Microbial enrichment agent of paraprenella, enrichment culture medium and application of microbial enrichment agent and enrichment culture medium of paraprenella

Abstract

The invention discloses an enrichment medium for a paraprevotella, an enrichment medium for the paraprevotella and application of the enrichment medium for the paraprevotella. The enrichment medium comprises the following components: a mango extract, a blueberry extract, an apple extract and a green tea extract. The enrichment culture medium comprises the enrichment agent. The invention further discloses application of the enrichment medium and the enrichment medium in separation or identification of the paraprevotella. The invention further discloses application of the enrichment medium and the enrichment medium in separation or identification of the paraprevotella. The enrichment medium and the culture medium provided by the invention have the advantages that the separation culture efficiency of the Paraprevotella is obviously improved, and a foundation is provided for further exploring the biological characteristics and developing the potential value of the Paraprevotella.

Description

Technical Field

[0001] The present application belongs to the technical field of microbial detection, and specifically relates to a bacterial enrichment agent, bacterial enrichment culture medium and application of Paraprevotella. Background Art

[0002] Paraprevotella belongs to the phylum Bacteroidetes, class Bacteroidetes, orders Bacteroidetes, family Prevotellaceae, and family Paraprevotella. Paraprevotella is primarily found in the intestinal microbiota of humans and animals and is closely associated with host metabolism and health. This species may be involved in the degradation of polysaccharides and the production of short-chain fatty acids (such as acetate and propionate).

[0003] Paraprevotella is often considered a bacterium associated with a healthy plant-based diet, acting as a probiotic in the human body. A decrease in Paraprevotella abundance is associated with certain diseases. In-depth research on Paraprevotella may help develop novel probiotic formulations, potentially offering new strategies for maintaining intestinal microecological balance and immune regulation.

[0004] Currently, it is difficult to isolate and culture Paraprevotella from human fecal samples. The existing isolation and culture protocols for Paraprevotella are inefficient, which seriously restricts the research on its functional properties and application potential. Therefore, it is necessary to improve this. Summary of the Invention

[0005] In response to the above technical problems, the present application provides a Paraprevotella enrichment agent, a culture medium and applications thereof, so as to improve the isolation and culture efficiency of Paraprevotella.

[0006] In a first aspect, the present application provides a bacterial growth agent for Paraprevotella, the components of which include: mango extract, blueberry extract, apple extract and green tea extract, wherein the content of mangiferin in the mango extract is greater than or equal to 25wt%, the content of proanthocyanidins in the blueberry extract is greater than or equal to 25wt%, the content of total apple polyphenols in the apple extract is greater than or equal to 10wt%, and the content of tea polyphenols in the green tea extract is greater than or equal to 30wt%.

[0007] In one possible implementation, the components of the bactericidal agent include, by weight, 2 to 6 parts of mango extract, 1 to 3 parts of blueberry extract, 1 to 4 parts of apple extract, and 0.3 to 1 part of green tea extract.

[0008] Furthermore, the components of the bactericidal agent include: in parts by weight, 2.5 to 4.5 parts of mango extract, 1.5 to 2.5 parts of blueberry extract, 2 to 3 parts of apple extract, and 0.4 to 0.8 parts of green tea extract.

[0009] Furthermore, the components of the bactericidal agent include: 3.5 parts of mango extract, 2.5 parts of blueberry extract, 2.5 parts of apple extract, and 0.4 parts of green tea extract, in parts by weight.

[0010] In a second aspect, the present application provides a microbial culture medium for Paraprevotella , which comprises the bacterial enrichment agent described in the first aspect.

[0011] In one possible implementation, the concentration of the bacteriostatic agent in the microbial culture medium is 0.43-1.40 g / L, and the concentrations of the components of the bacteriostatic agent in the microbial culture medium are: mango extract 0.2-0.6 g / L, blueberry extract 0.1-0.3 g / L, apple extract 0.1-0.4 g / L, and green tea extract 0.03-0.1 g / L.

[0012] In a third aspect, a method for improving the isolation and culture efficiency of Paraprevotella is provided, comprising the following steps:

[0013] Prepare raw materials according to the components of the bacteriostatic agent described in the first aspect, wherein the raw materials include sterilized mango extract, sterilized blueberry extract, sterilized apple extract, and sterilized green tea extract;

[0014] adding the mango extract, blueberry extract, apple extract and green tea extract to a basic liquid culture medium to obtain an enrichment liquid culture medium;

[0015] preparing a buffer, homogenizing a human stool sample with the buffer, and then filtering to obtain an inoculum;

[0016] adding the inoculum to the enrichment-liquid culture medium for fermentation under constant temperature and anaerobic conditions to obtain an enrichment culture;

[0017] Extracting genomic DNA from the enriched culture, amplifying it by PCR, and sequencing it to obtain an enriched culture containing Paraprevotella;

[0018] The enriched culture is diluted and then spread on a solid culture medium. After fermentation and culture, a single colony is obtained. The single colony is identified to obtain Paraprevotella.

[0019] In one possible implementation, the content of human fecal sample in the inoculum is 0.3-0.4 g / mL, the volume ratio of the inoculum to the enrichment-liquid culture medium is (0.5-1.5): (8.5-9.5); the dilution factor of the enrichment culture is 1×10 5 Times ~ 1×10 6 times.

[0020] In a fourth aspect, the present application provides use of the bacterial enrichment agent as described in the first aspect in the isolation or identification of Paraprevotella.

[0021] In a fifth aspect, the present application provides use of the culture medium as described in the second aspect in the isolation or identification of Paraprevotella.

[0022] The beneficial effects of this application are as follows:

[0023] The present application provides a Paraprevotella enrichment agent, a culture medium, and applications, which can increase the relative abundance of Paraprevotella from 0.00348 to 0.0887. Compared with the existing technology, the probability of obtaining the target strain is increased by nearly 25 times, significantly improving the efficiency of the value-added culture of Paraprevotella, reducing a large amount of repeated identification work and financial expenditure, and providing strong support and solid guarantee for the subsequent in-depth exploration of the biological characteristics of Paraprevotella and the development of its potential value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the limiting dilution plot of Paraprevotella;

[0025] Figure 2 This is a stacked diagram of Genus species distribution. DETAILED DESCRIPTION

[0026] The content of this application is further described below with reference to specific embodiments, but the content of this application is not limited thereto.

[0027] At present, there is little research on the mechanism of action, principle of action, functional properties and application value of intestinal microorganisms, and progress is slow. Since commonly used target bacteria (such as Paraprevotella clara) can generally be purchased directly on the market, when a specific target bacteria needs to be proliferated and cultured to facilitate subsequent systematic analysis of the metabolic mechanism and industrial development of the target bacteria, the existing technology generally directly cultivates the target bacteria on the market to reduce R&D costs.

[0028] However, the target bacteria available for purchase on the market are generally only one or several specific species (such as Paraprevotella clara) in a specific genus (such as Paraprevotella), and other known species in the specific genus (such as Paraprevotella) are difficult to purchase.

[0029] Currently, there are some commonly used probiotics on the market that are based on bacterial species (target bacteria, such as Paraprevotella clara), but there are few studies on probiotics in intestinal microorganisms that are based on bacterial genus (such as Paraprevotella).

[0030] Human fecal samples contain a certain amount of Paraprevotella. Currently, it is difficult to isolate and culture Paraprevotella in human fecal samples. When it is necessary to culture, isolate and identify probiotics (such as Paraprevotella) in intestinal microorganisms based on the genus, the experimental steps are as follows: 1) prepare solid culture medium (such as LB agar medium, GAM agar medium, MRS agar medium, etc.); 2) prepare buffer (such as saline, carbonate buffer, phosphate buffer, etc.); 3) collect and homogenize the sample, wash and filter it with buffer to obtain a filtrate; 4) limit dilution (such as 10 times, 100 times, 1×1010 times, etc.) of the filtrate obtained in step 3) to different concentration, and then spread them onto solid culture medium; 5) Ferment in vitro at a constant temperature for 48-72 hours to obtain a culture; 6) Collect single colonies and store them for future use; 7) Extract genomic DNA from the stored samples; 8) Amplify the full-length 16S gene sequence of the sample using a commercial kit and PCR technology; 9) Perform Sanger sequencing analysis on the obtained gene sequence; 10) Compare the gene information using the public BLAST database to determine whether it is the target bacterial genus (similarity > 98.7%); 11) If the identification result is negative, repeat steps 7)-10) until the target strain is obtained.

[0031] Typically, in step 4), the limiting dilution should be 1×10 7 times to 1×10 10 Therefore, the traditional culture method of Paraprevotella is inefficient and needs to be improved.

[0032] In response to the above problems, the present invention first studies the commercially available Paraprevotella clara (single-factor variable experiment) to preliminarily determine the material components that have a proliferation-promoting effect on Paraprevotella clara; on this basis, the present invention further studies the effects of the above-mentioned determined material components when used in combination on the proliferation efficiency of Paraprevotella clara (multi-factor orthogonal experiment) to determine the appropriate dosage ratio and optimal ratio of the above-mentioned material components; then, based on the inoculum obtained from human fecal samples and the optimal ratio of material components, the present application isolates, cultures and identifies Paraprevotella (with the genus as the research unit), and verifies that the bacterial enhancement agent described in the present invention and its corresponding microbial culture medium have a good proliferation-promoting effect on Paraprevotella (with the genus as the research unit).

[0033] Specifically, the present application provides a Paraprevotella enrichment agent, enrichment culture medium and application.

[0034] In a first aspect, the present application provides a bacterial growth agent for Paraprevotella, the components of which include: mango extract, blueberry extract, apple extract and green tea extract, wherein the content of mangiferin in the mango extract is greater than or equal to 25wt%, the content of proanthocyanidins in the blueberry extract is greater than or equal to 25wt%, the content of total apple polyphenols in the apple extract is greater than or equal to 10wt%, and the content of tea polyphenols in the green tea extract is greater than or equal to 30wt%.

[0035] In one possible implementation, the components of the bactericidal agent include, by weight, 2 to 6 parts of mango extract, 1 to 3 parts of blueberry extract, 1 to 4 parts of apple extract, and 0.3 to 1 part of green tea extract.

[0036] Furthermore, the components of the bactericidal agent include: in parts by weight, 2.5 to 4.5 parts of mango extract, 1.5 to 2.5 parts of blueberry extract, 2 to 3 parts of apple extract, and 0.4 to 0.8 parts of green tea extract.

[0037] Furthermore, the components of the bactericidal agent include: 3.5 parts of mango extract, 2.5 parts of blueberry extract, 2.5 parts of apple extract, and 0.4 parts of green tea extract, in parts by weight.

[0038] It should be noted that the weight percentages of the extracts are all based on the solid content. In the specific embodiments, the actual dosage can be converted according to the form of the extract. Generally speaking, the dosage of the active ingredient is ensured to be within the corresponding range.

[0039] In a second aspect, the present application provides a microbial culture medium for Paraprevotella , which comprises the bacterial enrichment agent described in the first aspect.

[0040] In one possible implementation, the concentration of the bacteriostatic agent in the microbial culture medium is 0.43-1.40 g / L, and the concentrations of the components of the bacteriostatic agent in the microbial culture medium are: mango extract 0.2-0.6 g / L, blueberry extract 0.1-0.3 g / L, apple extract 0.1-0.4 g / L, and green tea extract 0.03-0.1 g / L.

[0041] In a third aspect, a method for improving the isolation and culture efficiency of Paraprevotella is provided, comprising the following steps:

[0042] Prepare raw materials according to the components of the bacteriostatic agent described in the first aspect, wherein the raw materials include sterilized mango extract, sterilized blueberry extract, sterilized apple extract, and sterilized green tea extract;

[0043] adding the mango extract, blueberry extract, apple extract and green tea extract to a basic liquid culture medium to obtain an enrichment liquid culture medium;

[0044] preparing a buffer, homogenizing Paraprevotella with the buffer, and then filtering to obtain an inoculum;

[0045] adding the inoculum to the enrichment-liquid culture medium for fermentation under constant temperature and anaerobic conditions to obtain an enrichment culture;

[0046] Extracting genomic DNA from the enriched culture, amplifying it by PCR, and sequencing it to obtain an enriched culture containing Paraprevotella;

[0047] The enriched culture is diluted and then spread on a solid culture medium. After fermentation and culture, a single colony is obtained. The single colony is identified to obtain Paraprevotella.

[0048] In one possible implementation, the human fecal sample content is 0.3-0.4 g / mL; the volume ratio of the inoculum to the enrichment-liquid culture medium is (0.5-1.5): (8.5-9.5); the dilution factor of the enrichment culture is 1×10 5 Times ~ 1×10 6 times.

[0049] In a fourth aspect, the present application provides use of the bacterial enrichment agent as described in the first aspect in the isolation or identification of Paraprevotella.

[0050] In a fifth aspect, the present application provides use of the culture medium as described in the second aspect in the isolation or identification of Paraprevotella.

[0051] The content of this application is further described below with reference to more specific embodiments.

[0052] The relevant raw materials in the following embodiments are described in a unified manner:

[0053] 1. The type strain is DSM 19731ᵀ, which is also known as Paraprevotella clara and was purchased from the DSMZ collection in Germany.

[0054] 2. Various food-grade plant extracts were purchased from Shaanxi Ruilin Biotechnology Co., Ltd. Mango extract is powdered mangiferin extracted from mango fruit; blueberry extract is powdered proanthocyanidins extracted from blueberries; apple extract is powdered total apple polyphenols extracted from unripe apples; and green tea extract is powdered tea polyphenols extracted from tea leaves.

[0055] 3. The basal culture medium was purchased from Huankai Biotechnology Co., Ltd.

[0056] 4. DNA molecular manipulation kits were purchased from Beijing Qingke Biotechnology Co., Ltd.

[0057] Example 1 Components of the Bacterial Enhancement Agent and Method for Isolating and Proliferating Paraprevotella

[0058] 1.1. Components of Paraprevotella enrichment agents

[0059] The components of the bioaccumulator include:

[0060] 1) Mango extract (25wt% mangiferin), irradiated sterilized.

[0061] 2) Blueberry extract (25wt% proanthocyanidins), irradiated sterilized.

[0062] 3) Apple extract (10 wt% total apple polyphenols), irradiated sterilized.

[0063] 4) Green tea extract (30wt% tea polyphenols), irradiated sterilized.

[0064] The content range of each component of the bacteriostatic agent in the culture medium is:

[0065] 1) 0.2-0.6 g / L mango extract;

[0066] 2) 0.1-0.3 g / L blueberry extract;

[0067] 3) 0.1-0.4 g / L apple extract;

[0068] 3) 0.03-0.1 g / L green tea extract.

[0069] 1.2. Preparation of enrichment medium

[0070] Here are the steps:

[0071] (1) Add the bacterial growth agent in 1.1 to the basal liquid culture medium.

[0072] (2) Buffer preparation: Prepare 9% NaCl saline buffer.

[0073] 1.3 Application: Inoculation, Isolation, Proliferation and Identification

[0074] Here are the steps:

[0075] (1) Sample processing and inoculation: The fecal sample is homogenized with a buffer solution and then filtered to obtain an inoculum. 1 mL of the inoculum is added to 9 mL of enrichment medium. The fecal sample in this step is a human fecal sample, and the concentration of the human fecal sample in the inoculum is 0.3-0.4 g / mL (preferably 0.35 g / mL). The above-mentioned method for obtaining the sample to be separated is a conventional technical solution in this field and will not be described in detail here.

[0076] (2) Anaerobic enrichment culture: Anaerobic culture was performed at 37°C for 24 hours, and the culture was collected and genomic DNA was extracted.

[0077] (3) Target bacteria screening: The relative abundance of Paraprevotella was analyzed by 16S rDNA V3-V4 region amplification (PCR) and Illumina sequencing.

[0078] (4) Select high-abundance samples and perform multiple rounds of enrichment.

[0079] (5) Isolation of single colonies: Dilute the final culture 10n-fold in a gradient, spread on solid culture medium, culture aerobically at 37°C for 72 hours, and pick single colonies.

[0080] (6) Strain identification: Single colony DNA was extracted, full-length 16S rDNA was amplified and Sanger sequencing was completed.

[0081] (7) The target bacterial genus was confirmed through BLAST comparison (similarity > 98.7%).

[0082] Example 2 Single-factor experiment and multi-factor orthogonal experiment of Paraprevotella clara

[0083] 2.1 Single-factor experiment

[0084] Bacteria species: Paraprevotella clara;

[0085] Basic culture medium: GAM liquid medium; dilution factor: 10 7 ;

[0086] Culture conditions: anaerobic, 37°C;

[0087] Variables: 1) mango extract, 2) blueberry extract, 3) apple extract, 4) green tea extract.

[0088] The experimental steps are as follows:

[0089] (1) Weigh a certain amount of single-factor components respectively, and prepare solutions of different concentrations with sterile water for later use.

[0090] (2) Add the solution of step (2) to the sterilized GAM liquid culture medium to obtain culture medium with different components, and mark the groups separately.

[0091] (3) Aseptically inoculate live Paraprevotella clara into the culture media of different groups in step (2), and culture them in an anaerobic incubator at 37°C for 24 to 48 hours.

[0092] (4) The growth of the blank control group was observed at 24 h, 36 h, and 48 h. The growth of the blank control group tended to be stable at 36 h, so the cells were transferred to GAM solid culture medium at 36 h and plated for counting.

[0093] Among them, this embodiment also sets up three groups of blank controls (using only GAM liquid culture medium), and the average number of colonies cultured in the three groups of blank controls is 14.33. Among them, the experimental method of designing blank controls is a conventional technical means in this field and will not be described in detail here.

[0094] Results analysis: The 36-hour culture results were analyzed. The grouping, dosage of each component, concentration of each component and culture results are shown in Table 1.

[0095] Table 1 Univariate variables and results

[0096]

[0097] As can be seen from Table 1 above, each single component of the mango extract, blueberry extract, apple extract and green tea extract of the present application can enable Paraprevotella to achieve bacterial proliferation function. After a certain mass fraction of mango extract, blueberry extract, apple extract and green tea extract are respectively configured into a microbial culture medium of a specific concentration, the four single bacterial enrichment agent components all have a promoting effect on the proliferation of Paraprevotella.

[0098] At the same time, it can be seen from Table 1 above that among the four single components of the bacterial enhancement agent, mango extract has the best effect on promoting the proliferation of Paraprevotella clara. The optimal single dosage of mango extract is 0.6 g / L, the optimal single dosage of blueberry extract is 0.2 g / L, the optimal single dosage of apple extract is 0.4 g / L, and the optimal single dosage of green tea extract is 0.03 g / L.

[0099] 2.2 Multi-factor orthogonal experiment

[0100] Bacteria species: Paraprevotella clara;

[0101] Basic culture medium: GAM solid medium;

[0102] Culture conditions: anaerobic, 37°C;

[0103] Dilution factor: 10 7 ;

[0104] Variables: 1) mango extract, 2) blueberry extract, 3) apple extract, 4) green tea extract.

[0105] In this example, Paraprevotella clara was used as the research object, and four factors and three levels of L9 (3 4) was used to investigate the effects of different concentrations of mango extract, blueberry extract, apple extract, and green tea extract on the proliferation of Paraprevotella clara. The specific steps are as follows:

[0106] (1) Weigh a certain amount of multi-factor variable components (refer to Table 2) and prepare different concentrations of solutions with sterile water. The solutions of each group are shown in Table 2 and set aside.

[0107] (2) Add the solution from step (1) to the sterilized GAM solid culture medium and mark the groups.

[0108] In this step, the bactericidal component described in step (1) above is added to the GAM solid culture medium to prepare a solution of a certain concentration for standby use. For example, referring to Experiment 3 in Table 2, 0.25 g of mango extract, 0.25 g of blueberry extract, 0.3 g of apple extract and 0.08 g of green tea extract are added to 1 L of sterile water to form the components described in Experiment 3 in Table 2: 0.25 g / L mango extract, 0.25 g / L blueberry extract, 0.3 g / L apple extract and 0.08 g / L green tea extract. At this time, the number of colonies is 25.

[0109] (3) According to the different groups in step (2), the live bacteria of Paraprevotella clara were aseptically inoculated into the culture medium, and then diluted to 10 7 After doubling, place the cells in an anaerobic incubator and culture at 37°C for 24 to 48 hours.

[0110] (4) Observe at 24h, 36h, and 48h respectively, and culture Paraprevotella clara to the logarithmic growth phase (OD 600 ≈1.0) was transferred to GAM solid medium and counted on a plate.

[0111] The grouping is shown in Table 2.

[0112] Table 2 Factors and levels of orthogonal experiment

[0113]

[0114] Table 3 Orthogonal experiment results

[0115]

[0116] In the above table, k1, k2, k3 and R are the average number of colonies at level 1, the average number of colonies at level 2, the average number of colonies at level 3 and the range, respectively. 4) The manner and method for obtaining the experimental results recorded in the above Table 3 by orthogonal experiments and the contents recorded in Table 2 are conventional means in this field and will not be described in detail here.

[0117] At the same time, in order to make the experimental data of the present invention clearer and easier to understand, the present application explains the data in the orthogonal experiment of Table 3 above:

[0118] For example, in the four-factor three-level L9 (3 4 ), the number 69 means: in the 9th group of experiments, the mango extract used the concentration corresponding to level 3 (that is, the mango extract was 0.45 g / L), the blueberry extract used the concentration corresponding to level 3 (that is, the blueberry extract was 0.25 g / L), the apple extract used the concentration corresponding to level 2 (that is, the apple extract was 0.25 g / L), and the green tea extract used the concentration corresponding to level 1 (that is, the green tea extract was 0.04 g / L). At this time, the number of colonies of Paraprevotella clara 27340 was 69.

[0119] For example, in the four-factor three-level L9(3 4 ), the number 52 means that when the mango extract uses the concentration value corresponding to level 2 (that is, the mango extract is 0.4 g / L), the average colony count of Paraprevotella clara 27340 is 52.

[0120] For example, in the four-factor three-level L9(3 4 ), the number 17.67 means: the difference between the highest and lowest colony counts of apple extract, that is, the difference between the average colony count when the concentration value corresponding to level 2 (0.25 g / L for apple extract) is used and the average colony count when the concentration value corresponding to level 3 (0.3 g / L for apple extract) is used.

[0121] Combined with the four factors and three levels of L9(3 4 ) of the orthogonal experiment and the experimental results can be seen from Tables 2 and 3 above:

[0122] (1) When the four components described in the microbial growth agent of this application are used together, apple extract has the best effect on promoting the proliferation of the target strain (Paraprevotella clara), followed by mango extract, blueberry extract and green tea extract;

[0123] (2) The range of mango extract is the largest, so when mango extract is used in different dosages, its effect on promoting the proliferation of the target strain will also have significant differences; the range of green tea extract is the smallest, so when green tea extract is used in different dosages, its effect on promoting the proliferation of the target strain will have little difference.

[0124] (3) As shown in Tables 2 and 3, when the target strain (Paraprevotella clara) was cultured using the components described in this application, the culture was diluted to 1×10 6 After the four factors and three levels L9(3 4 ) of the 9 experimental schemes of the orthogonal experiment, the highest colony number of the target strain can be cultured to 69;

[0125] (4) When the four single components in this application are used together to culture the target bacteria, the optimal combination is 0.35 g / L mango extract, 0.25 g / L blueberry extract, 0.25 g / L apple extract, and 0.04 g / L green tea extract.

[0126] It should be noted that the optimal solution of the present invention is not from experimental groups 1 to 9, but from the data recorded in k1, k2 and k3 in Table 3, that is, the optimal combination of the present invention is the four-factor three-level L9 (3 4 ) in the orthogonal experiment. For example, the optimal dosage of mango extract is the best data among k1, k2 and k3 (that is, the data represented by k2), that is, the optimal dosage of mango extract is the concentration corresponding to level 2 (that is, 0.35 g / L), not the data corresponding to experimental group 9; similarly, the optimal dosage of blueberry extract is the concentration corresponding to level 3 (0.25 g / L), the optimal dosage of apple extract is the concentration corresponding to level 2 (0.25 g / L), and the optimal dosage of green tea extract is the concentration corresponding to level 1 (0.04 g / L).

[0127] Example 3: Inoculation, culture, isolation and identification of Paraprevotella

[0128] 3.1. Existing protocols for inoculation, culture, isolation and identification of Paraprevotella:

[0129] Objective: To investigate the effects of existing protocols on the proliferation of Paraprevotella;

[0130] Bacteria species: ParaprevotellaParaprevotella clara;

[0131] Basic culture medium: GAM solid culture medium; culture conditions: dilution factor 10 7 times, anaerobic, 37°C;

[0132] Variable: Paraprevotella was cultured using only basal medium without mango extract, blueberry extract, apple extract, and green tea extract.

[0133] Experimental steps:

[0134] (1) Weigh 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride in sequence, dissolve them, add 800 mL of deionized water, stir until completely dissolved, adjust the pH of the solution to 7.0, then make up the volume, aliquot, sterilize, and cool to room temperature to obtain the basal culture medium;

[0135] (2) Prepare 9% NaCl saline buffer and add it to the basal culture medium to obtain fresh culture medium;

[0136] (3) Collect fecal samples, homogenize them, wash and filter them with buffer, inoculate them into 5 mL of fresh culture medium, and culture them anaerobically at 37°C for 12 to 16 hours until the logarithmic growth phase (OD 600 ≈1.0), and the inoculum was obtained;

[0137] (4) Transfer 10% of the inoculum to 50 mL of fresh culture medium and incubate at 37°C in an anaerobic environment for 20 hours;

[0138] (5) Collect the cells by centrifugation (4000 × g, 10 min) and resuspend them in sterile PBS (containing 5% glycerol) to obtain a solution containing Paraprevotella for subsequent use or storage;

[0139] (6) extracting genomic DNA from the preserved bacterial sample;

[0140] (7) Using a commercial 16S rDNA kit and PCR technology to amplify the V3-V4 region of the sample;

[0141] (8) The obtained amplified fragments were sequenced and analyzed by Illumina;

[0142] (9) Screening the enrichment culture with a high proportion of Paraprevotella, repeating steps 4 to 5 to further increase the proportion of Paraprevotella in the enrichment culture, and obtaining the final culture;

[0143] (10) The final culture obtained is diluted in multiple ratios and then coated on solid culture medium;

[0144] (11) Ferment the culture in vitro under constant temperature conditions for 48 h, collect single colonies, and store them for future use;

[0145] (12) Amplify the full-length 16S rRNA gene using a commercial kit and PCR;

[0146] (13) Genes obtained by Sanger sequencing;

[0147] (14) Confirm the target bacterial genus by BLAST comparison with public databases (similarity ≥ 98.7%);

[0148] (15) If the identification result is negative, all steps need to be repeated until the target strain is obtained.

[0149] In the above steps, the experimental steps of 16S amplicon sequencing are:

[0150] S1. Extract total DNA from the sample using a DNA extraction kit according to the instructions. Perform PCR amplification using the extracted DNA as a template, as shown in Table 4:

[0151]

[0152] PCR reaction conditions: Initial denaturation: 95°C for 10 minutes. Amplification: 25 cycles: denaturation: 94°C for 30 seconds, annealing: 60°C for 90 seconds, extension: 72°C for 30 seconds. Final extension: 72°C for 10 minutes. Storage at 4°C.

[0153] S2. The amplified PCR products were recovered, end-repaired, A-tailed, and connected to sequencing adapters, as shown in Table 5.

[0154]

[0155] Reaction conditions: pre-denaturation at 98°C for 45 s; 8 cycles of 98°C for 15 s, 60°C for 30 s, and 72°C for 30 s; extension at 72°C for 10 min, and storage at 4°C.

[0156] S3. The constructed amplicon library was sequenced using the Illumina Miseq platform and the PE250 sequencing strategy.

[0157] S4. Off-machine data processing to remove low-quality sequences, adapter sequences, etc.

[0158] According to this plan, refer to Figure 1 For the control group, the limiting dilution was 1×10 5 Bitu, Figure 1 The dilution multiples from left to right are 1×10, 1×10 2 , 1×10 3 , 1×104 , 1×10 5 The ideal result is that the number of single colonies on the entire plate of the dilution plate method is between 30 and 300. Figure 2 As shown, the relative abundance of Paraprevotella species in the control group was 0.3%.

[0159] The limiting dilution of the control group should reach 1×10 7 The control scheme estimates the probability of obtaining the target strain by selecting a single colony to be approximately 0.3%. Reproducible identification requires approximately 300 replicates (95% confidence interval). Therefore, the control scheme has the disadvantages of a low probability of obtaining the target strain in a single experiment, a high workload, and high costs.

[0160] 3.2. Inoculation, culture, isolation, and identification of Paraprevotella using the newly added bacterial formulation

[0161] Objective: To investigate the effect of a new bacterial agent formula on the proliferation of Paraprevotella.

[0162] Bacteria species: Paraprevotella Paraprevotella clara.

[0163] Basic culture medium: GAM solid medium.

[0164] Culture conditions: dilution factor 10 7 times, anaerobic, 37°C.

[0165] Variable: Paraprevotella was cultured using an additional inoculum formulation, which included:

[0166] 1) The concentration of mango extract in the microbial culture medium is 0.35 g / L (the same below), the content of mangiferin in the mango extract is greater than or equal to 25 wt%, and the extract is sterilized by irradiation;

[0167] 2) The concentration of blueberry extract is 0.25 g / L, the proanthocyanidin content in the blueberry extract is greater than or equal to 25 wt%, and the extract is irradiated and sterilized;

[0168] 3) The apple extract has a concentration of 0.25 g / L, the content of total apple polyphenols in the apple extract is greater than or equal to 10 wt%, and the extract is sterilized by irradiation;

[0169] 4) The concentration of green tea extract is 0.04 g / L, the content of tea polyphenols in the green tea extract is greater than or equal to 30 wt%, and the extract is irradiated and sterilized.

[0170] Experimental steps:

[0171] (1) Prepare the extract of the enrichment agent formulation according to the above concentration;

[0172] (2) Weigh 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride in sequence, dissolve them, add 800 mL of deionized water, stir until completely dissolved, adjust the pH of the solution to 7.0, then make up the volume, aliquot, sterilize, and cool to room temperature to obtain the basal culture medium;

[0173] (3) The components of the microbial growth agent in step (1) above are added to the basal culture medium according to the concentration ratio to obtain a culture medium containing the microbial growth agent (also referred to as a microbial culture medium); at this time, the concentrations of the components in the microbial culture medium include: mango extract is 0.45 g / L; blueberry extract is 0.25 g / L; apple extract is 0.25 g / L; and green tea extract is 0.04 g / L.

[0174] (4) preparing a 9% NaCl physiological saline buffer solution, and then adding it to the culture medium containing the bacterial growth agent obtained in step (3) to obtain a fresh culture medium;

[0175] (5) Collect fecal samples, homogenize them, wash and filter them with buffer, inoculate them into 5 mL of fresh culture medium, and culture them anaerobically at 37°C for 12 to 16 hours until the logarithmic growth phase (OD 600 ≈1.0), to obtain an inoculum; wherein, the stool sample in this step is a human stool sample, and the concentration of the human stool sample in the inoculum is 0.3-0.4 g / mL (preferably 0.35 g / mL). The above scheme for obtaining the sample to be separated is a conventional technical scheme in the art and will not be described in detail here;

[0176] (6) Transfer the inoculum from step (5) to 50 mL of fresh culture medium (containing a microbial growth agent) at a 1% inoculum volume and incubate at 37°C in an anaerobic environment for 20 hours;

[0177] (7) Collect the cells by centrifugation (4000 × g, 10 min) and resuspend them in sterile PBS (containing 5% glycerol) to obtain a cell sample containing Paraprevotella (hereinafter referred to as the cell sample) for subsequent application or storage;

[0178] (8) extracting genomic DNA from the preserved bacterial sample;

[0179] (9) Use a commercial 16S rDNA kit and PCR technology to amplify the V3-V4 region of the sample;

[0180] (10) The obtained amplified fragments were subjected to Illumina sequencing analysis;

[0181] (11) Screening the enrichment culture with a high proportion of Paraprevotella, repeating steps 4 to 5 to further increase the proportion of Paraprevotella in the enrichment culture, and obtaining the final culture;

[0182] (12) The final culture obtained is diluted in multiple ratios and then coated on solid culture medium;

[0183] (13) Ferment the culture in vitro under constant temperature conditions for 48 h, collect single colonies, and store them for future use;

[0184] (14) Amplify the full-length 16S rRNA gene using a commercial kit and PCR;

[0185] (15) Perform Sanger sequencing on the obtained genes;

[0186] (16) Confirm the target genus by BLAST comparison with public databases (similarity ≥ 98.7%);

[0187] (17) If the identification result is negative, all steps need to be repeated until the target strain is obtained.

[0188] In the above steps, the experimental steps of 16S amplicon sequencing are:

[0189] S1. Extract total DNA from the sample using a DNA extraction kit according to the instructions. Perform PCR amplification using the extracted DNA as a template, as shown in Table 6:

[0190]

[0191] PCR reaction conditions: Initial denaturation: 95°C for 10 minutes. Amplification: 25 cycles: denaturation: 94°C for 30 seconds, annealing: 60°C for 90 seconds, extension: 72°C for 30 seconds. Final extension: 72°C for 10 minutes. Storage at 4°C.

[0192] S2. The amplified PCR products were recovered, end-repaired, A-tailed, and sequenced adapters were connected, as shown in Table 7.

[0193]

[0194] Reaction conditions: pre-denaturation at 98°C for 45 s; 8 cycles of 98°C for 15 s, 60°C for 30 s, and 72°C for 30 s; extension at 72°C for 10 min, and storage at 4°C.

[0195] S3. The constructed amplicon library was sequenced using the Illumina Miseq platform and the PE250 sequencing strategy.

[0196] S4. Off-machine data processing to remove low-quality sequences, adapter sequences, etc.

[0197] Following the above steps, the relative abundance of Paraprevotella species in Example 3 was approximately 0.0887. The estimated probability of obtaining the target strain by selecting a single colony was approximately 8.8%, and approximately 12 repeated identifications (95% confidence interval) were required to obtain the target strain. Therefore, the bacterial enrichment agent provided by the present invention can significantly increase the proportion of viable Paraprevotella bacteria during the isolation and culture of human fecal samples (intestinal microbial communities), effectively improving the isolation and culture efficiency, thereby saving a large amount of repeated identification work and funds for Paraprevotella species.

[0198] In actual work, in order to further illustrate the technical solution of the present application, the staff of the present application used bioinformatics software to analyze the data processed by Comparative Example 1 and Example 3, and obtained Table 8 of relative abundance of species.

[0199] Table 8 Species relative abundance

[0200]

[0201]

[0202]

[0203] Note: 1.83000000E-05 means 1.83×10 -5 ; 2. The existing scheme refers to the separation scheme that does not add bactericidal agents and only uses basic culture medium.

[0204] 3.3 Results and Discussion

[0205] Generally speaking, limiting dilution is usually to 1x10 7 times to 1x10 10 The dilution plate method generally requires that the number of single colonies on the entire plate be between 30 and 300 for an ideal result. If it is lower or higher than the range, it will affect the statistical accuracy. Figure 1 Limiting dilution 1x10 7 times (for reference).

[0206] With existing protocols, the probability of identifying the target strain from a single colony is approximately 0.3%. This requires approximately 300 re-identifications of a single colony sample. Therefore, the current approach suffers from a low probability of identifying the target strain in a single experiment, as well as repetitive work and high costs.

[0207] For this scheme, see Figure 2As shown in Table 8, the relative abundance of viable Paraprevotella after screening according to the steps of this protocol was about 0.0887, and the single colony sample needed to be repeatedly identified about 12 times to obtain the target strain.

[0208] Combined with the above data results, it can be seen that the bacterial enrichment agent provided in this example can significantly increase the proportion of viable Paraprevotella bacteria during the isolation and culture of human fecal samples (intestinal microbial communities), increasing the selection probability by 25 times compared with the existing scheme (one single colony is enough to obtain the target strain), saving a lot of repeated identification work and funds for Paraprevotella species, thereby effectively improving its isolation and culture efficiency, and laying a solid foundation for subsequent related research.

[0209] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent replacements and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention should be included in the scope of protection of the invention.

Claims

1. A fungicide for Paraprevotella, characterized in that The components include mango extract, blueberry extract, apple extract and green tea extract. The mango extract has a mangoside content of greater than or equal to 25wt%, the blueberry extract has a proanthocyanidin content of greater than or equal to 25wt%, the apple extract has a total apple polyphenol content of greater than or equal to 10wt%, and the green tea extract has a tea polyphenol content of greater than or equal to 30wt%.

2. The microbial enhancement agent according to claim 1, characterized in that Calculated by weight, the components of the bactericidal agent include: 2 to 6 parts of mango extract, 1 to 3 parts of blueberry extract, 1 to 4 parts of apple extract, and 0.3 to 1 part of green tea extract.

3. The microbial enhancement agent according to claim 2, characterized in that Calculated by weight, the components of the bactericidal agent include: 2.5 to 4.5 parts of mango extract, 1.5 to 2.5 parts of blueberry extract, 2 to 3 parts of apple extract, and 0.4 to 0.8 parts of green tea extract.

4. The microbial enhancement agent according to claim 3, characterized in that Calculated by weight, the components of the bactericidal agent include: 3.5 parts of mango extract, 2.5 parts of blueberry extract, 2.5 parts of apple extract, and 0.4 parts of green tea extract.

5. A microbial culture medium for Paraprevotella, characterized in that: The microbial culture medium comprises the bacterial growth agent according to any one of claims 1 to 4.

6. The microbial culture medium according to claim 5, characterized in that The concentration of the bacteriostatic agent in the microbial culture medium is 0.43-1.40 g / L, and the concentrations of the components of the bacteriostatic agent in the microbial culture medium are: mango extract 0.2-0.6 g / L, blueberry extract 0.1-0.3 g / L, apple extract 0.1-0.4 g / L, and green tea extract 0.03-0.1 g / L.

7. A method for improving the isolation and culture efficiency of Paraprevotella, characterized in that: The steps include: Prepare raw materials according to the components of the bacteriostatic agent according to any one of claims 1 to 4, wherein the raw materials include sterilized mango extract, sterilized blueberry extract, sterilized apple extract and sterilized green tea extract; adding the mango extract, blueberry extract, apple extract and green tea extract to a basic liquid culture medium to obtain an enrichment liquid culture medium; A buffer solution is prepared, and a human fecal sample is homogenized with the buffer solution, and then filtered to obtain an inoculum; adding the inoculum to the enrichment-liquid culture medium for fermentation under constant temperature and anaerobic conditions to obtain an enrichment culture; Extracting genomic DNA from the enriched culture, amplifying it by PCR, and sequencing it to obtain an enriched culture containing Paraprevotella; The enriched culture is diluted and then spread on a solid culture medium. After fermentation and culture, a single colony is obtained. The single colony is identified to obtain Paraprevotella.

8. The method according to claim 7, characterized in that The content of human fecal sample in the inoculum is 0.3-0.4 g / mL, and the volume ratio of the inoculum to the enrichment-liquid culture medium is (0.5-1.5): (8.5-9.5); the dilution factor of the enrichment culture is 1×10 5 Times ~ 1×10 6 times.

9. Use of the bacterial enrichment agent according to any one of claims 1 to 4 in inoculating, isolating or identifying Paraprevotella.

10. Use of the microbial culture medium according to any one of claims 5 to 6 in inoculating, isolating or identifying Paraprevotella.

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