Bacterium increasing agent of Blauria, microbial culture medium and application

Abstract

The invention discloses a bacterium increasing agent of Blauria, a microbial culture medium and application, and belongs to the technical field of microbial isolated culture. The enrichment medium comprises the following components: rhizoma anemarrhenae extract, seaweed extract, pine bark extract and resistant starch. According to the invention, the rhizoma anemarrhenae extract, the seaweed extract, the pine bark extract and the resistant starch are combined, so that the obtained enrichment medium can promote the proliferation of Blauria; therefore, the bacterium increasing agent has a relatively good application prospect in promoting the proliferation of the Blauria and / or efficiently separating the Blauria.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial separation and culture, and particularly relates to a Blautia bacteria enrichment agent, a microbial culture medium and applications. Background Art

[0002] Blautia is a newly discovered genus of intestinal anaerobic bacteria with probiotic properties. In-depth research on it will help develop new probiotic preparations and provide new strategies for maintaining the balance of human intestinal microecology and immune regulation.

[0003] Human fecal samples contain a certain amount of Blautia. Currently, it is difficult to isolate and culture Blautia from human fecal samples, which seriously restricts the research on its functional properties and application potential. It has the following limitations:

[0004] 1) Blautia is a strict anaerobe and is sensitive to oxygen, so traditional culture media are difficult to meet its growth requirements.

[0005] 2) The intestinal microbiome is complex. Blautia is relatively low in abundance in natural samples and competes for nutrients with other anaerobic bacteria (such as Bacteroidetes and Clostridium). Conventional enrichment methods make it difficult to selectively enrich target strains.

[0006] 3) Traditional culture media (such as basic anaerobic culture media) have an isolation and culture efficiency of less than 3% for Blautia in the intestine, which seriously restricts the research on its functional mechanism and the development of potential probiotic applications. Summary of the Invention

[0007] The present invention aims to provide a Blautia enrichment agent, a microbial culture medium and applications thereof, aiming to solve the problems of low proliferation capacity and low separation efficiency of wild Blautia strains in the isolation and culture process in the prior art.

[0008] In a first aspect, the present invention provides a Blautia bacteria enhancement agent, the components of the enhancement agent including: Anemarrhena asphodeloides extract, seaweed extract, pine bark extract, and resistant starch; wherein the Anemarrhena asphodeloides extract is water-soluble, with a specification of at least 10:1; the seaweed extract is water-soluble, with a specification of at least 10:1; the pine bark extract is water-soluble, with a specification of at least 10:1; and the resistant starch is type 2, with a specification of at least 99%.

[0009] In the microbial enhancer provided by the present invention, the inventors have found that after combining the extract of Anemarrhena asphodeloides, seaweed extract, pine bark extract and resistant starch, the resulting microbial enhancer can promote the proliferation of Blautia; therefore, the microbial enhancer has good application prospects in promoting the proliferation of Blautia and / or efficiently isolating Blautia.

[0010] In some embodiments, the components of the microbial enhancer include, by weight, 0.25-1.5 parts of Anemarrhena asphodeloides extract, 0.25-1.5 parts of seaweed extract, 0.25-1.5 parts of pine bark extract, and 1-4 parts of resistant starch.

[0011] In some embodiments, the components of the microbial enhancer include, by weight, 0.4-1.2 parts of Anemarrhena asphodeloides extract, 0.4-1.2 parts of seaweed extract, 0.4-1.2 parts of pine bark extract, and 1-3 parts of resistant starch.

[0012] In some embodiments, the components of the microbial enhancer include, by weight, 0.8 parts of Anemarrhena asphodeloides extract, 0.4 parts of seaweed extract, 0.8 parts of pine bark extract, and 3 parts of resistant starch.

[0013] In a second aspect, the present invention provides a microbial culture medium for improving the proliferation efficiency of Blautia, comprising any one of the above-mentioned bacterial enhancement agents.

[0014] In some embodiments, the mass volume ratio of the microbial growth agent to the culture medium in the microbial culture medium is 1.75-8.5 g / L, and the final concentrations of the components of the microbial growth agent in the culture medium include: 0.25-1.5 g / L of Anemarrhena asphodeloides extract, 0.25-1.5 g / L of seaweed extract, 0.25-1.5 g / L of pine bark extract, and 1-4 g / L of resistant starch.

[0015] In a third aspect, the present invention provides a method for improving the isolation and culture efficiency of Blautia, which comprises the following steps: preparing raw materials according to the components in any of the above-mentioned enrichment agents, the raw materials comprising sterilized Anemarrhena extract, sterilized seaweed extract, sterilized pine bark extract and sterilized resistant starch; adding the Anemarrhena extract, seaweed extract, pine bark extract and resistant starch to a basic liquid culture medium to obtain an enrichment-liquid culture medium; preparing a carbonate-phosphate buffer, homogenizing a human fecal sample in a potassium phosphate buffer, and then filtering to obtain an inoculum; adding the inoculum to the enrichment-liquid culture medium for fermentation and culture under constant temperature and anaerobic conditions to obtain an enrichment culture; extracting the genomic DNA of the enrichment culture, amplifying and sequencing it by PCR, and screening to obtain an enrichment culture containing Blautia; diluting the enrichment culture and then spreading it on a solid culture medium, obtaining a single colony after fermentation and culture, and identifying the single colony to obtain Blautia.

[0016] In some embodiments, 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.

[0017] In a fourth aspect, the present invention provides use of any one of the above-mentioned bacterial enrichment agents in inoculating, isolating or identifying Blautia.

[0018] In a fifth aspect, the present invention provides use of the above-mentioned microbial culture medium in inoculating, isolating or identifying Blautia.

[0019] The beneficial effects of the present invention are:

[0020] (1) The present invention first identified four groups of substances that have a proliferation-promoting effect on Blautia from a large number of known substances, including: mother extract, seaweed extract, pine bark extract and resistant starch.

[0021] On this basis, the present invention further combines Anemarrhena asphodeloides extract, seaweed extract, pine bark extract and resistant starch to obtain a compound formula that has a good proliferation-promoting effect on Blautia; therefore, the bacterial enhancement agent of the present invention has good application prospects in promoting the proliferation of Blautia and / or efficiently isolating Blautia.

[0022] Furthermore, this program improves the isolation and culture efficiency of wild Blautia strains by optimizing the formulation of the enrichment agent and culture conditions, providing a solid foundation for further exploring its biological characteristics and developing its potential value.

[0023] (2) The microbial culture medium of the present invention can provide a suitable environment for the growth of Blautia, reduce the redox potential in the culture environment, and create an anaerobic condition for Blautia that is closer to its natural living environment, thereby facilitating the survival and growth of Blautia.

[0024] (3) Traditional methods mainly rely on selective culture media to enrich target bacteria. The present invention first performs Illumina sequencing on the culture, and screens out cultures with a high proportion of Blautia based on the sequencing results. Then, repeated subculture is performed to further increase the proportion of Blautia in the culture. This method is based on molecular-level monitoring and can understand the composition changes of the microbial community in the culture in real time, which is more scientific and efficient.

[0025] (4) Conventional microbial identification methods usually use only one sequencing technology. The present invention first uses Illumina sequencing to analyze the V3-V4 region to obtain a preliminary understanding of the composition of the microbial community, and then uses Sanger sequencing to identify the full-length 16S rDNA. This method combines two different sequencing technologies to complement each other and ensure the accuracy of the identification results. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] To more clearly illustrate the technical solution of this application, the following is a brief introduction to the drawings used in the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be derived from these drawings without inventive effort.

[0027] Figure 1 This is a stacked diagram of species distribution in Test Example 1 of the present invention. DETAILED DESCRIPTION

[0028] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention; the terms "including" and "having" and any variations thereof in the specification and claims of the present invention and the above-mentioned drawings are intended to cover non-exclusive inclusions.

[0030] In the description of the embodiments of the present invention, technical terms such as "first" and "second" are used solely to distinguish between different objects and should not be understood to indicate or imply relative importance or to implicitly specify the quantity, specific order, or primary and secondary relationship of the technical features indicated. In the description of the embodiments of the present invention, "plurality" means more than two, unless otherwise specifically defined.

[0031] References herein to "embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments may be included in at least one embodiment of the present application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor does it constitute an independent or alternative embodiment that is mutually exclusive of other embodiments. It is understood, both explicitly and implicitly, by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0032] In the description of the embodiments of the present invention, the term "and / or" is simply a description of the association relationship between associated objects, indicating that three relationships can exist. For example, A and / or B can represent the following three situations: A exists alone, A and B exists simultaneously, and B exists alone. In addition, the character " / " in this document generally indicates that the associated objects are in an "or" relationship.

[0033] Experimental procedures in the examples, where specific conditions are not specified, were generally performed in accordance with conventional methods in molecular biology, including but not limited to those described in M.R. Green's Molecular Cloning: A Laboratory Manual and Robert F. Weaver's Molecular Biology, or according to the recommendations of kit and instrument manufacturers. Unless otherwise specified, reagents and biological materials used in the examples were commercially available.

[0034] In the present invention, in actual operation, the sterilization method of the present invention can be irradiation sterilization or filtration sterilization. People can choose a suitable sterilization method according to their needs. The model strain Blautia producta (Prevot) Liu et al.-27340 was purchased from the American Type Culture Collection (ATCC); the Anemarrhena extract, seaweed extract, pine bark extract, and resistant starch were all purchased from Shaanxi Ruilin Biotechnology Co., Ltd.; GAM culture medium was purchased from Huankai Biotechnology Co., Ltd.; DNA extraction kits, PCR amplification kits, and library construction kits were all purchased from Thermo Fisher Scientific (China) Co., Ltd. (Thermofisher).

[0035] Currently, research on the mechanisms, principles, functional properties, and application value of intestinal microorganisms is limited and progress has been slow. Since commonly used target bacteria (such as Blautia ATCC27340) are generally commercially available, when a specific target bacteria needs to be proliferated and cultured to facilitate subsequent systematic analysis of its metabolic mechanisms and industrial development, existing technologies generally cultivate the commercially available target bacteria directly to reduce R&D costs. However, commercially available target bacteria are generally limited to one or several specific species (such as Blautia) within a specific genus (such as Blautia), while other known species within this genus (such as Blautia) are difficult to obtain.

[0036] Currently, there are some commonly used probiotics (target bacteria) on the market that are based on species, but there are few studies on probiotics in intestinal microorganisms that are based on genus (such as Blautia).

[0037] Human fecal samples contain a certain amount of Blautia. Currently, it is difficult to isolate and culture Blautia in human fecal samples. When it is necessary to culture, isolate and identify probiotics (such as Blautia) 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 physiological 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 concentrations, and then The samples were then spread onto solid culture medium; 5) culture was obtained by in vitro fermentation at a constant temperature for 48-72 hours; 6) single colonies were collected and stored for future use; 7) genomic DNA was extracted from the stored samples; 8) full-length 16S gene sequences of the samples were amplified using a commercial kit and PCR technology; 9) the obtained gene sequences were subjected to Sanger sequencing analysis; 10) gene information was compared using the public BLAST database to determine whether it was the target bacterial genus (similarity > 98.7%); 11) if the identification result was negative, steps 7)-10) were repeated until the target strain was obtained.

[0038] Typically, in step 4), the limiting dilution should be 1×10 7 times to 1×10 10 Therefore, the traditional culture method of Blautia is inefficient and needs to be improved.

[0039] In response to the above problems, the present invention first studies the commercially available model strain Blautia ATCC27340 (single-factor variable experiment) to preliminarily determine the material components that have a proliferation-promoting effect on the model strain Blautia ATCC27340; 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 the model strain Blautia ATCC27340 (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 Blautia (taking 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 Blautia (taking the genus as the research unit).

[0040] Specifically: To solve the problems of low proliferation ability and low separation efficiency of wild Blautia strains during isolation and culture, the present invention provides a Blautia enrichment agent, a microbial culture medium, and applications.

[0041] In a first aspect, an embodiment of the present invention provides a Blautia bacteria enhancement agent, wherein the components of the enhancement agent include: Anemarrhena asphodeloides extract, seaweed extract, pine bark extract, and resistant starch.

[0042] In the technical solution of the embodiment of the present invention, by combining the Anemarrhena asphodeloides extract, the seaweed extract, the pine bark extract and the resistant starch, the obtained bacterial growth agent can promote the proliferation of Blautia and is conducive to the efficient separation of Blautia.

[0043] Among them, the Anemarrhena extract is water-soluble, with a specification of 10:1 and irradiation sterilization; the seaweed extract is water-soluble, with a specification of 10:1 and irradiation sterilization; the pine bark extract is water-soluble, with a specification of 10:1 and irradiation sterilization; the resistant starch is type 2, with a specification of 99% and irradiation sterilization; the above-mentioned Anemarrhena extract, seaweed extract, pine bark extract and resistant starch were all purchased from Shaanxi Ruilin Biotechnology Co., Ltd.

[0044] Furthermore, in some embodiments, the components of the microbial enhancer include, by weight, 0.25-1.5 parts of Anemarrhena asphodeloides extract, 0.25-1.5 parts of seaweed extract, 0.25-1.5 parts of pine bark extract, and 1-4 parts of resistant starch.

[0045] In the technical solution provided in the embodiment of the present invention, the bacterial enhancement agent obtained by combining specific mass fractions of Anemarrhena asphodeloides extract, seaweed extract, pine bark extract and resistant starch can further promote the proliferation of Blautia and is more conducive to the efficient separation of Blautia.

[0046] In the bactericidal agent provided in the embodiments of the present invention, the mass fraction of the Anemarrhena asphodeloides extract can be, for example, 0.25 parts, 0.4 parts, 0.6 parts, 0.8 parts, 1 parts, 1.2 parts, 1.5 parts or other values ​​within this range, the mass fraction of the seaweed extract can be, for example, 0.25 parts, 0.4 parts, 0.6 parts, 0.8 parts, 1 parts, 1.2 parts, 1.5 parts or other values ​​within this range, the mass fraction of the pine bark extract can be, for example, 0.25 parts, 0.4 parts, 0.6 parts, 0.8 parts, 1 parts, 1.2 parts, 1.5 parts or other values ​​within this range, and the mass fraction of the resistant starch can be, for example, 1 parts, 1.5 parts, 2 parts, 2.5 parts, 3 parts, 3.5 parts, 4 parts or other values ​​within this range.

[0047] Furthermore, in some embodiments, the components of the microbial enhancer include, by weight, 0.4-1.2 parts of Anemarrhena asphodeloides extract, 0.4-1.2 parts of seaweed extract, 0.4-1.2 parts of pine bark extract, and 1-3 parts of resistant starch.

[0048] In the technical solution provided in the embodiment of the present invention, the bacterial enhancement agent obtained by optimizing the mass fractions of Anemarrhena asphodeloides extract, seaweed extract, pine bark extract and resistant starch and combining them can significantly promote the proliferation efficiency of Blautia and is more conducive to the efficient separation of Blautia.

[0049] Furthermore, in some embodiments, the components of the microbial enhancer include, by weight, 0.8 parts of Anemarrhena asphodeloides extract, 0.4 parts of seaweed extract, 0.8 parts of pine bark extract, and 3 parts of resistant starch.

[0050] In the technical solution provided in the embodiments of the present invention, the mass fractions of Anemarrhena extract, seaweed extract, pine bark extract and resistant starch are optimized by using orthogonal experiments. The bacterial enhancement agent obtained by combining the optimal mass fractions of Anemarrhena extract, seaweed extract, pine bark extract and resistant starch has the best promoting effect on the proliferation of Blautia and can best and most efficiently separate Blautia.

[0051] In a second aspect, an embodiment of the present invention provides a microbial culture medium for improving the proliferation efficiency of Blautia, comprising any one of the above-mentioned bacterial enhancement agents.

[0052] In the microbial culture medium provided in the embodiment of the present invention, after adding the above-mentioned bacterial enhancement agent to the basic culture medium, the obtained microbial culture medium can promote the proliferation efficiency of Blautia and can efficiently separate Blautia.

[0053] Furthermore, in some embodiments, the mass volume ratio of the microbial growth agent to the culture medium in the microbial culture medium is 1.75-8.5 g / L, and the final concentrations of the components of the microbial growth agent in the culture medium include: 0.25-1.5 g / L of Anemarrhena asphodeloides extract, 0.25-1.5 g / L of seaweed extract, 0.25-1.5 g / L of pine bark extract, and 1-4 g / L of resistant starch.

[0054] In the technical solution provided in the embodiments of the present invention, the bacterial enhancement agent obtained by optimizing and combining the final concentrations of Anemarrhena asphodeloides extract, seaweed extract, pine bark extract and resistant starch in the culture medium can significantly promote the proliferation efficiency of Blautia and is more conducive to the efficient separation of Blautia.

[0055] In the microbial culture medium provided by the embodiments of the present invention, the final concentration of the Anemarrhena asphodeloides extract can be, for example, 0.25 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, 1 g / L, 1.2 g / L, 1.5 g / L or other values ​​within this range; the final concentration of the seaweed extract can be, for example, 0.25 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, 1 g / L, 1.2 g / L, 1.5 g / L or other values ​​within this range; the final concentration of the pine bark extract can be, for example, 0.25 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, 1 g / L, 1.2 g / L, 1.5 g / L or other values ​​within this range; and the final concentration of the resistant starch can be, for example, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L or other values ​​within this range.

[0056] In the technical solution provided in the embodiment of the present invention, the culture medium is used to meet the nutritional requirements of Blautia proliferation.

[0057] It is understood that the culture medium can be selected from conventional culture media in the prior art according to actual use needs, as long as it can meet the nutritional requirements of Blautia proliferation. The culture medium in the present invention preferably includes at least one of LB medium, GAM medium, and MRS medium.

[0058] In a third aspect, an embodiment of the present invention provides a method for improving the isolation and culture efficiency of Blautia, comprising the following steps: preparing raw materials according to the components of any of the above-mentioned microbial enhancers, the raw materials comprising a sterilized extract of Anemarrhena rhizome, a sterilized seaweed extract, a sterilized pine bark extract, and a sterilized resistant starch; adding the extract of Anemarrhena rhizome, the seaweed extract, the pine bark extract, and the resistant starch to a basic liquid culture medium to obtain a microbial enhancer-liquid culture medium; preparing a carbonate-phosphate buffer solution; Blautia is homogenized with a carbonate-phosphate buffer solution and then filtered to obtain an inoculum; the inoculum is added to an enrichment-liquid culture medium for fermentation culture under constant temperature and anaerobic conditions to obtain an enrichment culture; genomic DNA of the enrichment culture is extracted, and after PCR amplification and sequencing, an enrichment culture containing Blautia is screened; the enrichment culture is diluted and then spread on a solid culture medium, and after fermentation culture, a single colony is obtained, and the single colony is identified to obtain Blautia.

[0059] In the method provided in the embodiment of the present invention, by adopting the enrichment-liquid culture medium of the present invention, it can promote the proliferation of Blautia, thereby shortening the process of Blautia isolation and culture, and significantly improving the efficiency of Blautia isolation and culture, solving the problems of low strain proliferation ability and low separation efficiency of Blautia wild strains in the isolation and culture process in the prior art, avoiding the need for a large number of repeated screening and identification steps in traditional methods, and saving separation costs; in addition, the method is simple to operate and the raw materials are cheap and easily available, and therefore is suitable for industrial large-scale production applications.

[0060] Furthermore, in some embodiments, 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.

[0061] In the technical solution provided in the embodiment of the present invention, the volume ratio of the inoculum to the enrichment-liquid culture medium is controlled within a specific range, which is conducive to the rapid proliferation of the inoculum; the dilution of the applied bacterial liquid is controlled within a smaller multiple, which can reduce the number of dilutions and save manpower and material resources.

[0062] In the present invention, the volume ratio of the inoculum to the enrichment-liquid culture medium can be, for example, 0.5:8.5, 0.5:9, 0.5:9.5, 1:8.5, 1:9, 1:9.5, 1.5:8.5, 1.5:9, 1.5:9.5 or other ratios within the range; the dilution multiple of the enrichment culture can be, for example, 1×10 5 times, 1.2×10 5 times, 1.4×10 5 times, 1.6×10 5 times, 1.8×10 5 times, 1×10 6 times or other multiples within this range.

[0063] In a fourth aspect, embodiments of the present invention provide use of any of the aforementioned bacterial enrichment agents in inoculating, isolating, or identifying Blautia.

[0064] In the application provided by the embodiment of the present invention, by using the bacterial enhancement agent of the present invention, it can promote the proliferation of Blautia, and therefore has a good application prospect in the efficient separation of Blautia.

[0065] In a fifth aspect, embodiments of the present invention provide use of the above-mentioned microbial culture medium in inoculating, isolating, or identifying Blautia.

[0066] In the application provided by the embodiment of the present invention, by using the microbial culture medium of the present invention, it can promote the proliferation of Blautia, and therefore has good application prospects in the efficient separation of Blautia.

[0067] Some specific embodiments are listed below. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0068] Example 1 Effect of a single-component bacterial enrichment agent on the proliferation of Blautia

[0069] In this example, the model strain Blautia ATCC27340 was used as the research object to explore the effects of Anemarrhena Rhizoma extract, seaweed extract, pine bark extract, and resistant starch on the proliferation of Blautia.

[0070] This example uses the extraction of Anemarrhena as an example to illustrate the effect of a single bacterial species on the proliferation of the model strain Blautia ATCC27340. Specifically, the method includes the following steps:

[0071] 1) Prepare solutions of certain concentrations of Anemarrhena asphodeloides extract (0.25 parts, 1 part, and 1.5 parts) using sterile water;

[0072] 2) Add the Anemarrhena extract solution from step 1) to the sterilized GAM liquid culture medium to achieve final concentrations of the Anemarrhena extract of 0.25 g / L, 1 g / L, and 1.5 g / L, respectively. Meanwhile, use the GAM liquid culture medium without the Anemarrhena extract solution as a blank control, and mark the groups accordingly;

[0073] 3) OD 600nm The Blautia bacterial solution with a pH value of 0.6-0.8 was inoculated into the culture medium in step 2) at an inoculum size of 1%, and the culture was cultured in an anaerobic incubator at 37°C for 24 hours to obtain a culture;

[0074] 4) Dilute the culture in step 3) to 1×10 7 After the culture was doubled, 0.25 mL of the bacterial solution was spread on GAM solid culture medium, and then placed in an anaerobic incubator and cultured at 37°C for 48 h. The plate count was then performed. The results are shown in Table 1 below.

[0075] Similarly, the Anemarrhena extract in the above method was replaced with seaweed extract, pine bark extract and resistant starch, and the final concentrations of seaweed extract and pine bark extract were 0.25 g / L, 1 g / L and 1.5 g / L, respectively, and the final concentrations of resistant starch were 1 g / L, 2 g / L and 4 g / L, respectively; then, the plate count was performed after treatment according to the above method, and the results are shown in Table 1 below.

[0076] In actual work, this example also used a blank control to conduct three parallel experiments (using only GAM liquid culture medium). The average number of colonies cultured in the blank control group was 22.33. Among them, the experimental method of designing a blank control is a conventional technical means in this field and will not be described in detail here.

[0077] Table 1 Effects of single-component bacterial growth agents on the proliferation of Blautia

[0078]

[0079]

[0080] The data in Table 1 show that microbial culture media containing a specific concentration of Anemarrhena extract, seaweed extract, pine bark extract, and resistant starch in certain mass fractions all promote the proliferation of Blautia. Furthermore, in the aforementioned experiments, the number of colonies in the four single components at low concentrations was lower than that in the control group. This is reasonable, as the experimental groups do not necessarily all have a larger number than the control group. It is possible that the four single components of the present application, at low concentrations, were not as effective in promoting the proliferation of Blautia as the GAM liquid culture medium.

[0081] The data in Example 1 show that adding 0.25-1.5 parts of Anemarrhena extract, 0.25-1.5 parts of seaweed extract, 0.25-1.5 parts of pine bark extract, and 1-4 parts of resistant starch to the GAM liquid culture medium respectively to obtain a microbial culture medium containing 0.25-1.5 g / L of Anemarrhena extract, 0.25-1.5 g / L of seaweed extract, 0.25-1.5 g / L of pine bark extract, and 1-4 g / L of resistant starch can promote the proliferation of Blautia.

[0082] At the same time, it can be seen from Table 1 above that among the four single components of the Blautia bacteria enhancement agent, seaweed extract has the best effect on promoting the proliferation of the model strain Blautia 27340, the optimal single dosage of Anemarrhena asphodeloides extract is 1 g / L, the optimal single dosage of seaweed extract is 1 g / L, the optimal single dosage of pine bark extract is 1.5 g / L, and the optimal single dosage of resistant starch is 2 g / L.

[0083] Example 2 Effect of a composite bacterial growth agent on the proliferation of Blautia

[0084] In order to further determine the effects of the combination of different concentrations of the above-mentioned Anemarrhena extract, seaweed extract, pine bark extract, and resistant starch on the proliferation of Blautia, in this example, the model strain Blautia 27340 was used as the research object, and the effects of different concentrations of the combination of Anemarrhena extract, seaweed extract, pine bark extract, and resistant starch on the proliferation of Blautia were explored.

[0085] Specifically, the steps include:

[0086] 1) First, design the factors and levels table for the orthogonal experiment (as shown in Table 2 below). Then, use sterile water to prepare solutions of certain concentrations of Anemarrhena rhizome extract, seaweed extract, pine bark extract, and resistant starch.

[0087] 2) In nine orthogonal design experiments, different concentrations of Anemarrhena extract, seaweed extract, pine bark extract, and resistant starch were added to sterilized GAM liquid culture medium and marked in groups.

[0088] 3) OD 600nm The inoculum with a value of 0.6-0.8 was inoculated into the culture medium in step 2) at an inoculum size of 1%, and the culture medium was placed in an anaerobic incubator and cultured at a constant temperature of 37°C for 24 hours to obtain a culture;

[0089] 4) Dilute the culture in step 3) to 1×10 6 After the culture was doubled, 0.25 mL of the bacterial solution was spread on GAM solid culture medium, and then placed in an anaerobic incubator and cultured at 37°C for 48 h before plate counting. The results of the orthogonal experiment are shown in Table 3 below.

[0090] Table 2 Factors and levels of orthogonal experiment

[0091]

[0092] Table 3 Orthogonal experiment results

[0093]

[0094] 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.

[0095] 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:

[0096] For example, in the four-factor three-level L9 (3 4 ), the number 85 means: in the fourth group of experiments, the Anemarrhena asphodeloides extract adopted the concentration corresponding to level 2 (that is, the Anemarrhena asphodeloides extract was 0.8 g / L), the seaweed extract adopted the concentration corresponding to level 1 (that is, the seaweed extract was 0.4 g / L), the pine bark extract adopted the concentration corresponding to level 2 (that is, the pine bark extract was 0.8 g / L), and the resistant starch adopted the concentration corresponding to level 3 (that is, the resistant starch was 3 g / L). At this time, the number of colonies of the model strain Blautia 27340 was 85.

[0097] For example, in the four-factor three-level L9(3 4 ), the number 26.33 means that when the seaweed extract adopts the concentration value corresponding to level 2 (that is, the seaweed extract is 0.8g / L), the average colony number of the model strain Blautia27340 is 26.33.

[0098] For example, in the four-factor three-level L9(3 4 ), the number 19.67 means: the difference between the highest and lowest colony counts of pine bark extract, that is, the difference between the average colony count when the concentration value corresponding to level 3 (pine bark extract is 1.2 g / L) is used and the average colony count when the concentration value corresponding to level 1 (pine bark extract is 0.4 g / L) is used.

[0099] 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:

[0100] (1) When the four components described in the microbial enhancer of the present application are used together, the pine bark extract has the best effect on promoting the proliferation of the model strain Blautia 27340, followed by the Anemarrhena extract, seaweed extract and resistant starch; the seaweed extract has the largest range, so when the seaweed extract is used in different dosages, its effect on promoting the proliferation of the model strain Blautia 27340 will also have significant differences; the Anemarrhena extract has the smallest range, so when the Anemarrhena extract is used in different dosages, its effect on promoting the proliferation of the model strain Blautia 27340 will have little difference.

[0101] (2) As shown in Tables 2 and 3, when the components described in this application were used to culture Blautia, the culture was diluted to 1×10 6 After the four factors and three levels L9(3 4 ) in the 9 experimental schemes of the orthogonal experiment, the highest number of Blautia colonies that could be cultured was 85;

[0102] (3) It can be seen from Tables 2 and 3 that when the four single components in this application are used together to culture Blautia, the optimal combination is 0.8 g / L of Anemarrhena extract, 0.4 g / L of seaweed extract, 0.8 g / L of pine bark extract, and 3 g / L of resistant starch.

[0103] Application Test Example 1

[0104] The method for improving the isolation and culture efficiency of Blautia provided by the present invention is used to isolate and culture Blautia in human fecal samples, specifically comprising the following steps:

[0105] S1. Homogenize the sample to be separated with a buffer solution (carbonate-phosphate buffer, 1:3, W / V), and then filter to obtain an inoculum. The sample to be separated 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 scheme for obtaining the sample to be separated is a conventional technical scheme in the art and is not described in detail here.

[0106] S2. 1 mL of the inoculum obtained in step S1 was added to 9 mL of sterilized GAM liquid medium (containing 0.8 g / L of Anemarrhena asphodeloides extract, 0.4 g / L of seaweed extract, 0.8 g / L of pine bark extract, and 3 g / L of resistant starch), and the mixture was placed in an anaerobic incubator and cultured at 37° C. for 24 h to obtain an enriched culture;

[0107] S3. Extracting genomic DNA from the enriched culture obtained in step S2, amplifying the V3-V4 region of the sample by PCR, and sequencing the fragments by Illumina to obtain the enriched culture containing Blautia;

[0108] S4, dilute the enriched culture containing Blautia obtained in step S3 to 1×10 6The sample was amplified at 100 μg / ml and then spread on GAM solid culture medium. After that, the sample was placed in an anaerobic incubator and cultured at 37°C for 48 h to obtain a single colony. The single colony was identified (the full-length 16S gene sequence of the sample was first amplified, followed by Sanger sequencing analysis), and Blautia was obtained (gene information was compared using the public BLAST database, and the similarity was >98.7%).

[0109] Among them, in step S3, the species distribution stacking diagram of the enrichment culture containing Blautia and the human fecal sample directly sequenced by Illumina is as follows: Figure 1 As shown, that is Figure 1 The source is the analysis data (otu_table.g_relative.mat.xls) obtained from the Illumina sequencing source file using bioinformatics software.

[0110] from Figure 1 It can be seen that after cultivation using the method of the present invention, the relative abundance of Blautia in the sample was significantly increased from 2.78% in the original sample to 19.88%, which facilitates the subsequent rapid and efficient separation of Blautia, saves repeated identification work and funds for Blautia, and significantly improves the isolation and cultivation efficiency of Blautia.

[0111] Application Test Example 2

[0112] The method for improving the isolation and culture efficiency of Blautia provided by the present invention is used to isolate and culture Blautia in fecal samples, specifically comprising the following steps:

[0113] S1. Homogenize the sample to be separated with a buffer solution (carbonate-phosphate buffer, 1:3, W / V), and then filter to obtain an inoculum. The sample to be separated 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 scheme for obtaining the sample to be separated is a conventional technical scheme in the art and is not described in detail here.

[0114] S2. 1 mL of the inoculum obtained in step S1 was added to 9 mL of sterilized GAM liquid medium (containing 0.8 g / L of Anemarrhena asphodeloides extract, 0.4 g / L of seaweed extract, 0.8 g / L of pine bark extract, and 3 g / L of resistant starch), and the mixture was placed in an anaerobic incubator and cultured at 37° C. for 24 h to obtain an enriched culture;

[0115] S3. Extracting genomic DNA from the enriched culture obtained in step S2, amplifying the V3-V4 region of the sample by PCR, and sequencing the fragments by Illumina to obtain the enriched culture containing Blautia;

[0116] S4, dilute the enriched culture containing Blautia obtained in step S3 to 1×10 6 The sample was amplified at 100 μg / ml and then spread on GAM solid culture medium. After that, the sample was placed in an anaerobic incubator and cultured at 37°C for 48 h to obtain a single colony. The single colony was identified (the full-length 16S gene sequence of the sample was first amplified, followed by Sanger sequencing analysis), and Blautia was obtained (gene information was compared using the public BLAST database, and the similarity was >98.7%).

[0117] Among them, in step S3, the specific method is as follows:

[0118] S31. Use a DNA extraction kit according to the instructions to extract total DNA from the sample;

[0119] S32. Using the extracted sample DNA as a template, perform PCR amplification using universal primers according to the reaction system in Table 4 below;

[0120] The nucleotide sequence of the universal primer is as follows:

[0121] 341F: 5'-CCTACGGGNGGCWGCAG-3' (SEQ ID NO: 1);

[0122] 806R: 5'-GGACTACNVGGGTWTCTAAT-3' (SEQ ID NO: 2);

[0123]

[0124] PCR reaction conditions: pre-denaturation at 95°C for 3 min; 25 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 15 s, starting from the second step; extension at 72°C for 5 min, and storage at 4°C.

[0125] S33, recovering the PCR product amplified in step S32 as a template, performing end repair, adding A tails, and ligating sequencing adapters according to the reaction system in Table 5 below to obtain an amplicon;

[0126]

[0127] 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.

[0128] S34, sequencing the amplicon library constructed in step S33 using an Illumina Miseq sequencing platform and a PE250 sequencing strategy;

[0129] S35. Data was processed offline to remove low-quality sequences and adapter sequences. The processed data were analyzed for relative abundance using bioinformatics software. The results are shown in Table 6 below.

[0130]

[0131]

[0132]

[0133] Note: 1. Note: E is used to represent the power of 10. For example, 6.89E-02 means 6.89×10 -2 ;2. The experimental control group refers to the isolation scheme using only the basic culture medium without adding the bacterial growth agent;

[0134] from Figure 1 As can be seen from Table 6, after cultivation using the method of the present invention, the relative abundance of Blautia in the sample was significantly increased from 0.0278 in the original sample to 0.199; the present application can be used to quickly and efficiently isolate Blautia, saving the repeated identification work and funds of Blautia, and significantly improving the isolation and cultivation efficiency of Blautia.

[0135] In summary, the present invention can promote the proliferation of Blautia by using specific concentrations of Anemarrhena extract, seaweed extract, pine bark extract and resistant starch alone as bacterial growth agents; further, when specific concentrations of Anemarrhena extract, seaweed extract, pine bark extract and resistant starch are combined, the resulting bacterial growth agent can further promote the proliferation of Blautia.

[0136] It should be noted that the microbial culture medium of the present application can also be called an enrichment medium. How to achieve the isolation and cultivation of bacterial species is a conventional technical means in this field. The present application mainly uses a new enrichment agent and its corresponding enrichment medium to isolate and culture Blautia genus, so as to detect the effect of the new enrichment agent and its corresponding enrichment medium on the proliferation of Blautia genus, and on this basis complete the proliferation culture, isolation and identification of Blautia genus.

[0137] The above-described embodiments merely illustrate the implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A bacterial growth agent for Blautia, characterized in that: The components of the microbial enhancer include: Anemarrhena asphodeloides extract, seaweed extract, pine bark extract, and resistant starch; wherein the Anemarrhena asphodeloides extract is water-soluble, with a specification of at least 10:1; the seaweed extract is water-soluble, with a specification of at least 10:1; the pine bark extract is water-soluble, with a specification of at least 10:1; and the resistant starch is type 2, with a specification of at least 99%.

2. The microbial enhancement agent according to claim 1, characterized in that Calculated by weight, the components of the bactericidal agent include: 0.25-1.5 parts of Anemarrhena asphodeloides extract, 0.25-1.5 parts of seaweed extract, 0.25-1.5 parts of pine bark extract, and 1-4 parts of resistant starch.

3. The microbial enhancement agent according to claim 2, characterized in that Calculated by weight, the components of the bactericidal agent include: 0.4 to 1.2 parts of Anemarrhena asphodeloides extract, 0.4 to 1.2 parts of seaweed extract, 0.4 to 1.2 parts of pine bark extract, and 1 to 3 parts of resistant starch.

4. The microbial enhancement agent according to claim 3, characterized in that Calculated by weight, the components of the bactericidal agent include: 0.8 parts of Anemarrhena asphodeloides extract, 0.4 parts of seaweed extract, 0.8 parts of pine bark extract, and 3 parts of resistant starch.

5. A microbial culture medium for improving the proliferation efficiency of Blautia, 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 mass volume ratio of the microbial growth agent to the culture medium in the microbial culture medium is 1.75-8.5 g / L, and the final concentrations of the components of the microbial growth agent in the culture medium include: 0.25-1.5 g / L of Anemarrhena asphodeloides extract, 0.25-1.5 g / L of seaweed extract, 0.25-1.5 g / L of pine bark extract, and 1-4 g / L of resistant starch.

7. A method for improving the isolation and culture efficiency of Blautia, characterized in that: The steps include: Prepare raw materials according to the components of the microbial enhancer according to any one of claims 1 to 4, wherein the raw materials include a sterilized extract of Anemarrhena asphodeloides, a sterilized seaweed extract, a sterilized pine bark extract, and a sterilized resistant starch; adding the Anemarrhena asphodeloides extract, seaweed extract, pine bark extract and resistant starch to a basic liquid culture medium to obtain a bacterial enrichment-liquid culture medium; Prepare carbonate-phosphate buffer, homogenize the human fecal sample in potassium phosphate buffer, and then filter to obtain an inoculum; adding the inoculum to the enrichment-liquid culture medium for fermentation culture 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 Blautia; 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 Blautia.

8. The method according to claim 7, characterized in that The content of human fecal sample in the inoculum is 0.3~0.4g / 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 multiple of the enrichment culture is 1×105 times to 1×10 6 times.

9. Use of the bacterial enhancement agent according to any one of claims 1 to 4 in inoculation, isolation or identification of Blautia.

10. Use of the microbial culture medium according to claim 5 or 6 in inoculation, isolation or identification of Blautia.

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