CN117467542A - Intestinal strain screening culture medium based on improved PYG and preparation method thereof - Google Patents
Intestinal strain screening culture medium based on improved PYG and preparation method thereof Download PDFInfo
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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
- C12N1/02—Separating microorganisms from their culture media
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- 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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- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/045—Culture media therefor
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention relates to an intestinal strain screening culture medium based on improved PYG and a preparation method thereof, wherein the culture medium is composed of an original PYG culture medium and four different components: soluble starch, pectin, galactose and glucose granules; the four components are combined with the original PYG culture medium to form an improved PYG culture medium, the concentration of carbon sources and nitrogen sources in the culture medium is improved, microorganisms and growth factors are increased, the proliferation of bifidobacteria is promoted, and the probability of obtaining bifidobacteria through screening is improved.
Description
Technical Field
The invention relates to the technical field of microorganism screening, in particular to a bifidobacterium probiotic separation culture medium, a preparation method and application thereof, and specifically relates to an intestinal strain screening culture medium based on improved PYG and a preparation method thereof.
Background
The intestinal tract is the largest digestive organ of human body, and in healthy adults, the strain of the digestive tract is mainly distributed in large intestine, cecum and colon segments to form a complex and dynamic ecological system, the human intestinal tract has the largest microorganism number and population, and the intestinal tract contains 1000-1150 about 100 ten thousand bacteria, which is about 10 times of the cell number of the human body. It is colonized and constantly exposed to a variety of microorganisms, including bifidobacteria, lactobacilli, escherichia coli, enterococci, clostridium perfringens, pseudomonas, and the like.
Wherein bifidobacteria are the predominant normal host bacteria in the adult intestinal tract. In the burjie bacterial identification handbook, bifidobacteria obtained based on the 16S rDNA sequencing technology are divided into 34 different species including bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium animalis, bifidobacterium breve, bifidobacterium infantis (bifidobacterium longum subspecies infantis), bifidobacterium lactis (bifidobacterium animalis subspecies), bifidobacterium longum, bifidobacterium pseudocatenulatum, bifidobacterium thermophilum, bifidobacterium acidophilus and the like. Bifidobacteria have a variety of probiotic functions such as: improving intestinal diseases caused by immune system disorder, and maintaining intestinal flora balance; scavenging free radicals, relieving oxidative damage of organism, and delaying aging; secretion of beta-galactosidase, and alleviation of lactose intolerance; inhibiting growth of putrefying bacteria and decomposing carcinogen to achieve anticancer (such as colon cancer); reducing serum cholesterol, and preventing atherosclerosis; reducing inflammatory response caused by tumor necrosis factor TNF-alpha and lipopolysaccharide; raise the serum calcium ion concentration of type 2 diabetes patient and reduce the level of glutamic pyruvic transaminase, etc. Bifidobacteria have been added as probiotics to dairy products for many years and fermented milk is also the most widely used and mature product of bifidobacteria. Due to the lack of human intestinal microbial strain resources, the application and research of intestinal microorganisms are greatly limited. The basic PYG culture medium is easier to screen and obtain harmful strains such as high-ratio escherichia and shigella, and the probiotics for human intestinal tracts are harder to screen and obtain. The use of bifidobacteria in products is numerous, but lacks a selective medium, in particular on the basis of PYG medium, and therefore improvements are needed.
Disclosure of Invention
In order to solve the problems, the primary object of the invention is to provide an intestinal strain screening culture medium based on improved PYG and a preparation method thereof, wherein the culture medium and the preparation method are based on the PYG culture medium, high-purity pectin, galactose, glucose granules and soluble starch are added to promote the growth of strains, and the obtained separation culture medium improves the separation type and quantity of probiotics of bifidobacterium and the quantity of the separated strains of bifidobacterium.
Another object of the present invention is to provide an improved PYG-based intestinal strain screening medium and a preparation method thereof, wherein the medium is capable of supplementing a large amount of harmful intestinal strains such as escherichia and shigella in the PYG medium screening process, and is difficult to screen for defects of potentially beneficial intestinal functions to human health, and basically incapable of screening harmful intestinal strains such as escherichia and shigella.
In order to achieve the above purpose, the invention adopts the following technical scheme:
an improved PYG-based intestinal strain screening medium expressed in terms of concentration comprising the following components:
solution 1:
solution 2:
further, the intestinal strain screening medium for improving the PYG further comprises a solution 3:
(3) 200-400 mu L/L gentamycin sulfate
(4) 200-400 mu L/L of ampicillin sodium.
The culture medium consists of four different components in the original PYG culture medium and the solution 2, namely soluble starch, pectin, galactose and glucose granules; the soluble starch is a starch derivative prepared by treating starch by an oxidant, acid, glycerol, enzyme or other methods, and can be used as a carbon source, a nitrogen source, vitamins and growth factors for the growth of microorganisms; pectin is a complex dietary fiber and prebiotic found in fruits and vegetables, and studies have shown that the consumption of fiber in fruits is closely related to the large number of pectolytic bacteria in the intestinal tract of healthy adults; galactose can selectively promote the proliferation of beneficial bacteria such as bifidobacteria and the like inherent in human intestinal tracts, thereby achieving the effect of assisting in regulating intestinal flora; the glucose concentration is increased on the basis of the original PYG culture medium, because glucose not only contains various plant chemicals but also is a good source of dietary fibers, can provide energy and supply energy, can also be used as a carbon source, a nitrogen source and a growth factor, maintains the abundance of intestinal strains, and has a certain effect on the separation of the intestinal strains. In conclusion, the four components are combined with the original PYG culture medium to form an improved PYG culture medium, the concentration of a carbon source and a nitrogen source in the culture medium is improved, microorganisms and growth factors are increased, the proliferation of bifidobacteria is promoted, and the probability of obtaining bifidobacteria through screening is improved.
The original PYG culture medium is expressed according to concentration and comprises a solution 1, a solution 2 and a solid culture medium, and specifically comprises the following components:
solution 1:
solution 2:
(1) 0.1% vitamin K1. Mu.L
(2) Heme solution (5 mg/mL) 5mL
pH value is 5.9-6.1
Solid culture medium agar 15.00-16.00g/L.
The preparation method of the original PYG culture medium comprises the following steps: firstly, about 0.008g of calcium chloride is weighed and put into a glass beaker filled with 200mL of ultrapure water to be fully dissolved, and then each component in the solution 2 is respectively dissolved in the calcium chloride solution for standby. The components of solution 1 were weighed out and placed in a 1.5L beaker for further use. Uniformly mixing the component solutions, regulating the pH to 5.9-6.1, and then fixing the volume to 1L; if the solid culture medium is prepared, 7.5g of agar is weighed into a 1L conical flask, 500mL of culture solution with constant volume is added, and the culture solution is sterilized in an autoclave at 121 ℃ for 20min, and if the liquid culture medium is prepared, agar is not needed. Adding solution 2 into each 500mL culture medium after sterilizing and cooling to 50deg.C, mixing, and pouring into flat plate or packaging into blue mouth bottle.
The preparation method of the intestinal strain screening culture medium based on the improved PYG, which is realized by the invention, comprises the following steps:
step (1), preparing an improved PYG culture medium; preparing a culture solution for improving the PYG culture medium according to the components; if the solid culture medium is prepared, agar is respectively weighed and added into the culture solution, the culture solution is sterilized in an autoclave, and if the liquid culture medium is prepared, agar is not needed to be added;
further, cooling after sterilization, adding the solution 3 into the culture solution, uniformly mixing, pouring into a flat plate or split charging into a blue mouth bottle for standby.
Preparing an improved PYG culture medium according to the components and fixing the volume of the culture medium to 1L; if the solid culture medium is prepared, respectively weighing 7.5g of agar in a 1L conical flask, adding 500mL of culture solution with constant volume, sterilizing in an autoclave at 121 ℃ for 20min, and if the liquid culture medium is prepared, agar is not needed to be added; adding solution 3 into each 500mL culture solution after sterilizing and cooling to 50deg.C, mixing, and pouring into flat plate or packaging into blue mouth bottle.
Step (2) sample acquisition and pretreatment: providing a fecal sample from a healthy human body; performing high-throughput sequencing on the obtained fresh fecal sample by using a 16S amplicon, and analyzing the flora richness of the fecal sample through biological information;
step (3) high-flux enrichment culture: carrying out gradient dilution on the human body excrement sample pretreated in the step (2), carrying out living bacteria detection by using a flow cytometer, carrying out limiting dilution according to the result, and then carrying out high-throughput screening enrichment culture for 48-72h;
and (4) identifying strains: picking the bacterial liquid obtained by culturing in the step (3), extracting DNA, amplifying the 16S gene sequence by PCR, adding an improved PYG culture medium, placing the culture medium into an anaerobic incubator for continuous culture, checking the quality of sequenced peak images, comparing the sequences through an Ez website, and summarizing and sorting the comparison results;
further, 900 mu L of an improved PYG culture medium is added, the mixture is placed into an anaerobic incubator for continuous culture, the quality of sequenced peak images is checked, and sequence comparison is carried out through an Ez website, and the comparison results are summarized and arranged;
and (5) strain preservation:
and (3) screening the strain numbers to be preserved according to the strain identification result obtained by comparing the step (4), preserving the bacterial liquid cultured by the 96 deep-hole plate in the step (4) according to the corresponding numbers, and verifying whether the strain is polluted by using a flat plate streak.
Compared with the prior art, the invention has the beneficial effects that:
experiments prove that compared with the original PYG culture medium, the probability of obtaining the Escherichia coli and the Shigella flexneri by screening the modified PYG culture medium is reduced from 55.18% to about 0%.
Experiments prove that the probability of obtaining bifidobacterium by screening the modified PYG culture medium is increased from 1.72% to about 77.78% compared with the PYG culture medium.
The improved PYG culture medium is improved on the basis of the original PYG culture medium, and is simple in preparation and not easy to be infected.
The improved PYG culture medium is easy to screen potential functional strains beneficial to human health, and meanwhile, harmful intestinal strains such as escherichia coli, shigella flexneri and the like can not be screened basically, so that abundant strain resources are provided for development of microecological medicaments.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a statistical schematic of isolated strains of original PYG medium.
FIG. 2 is a statistical representation of isolated strains of modified PYG medium of example 2 of the present invention.
FIG. 3 shows the percentage of the modified PYG medium of example 2 of the present invention to the total strains obtained by screening the original PYG medium for Escherichia and Shigella.
FIG. 4 shows the percentage of probiotics such as Bifidobacterium adolescentis, bifidobacterium bifidum, longum subsp.longum obtained by screening the modified PYG medium and the original PYG medium of example 2 of the present invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The invention discloses an improved PYG-based intestinal strain screening culture medium, which is expressed according to concentration and comprises the following components:
solution 1:
solution 2:
solution 3:
example 1.
The present example provides an improved medium: based on a PYG culture medium, adding soluble starch, pectin, sodium thiosulfate and riboflavin, wherein in the embodiment, the soluble starch and the pectin are added in an amount of 2g/mL respectively, and the pectin and the sodium thiosulfate are added in an amount of 0.01g/mL respectively to increase the ratio of the separated Bifidobacterium, the culture medium is named as PYG-1, and the embodiment shows that the PYG-1 culture medium has a promoting effect on the separated Bifidobacterium probiotics, and the specific steps are as follows:
(1) Preparing raw materials: 19.00-20.00g/L of bacteriological peptone, 4.10-5.20g/L of glucose, 8.10-10.50g/L of yeast extract powder, 0.75-0.8g/L of sodium chloride, 0.35-0.5g/L of cysteine hydrochloride, 0.0075-0.008g/L of magnesium sulfate, 0.035-0.04g/L of dipotassium hydrogen phosphate, 0.035-0.04g/L of potassium dihydrogen phosphate, 0.35-0.4g/L of sodium bicarbonate, 0.007-0.008g/L of calcium chloride, 2g/mL of soluble starch and 2g/mL of high-purity pectin in solution (2);
(2) Preparing a culture medium:
A. firstly, about 0.008g of calcium chloride is weighed and put into a glass beaker filled with 200mL of ultrapure water for complete dissolution, and then the other components of the solution (1) are respectively dissolved in the calcium chloride solution for standby.
B. The accuracy is that: the components of the soluble starch and pectin of solution (2) were weighed out and placed in a 1.5L beaker for use.
C. Mixing the solutions obtained in the step A, B uniformly, regulating the pH to 5.9-6.1, and then fixing the volume to 1L; if the solid culture medium is prepared, 7.5g of agar is weighed into a 1L conical flask, 500mL of culture solution with constant volume is added, and the culture solution is sterilized in an autoclave at 121 ℃ for 20min, and if the liquid culture medium is prepared, agar is not needed. After sterilization, cooling to 50 ℃, uniformly mixing every 500mL of culture medium, pouring into a flat plate or sub-packaging into blue-mouth bottles for standby.
(3) Sample acquisition and pretreatment: providing a fecal sample from a healthy human body; performing high-throughput sequencing on the obtained fresh fecal sample by using a 16S amplicon, and analyzing the flora richness of the fecal sample through biological information;
(4) High-flux enrichment culture: the human body excrement sample pretreated in the step (3) is prepared according to the following weight ratio of 1g:1000 mu L of physiological saline is added, and the mixture is vibrated and mixed uniformly.200 mu L of fecal bacterial suspension physiological saline is sucked for gradient dilution 10 1 Multiple of 10 2 Multiple of 10 3 Multiple of 10 4 Multiple of 10 5 Double sum 10 6 And the dilution numbers are (1), (2), (3), (4), (5) and (6), and each dilution tube needs to be mixed uniformly by shaking. Taking the tube (2), adding samples according to the using method of the flow type dye, and carrying out light-shielding treatment for 15min. Flow cytometry was used to count viable bacteria using the formula A×a×10 7 ÷4.5＝k×10 b And calculating to obtain k and b values. The treatment of Table 1 below was performed using 2mL centrifuge tubes numbered b-3, 4 15mL centrifuge tubes labeled blank, 4, 2, and 1, respectively. Dilution with saline limit was continued to 4, 2 and 1 bacteria per well, respectively. Respectively adding samples into a 96-well plate by using a row gun, sealing by using a sealing film, and culturing in an anaerobic box at 37 ℃ for 48-72h;
(5) And (3) strain identification: culturing to obtain bacterial liquid in the step (4), picking bacteria according to colony morphology, size and the like, placing 100 mu L of bacterial liquid in 96 holes, extracting genome DNA by using a bacterial genome DNA rapid extraction Kit (T5 Direct PCR Kit), and taking the extracted genome DNA as a template.
The PCR reaction system was prepared to amplify 16SDNA sequences of the strain, 2X Taq Plus Master Mix (Optimaceae, china) 15. Mu.L, 1.2. Mu.L of the upstream primer 27F (5'AGAGTTTGATCCTGGCTCAG 3'), 1.2. Mu.L of the downstream primer 1492R (5'TACGGCTACCTTGTTACGACTT 3'), 10.6. Mu.L of sterilized water and 2. Mu.L of the strain DNA template. The mixture was put into a P2CR instrument to react, and the mixture was pre-denatured at 98℃for 3min and then subjected to 38 cycles (denaturation at 98℃for 10s, renaturation at 65℃for 10s, extension at 72℃for 20 s), followed by thorough extension at 72℃for 3min.
The PCR products were purified and then sequenced for one generation, and the sequencing results were placed in the 16S ribosomal RNA sequences (Bacteria and Archaea) database of the Ez website for sequence alignment. Summarizing and sorting the comparison results, and co-separating to obtain 18 microorganisms from 4 genera;
(6) And (3) strain preservation: and (3) screening the strain numbers to be preserved according to the strain identification result obtained by comparing the step (5), and preserving the bacterial liquid cultured by the 96 deep-hole plate in the step (5) according to the corresponding numbers. Meanwhile, in order to ensure that the bacterial liquid is free from environmental pollution, a part of the preserved bacterial liquid is dipped in an inoculating loop to carry out plate streaking for verification.
Pectin added to the PYG-1 culture medium used in the example is an optimal growth factor of intestinal beneficial bacteria, promotes the propagation of bifidobacteria and lactobacillus, and inhibits the growth of harmful bacteria in intestinal tracts, thereby being beneficial to exerting the normal functions of normal intestinal bacteria on shielding, nutrition and immunity; starch can increase the abundance of bifidobacteria and increase the yield of short chain fatty acids; the Bifidobacterium content obtained by separation from the separation results of this example was 61.11%.
Example 2.
In the embodiment, galactose and glucose are further added on the basis of a PYG-1 culture medium, the galactose and the glucose are respectively added in an amount of 2g/mL, the culture medium is named as modified PYG, and the embodiment carries out high-flux separation culture on fecal samples of 4 healthy adults by using an original PYG culture medium and a modified PYG culture medium, wherein the proportion of Bifidobacterium obtained by separation is respectively up to 1.72% and 77.78%, and the specific steps are as follows:
(1) Preparing raw materials: 19.00-20.00g/L of bacteriological peptone, 4.10-5.20g/L of glucose, 8.10-10.50g/L of yeast extract powder, 0.75-0.8g/L of sodium chloride, 0.35-0.5g/L of cysteine hydrochloride, 0.0075-0.008g/L of magnesium sulfate, 0.035-0.04g/L of dipotassium phosphate, 0.035-0.04g/L of potassium dihydrogen phosphate, 0.35-0.4g/L of sodium bicarbonate, 0.007-0.008g/L of calcium chloride, 2g/mL of soluble starch, 2g/mL of galactose, 2g/mL of glucose granule and 2g/mL of high-purity pectin, 200-400 mu L of gentamycin sulfate and 200-400 mu L of ampicillin sodium;
(2) Preparing a culture medium:
A. firstly, about 0.008g of calcium chloride is weighed and put into a glass beaker filled with 200mL of ultrapure water for complete dissolution, and then the other components of the solution (1) are respectively dissolved in the calcium chloride solution for standby.
B. The accuracy is that: weighing the components in the soluble starch, galactose, glucose granule and high-purity pectin of the solution (2) and a 1.5L beaker for standby.
C. Mixing the solutions obtained in the step A, B uniformly, regulating the pH to 5.9-6.1, and then fixing the volume to 1L; if the solid culture medium is prepared, 7.5g of agar is weighed into a 1L conical flask, 500mL of culture solution with constant volume is added, and the culture solution is sterilized in an autoclave at 121 ℃ for 20min, and if the liquid culture medium is prepared, agar is not needed. And C, after sterilizing and cooling to 50 ℃, adding the solution in the step C into each 500mL of culture medium, uniformly mixing, pouring into a flat plate or split charging into a blue-mouth bottle for standby.
The original PYG medium is described above.
(3) Sample acquisition and pretreatment: providing a fecal sample from a healthy human body; the obtained fresh fecal sample is subjected to high-throughput sequencing of the 16S amplicon, and the flora richness of the fecal sample is analyzed through biological information.
(4) High-flux enrichment culture: the stool sample after pretreatment was mixed at 1g:1000 mu L of physiological saline is added, and the mixture is vibrated and mixed uniformly. 200 mu L of fecal bacterial suspension physiological saline is sucked for gradient dilution 10 1 Multiple of 10 2 Multiple of 10 3 Multiple of 10 4 Multiple of 10 5 Double sum 10 6 And the dilution numbers are (1), (2), (3), (4), (5) and (6), and each dilution tube needs to be mixed uniformly by shaking. Taking the tube (2), adding samples according to the using method of the flow type dye, and carrying out light-shielding treatment for 15min. Flow cytometry was used to count viable bacteria using the formula A×a×10 7 ÷4.5＝k×10 b And calculating to obtain k and b values. The treatment of Table 1 below was performed using 2mL centrifuge tubes numbered b-3, 4 15mL centrifuge tubes labeled blank, 4, 2, and 1, respectively. Dilution with saline limit was continued to 4, 2 and 1 bacteria per well, respectively. Respectively adding samples into 96-well plates by using a row gun, sealing by using a sealing film, and culturing in an anaerobic box at 37 ℃ for 48-72h.
(5) And (3) strain identification: culturing to obtain bacterial liquid in the step (4), picking bacteria according to colony morphology, size and the like, placing 100 mu L of bacterial liquid in a 96-well plate, extracting genome DNA from the bacterial liquid obtained by enrichment culture by using a bacterial genome DNA rapid extraction Kit (T5 Direct PCR Kit), and taking the extracted genome DNA as a template.
The PCR reaction system was prepared to amplify 16SDNA sequences of the strain, 2X Taq Plus Master Mix (Optimaceae, china) 15. Mu.L, 1.2. Mu.L of the upstream primer 27F (5'AGAGTTTGATCCTGGCTCAG 3'), 1.2. Mu.L of the downstream primer 1492R (5'TACGGCTACCTTGTTACGACTT 3'), 10.6. Mu.L of sterilized water and 2. Mu.L of the strain DNA template. The mixture was put into a P2CR instrument to react, and the mixture was pre-denatured at 98℃for 3min and then subjected to 38 cycles (denaturation at 98℃for 10s, renaturation at 65℃for 10s, extension at 72℃for 20 s), followed by thorough extension at 72℃for 3min.
The PCR products were purified and then sequenced for one generation, and the sequencing results were placed in the 16S ribosomal RNA sequences (Bacteria and Archaea) database of the Ez website for sequence alignment. Co-isolation gives microorganisms from 6 genera, see in particular FIG. 1;
(6) And (3) strain preservation: and (3) screening the strain numbers to be preserved according to the strain identification result obtained by comparing the step (5), and preserving the bacterial liquid cultured by the 96 deep-hole plate in the step (5) according to the corresponding numbers. Meanwhile, in order to ensure that the bacterial liquid is free from environmental pollution, a part of the preserved bacterial liquid is dipped in an inoculating loop to carry out plate streaking for verification.
Galactose and glucose medicinal granules are added on the basis of a PYG-1 culture medium, wherein galactose can selectively promote proliferation of beneficial bacteria such as bifidobacteria inherent in human intestinal tracts, so that the effects of assisting in regulating intestinal flora, preventing constipation and the like are achieved; the glucose concentration is increased on the basis of the original PYG culture medium, because the glucose can not only provide energy and supply energy, but also can be used as a carbon source, a nitrogen source and a growth factor, and has a certain effect on probiotic separation.
And (3) performing intestinal strain isolation culture on the 4 fecal samples by utilizing the improved and original PYG, and co-isolation culturing to obtain strain 103. Wherein the original PYG culture medium is separated to obtain 11 58 intestinal strains as shown in figure 1, the modified PYG culture medium is separated to obtain 6 45 intestinal strains as shown in figure 2, and the modified PYG culture medium contains bifidobacterium probiotics with beneficial functions reported in the related literature, such as: bifidobacterium adolescentis, bifidobacterium longum subsp.longum, bifidobacterium bifidum, bifidobacterium pseudocatenulatum, etc. Example results show that the isolated Bifidobacterium from the sample is much higher than the original PYG medium and also increased by 16.67% over the PYG-1 medium.
Performing data analysis on strains obtained by screening 4 fecal samples in an original PYG culture medium and a modified PYG culture medium, wherein the original PYG screening is performed to obtain strains of the genus Escherichia and the genus Shigella which have no obvious functions or are harmful, the ratio of the genus Escherichia and the genus Shigella is 55.18%, the modified PYG screening is performed to obtain strains of the genus Escherichia and the genus Shigella which have no obvious functions or are harmful, the ratio of the strains of the genus Escherichia and the genus Shigella which are basically not obtained is 0%, the ratio of the strains of the genus Bacillus which are obtained by screening the modified PYG culture medium is 77.78%, the ratio of the strains of the genus Bifidobacterium which are obtained by screening the modified PYG culture medium is 76.06% higher than that of the strains obtained by screening the original PYG culture medium, and the number of the strains obtained by screening the modified PYG culture medium is less than that of the strains of the genus Bacillus which are used for screening the strain of the modified PYG culture medium, as shown in FIG. 3; bifidobacterium adolescentis Bifidobacterium adolescentis was not obtained in the original PYG screening process, whereas bifidobacterium adolescentis Bifidobacterium adolescentis was obtained in the modified PYG medium screening at a ratio of 8.89%.
As shown in fig. 4. The percentage of the probiotics such as Bifidobacterium adolescentis, bifidobacterium bifidum, longum subsp.longum and the like obtained by screening the modified PYG culture medium and the original PYG culture medium is increased from 1.72% to 77.98%.
The improved PYG culture medium can reduce the generation of a large number of nonfunctional or harmful strains such as Escherichia fergusonii, escherichia hominis and Shigella flexneri existing in human intestinal tracts in the screening process of intestinal flora, and the increase of bifidobacterium strains can be obtained by screening, for example: the use of the culture medium provides powerful technical support for the construction of the Chinese human intestinal strain resource library, provides powerful support for the construction of the national human intestinal strain resource library, provides help for the development of microecological medicaments, and has important social and economic significance.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (6)
1. An improved PYG-based intestinal strain screening medium, characterized in that the improved PYG-based intestinal strain screening medium is expressed in terms of concentration and comprises the following components:
solution 1:
solution 2:
2. the modified PYG-based intestinal strain screening medium according to claim 1, further comprising solution 3:
(1) 200-400 mu L/L gentamycin sulfate
(2) 200-400 mu L/L of ampicillin sodium.
3. The method for preparing an improved PYG-based intestinal strain screening medium according to claim 1, characterized by comprising the steps of:
step (1), preparing an improved PYG culture medium; preparing a culture solution for improving the PYG culture medium according to the components; if the solid culture medium is prepared, agar is respectively weighed and added into the culture solution, the culture solution is sterilized in an autoclave, and if the liquid culture medium is prepared, agar is not needed to be added;
step (2) sample acquisition and pretreatment: providing a fecal sample from a healthy human body; performing high-throughput sequencing on the obtained fresh fecal sample by using a 16S amplicon, and analyzing the flora richness of the fecal sample through biological information;
step (3) high-flux enrichment culture: carrying out gradient dilution on the human body excrement sample pretreated in the step (2), carrying out living bacteria detection by using a flow cytometer, carrying out limiting dilution according to the result, and then carrying out high-throughput screening enrichment culture for 48-72h;
and (4) identifying strains: picking the bacterial liquid obtained by culturing in the step (3), extracting DNA, amplifying the 16S gene sequence by PCR, adding an improved PYG culture medium, placing the culture medium into an anaerobic incubator for continuous culture, checking the quality of sequenced peak images, comparing the sequences through an Ez website, and summarizing and sorting the comparison results;
and (5) strain preservation:
and (3) screening the strain numbers to be preserved according to the strain identification result obtained by comparing the step (4), preserving the bacterial liquid cultured by the 96 deep-hole plate in the step (4) according to the corresponding numbers, and verifying whether the strain is polluted by using a flat plate streak.
4. A process for separating and screening spore flora from intestinal flora according to claims 2 and 3, wherein in step (1), the bacterial flora is cooled after sterilization, solution 3 is added into the culture solution, and the culture solution is poured into flat plates or split-packed into blue-mouth bottles for standby.
5. The process for separating and screening a spore flora from an intestinal flora according to claim 4, wherein in step (1), the preparation of the modified PYG medium is performed according to the above components and the volume of the culture solution is fixed to 1L; if the solid culture medium is prepared, respectively weighing 7.5g of agar in a 1L conical flask, adding 500mL of culture solution with constant volume, sterilizing in an autoclave at 121 ℃ for 20min, and if the liquid culture medium is prepared, agar is not needed to be added; adding solution 3 into each 500mL culture solution after sterilizing and cooling to 50deg.C, mixing, and pouring into flat plate or packaging into blue mouth bottle.
6. A process for separating and screening a spore flora from the intestinal flora according to claim 3, wherein in step (4) the strain is identified: picking up bacterial liquid obtained by culturing in the step (3), extracting DNA, amplifying a 16S gene sequence by PCR, adding 900 mu L of an improved PYG culture medium, placing into an anaerobic incubator for continuous culture, checking the quality of sequenced peak images, carrying out sequence comparison through Ez websites, and summarizing and sorting the comparison results.
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| CN117736940B (en) * | 2024-02-18 | 2024-04-23 | 广州同康生物科技有限公司 | Bifidobacterium longum subspecies BN08 and its progeny for improving intestinal health |
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