CN116926158A - Method for in vitro systematic evaluation of enterococcus faecium - Google Patents
Method for in vitro systematic evaluation of enterococcus faecium Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 28
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- 230000009897 systematic effect Effects 0.000 title claims description 12
- 238000012360 testing method Methods 0.000 claims abstract description 80
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- 235000011389 fruit/vegetable juice Nutrition 0.000 claims abstract description 19
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- 239000003085 diluting agent Substances 0.000 claims abstract description 13
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- 241000607142 Salmonella Species 0.000 claims description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 230000001580 bacterial effect Effects 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 9
- 210000002249 digestive system Anatomy 0.000 claims description 7
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 6
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- 238000002360 preparation method Methods 0.000 claims description 5
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- 102000057297 Pepsin A Human genes 0.000 claims description 4
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- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 4
- 238000007865 diluting Methods 0.000 claims description 4
- 229940111202 pepsin Drugs 0.000 claims description 4
- 239000008363 phosphate buffer Substances 0.000 claims description 4
- 238000005303 weighing Methods 0.000 claims description 4
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- 239000010871 livestock manure Substances 0.000 abstract description 3
- 238000000528 statistical test Methods 0.000 abstract 1
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 16
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- 241000194033 Enterococcus Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The application provides a method for systematically evaluating enterococcus faecium in vitro, which comprises the following steps: adding simulated gastric juice into a transparent container filled with sterilized animal feed, adding sterilized paraffin to form an anaerobic culture system, setting test groups S1 and S2 added with physiological saline, and setting test groups S3 and S4 respectively supplemented with enterococcus faecium sample diluent; adding simulated intestinal juice and phosphate buffer solution containing bovine choline below the sterilized paraffin liquid level of gastric digestion liquid of S1, S2, S3, and S4, and supplementing animal manure to S2 and S4Culturing the fecal flora suspension in a shaking way; comparing with S1, testing and counting the appearance change, pH value change and OD of the intestinal digestive juice of the test group S3 600nm The acid resistance, enzyme resistance, bile salt resistance and growth metabolism of enterococcus faecium are evaluated according to the change and the change of the organic acid content; and comparing S2, testing the number of different types of flora in the intestinal digestive juice of the statistical test group S4, and evaluating the influence of enterococcus faecium on other flora, thereby realizing the rapid system evaluation of enterococcus faecium.
Description
Technical Field
The application relates to the technical field of microorganisms, in particular to a method for systematically evaluating enterococcus faecium in vitro.
Background
Enterococcus faecium is a probiotic which can be colonized in the intestinal tract of animals, is an aerobic or facultative anaerobic bacterium, is usually colonized in the ileum and colon of the host, and has a fast growth rate. Enterococcus faecium has been listed by the agricultural rural division No. 318 bulletin as one of fifteen directly feedable feed grade microbial species.
At present, performance evaluation of enterococcus faecium is mostly focused on indexes such as acid resistance, bile salt resistance, digestive enzyme resistance and the like, but the conventional evaluation method utilizes a fermentation medium for evaluation, so that the growth and metabolism conditions of enterococcus faecium in animal intestinal tracts and the influence on intestinal flora cannot be truly reflected.
Disclosure of Invention
Based on this, it is necessary to provide a method for systematically evaluating enterococcus faecium in vitro, which can relatively truly evaluate the growth metabolic capacity of enterococcus faecium and the effect of the enterococcus faecium on the intestinal flora.
The application adopts the following technical scheme:
the application provides a method for systematically evaluating enterococcus faecium in vitro, which comprises the following steps:
respectively preparing enterococcus faecium sample diluent and sterilized animal feed;
gastric digestion was simulated: adding simulated gastric juice and physiological saline with pH of 2.0 into a transparent container for containing sterilized animal feed, wherein the simulated gastric juice contains pepsin, and then adding sterilized paraffin to form an anaerobic culture system, and repeating the steps in parallel, wherein the test groups S1 and S2 are respectively marked; adding simulated gastric juice and enterococcus faecium sample diluent into a transparent container for containing sterilized animal feed, adding sterilized paraffin to form an anaerobic culture system, repeating in parallel, and respectively marking as test groups S3 and S4; respectively placing samples of test groups S1, S2, S3 and S4 in a constant-temperature water bath shaking table at 37-45 ℃ for shaking culture to obtain gastric digestion liquid;
intestinal digestion was simulated: adding simulated intestinal juice and phosphate buffer solution containing bovine choline below the sterilized paraffin liquid level of the gastric digestion liquid of the test groups S1 and S3, wherein the simulated intestinal juice contains trypsin, chymotrypsin and amylase, controlling the pH value of the mixed liquid to be 6.4-6.6, and carrying out shaking culture in a constant-temperature water bath shaking table at 37-45 ℃; adding simulated intestinal juice, phosphoric acid buffer solution and animal faeces flora suspension below the paraffin liquid level of the gastric digestion liquid of the test groups S2 and S4, controlling the pH value of the mixed liquid to be 6.4-6.6, and carrying out shaking culture in a constant-temperature water bath shaking table at 37-45 ℃;
system evaluation:
comparing the test group S1, and testing and counting the appearance change, pH value change and OD of the intestinal digestive juice of the test group S3 600nm The acid resistance, enzyme resistance, bile salt resistance and growth metabolism of enterococcus faecium are evaluated according to the change and the change of the organic acid content; and comparing the test group S2, testing and counting the quantity of escherichia coli and salmonella in the intestinal digestive juice of the test group S4, and evaluating the influence of enterococcus faecium on other bacterial groups.
In some embodiments, the animal feed is selected from broiler feeds, the composition comprising 58% -65% corn, 25% -35% soybean meal, and a small amount of soybean oil and broiler premix.
In some of these embodiments, in test groups S1, S2, the mass to volume ratio of sterilized animal feed to simulated gastric fluid and physiological saline is 1g:10mL:0.1mL; in test groups S3 and S4, the mass to volume ratio of the sterilized animal feed to the simulated gastric fluid and the enterococcus faecium sample diluent is 1g:10mL:0.1mL, and the addition amount of enterococcus faecium is 0.01 hundred million/g animal feed.
In some of these embodiments, in test groups S1, S2, the mass to volume ratio of sterilized animal feed to simulated intestinal fluid and phosphate buffer containing bovine choline is 1g:2ml:8ml; in test groups S3 and S4, the mass-to-volume ratio of the sterilized animal feed to the simulated intestinal fluid, the phosphate buffer solution containing bovine choline and the fecal flora suspension was 1g:2mL:8mL:2mL, and the concentration of bovine choline in the simulated digestive system was 0.03%.
In some of these embodiments, the simulated gastric fluid is prepared by the steps of: 4.43mg pepsin (enzyme activity 700U/mg) was dissolved in every 10mL of HCL solution at pH 2.0.
In some embodiments, the simulated intestinal fluid is prepared by the steps of: 9.57mg trypsin (enzyme activity 100U/mg), 5.28mg chymotrypsin (enzyme activity 40U/mg) and 0.39mL amylase (enzyme activity 20000U/mL) were dissolved in every 2mL deionized water.
In some embodiments, the phosphate buffer of bovine choline is prepared by the steps of: in every 100mL deionized water, 0.75g Niu Danjian, 0.4675g anhydrous disodium hydrogen phosphate, 2.0045g anhydrous sodium dihydrogen phosphate, 0.5565g sodium chloride and 0.1545g potassium chloride were dissolved, and the pH of the solution was adjusted to 6.60 with sodium hydroxide.
In some of these embodiments, the sterilized animal feed is prepared by the steps of: weighing animal feed sample, placing into glass tube, sealing, sterilizing in 121 deg.C autoclave for 30min, oven drying, and cooling.
In some embodiments, the enterococcus faecium sample diluent is prepared by the steps of: weighing 1g of enterococcus faecium sample with viable count of 2000 hundred million/g, adding into 99mL sterilized 0.85% physiological saline, sealing with cotton plug, packaging with kraft paper, placing into a shaking table, and extracting under shaking at room temperature for 30min; and then taking the extracted bacterial suspension, and gradually diluting by 200 times until the final concentration of bacterial liquid is 0.1 hundred million/mL to obtain enterococcus faecium sample diluent.
Compared with the prior art, the application has the core advantages that:
1) The method for systematically evaluating enterococcus faecium in vitro provided by the application can be used for linking the gastric and intestinal digestion processes by simulating the pH value, digestive enzyme environment, buffer salt system and the like in the gastrointestinal digestion environment by using animal feed and paraffin sealing conditions, can be used for directly evaluating and screening enterococcus faecium which can resist acid, enzyme and bile salt, and can also systematically evaluate the growth metabolism condition of enterococcus faecium in the simulated digestion environment and the influence effect on the growth of other bacterial groups.
2) The method adopts the animal feed and digestive enzyme compound system to replace the conventional culture medium, provides nutrition growth conditions for enterococcus faecium, is close to nutrition supply in the actual animal feeding environment, and can simulate the growth and metabolism conditions of enterococcus faecium in the animal digestive tract nutrition environment to the greatest extent.
Drawings
FIG. 1 is a photograph showing the appearance of intestinal digestive juice products in test group one and test three in example 1.
Detailed Description
The present application will be described in further detail with reference to specific examples so as to more clearly understand the present application by those skilled in the art. The following examples are given for illustration of the application only and are not intended to limit the scope of the application. All other embodiments obtained by those skilled in the art without creative efforts are within the protection scope of the present application based on the specific embodiments of the present application. In the examples of the present application, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise; in the embodiments of the present application, unless specifically indicated, all technical means used are conventional means well known to those skilled in the art.
Key test materials:
enterococcus faecium: commercial enterococcus faecium products.
Broiler feed: commercially available contains 62% corn, 33% soybean meal, 2.5% soybean oil, 2.5% broiler premix.
The preparation method of the simulated intestinal juice comprises the following steps: to pure water, 9.57mg of trypsin (enzyme activity 100U/mg), 5.28 chymotrypsin (enzyme activity 40U/mg) and 0.39mL of amylase (enzyme activity 20000U/mg) were added.
The preparation method of the phosphate buffer solution comprises the following steps: 0.75g of bovine choline, 0.4675g of anhydrous disodium hydrogen phosphate, 2.0045g of anhydrous sodium dihydrogen phosphate, 0.5565g of sodium chloride and 0.1545g of potassium chloride are weighed into a beaker, 100mL of deionized water is added for dissolution, and the pH of the solution is adjusted to 6.60 by sodium hydroxide. Wherein, the concentration of the bovine choline in the simulated digestive system is 0.03 percent.
The preparation method of the fecal flora suspension comprises the following steps: taking a proper amount of broiler manure sample (the specific source is that manure is collected from a conventional broiler raising experiment, and the broiler is fed with conventional broiler feed without antibiotics or other functional additives), adding 0.85% physiological saline to obtain a total weight of 1:50 Mixing and diluting (m/V), vortex mixing until no particle precipitate exists, centrifuging at 2000r/min for 5min, removing impurities, and taking the upper turbid liquid as fecal flora suspension.
Example 1
The embodiment provides a method for systematically evaluating enterococcus faecium in vitro, which comprises the following steps:
s1, preparing enterococcus faecium sample diluent:
1g of enterococcus faecium sample (2000 hundred million/g of viable bacteria) was weighed, added to 99mL of sterilized 0.85% physiological saline, sealed with a cotton plug, wrapped with kraft paper, placed in a shaker, and extracted by shaking at room temperature for 30min. And then taking the extracted bacterial suspension, and gradually diluting by 200 times until the final concentration of bacterial liquid is 0.1 hundred million/mL to obtain enterococcus faecium sample diluent.
S2, preparing sterilized broiler feed:
1g of broiler feed sample is weighed and placed in a 50mL glass tube respectively, a bottle cap is plugged, the broiler feed sample is sterilized in an autoclave at 121 ℃ for 30min, and the broiler feed sample is placed in an oven for drying after sterilization, and is cooled for standby.
S3, simulating gastric digestion:
respectively adding 10mL of simulated gastric fluid and 0.1mL of 0.85% physiological saline into 50mL of glass tubes filled with 1g of sterilized broiler feed, and adding sterilized paraffin to form an anaerobic culture system, and marking as a first test group and a second test group, wherein each test group is parallel to 3;
respectively adding 10mL of simulated gastric fluid and 0.1mL of enterococcus faecium sample diluent into a 50mL glass tube filled with 1g of sterilized broiler feed, and adding sterilized paraffin to form an anaerobic culture system, wherein the anaerobic culture system is marked as a test group III and a test group IV, and each group of tests are parallel to 3;
and (3) placing the anaerobic culture systems of the first test group, the second test group, the third test group and the fourth test group in a constant-temperature water bath shaking table at 41 ℃ for shake culture for 4 hours to obtain gastric digestion liquid. All operations are performed in an ultra-clean bench to ensure a sterile environment.
S4, simulating intestinal digestion:
after the step S3 is finished, 2mL of simulated intestinal fluid and 8mL of phosphate buffer solution are respectively added into the gastric digestive tract bacteria fluid of each test group I and test group III in an ultra clean bench. To each of the second and fourth gastric digestive tract solutions were added 2mL of simulated intestinal fluid, 8mL of phosphate buffer solution, and 2mL of fecal flora suspension. The simulated intestinal juice and the phosphate buffer solution are added below the liquid level of the paraffin, and the pH of the intestinal juice solution is about 6.50 (the pH environment of the intestinal tracts of the broiler chickens).
After the reagent is added, a bottle cap is plugged, and the mixture is subjected to shaking culture in a constant-temperature water bath shaking table at 41 ℃ for 15 hours to obtain intestinal digestion liquid.
Evaluation of faecium intestine by test group one and test threeGrowth metabolism of cocci: taking samples of the first test group and the third test group respectively for appearance observation record, pH value and OD 600nm And measuring the organic acid by adopting high performance liquid chromatography.
Appearance pairs of test group one, test group three samples such as shown in fig. 1: the 3 replicate sample solutions of test group one were clear and transparent, and the 3 replicate sample solutions of test group three were cloudy.
OD of test group I, test group III intestinal tract digestive juice sample 600nm The results of the pH value of the fermentation broth, the lactic acid content and the acetic acid content of the organic acid are shown in the following table:
OD of test group sample 600nm pH value, lactic acid and acetic acid content in organic acid determination
As can be seen from the above table: in the simulated digestive tract environment, the method of the embodiment can evaluate and screen enterococcus faecium which can resist gastric acid, gastric enzyme, intestinal phase enzyme and bile salt, and the enterococcus faecium survives in the simulated digestive tract environment, so that nutrient substances generated by the feed after the effect of digestive enzyme can be used for self growth and are subjected to growth metabolism, the pH value of the intestinal tract is reduced by acid production, and lactic acid is mainly produced.
The effect of enterococcus faecium on the intestinal flora was evaluated by test group two and test four:
samples of test group two and test group four were taken for E.coli and Salmonella counts. The method comprises the following specific steps: and respectively carrying out progressive dilution on the sample liquid of the test group II and the sample liquid of the test group IV, respectively sucking 100 mu L of diluted sample liquid, uniformly coating the sample liquid on the escherichia coli and salmonella culture medium, wherein each dilution multiple is 2, culturing the sample liquid in a constant temperature incubator at 37 ℃ for 24 hours after coating, and observing and counting the growth conditions of the escherichia coli and salmonella. Wherein, the escherichia coli is cultivated and counted by adopting an eosin-methylene blue culture medium, and the salmonella is counted by adopting a bismuth sulfite agar culture medium.
Detection counts of E.coli and Salmonella in samples of test group two and test group four
The results of the above examples show that the systematic method for rapidly evaluating enterococcus faecium can be used for rapidly evaluating whether the screened enterococcus faecium has acid resistance, enzyme resistance and bile salt resistance effects, can comprehensively evaluate the growth metabolism, acid production effect and beneficial effect on intestinal flora of enterococcus faecium in the simulated gastrointestinal environment of animals, can greatly save animal test cost, and provides a convenient and rapid method for systematically evaluating the effect of enterococcus faecium.
Comparative example 1
This comparative example provides a method for evaluating enterococcus faecium, which is essentially the same as example 1, except that:
the method does not comprise the step S2 of preparing sterilized broiler feed and the step of adding sterilized broiler feed in the step S3 of simulating gastrointestinal digestion, and directly utilizes simulated gastric juice or physiological saline for evaluation.
OD of intestinal digestive juice samples of test group I and test group III 600nm The results of the pH value of the fermentation broth, the lactic acid content and the acetic acid content of the organic acid are shown in the following table:
OD of test group sample 600nm pH value, lactic acid and acetic acid content in organic acid determination
As can be seen from the above table, the feed can not provide nutrition for enterococcus faecium growth without adding sterilized broiler feed, so OD 600nm The pH value of the fermentation liquor is maintained at the initial value of the sample liquor, and the content of the organic acid lactic acid and acetic acid is not increased.
Detection counts of E.coli and Salmonella in samples of test group two and test group four
Example 1 used a digestive system containing animal feed, as compared to comparative example 1 which used a digestive system containing no animal feed. In comparative example 1, the fecal flora was gradually killed during the culture without the feed, and enterococcus faecium was not grown under the condition of lack of nutrition, and the beneficial effect on the intestinal flora could not be evaluated.
Comparative example 2
This comparative example provides a method for evaluating enterococcus faecium, which is essentially the same as example 1, except that:
the method does not comprise the step S2 of preparing sterilized broiler feed and the step S3 of adding sterilized broiler feed in the step of simulating gastrointestinal digestion, wherein a fermentation medium (1% yeast extract, 2% peptone and 2% glucose) is directly adopted to replace the sterilized broiler feed, and then simulated gastric juice or physiological saline is added for evaluation.
OD of intestinal digestive juice samples of test group I and test group III 600nm The results of the pH value of the fermentation broth, the lactic acid content and the acetic acid content of the organic acid are shown in the following table:
OD of test group sample 600nm pH value, lactic acid and acetic acid content in organic acid determination
As can be seen from the results of the table, enterococcus faecium can grow well in the fermentation medium, and the nutrition provided by the fermentation medium is more direct and rich, so that the enterococcus faecium grows more in quantity and produces acid.
However, the comparative example directly provides nutrition by a culture medium, and the buffer systems of different culture mediums are different, so that the acid production conditions of the cultured sample liquid are different, and the nutrient utilization and the growth metabolism conditions of enterococcus faecium in an animal body cannot be truly simulated.
Detection counts of E.coli and Salmonella in samples of test group two and test group four
Example 1 used a digestive system containing animal feed, as compared to comparative example 2 which used a digestive system containing no animal feed. As is clear from the above table, E.coli and Salmonella were inhibited after enterococcus faecium had grown in the medium, but the inhibition effect was slightly different from that of example 1 depending on the culture system.
In the embodiment 1 of the application, the animal feed and digestive enzyme compound system is taken as a nutrition system to evaluate enterococcus with more authenticity and accuracy.
It should be noted that the above examples are only for further illustrating and describing the technical solution of the present application, and are not intended to limit the technical solution of the present application, and the method of the present application is only a preferred embodiment and is not intended to limit the scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (9)
1. A method for the in vitro systematic evaluation of enterococcus faecium comprising the steps of:
respectively preparing enterococcus faecium sample diluent and sterilized animal feed;
gastric digestion was simulated: adding simulated gastric juice and physiological saline with pH of 2.0 into a transparent container for containing sterilized animal feed, wherein the simulated gastric juice contains pepsin, and then adding sterilized paraffin to form an anaerobic culture system, and repeating the steps in parallel, wherein the test groups S1 and S2 are respectively marked; adding simulated gastric juice and enterococcus faecium sample diluent into a transparent container for containing sterilized animal feed, adding sterilized paraffin to form an anaerobic culture system, repeating in parallel, and respectively marking as test groups S3 and S4; respectively placing samples of test groups S1, S2, S3 and S4 in a constant-temperature water bath shaking table at 37-45 ℃ for shaking culture to obtain gastric digestion liquid;
intestinal digestion was simulated: adding simulated intestinal juice and phosphate buffer solution containing bovine choline below the sterilized paraffin liquid level of the gastric digestion liquid of the test groups S1 and S3, wherein the simulated intestinal juice contains trypsin, chymotrypsin and amylase, controlling the pH value of the mixed liquid to be 6.4-6.6, and carrying out shaking culture in a constant-temperature water bath shaking table at 37-45 ℃; adding simulated intestinal juice, phosphoric acid buffer solution and animal faeces flora suspension below the paraffin liquid level of the gastric digestion liquid of the test groups S2 and S4, controlling the pH value of the mixed liquid to be 6.4-6.6, and carrying out shaking culture in a constant-temperature water bath shaking table at 37-45 ℃;
system evaluation:
comparing the test group S1, and testing and counting the appearance change, pH value change and OD of the intestinal digestive juice of the test group S3 600nm The acid resistance, enzyme resistance, bile salt resistance and growth metabolism of enterococcus faecium are evaluated according to the change and the change of the organic acid content;
and comparing the test group S2, testing and counting the quantity of escherichia coli and salmonella in the intestinal digestive juice of the test group S4, and evaluating the influence of enterococcus faecium on other bacterial groups.
2. The method for the in vitro systematic evaluation of enterococcus faecium according to claim 1, wherein the animal feed is selected from the group consisting of broiler feed, composition comprising corn, soybean meal, soybean oil and premix.
3. The method for the in vitro systematic evaluation of enterococcus faecium according to claim 1, wherein in the test groups S1, S2 the mass to volume ratio of sterilized animal feed to simulated gastric fluid and physiological saline is 1g:10ml:0.1ml;
in test groups S3 and S4, the mass to volume ratio of the sterilized animal feed to the simulated gastric fluid and the enterococcus faecium sample diluent is 1g:10mL:0.1mL, and the addition amount of enterococcus faecium is 0.01 hundred million/g animal feed.
4. The method for the in vitro systematic evaluation of enterococcus faecium according to claim 3, wherein in the test groups S1, S2 the mass to volume ratio of sterilized animal feed to simulated intestinal fluid and phosphate buffer containing bovine choline is 1g:2ml:8ml;
in test groups S3 and S4, the mass-to-volume ratio of the sterilized animal feed to the simulated intestinal fluid, the phosphate buffer solution containing bovine choline and the fecal flora suspension was 1g:2mL:8mL:2mL, and the concentration of bovine choline in the simulated digestive system was 0.03%.
5. The method for the in vitro systematic evaluation of enterococcus faecium according to claim 3 or 4, wherein said simulated gastric fluid is prepared by the steps of: 4.43mg pepsin (enzyme activity 700U/mg) was dissolved in every 10mL of HCL solution at pH 2.0.
6. The method for the in vitro systematic evaluation of enterococcus faecium according to claim 3 or 4, wherein said simulated intestinal fluid is prepared by the steps of: 9.57mg trypsin (enzyme activity 100U/mg), 5.28mg chymotrypsin (enzyme activity 40U/mg) and 0.39mL amylase (enzyme activity 20000U/mL) were dissolved in every 2mL deionized water.
7. The method for the in vitro systematic evaluation of enterococcus faecium according to claim 3 or 4, wherein the step of preparing the phosphate buffer of bovine choline is: in every 100mL deionized water, 0.75g Niu Danjian, 0.4675g anhydrous disodium hydrogen phosphate, 2.0045g anhydrous sodium dihydrogen phosphate, 0.5565g sodium chloride and 0.1545g potassium chloride were dissolved, and the pH of the solution was adjusted to 6.60 with sodium hydroxide.
8. The method for the in vitro systematic assessment of enterococcus faecium according to claim 1 or 2, wherein the preparation steps of said sterilized animal feed are:
weighing animal feed sample, placing into glass tube, sealing, sterilizing in 121 deg.C autoclave for 30min, oven drying, and cooling.
9. The method for the in vitro systematic assessment of enterococcus faecium according to claim 1 or 2, wherein the preparation steps of the enterococcus faecium sample dilution are as follows:
weighing 1g of enterococcus faecium sample with viable count of 2000 hundred million/g, adding into 99mL sterilized 0.85% physiological saline, sealing with cotton plug, packaging with kraft paper, placing into a shaking table, and extracting under shaking at room temperature for 30min; and then taking the extracted bacterial suspension, and gradually diluting by 200 times until the final concentration of bacterial liquid is 0.1 hundred million/mL to obtain enterococcus faecium sample diluent.
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