Screening medium for precise gut microbiota transplantation, preparation method thereof and method for enriching enterobacteriales bacteria

Abstract

This invention belongs to the field of microbial technology, and particularly relates to a screening culture medium for precise intestinal flora transplantation, its preparation method, and a method for enriching Enterobacteriaceae bacteria. The application of the composition provided by this invention in the preparation of intestinal flora screening culture media, through the synergistic effect of the basic nutritional conditions provided by the oligofructose, chlorogenic acid, and CDC anaerobic agar basal medium, enables effective screening and enrichment of Enterobacteriaceae bacteria in fecal samples under in vitro culture conditions, showing excellent application prospects in the isolation, purification, and functional studies of Enterobacteriaceae bacteria.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and particularly relates to a screening culture medium for precise intestinal flora transplantation, its preparation method, and a method for enriching Enterobacteriaceae bacteria. Background Technology

[0002] Enterobacteriaceae is an extremely important taxonomic unit within the domain Bacteria, encompassing numerous families and genera closely related to human health, disease, and environmental ecology, such as the genus Escherichia (…). Escherichia Enterobacteriaceae, etc. The bacteria included in the order Enterobacteriaceae are mostly core members of the mammalian gut microbiota, playing important roles in host nutrition and metabolism, immune regulation, etc.; at the same time, some members are also important opportunistic pathogens, closely related to intestinal and extraintestinal infections.

[0003] Therefore, developing culture techniques capable of effectively enriching Enterobacteriaceae is of significant application value for intestinal microbiome analysis, pathogen detection, disease diagnosis, and probiotic resource discovery. However, traditional anaerobic agar basal media lack specific nutrient supply or selective inhibitors for Enterobacteriaceae in their composition, resulting in insufficient enrichment capacity for Enterobacteriaceae and limiting their ability to efficiently screen Enterobacteriaceae from complex bacterial communities. Summary of the Invention

[0004] The primary objective of this invention is to address the problems of insufficient enrichment capacity and low bacterial screening efficiency in the application of existing anaerobic agar basal culture media for screening Enterobacteriaceae, and to provide the application of the composition in the preparation of intestinal flora screening culture media.

[0005] A second objective of this invention is to provide a screening culture medium.

[0006] A third objective of this invention is to provide a method for preparing the above-mentioned screening culture medium.

[0007] The fourth objective of this invention is to provide a method for enriching Enterobacteriaceae in fecal samples under in vitro culture conditions.

[0008] Specifically, in the application of the composition provided by the present invention in the preparation of intestinal flora screening culture medium, the composition comprises chlorogenic acid and fructooligosaccharides in a mass ratio of (0.01~1):(5~20), and the intestinal flora screening culture medium comprises CDC anaerobic agar basal medium.

[0009] Furthermore, the degree of polymerization of the oligofructose is 3 to 7.

[0010] Furthermore, the gut microbiota screening medium is used to enrich and / or purify Enterobacteriaceae bacteria in the gut microbiota.

[0011] The screening culture medium provided by this invention includes 0.05 g / L to 0.5 g / L chlorogenic acid, 1 g / L to 20 g / L fructooligosaccharides, and 35 to 45 g / L CDC anaerobic agar basal medium.

[0012] Furthermore, the degree of polymerization of the oligofructose is 3 to 7.

[0013] The method for preparing the screening culture medium provided by the present invention includes: taking the CDC anaerobic agar basal culture medium, fructooligosaccharide and water, mixing them and sterilizing them; then adding the chlorogenic acid, heme chloride and vitamin K1 and mixing them to obtain the screening culture medium.

[0014] Furthermore, the sterilization process is carried out at a temperature of 115℃~130℃, a pressure of 0.10kPa~0.22kPa, and a time of 10min~20min.

[0015] The present invention provides a method for enriching Enterobacteriaceae in fecal samples under in vitro culture conditions, comprising: taking diluted fecal samples and inoculating them into the above-mentioned screening medium for screening culture to obtain purified colonies.

[0016] Furthermore, the fecal sample was diluted by a factor of 10. -7 ~10 -6 The screening and culture were carried out in an anaerobic environment.

[0017] Beneficial effects: The application of the composition provided by this invention in the preparation of intestinal flora screening culture medium, the synergistic effect of the basic nutritional conditions provided by the oligofructose, chlorogenic acid and CDC anaerobic agar basal culture medium, can achieve effective screening and enrichment of Enterobacteriaceae in fecal samples under in vitro culture conditions, and has excellent application prospects in the isolation, purification and functional study of Enterobacteriaceae.

[0018] The reasons for the aforementioned technical effects of this composition are speculated to be as follows: Under the basic nutritional conditions provided by CDC anaerobic agar basal medium and in the presence of chlorogenic acid, the oligofructose can effectively improve the metabolic level of Enterobacteriaceae and promote their proliferation. Furthermore, the oligofructose can stimulate and enhance the hydrolytic metabolism of chlorogenic acid by Enterobacteriaceae, thereby increasing the growth-promoting effect of chlorogenic acid on Enterobacteriaceae. As a growth-promoting factor for Enterobacteriaceae, chlorogenic acid can also promote the anaerobic decomposition and metabolism of oligofructose by Enterobacteriaceae, enhancing the growth-promoting effect of oligofructose on Enterobacteriaceae. It promotes metabolism and growth, enhances the bacteria's tolerance to fluctuations in the physicochemical environment, and effectively inhibits the growth and proliferation of pathogenic bacteria. Through the synergistic effect of the basic nutrients provided by fructooligosaccharides, chlorogenic acid, and CDC anaerobic agar basal medium, Enterobacteriaceae exhibit excellent metabolic competitive advantages under in vitro culture conditions, effectively improving their metabolic efficiency and proliferation capacity. This enables the directional proliferation of Enterobacteriaceae, resulting in excellent enrichment of Enterobacteriaceae. It shows great promise for the preparation of screening media for the isolation, purification, and functional studies of Enterobacteriaceae. Attached Figure Description

[0019] Figure 1 The diagram shows the results of identifying the bacterial community structure of purified colonies, provided as a test example of the present invention. Detailed Implementation

[0020] This invention provides the application of the composition in the preparation of a screening medium for intestinal flora, wherein the basal medium used in the screening medium is CDC anaerobic agar basal medium. Specifically, the composition comprises chlorogenic acid and fructooligosaccharides in a mass ratio of (0.01~1):(5~20), specifically 0.01:5, 0.01:10, 0.01:20, 0.1:5, 0.1:20, 0.5:20, 1:5, 1:20, or any value between them. In this case, the chlorogenic acid, fructooligosaccharides, and the basal nutritional conditions provided by the CDC anaerobic agar basal medium work synergistically to give Enterobacteriaceae a metabolic competitive advantage under in vitro culture conditions. This allows for rapid screening and enrichment of Enterobacteriaceae in fecal samples under in vitro culture conditions, demonstrating excellent application prospects.

[0021] In this invention, the oligofructose refers to a short-chain carbohydrate composed of fructose molecules linked by β-glycosidic bonds, and its degree of polymerization is preferably 3 to 7, specifically 3, 4, 5, 6 or 7.

[0022] In this invention, the gut microbiota screening medium is specifically used to enrich and / or purify Enterobacteriaceae bacteria in the gut microbiota. The screening targets of the gut microbiota screening medium can be, but are not limited to, one or more of the following: unpurified fecal samples, unpurified but diluted fecal samples, gut microbiota sludge obtained from purified fecal samples, and gut microbiota suspension obtained from purified and diluted fecal samples.

[0023] The screening culture medium provided by this invention specifically includes: chlorogenic acid, fructooligosaccharides, and a basal culture medium. The concentration of chlorogenic acid is preferably 0.05 g / L to 0.5 g / L, specifically 0.05 g / L, 0.08 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, or any value between them; the concentration of fructooligosaccharides is preferably 1 g / L to 20 g / L, specifically 1 g / L, 1.2 g / L, 1.5 g / L, 2 g / L, or 1 g / L. The concentration of the CDC anaerobic agar basal medium is preferably 35 g / L to 45 g / L, specifically 35 g / L, 38 g / L, 40 g / L, 40.5 g / L, 42 g / L, 44 g / L, 45 g / L, or any value between them. The CDC anaerobic agar basal medium is a commonly used reagent in existing microbial culture. It can be prepared in-house or obtained by purchasing commercially available products. Those skilled in the art can make appropriate choices according to actual needs, and this invention does not impose any particular limitations.

[0024] The method for preparing the screening medium provided by the present invention specifically includes: taking the CDC anaerobic agar basal medium, fructooligosaccharide and water, mixing them and sterilizing them; then adding the chlorogenic acid, heme chloride and vitamin K1 and mixing them to obtain the screening medium.

[0025] In this invention, the method of mixing the CDC anaerobic agar basal medium, fructooligosaccharides and water can be any of the existing options, as long as it can achieve full dissolution and mixing of the CDC anaerobic agar basal medium and fructooligosaccharides in water. This invention does not impose any particular limitation.

[0026] In this invention, the sterilization conditions specifically include a temperature preferably between 115°C and 130°C, specifically 115°C, 118°C, 120°C, 121°C, 125°C, 129°C, 130°C, or any value between them; a pressure preferably between 0.1 kPa and 0.22 kPa, specifically 0.1 kPa, 0.11 kPa, 0.12 kPa, 0.15 kPa, 0.18 kPa, 0.2 kPa, 0.22 kPa, or any value between them; and a time preferably between 10 min and 20 min, specifically 10 min, 12.5 min, 14 min, 15 min, 18 min, 20 min, or any value between them.

[0027] The method for enriching Enterobacteriaceae in fecal samples under in vitro culture conditions provided by the present invention specifically includes: taking diluted fecal samples and inoculating them into the above-mentioned screening medium for screening culture to obtain purified colonies.

[0028] In this invention, the preferred dilution factor for the fecal sample is 10. -7 ~10 -6 Specifically, it can be 10 -7 1.2×10 -7 2×10 -7 4×10 -7 8×10 -7 9×10 -7 10 -6 Or any value between them. At this point, the amount of fecal sample inoculated in the screening medium and the number of microorganisms it contains are appropriate, which is conducive to achieving high-efficiency screening and enrichment of Enterobacteriaceae in fecal samples.

[0029] In this invention, the inoculation of diluted fecal samples into the screening medium can be any of the existing options, limited to the extent that the fecal sample can be loaded onto the screening medium, and this invention does not impose any particular limitation.

[0030] In this invention, the screening culture refers to the culture process of targeted screening and enrichment of Enterobacteriaceae, which can be any of the existing options. The key to achieving screening lies in the introduction of the screening culture medium. This invention does not impose any particular limitations on the methods and conditions used in the screening culture.

[0031] In some specific embodiments, the screening culture is carried out in an anaerobic environment, which is the culture condition conventionally used in existing anaerobic bacterial culture technology. Those skilled in the art can make adaptive designs according to actual needs, and the present invention does not impose any particular limitations on it.

[0032] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0033] Example 1 This embodiment illustrates the preparation of a screening culture medium, specifically including: mixing CDC anaerobic agar base powder (Solebio Biotechnology Co., Ltd., catalog number 2503140010), fructooligosaccharides (Shanghai Yuanye Technology Co., Ltd., catalog number KS407435) with distilled water, stirring and heating to boiling until completely dissolved, sterilizing at 121℃ and 0.11 kPa for 15 min, cooling to 50℃, adding heme chloride, vitamin K1, and chlorogenic acid, mixing thoroughly, pouring into plates, cooling and solidifying to obtain the screening culture medium. The specific amounts of each substance added are shown in Table 1.

[0034] Examples 2-3 Examples 2 and 3 use the method provided in Example 1 to prepare screening culture media. The difference is that the concentrations of fructooligosaccharides and chlorogenic acid in the screening culture media are different, as shown in Table 1. Other conditions are the same, and the screening culture media are obtained.

[0035] Table 1.

[0036] Comparative Example 1 The comparative example uses the method provided in Example 1 to prepare the comparative screening medium. The difference is that the screening medium is prepared by replacing the CDC anaerobic agar base powder with an equal amount of MRS anaerobic agar base powder (Guangdong Huankai Microbial Technology Co., Ltd., catalog number 251105A31). All other conditions are the same to obtain the comparative screening medium.

[0037] Comparative Example 2 The comparative example uses the method provided in Example 1 to prepare the comparative screening medium, except that chlorogenic acid and fructooligosaccharides are not added to the comparative screening medium, while other conditions are the same, to obtain the comparative screening medium.

[0038] Comparative Example 3 The comparative example uses the method provided in Example 1 to prepare the comparative screening medium, except that no oligofructose is added to the comparative screening medium, and all other conditions are the same, to obtain the comparative screening medium.

[0039] Comparative Example 4 The comparative example uses the method provided in Example 1 to prepare the comparative screening medium, except that chlorogenic acid is not added to the comparative screening medium, while other conditions are the same, to obtain the comparative screening medium.

[0040] Comparative Example 5 The comparative example uses the method provided in Example 1 to prepare the comparative screening medium, except that galactooligosaccharides are added in the same amount instead of fructooligosaccharides, while other conditions are the same, to obtain the comparative screening medium.

[0041] Test case This test example illustrates the relevant performance of the screening medium in the above embodiments, and uses the comparative screening medium provided in the comparative example as a control. The test specifically includes: 1. Preparation of the bacterial suspension to be screened: Take 0.1g of fecal samples I~III (batch numbers 32125012301MS [denoted as 01MS], 32125012302MS [denoted as 02MS], and 32125012303MS [denoted as 03MS] respectively) and mix them with 900μL of sterile physiological saline by vortexing to obtain 10 -1 The diluent was then diluted with sterile saline, and the above steps were repeated to obtain 10- 7 Diluent, also known as bacterial suspensions I to III to be screened.

[0042] 2. Screening culture of bacterial suspensions to be screened: Take 200 μL of bacterial suspension to be screened and spread it evenly on each screening medium or control screening medium. Incubate in an anaerobic workstation for 48 h. Pick colonies and dilute them according to "1. Preparation of bacterial suspension to be screened". Repeat the above culture operation twice to obtain purified colonies. Each group has 3 replicates.

[0043] 3. Detection of bacterial community structure of bacterial suspension and purified colony: (1) Select a single colony directly as a PCR template; use the primers shown in Table 2 to perform PCR amplification on the DNA of each colony to obtain PCR amplification products.

[0044] Table 2.

[0045] The PCR amplification reaction system consisted of: 15 μL of 2×Taq PCR Master MIX (Qingke Biotechnology, China), 1.2 μL of upstream primer 27F, 1.2 μL of downstream primer 1492R, 2 μL of colony DNA, and 10.6 μL of sterile water. The PCR amplification reaction program included: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 65℃ annealing for 10 s, 72℃ extension for 20 s, for 38 cycles; and 72℃ complete extension for 3 min.

[0046] (2) The PCR amplification products were subjected to first-generation sequencing. The sequencing results were compared with the 16S ribosomal RNAsequences (Bacteria and Archaea) database on the Ez website to calculate the relative abundance at the target level. The results are shown in Table 3 and Figure 1 As shown in Table 3, which presents the average results of three parallel tests of fecal sample I.

[0047] Table 3.

[0048] From Table 3 and Figure 1 The results show that, compared with comparative examples 1-5, the screening medium provided by the present invention can effectively enrich Enterobacteriaceae in fecal microbiota and has good applicability to fecal microbiota from different sources.

[0049] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. Use of a composition for the preparation of an intestinal flora screening medium, characterized in that, The composition comprises chlorogenic acid and fructooligosaccharides in a mass ratio of (0.01~1):(5~20), and the intestinal flora screening medium comprises CDC anaerobic agar basal medium.

2. Use according to claim 1, characterized in that, The degree of polymerization of the oligofructose is 3 to 7.

3. Use according to claim 1, characterized in that, The intestinal flora screening medium is used to enrich and / or purify Enterobacteriaceae bacteria in the intestinal flora.

4. A screening culture medium, characterized in that, The screening medium includes 0.05 g / L to 0.5 g / L chlorogenic acid, 1 g / L to 20 g / L fructooligosaccharides, and 35 g / L to 45 g / L CDC anaerobic agar basal medium.

5. The screening culture medium according to claim 4, characterized in that, The degree of polymerization of the oligofructose is 3 to 7.

6. The method for preparing the screening culture medium according to claim 4 or 5, characterized in that, The preparation method includes: taking the CDC anaerobic agar basal medium, oligofructose and water, mixing them, and sterilizing them; then adding the chlorogenic acid, heme chloride and vitamin K1 and mixing them to obtain the screening medium.

7. The method for preparing the screening culture medium according to claim 6, characterized in that, The sterilization process is carried out at a temperature of 115℃~130℃, a pressure of 0.1kPa~0.22kPa, and a time of 10min~20min.

8. A method for enriching Enterobacteriaceae in fecal samples under in vitro culture conditions, characterized in that, The method includes: taking a diluted fecal sample and inoculating it into the screening medium described in claim 4 or 5 for screening culture to obtain purified colonies.

9. The method for enriching Enterobacteriaceae in fecal samples under in vitro culture conditions according to claim 8, characterized in that, The dilution factor of the fecal sample is 10 -7 ~10 -6 The screening is performed in an anaerobic environment.

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