Enrichment culture of Bifidobacterium ① In a 5 ml vial, sequentially add 1 / 2 pore volume of the YCFA anaerobic medium in Example 1, and then add 1 / 20 pore volume of the supernatant of the fermentation broth in Example 2 above; ② Add 1 / 20 - 1 / 10 volume of silicone oil, oil-soluble antioxidant, and liquid paraffin respectively, and let it naturally stratify to block oxygen. The oil-soluble antioxidant can be vitamin E and unsaturated fatty acids; ③ Add 1 / 20 - 1 / 10 volume of 1% cysteine solution, seal the cap, and place it in the refrigerator or at room temperature for more than 12 hours, waiting for the red color of the oxygen indicator to fade; ④ Take a total of 1 g of fresh feces from multiple people, add it to 10 ml of basic medium solution containing 1% cysteine deoxidizer, shake and disperse it evenly, and let it stand for 3 - 5 min; ⑤ Add 1 / 20 volume of the supernatant of the mixed fecal bacteria liquid to the above vial, culture it in a 37 °C incubator, and after the bacteria grow, directly dispense it into 1 ml tubes and freeze it at -80 °C with a final concentration of 15% glycerol; Example 4. Extraction of intestinal bacteria nucleic acid ① Take 1 ml of the above intestinal bacteria liquid sample, centrifuge it at 4000 g for 5 min and retain the precipitate, add 95% zirconia grinding beads, add 500 ul of CTAB DNA extraction solution to the centrifuge tube with grinding beads, and grind it on a grinder for 5 min; ② Place it in a 65 °C water bath for incubation for 15 min, centrifuge it at 12000 g for 1 min; Take 400 ul of the supernatant into the adsorption column, and add 200 ul of absolute ethanol to the adsorption column, mix well; After centrifuging at 12000 g for 1 min, replace the collection tube; ③ Add 500 μl of 70% ethanol, centrifuge at 12,000 g for 1 min, replace the collection tube, and repeat this step once more; replace the collection tube again, place the washed adsorption column AC, drip 50 μl of elution buffer EB, centrifuge at 12,000 g for 1 min, collect the DNA after elution, and perform electrophoresis and imaging scanning on the extracted DNA sample; Example 5. High-throughput sequencing and analysis of the microbial community samples ① For the V4-V5 region of 16S rRNA, synthesize specific primers with barcodes for high-throughput sequencing. PCR uses TransGen AP221-02: TransStart Fastpfu DNA Polymerase; PCR instrument: ABI Model 9700. After mixing the PCR products, detect them by 2% agarose gel electrophoresis, and use the AxyPrep DNA Gel Extraction Kit (AXYGEN) to cut and recover the PCR products; ② After removing the primer adapter sequences and low-quality bases (Phred Quality Score = 20) from the raw sequencing data, splice the data. After removing non-specific amplification sequences and chimeras, obtain the effective sequence data for each sample. Use the Mothur software package to divide the 16S sequences into operational taxonomic units (OTUs) with a threshold of 97%. Use RDP classifier 2.2 to perform taxonomic analysis on the OTU representative sequences at the 97% similarity level, obtain the species classification information corresponding to each OTU, and statistically analyze the bacterial community composition of each sample at the levels of kingdom, phylum, class, order, family, and genus; The results after experimental tests are as follows: The microbial community proportions of only the basal medium (CK) and the basal medium containing Bifidobacterium enrichment (15B1) are shown in Table 1. With a 5% bacterial content as the dominant bacterium, the dominant bacterium Bifidobacterium did not appear in the CK group, and the content in the 15B1 group reached the dominant bacterium level, indicating that the 15B1 medium has successfully induced the Bifidobacterium genus; Table 1 The influence of the 15B1 group medium on the dominant genus composition and structure of the intestinal microbiota Example 6. Isolation and culture of Bifidobacterium strains ① Autoclave 15% agar alone. When it has cooled down to a temperature where it is not hot to the touch, add the pre-warmed liquid medium containing the enrichment prepared above, mix well, pour the mixture into petri dishes, let it air-dry in a laminar flow hood for 10 min, and place it in a ready-to-use anaerobic bag prepared in advance for spreading bacterial solutions (utility model patent product "A ready-to-use anaerobic bag for anaerobic plate separation and culture", patent number: ZL201920214535.7); ② After the color of the petri dish has faded, perform operations and spreading according to the requirements of the ready-to-use anaerobic bag, and incubate overnight at 37 °C; ③ Pick colonies and culture them according to the liquid medium in 1) above. Finally, add glycerol to the culture to a final concentration of 15% and store it at low temperature for future testing; Example 7. Molecular identification of intestinal bacteria ① Take 1 ml of the preserved intestinal bacteria liquid sample, centrifuge at 4000 g for 5 min and retain the precipitate. Add 95% zirconia grinding beads, add 500 μl of CTAB DNA extraction solution to the centrifuge tube with grinding beads, and grind on a MM400 grinder at 25 HZ for 5 min; ② Place it in a water bath at 65 °C for incubation for 15 min, and centrifuge at 12000 g for 1 min; Take 400 μl of the supernatant and transfer it to an adsorption column, and add 200 μl of absolute ethanol to the adsorption column, and mix well; After centrifuging at 12000 g for 1 min, replace the collection tube; ③ Add 500 μl of 70% ethanol, centrifuge at 12000 g for 1 min, replace the collection tube, and repeat this step once; Replace the collection tube again, place the washed adsorption column AC, drop 50 μl of elution buffer EB, centrifuge at 12000 g for 1 min in a centrifuge, collect the DNA after elution, and perform electrophoresis and imaging scanning on the extracted DNA sample; ④ Conduct PCR experiments according to the following formula: The reaction volume is 20 μl, 2 μl of 10X Taq Buffer, 1.6 μl of 2.5 mM dNTP mixture, 1 u of Taq, 0.8 μl of each 10 μM primer, the upstream primer is BifLP: AACGCCATGCTGACCGAT, the downstream primer BifRP is: AAGGCAGGTTGACGTCCAG, the template volume is 1 μl, and the rest is made up with pure water; The PCR reaction program is: 95 °C for 300 s; 94 °C for 15 s, 56 °C for 45 s, 72 °C for 45 s, for a total of 35 - 40 cycles; 72 °C for 300 s. The PCR instrument is a genepro Thermal Cycler (Bori), the electrophoresis device is a Beijing Liuyi horizontal electrophoresis device, the electrophoresis marker M is DL2000 (Takara), and the screenshot is as shown. Figure 1 The bacterial sample with a 277 bp band appearing is the Bifidobacterium bacterium.

Claims

1. A method for enriching and screening Bifidobacterium intestinal bacteria, characterized in that, The method includes the following steps: (1) Preparation of basic anaerobic medium: Prepare the components of the basic medium according to the growth rules of most intestinal bacteria and adjust the pH value. These media generally contain necessary sugars, proteins, vitamins, and some short-chain fatty acids. Add resazurin at a concentration of one-thousandth as an oxygen indicator, fill with nitrogen, and sterilize by autoclaving or filtration; separately prepare a cysteine solution with a concentration of more than 1%, fill with nitrogen, and sterilize by autoclaving or filtration, and use it before inoculation; (2) Preparation of special enrichment culture is carried out according to the following steps: ①Take out the preserved Pichia pastoris 15B1 strain from a -80 °C ultra-low temperature refrigerator ( Pichia fermentans ), (the preservation number of the strain at the China Center for Type Culture Collection is CGMCC No. 19317), inoculate it into PDB medium and shake the bacteria at 32 °C for 24 h; ② Centrifuge the obtained 15B1 bacterial liquid at 10,000 rpm for 1 min, and take the supernatant for standby; (3) Enrichment culture: ① Sequentially add 1 / 2 well volume of the basic anaerobic medium in step (1) to a 5-ml vial, and then add 1 / 20 well volume of the supernatant of the fermentation broth in step (2) above; ② Add 1 / 10 volume of silicone oil, oil-soluble antioxidant, and liquid paraffin respectively, and let it naturally stratify to block oxygen. The oil-soluble antioxidant can be vitamin E and unsaturated fatty acids; ③ Add 1 / 10 volume of 1% cysteine solution, seal the lid, and place it in the refrigerator or at room temperature for more than 12 hours, waiting for the red color of the oxygen indicator to fade; ④ Take a total of 1 g of fresh feces from multiple people, add it to 10 ml of basic medium solution containing 1% cysteine deoxidizer, shake and disperse it evenly, and let it stand for 3 - 5 min; ⑤ Add 1 / 20 volume of the supernatant of the mixed bacterial liquid to the above-mentioned vial, culture it in a 37°C incubator, and after the bacteria grow, directly dispense it into 1-ml tubes, add glycerol at a final concentration of 15%, and freeze and store it; (4) Extraction of intestinal bacteria nucleic acid: ① Take 1 ml of the above-mentioned intestinal bacterial liquid sample, centrifuge it at 4,000 g for 5 min and retain the precipitate, add 95% zirconia grinding beads, add 500 μl of CTAB DNA extraction solution to the centrifuge tube with grinding beads, and grind it on a grinder for 5 min; ② Place it in a 65°C water bath for incubation for 15 min, and centrifuge it at 12,000 g for 1 min; take 400 μl of the supernatant into an adsorption column, and add 200 μl of absolute ethanol to the adsorption column, and mix well; after centrifuging at 12,000 g for 1 min, replace the collection tube; ③ Add 500 μl of 70% ethanol, centrifuge it at 12,000 g for 1 min, replace the collection tube, and repeat this step once; replace the collection tube again, place the washed adsorption column AC, drop 50 μl of elution buffer EB, centrifuge it at 12,000 g for 1 min in a centrifuge, collect the DNA after elution, and perform electrophoresis and imaging scanning on the extracted DNA sample; (5) High-throughput sequencing and analysis: ① Design primers targeting the 16S rRNA v3 - v4 region for high-throughput sequencing, and estimate the proportion of each genus of bacteria by sequence alignment; ② Analyze the proportion of bacteria belonging to the genus Bifidobacterium in the sample. If it is less than 5%, take out the frozen tube and repeat the culture in step (3) and the nucleic acid extraction process in step (4); ③ If the proportion of bacteria belonging to the genus Bifidobacterium is greater than 5%, then continue with step (6) for strain isolation; (6) Strain isolation and culture: ① Prepare in advance a ready-to-use anaerobic bag for spreading bacterial liquid; ② Take 50 μl of the cryopreserved intestinal bacteria sample, dilute it 10 times, and spread it on the solid medium in the above step (2), then culture it at 37 °C for 1 - 2 days; ③ Pick colonies and perform anaerobic culture as described in the above step (3). After the bacteria grow, use them for subsequent nucleic acid extraction; (7) Molecular identification of intestinal bacteria: ① Extract the nucleic acids of single colony cultures one by one according to the intestinal bacteria nucleic acid extraction in the above step (4); ② Perform PCR experiments according to the following formula: The reaction volume is 20 μl, 2 μl of 10X Taq Buffer, 1.6 μl of 2.5 mM dNTP mixture, 1 u of Taq, 0.8 μl of each 10 μM primer. The upstream primer is BifLP: AACGCCATGCTGACCGAT, and the downstream primer BifRP is: AAGGCAGGTTGACGTCCAG. The template volume is 1 μl, and the rest is made up with pure water; The PCR reaction program is: 95 °C for 300 s; 94 °C for 15 s, 56 °C for 45 s, 72 °C for 45 s, for a total of 35 - 40 cycles; 72 °C for 300 s; ③ After the products are detected by electrophoresis, if there is a characteristic band of 277 bp, it is a positive colony. The bacteria to which this positive band belongs are bacteria of the genus Bifidobacterium.

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