CN113106031A - Selective culture medium for separating equol producing bacteria in human excrement - Google Patents
Selective culture medium for separating equol producing bacteria in human excrement Download PDFInfo
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- CN113106031A CN113106031A CN202110230351.1A CN202110230351A CN113106031A CN 113106031 A CN113106031 A CN 113106031A CN 202110230351 A CN202110230351 A CN 202110230351A CN 113106031 A CN113106031 A CN 113106031A
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- culture medium
- equol
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
Abstract
The invention belongs to the technical field of microbial culture, and discloses a selective culture medium for separating equol producing bacteria in human feces, which comprises a common anaerobic culture medium and antibiotics, wherein the common anaerobic culture medium contains: tryptone 10.0g,
10.0g of peptone, 3.0g of soybean peptone, 5.0g of yeast extract powder, 2.2g of beef powder, 1.2g of digestive serum powder, 3.0g of glucose, 2.5g of monopotassium phosphate, 3.0g of sodium chloride, 5.0g of soluble starch, 0.3g of L-cysteine, 4.0-6.0g of L-arginine, 0.05-0.1g of daidzein and 0.3g of sodium thioglycolate; the invention can carry out compound enrichment on target bacteria and inhibit part of non-target bacteria, so that the target bacteria can be effectively enriched; enriching and separating equol producing bacteria, researching intestinal flora biotransformation daidzein into equol to improve isoflavone bio-activityThe field of the learning effect has great value.
Description
Technical Field
The invention relates to the technical field of microbial culture, in particular to a selective culture medium for separating equol producing bacteria from human excrement.
Background
Equol is a final product obtained by transforming soybean isoflavone in a part of human bodies, and among all metabolites of soybean isoflavone, studies on equol activity are most reported, wherein the antioxidant and anticancer activities of equol are most concerned, and equol is reported to have the efficacies of relieving climacteric symptoms, preventing osteoporosis and resisting cardiovascular diseases. The equol is formed by converting daidzein through a specific flora, but the activity of the equol is 100 times of that of daidzein which is a precursor material of the equol. Therefore, the structure and properties of equol-producing bacteria in the intestinal tract are particularly important.
The culture of microorganisms is a technique for artificially growing and propagating microorganisms, and a selection medium is a medium designed to promote or inhibit certain organisms, and the culture medium can be used for screening out desired microorganisms from complex microorganisms to achieve the purpose of separation. The equol producing bacteria belong to the phylum of firmicutes and actinomycetes, and are almost strict anaerobes, and after the equol producing bacteria are screened, equol can be synthesized in vitro to prepare equol, so that equol can be used by non-equol producers, and the method has important significance in the aspect of human health care. Therefore, it is particularly important to select equol-producing bacteria from the intestinal flora.
Disclosure of Invention
The invention aims to solve the problems of low probability and large workload of screening equol-producing bacteria from complex intestinal flora in the prior art, and provides a selective culture medium for separating equol-producing bacteria from human feces according to different inhibition effects of different antibiotics on conversion of daidzein in human intestinal flora into equol.
The second object of the present invention is to provide a method for preparing the above selective medium.
The purpose of the invention is realized by the following technical scheme:
a selective culture medium for separating equol producing bacteria from human feces comprises a common anaerobic culture medium and antibiotics.
Preferably, said common anaerobic medium contains per liter: tryptone 10.0g,
10.0g of peptone, 3.0g of soybean peptone, 5.0g of yeast extract powder, 2.2g of beef powder, 1.2g of digested serum powder, 3.0g of glucose, 2.5g of monopotassium phosphate, 3.0g of sodium chloride, 5.0g of soluble starch, 0.3g of L-cysteine, 4.0-6.0g of L-arginine, 0.05-0.1g of daidzein, 0.3g of sodium thioglycolate and pH of 6.9-7.1.
Arginine is added into a culture medium during anaerobic culture, and the equol producing strain can ferment the arginine through an arginine dihydrolase way to obtain energy to promote growth. Adding some inhibiting factors (antibiotics) into the culture medium to kill part of non-target bacteria, so as to enrich the equol-producing bacteria, and rescreening the strain producing equol from the bacteria.
Preferably, the antibiotic is selected from ampicillin and chloramphenicol.
Preferably, the addition concentration of each antibiotic is 0.05-0.2 g/L.
More preferably, the antibiotic is ampicillin.
More preferably, the concentration of the antibiotic is 0.2 g/L.
The invention also provides a preparation method of the selective culture medium, which comprises the following steps:
(1) preparing a common anaerobic culture medium: tryptone 10.0g,
10.0g of peptone, 3.0g of soybean peptone, 5.0g of yeast extract powder, 2.2g of beef powder, 1.2g of digestive serum powder, 3.0g of glucose, 2.5g of monopotassium phosphate, 3.0g of sodium chloride, 5.0g of soluble starch, 0.3g of L-cysteine, 4.0-6.0g of L-arginine, 0.05-0.1g of daidzein and 0.3g of sodium thioglycolate; dissolving in 1L distilled water, heating, mixing, autoclaving at 121 deg.C for 20min, and cooling to 60 deg.C;
(2) adding antibiotics sterilized by a bacterial filter into the common anaerobic culture medium obtained in the step (1); the addition concentration of the antibiotic is 0.05-0.2 g/L.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses a selective culture medium for separating equol producing bacteria in human feces, which comprises a common anaerobic culture medium and antibiotics, wherein the common anaerobic culture medium contains: tryptone 10.0g,
10.0g of peptone, 3.0g of soybean peptone, 5.0g of yeast extract powder, 2.2g of beef powder, 1.2g of digestive serum powder, 3.0g of glucose, 2.5g of monopotassium phosphate, 3.0g of sodium chloride, 5.0g of soluble starch, 0.3g of L-cysteine, 4.0-6.0g of L-arginine, 0.05-0.1g of daidzein and 0.3g of sodium thioglycolate; the invention can carry out compound enrichment on target bacteria and inhibit part of non-target bacteria, and has smaller inhibition effect on the target bacteria than the non-target bacteria, so that the target bacteria can be effectively enriched; the preparation method of the culture medium is simple to operate, compared with the existing nutrient culture medium, the separation efficiency of the invention is greatly improved, and the invention has great value in the fields of enriching and separating equol producing bacteria, researching intestinal flora biotransformation of daidzein into equol and improving the biological action of soybean isoflavone.
Drawings
FIG. 1 shows the enrichment broth amplification curves at different dilution concentrations;
FIG. 2 shows a fluorescence quantitative PCR standard curve of live bacteria of the intestinal flora.
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1
The formula of the medium for separating equol-producing bacteria in human intestinal tract in the embodiment is as follows: tryptone 10.0g,
10.0g of peptone, 3.0g of soybean peptone, 5.0g of yeast extract powder, 2.2g of beef powder, 1.2g of digested serum powder, 3.0g of glucose, 2.5g of monopotassium phosphate, 3.0g of sodium chloride, 5.0g of soluble starch, 0.3g of L-cysteine, 4.0-6.0g of L-arginine, 0.05-0.1g of daidzein, 0.3g of sodium thioglycolate and 1000mL of distilled water, wherein the pH is 7.0 +/-0.1.
The stool sample in this example was taken from a healthy population with equol-producing ability, and immediately after sampling, 1g of the stool was diluted with Phosphate Buffered Saline (PBS) (pH 7.0, 0.2mol/L) by shaking in an anaerobic workstation to prepare a bacterial suspension.
The culture medium is prepared according to the following steps:
(1) weighing the components according to the formula, mixing tryptone,
Putting peptone, soybean peptone, yeast extract powder, beef powder, digestive serum powder, glucose, potassium dihydrogen phosphate, sodium chloride, soluble starch, L-cysteine, L-arginine, daidzein and sodium thioglycolate into a container, adjusting pH to 7.0, and sterilizing at 121 deg.C for 20 min;
(2) respectively dissolving ampicillin, chloramphenicol, ciprofloxacin and polymyxin in water, filtering and sterilizing;
(3) and (3) cooling the sterilized mixture obtained in the step (1) to 60 ℃, and respectively and uniformly mixing the antibiotic solutions obtained in the step (2) to obtain the culture medium.
Separation and screening:
and (3) sucking 100 mu L of bacterial suspension into a culture medium containing different antibiotics, and fermenting for 48 hours at 37 ℃ under an anaerobic condition. Mixing 1mL of fermentation liquid with 6mL of ethyl acetate, carrying out vortex oscillation for 60s, centrifuging at 8000rpm/min for 5min, evaporating the supernatant by using a rotary evaporator (normal temperature, 80rpm/min), adding 1mL of methanol for dissolution, and filtering by using a 0.22-micron nylon filter membrane to a sample injection bottle. Determining the chromatographic peak area of each component by using a high performance liquid chromatograph, and comparing with a standard curve to determine the content of daidzein and equol in the sample solution. The method for detecting the total number of the viable bacteria by using the azide propidium bromide combined fluorescence quantitative polymerase chain reaction (PMA-qPCR) established in the early stage of a laboratory is used for quickly quantifying the total number of the viable bacteria in the culture solution (the result is shown in the table 1g), in the table 1, a Control group is fermentation liquor for culturing for 0h, and GAM-Mod treatment is to add sterile water with the same volume as that of an antibiotic solution for culturing for 48 h. As can be seen from the results in table 1, ciprofloxacin and polymyxin were not suitable for screening of equol-producing bacteria, and ampicillin-treated group and chloramphenicol-treated group were suitable for screening of equol-producing bacteria, and ampicillin-treated group was effective for increasing the relative content of equol-producing bacteria.
TABLE 1 daidzein and equol content and viable count reduction by in vitro fermentation of fecal flora before and after antibiotic intervention
Note: the daidzein and equol contents and the number of colony forming units in the fermentation broth were the mean values ± standard deviation (n ═ 3); NT represents not detected.
TABLE 2 Total viable count of fecal sample flora after antibiotic intervention, Equol producing bacteria isolation Rate
The equol-containing culture solution was adjusted to 105、106、107Diluting and plating, fermenting at 37 deg.C under anaerobic condition for 48 hr, picking each colony on the plate, streaking and purifying, and re-screening strains with equol-producing ability with modified common anaerobic culture medium (results are shown in Table 2). The experimental results of antibiotic treatment show that polymyxin and ciprofloxacin can completely kill equol producing bacteria, clotrimazoleThe group of the antibiotics and the group of the ampicillin retained a part of the equol-producing bacteria; and effectively separating an equol-producing strain (the 16S rRNA sequence of the strain is 1260bp in total, and the NCBI gene bank is submitted with the registration number of MN560033), which indicates that the culture medium is suitable for separating equol-producing strains.
Claims (7)
1. A selective culture medium for separating equol producing bacteria from human feces is characterized by comprising a common anaerobic culture medium and antibiotics.
2. The selective culture medium of claim 1, wherein the common anaerobic culture medium comprises, per liter: tryptone 10.0g,
10.0g of peptone, 3.0g of soybean peptone, 5.0g of yeast extract powder, 2.2g of beef powder, 1.2g of digested serum powder, 3.0g of glucose, 2.5g of monopotassium phosphate, 3.0g of sodium chloride, 5.0g of soluble starch, 0.3g of L-cysteine, 4.0-6.0g of L-arginine, 0.05-0.1g of daidzein, 0.3g of sodium thioglycolate and pH of 6.9-7.1.
3. The selective culture medium of claim 2, wherein the antibiotic is selected from the group consisting of ampicillin and chloramphenicol.
4. The selective culture medium of claim 3, wherein each antibiotic is added at a concentration of 0.05-0.2 g/L.
5. The selective media of claim 4, wherein the antibiotic is ampicillin.
6. The selective culture medium of claim 5, wherein the antibiotic is at a concentration of 0.2 g/L.
7. A method for preparing the selective culture medium according to any one of claims 1 to 6, comprising the steps of:
(1) preparing a common anaerobic culture medium: tryptone 10.0g,
10.0g of peptone, 3.0g of soybean peptone, 5.0g of yeast extract powder, 2.2g of beef powder, 1.2g of digestive serum powder, 3.0g of glucose, 2.5g of monopotassium phosphate, 3.0g of sodium chloride, 5.0g of soluble starch, 0.3g of L-cysteine, 4.0-6.0g of L-arginine, 0.05-0.1g of daidzein and 0.3g of sodium thioglycolate; dissolving in 1L distilled water, heating, mixing, autoclaving at 121 deg.C for 20min, and cooling to 60 deg.C;
(2) adding antibiotics sterilized by a bacterial filter into the common anaerobic culture medium obtained in the step (1); the addition concentration of the antibiotic is 0.05-0.2 g/L.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102925378A (en) * | 2012-05-11 | 2013-02-13 | 华侨大学 | Proteus mirabilis strain and method for producing S-equol through daidzein conversion by using the same |
| JP2013128414A (en) * | 2011-12-20 | 2013-07-04 | Daicel Corp | Method for producing equol |
| CN105707899A (en) * | 2008-09-19 | 2016-06-29 | 大塚制药株式会社 | Fermentation Product Containing Equol-Producing Microorganism Having Maintained Equol-Producing Ability, And Method For Producing Same |
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2021
- 2021-03-02 CN CN202110230351.1A patent/CN113106031A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105707899A (en) * | 2008-09-19 | 2016-06-29 | 大塚制药株式会社 | Fermentation Product Containing Equol-Producing Microorganism Having Maintained Equol-Producing Ability, And Method For Producing Same |
| JP2013128414A (en) * | 2011-12-20 | 2013-07-04 | Daicel Corp | Method for producing equol |
| CN102925378A (en) * | 2012-05-11 | 2013-02-13 | 华侨大学 | Proteus mirabilis strain and method for producing S-equol through daidzein conversion by using the same |
Non-Patent Citations (4)
| Title |
|---|
| CHARLOTTE ATKINSON ET AL.: "In vitro incubation of human feces with daidzein and antibiotics suggests interindividual differences in the bacteria responsible for equol production", 《THE JOURNAL OF NUTRITION》 * |
| HIROKAZU TSUJI ET AL.: "Identification of an Enzyme System for Daidzein-to-Equol Conversionin Slackia sp. Strain NATTS", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
| 杨秀伟 等: "《中药成分代谢分析》", 30 September 2003 * |
| 郭远洋: "生物催化大豆苷元产S-雌马酚菌株筛选及其转化特性研究", 《中国优秀博硕士学位论文全文数据库(硕士) 工程科技Ⅰ辑》 * |
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