CN118048434B - Method for rapidly evaluating inhibition capability of antibacterial component to escherichia coli and application of method - Google Patents
Method for rapidly evaluating inhibition capability of antibacterial component to escherichia coli and application of method Download PDFInfo
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Abstract
The invention relates to a method for rapidly evaluating the inhibition capability of an antibacterial component to escherichia coli and application thereof. The method comprises the following steps: preparing a selective culture medium of the escherichia coli, sub-packaging, sterilizing and culturing for observation. By adopting the evaluation method combining the selective culture medium and the gas production observation method, the result of whether the antibacterial component to be detected has an inhibition effect on escherichia coli can be directly observed in 3 hours, and the efficiency is far higher than that of the method in the prior art. The invention further provides an evaluation method capable of developing color, namely bromocresol purple can be added into the selective culture medium as an indicator, the color development reaction and gas production reaction results are consistent, the evaluation method can be used as an auxiliary observation means, the results can be obtained rapidly and efficiently, the method is safe and simple, and the stability and accuracy of the detection evaluation result are improved.
Description
Technical Field
The invention relates to the field of feed component detection, in particular to a method for rapidly evaluating the inhibiting capability of an antibacterial component on escherichia coli and application thereof.
Background
Coli is one of the main causes of diarrhea in poultry farming and piglets. In actual production, the harm of escherichia coli in the feed to the health of livestock and poultry can be reduced by adding the feed additive component with the antibacterial capability to the escherichia coli. However, in theory, the components with antibacterial capability are more, and how to rapidly evaluate the antibacterial capability of various antibacterial components on escherichia coli is a difficult problem encountered by a cultivation terminal. Because of the limitation of the field experimental conditions used by feed factories or large farms and the higher requirement of the field antibacterial component detection efficiency, when products with antibacterial capacity are screened at the terminal, quick and visual antibacterial effect display and simple operation evaluation tests are often required to be carried out on the antibacterial capacity of the antibacterial components, the conventional common evaluation method is a flat plate culture method, which takes longer time, generally requires 12-24 hours, can obtain results only one night, is complex to operate and has higher professional requirements on testers. The existing evaluation method in production practice cannot meet the field production requirement, so that an evaluation method which is simple in operation and can rapidly and intuitively display the antibacterial effect of the screened antibacterial components is urgently needed.
At present, a rapid evaluation method is also reported for escherichia coli: the experimental process mainly comprises the steps of inoculating 1mL of quantitative escherichia coli indication bacteria liquid into a prepared sterilized LB culture medium test tube, adding an equal volume of to-be-tested sample (antimicrobial peptide, essential oil and the like) with each concentration, culturing for a proper time (3-6 h) according to proper conditions (temperature and rotating speed), measuring the change of cell density (OD 600) for a plurality of times by a spectrophotometer, calculating the antibacterial rate of the antimicrobial peptide and the like on the escherichia coli and other indication bacteria, and evaluating the inhibition effect. Although this method is faster than conventional plate culture methods, there are several serious problems in application. Firstly, the LB culture medium selected in the operation process is a universal culture medium, and the growth and propagation of mixed bacteria cannot be prevented in the operation mode, because the propagation of mixed bacteria can cause the increase of the light absorption value of the LB culture medium, and the error of the test result is larger. Secondly, in the test process, a spectrophotometer is required to measure the cell density of the culture solution of the escherichia coli for multiple times, leakage of indicator bacteria is easy to cause, and conditional pathogenic bacteria bring potential safety hazards to operators, for example, after the escherichia coli invades tissues and organs outside the intestinal tract, the conditional pathogenic bacteria can be formed, and the parenteral infection is caused; some serotypes of Escherichia coli have pathogenicity and can cause human gastroenteritis, diarrhea, abdominal pain, nausea, vomiting and other symptoms. Third, this method takes 3-6 hours, although faster than conventional plate culture methods, it still takes too much time for production. Fourth, the method has high requirements on the sample to be measured, and the light absorption value is interfered by the conditions such as water solubility, color shade, clarity and the like of the sample to be measured, so that the result is deviated.
In the prior art, lactose and an indicator are added into a culture medium to promote the growth of escherichia coli, and the rapid detection of escherichia coli is assisted by observing gas production and color change reaction, for example, the culture medium for rapid display detection of escherichia coli flora disclosed in China patent 202010121703.5 is characterized in that lactose and the indicator bromocresol purple and thiazole blue are added into a conventional LB culture medium to assist in rapid detection of escherichia coli by observing gas production and color change reaction, but the color change reaction is observed to be required to be cultured for 18-24 hours, and the gas production reaction is observed to be required to be cultured for 24-48 hours, so that the requirements of production practice cannot be met.
Therefore, various evaluation methods cannot meet the requirements in actual production at present, and in actual production, a qualitative judgment of the antibacterial ability of the antibacterial component to be tested to the escherichia coli can be accurately obtained in a short time (within 3 hours and visual observation) on an evaluation site, so that the inhibition ability of the product to the escherichia coli can be rapidly and intuitively evaluated.
Disclosure of Invention
In order to solve the problems, the invention provides a method for rapidly evaluating the inhibition capability of an antibacterial component to escherichia coli and application thereof.
The method for rapidly evaluating the inhibition capability of the antibacterial component to the escherichia coli provided by the invention comprises the following steps:
S1, preparing an escherichia coli selective medium: dissolving 20.0g of tryptone, 5.0g of sodium chloride, 5.0g of lactose, 2.75g of dipotassium hydrogen phosphate, 2.75g of monopotassium phosphate and 0.1g of sodium lauryl sulfate in distilled water, adjusting pH=7.0+/-0.2, and fixing the volume to 1L by using distilled water;
s2, split charging and sterilization: subpackaging the E.coli selective medium prepared in the step S1 into glass test tubes, adding a pouring tube of a Du' S small tube into each test tube, adding a silica gel plug for sealing, tightly winding and sealing by using a rubber band or a sealing film, and maintaining the pressure at 121 ℃ for 20min for sterilization;
s3, culturing and observing: 1ml of escherichia coli culture solution and 1ml of antibacterial component diluent are gently added into a test tube prepared by S2, the test tube is cultured for 3 hours in a water bath at 37 ℃, whether air bubbles appear in a Du Shixiao tube in the test tube is observed, and the antibacterial component without air bubbles is judged to have inhibition capability on escherichia coli.
Further, the E.coli selective medium prepared in S1 also contains 0.01g of bromocresol purple.
Further, the antibacterial component is an antibacterial peptide, an essential oil or a probiotic preparation.
Further, the probiotic preparation comprises a live bacterial preparation.
Further, the preparation method of the antibacterial component diluent comprises the following steps: 1g of the antibacterial component to be measured is weighed, dissolved in 100mL of sterile water, centrifuged at 4000rpm for 5min, and the supernatant is taken to prepare the antibacterial component diluent.
Further, the preparation method of the escherichia coli culture solution comprises the following steps: culturing Escherichia coli indicator bacteria in LB liquid medium at 37deg.C and 180rpm for 20 hr, and adjusting the concentration of Escherichia coli culture solution with LB liquid medium to make it consistent with 0.5 turbidity of the Mirabilitum.
Further, the preparation method of the escherichia coli indicator bacteria comprises the following steps: thawing the escherichia coli frozen stock solution at 4 ℃, dipping the escherichia coli frozen stock solution in an ultra-clean bench by using a sterile inoculating loop, streaking on an LB flat plate medium, culturing overnight at 37 ℃, selecting 2-3 single colonies, inoculating the single colonies into 100ml of LB liquid medium, culturing for 12-16 h by shaking at 37 ℃ and a shaking table at 100rpm after inoculating, and obtaining the escherichia coli frozen stock solution after culturing.
Further, the preparation method of the LB liquid medium comprises the following steps: 10g of tryptone, 5g of yeast extract powder and 10g of sodium chloride are dissolved in distilled water, the pH value is regulated to be 7.0+/-0.2 at 25 ℃, distilled water is used for fixing the volume to be 1L, and the pressure is maintained for 20min at 121 ℃ for sterilization.
Further, the LB plate medium is prepared by dissolving 10g of tryptone, 5g of yeast extract powder and 10g of sodium chloride in distilled water, adjusting the pH to 7.0+/-0.2 at 25 ℃, adding 17g of agar, uniformly stirring, fixing the volume to 1L by using distilled water, and maintaining the pressure at 121 ℃ for 20min for sterilization.
The method for rapidly evaluating the inhibition capability of the antibacterial component to the escherichia coli is applied to detection of feed components.
The beneficial effects of the invention are as follows:
1. The technical concept of the invention is to prepare a selective culture medium aiming at escherichia coli, then add escherichia coli bacterial liquid and an antibacterial component to be tested into the selective culture medium together, and judge whether the antibacterial component to be tested can inhibit escherichia coli by observing whether gas is produced or not. The selective culture medium adopted by the invention is prepared by removing yeast components in a conventional LB culture medium, adding selective components of dipotassium hydrogen phosphate, potassium dihydrogen phosphate and sodium lauryl sulfate, wherein the dipotassium hydrogen phosphate and the potassium dihydrogen phosphate have the functions of maintaining osmotic pressure, and the sodium lauryl sulfate has the function of inhibiting the growth of gram-positive bacteria, so that the selective culture of escherichia coli (gram-negative bacteria) is realized. If the antibacterial component to be detected has no inhibition effect, the escherichia coli can proliferate and metabolize gas production, and the Du Shixiao pipe adopted by the invention can rapidly show whether bubbles are generated or not, if so, the antibacterial component of the culture medium has no inhibition effect on the escherichia coli.
2. By adopting the evaluation method combining the selective culture medium and the gas production observation method, the result of whether the antibacterial component to be detected has an inhibition effect on escherichia coli can be directly observed in 3 hours, and the efficiency is far higher than that of the method in the prior art.
3. The invention further provides an evaluation method capable of developing color, namely bromocresol purple can be added into the selective culture medium as an indicator, and the color development reaction and the gas production reaction result are consistent and can be used as auxiliary observation means of the evaluation method.
4. According to the invention, the culture medium and the detection and evaluation method are optimized to form a novel detection and evaluation method, the method can better solve the problems existing in the existing method, the result can be obtained rapidly and efficiently, the method is safe and simple, and the stability and accuracy of the detection and evaluation result are improved.
Drawings
FIG. 1 is a graph showing the comparison of test results of example 1;
FIG. 2 is a graph showing the comparison of the test results of example 3;
FIG. 3 is a graph showing the comparison of the test results of example 4;
FIG. 4 is a graph showing the comparison of the test results of the control group of example 5;
FIG. 5 is a graph showing comparison of experimental results of experimental groups of example 5.
Detailed Description
The invention is further illustrated by the following examples.
Example 1: verifying whether the antibacterial effect of the antibacterial peptide sample can be rapidly observed by the method
1.1 Laboratory apparatus: an autoclave, a constant temperature incubator, a pipetting gun, a water bath, an electroceramic stove and an ultra-clean workbench;
1.2 experimental method:
1.2.1 preparation of selective Medium: dissolving 20.0g of tryptone, 5.0g of sodium chloride, 5.0g of lactose, 2.75g of dipotassium hydrogen phosphate, 2.75g of monopotassium phosphate and 0.1g of sodium lauryl sulfate in distilled water, adjusting pH=7.0+/-0.2, and fixing the volume to 1L by using distilled water; subpackaging the E.coli selective medium prepared in the step S1 into 18 x 180mm glass test tubes, 8ml each test tube, adding one Du Shixiao tube/inverted tube into each test tube after subpackaging, adding a silica gel plug for sealing, tightly winding and sealing by using a rubber band or a sealing film, and maintaining the pressure at 121 ℃ for 20min for sterilization.
1.2.2 Indicator bacteria culture:
Preparation of LB liquid Medium: 10g of tryptone, 5g of yeast extract powder and 10g of sodium chloride are dissolved in distilled water, the pH value is regulated to be 7.0+/-0.2 at 25 ℃, distilled water is used for fixing the volume to be 1L, and the pressure is maintained for 20min at 121 ℃ for sterilization.
Preparing LB plate culture medium, dissolving tryptone 10g, yeast extract 5g and sodium chloride 10g in distilled water, adjusting pH to 7.0+ -0.2 at 25deg.C, adding agar 17g, stirring uniformly, fixing volume to 1L with distilled water, maintaining pressure at 121deg.C for 20min, and sterilizing.
Preparation of E.coli indicator bacteria: thawing E.coli (ATCC 25922) frozen stock solution at 4 ℃, dipping the E.coli frozen stock solution in an ultra clean bench by a sterile inoculating loop, streaking on an LB flat plate medium, culturing overnight at 37 ℃, picking 2-3 single colonies, inoculating the single colonies into 100ml of LB liquid medium, culturing for 12-16 h at 37 ℃ by shaking by a shaking table at 100rpm after inoculating, and obtaining turbid culture solution after culturing.
Preparing an escherichia coli culture solution: culturing Escherichia coli indicator bacteria in LB liquid medium at 37deg.C and 180rpm for 20 hr, and adjusting the concentration of Escherichia coli culture solution with LB liquid medium to make it consistent with 0.5 turbidity of the Mirabilitum.
1.2.3 Preparation of antibacterial peptide dilutions: 1g of antibacterial peptide is weighed, dissolved in 100ML of sterile water, centrifuged at 4000rpm for 5min, and the supernatant is taken for standby to prepare 100-fold dilution of the antibacterial peptide.
1.2.4 Co-cultivation of antibacterial peptides with E.coli: and (3) respectively taking 1mL of the prepared escherichia coli culture solution and 1mL of the antibacterial peptide diluent, lightly adding the escherichia coli culture solution and the antibacterial peptide diluent into a selective culture medium test tube, carrying out water bath culture at 37 ℃ for 3 hours, observing whether bubbles and color changes occur in a pouring tube in the test tube, and simultaneously, preparing a blank control group without adding the antibacterial peptide. The experimental group at this time used the antimicrobial peptide at a concentration of 1/100 x 1/10=0.1%.
1.3 Experimental results are shown in table 1 and fig. 1: FIG. 1 is a photograph of a control group and an experimental group in a water bath at 37℃for 3 hours, a left test tube is a control group, and a right test tube is an experimental group.
Experimental results: by adopting the selective culture medium, the growth of mixed bacteria can be inhibited, the growth metabolism of the escherichia coli is promoted, bubbles produced in test tubes of a control group can be seen in 2h at the highest speed, and no bubbles are produced after the antibacterial peptide is added into an experimental group, so that the growth activity of the escherichia coli for fermenting lactose to produce gas under the condition is inhibited.
Example 2: detection of antibacterial peptide inhibition effect on colibacillus by turbidimetry
2.1 Experimental facility: constant temperature incubator, pipette, sterilizing pot, spectrophotometer.
2.2 Experimental method:
2.2.1LB liquid culture medium, LB plate culture medium and escherichia coli culture solution are prepared by the same method as in example 1, and antibacterial peptide diluent is prepared by the same method as in example 1, but supernatant liquid is required to be centrifugally obtained when the supernatant liquid is obtained, so that the preparation of the obtained sample supernatant liquid is ensured, and the influence on the subsequent absorbance detection is avoided.
2.2.2 Co-cultivation of the antibacterial peptide with E.coli: 9mL of escherichia coli culture solution and 1mL of antibacterial peptide diluent are taken and lightly added into a sterile test tube filled with LB liquid culture medium, and a sterile silica gel plug is covered. And a blank control group without adding antibacterial peptide is also prepared. The co-culture tube culture solution is put into a 37 ℃ water bath for 0-3 h, and the change of OD600 is measured every h by using a spectrophotometer. At this time, the concentration of the antimicrobial peptide used was 1/100×1/10=0.1%.
2.3 Experimental results are shown in Table 2, and the 3h antibacterial rate is calculated
A, A is empty: blank control was incubated for 3h light absorbance;
a void 0: blank control group 0h light absorption value;
sample a: adding the light absorption value of the sample group to be detected for 3 hours;
A sample 0: adding the light absorption value of the sample group to be detected for 0 h;
3h inhibition = (1.55-0.05) ×100/1.55=96.77%
It can be seen that the antibacterial peptide can be obtained by calculating by turbidimetry to substantially completely inhibit the growth of Escherichia coli in 3 hours.
As can be seen from Table 3, the method of the invention of example 1 can realize effective evaluation of the antibacterial peptide to inhibit colibacillus, the test result is basically consistent with that of the turbidimetry, but frequent pathogenic bacteria cell density test work is not required, the operation is simple, and the result is visual. When the method is adopted to test the inhibition effect of the antibacterial component on the escherichia coli, the experimental result can be observed with naked eyes, the method is simple and easy to implement, and the sample does not need to be subjected to sterilization treatment, when the turbidimetry is adopted to test the inhibition effect of the antibacterial component on the escherichia coli, the spectrophotometry is required to be used for measuring the light absorption value for a plurality of times on the culture solution containing the escherichia coli, and the detection result fluctuation is larger because the sterilization effect can influence the light absorption value, so that the sample is required to be subjected to sterilization treatment when the turbidimetry is adopted. Therefore, the method is simple and easy to operate, does not need to carry out degerming treatment on a sample, does not need to measure the absorbance value of the escherichia coli culture solution for multiple times, has stable test results, and can be used as a reliable and stable detection and evaluation method for rapidly evaluating the bacteriostasis performance of the escherichia coli by the antibacterial component.
Example 3: the method of the invention is compared with the accuracy of the rapid detection and evaluation method in the prior art
The selective medium of the present invention was compared with the medium of chinese patent 202010121703.5.
The preparation method of the selective medium, the antibacterial peptide diluent and the escherichia coli culture solution is the same as that of the example 1. To the selective medium was added bromocresol purple 0.01g as an indicator.
A comparative medium was prepared according to the medium preparation method described in China patent 202010121703.5: weighing each component according to a formula of 6g of yeast powder, 12g of tryptone, 8g of lactose, 9g of sodium chloride, 0.01 g of bromocresol purple and 0.003 g of thiazole blue, fixing the volume to 1000mL, preparing a culture medium, packaging into 18X 180mm glass test tubes, and sealing and sterilizing.
The experimental method comprises the following steps: 1mL of escherichia coli culture solution and 1mL of antibacterial peptide diluent are respectively and lightly added into a selective culture medium test tube (containing a pouring tube) and a comparison culture medium test tube of the invention, and water bath culture is carried out for 3 hours at 37 ℃, and whether bubbles appear in the pouring tube and whether the color changes in the test tube are observed.
The experimental results are shown in table 4 and fig. 2. FIG. 2 shows a selective medium of the present invention in the left tube and a comparative medium of the prior art in the right tube.
The thiazole blue used in the comparative medium was yellow or orange powder, poorly soluble in water. When in use, the bacterial strain can be reduced into water-insoluble blue-violet crystal formazan by living bacteria and deposited in cells, and bromocresol purple and thiazole blue are added into a liquid culture medium system (water is a solvent), the initial color is purple, and the bacterial strain grows to cause the pH change of the system to be acidic and then to be yellow.
As can be seen from the results in Table 4 and FIG. 2, the indicator of the selective medium of the invention has unchanged color and no gas production due to the use of the antibacterial peptide, the observed results of gas production and color are consistent with the theoretical results, the indicator of the comparative medium changes color, shows the growth of bacteria, and is inconsistent with the theoretical results, which shows that the selective medium of the invention has stronger specificity, can effectively eliminate the interference of the growth of other miscellaneous bacteria in the sample to be detected on the detection results, and has more accurate evaluation results.
Because most of the engineering bacteria for producing the antibacterial peptide at present are gram-positive bacteria such as bacillus subtilis and lichen, inaccurate evaluation results are easy to appear by adopting a comparison culture medium in the prior art (false positives, and misjudgment of results is caused by the growth of residual living bacteria in a product in a system). The selective culture medium adopted by the method is suitable for the growth of a work indication strain (escherichia coli), can inhibit the growth of other non-target bacteria, and can accurately evaluate the antibacterial performance of the antibacterial peptide.
Example 4 comparison of the timeliness of the method of the invention with the prior art rapid test evaluation method
The selective media of the present invention was compared to the brilliant green lactose bile salt broth (BGLB) employed in chinese patent 202010121703.5, which is commercially available (BGLB).
The preparation method of the selective medium and the escherichia coli culture solution of the invention is the same as that of the example 3.
The experimental method comprises the following steps: 1mL of the E.coli culture solution was gently added to the test tube (containing a pouring tube) and the test tube BGLB (containing a pouring tube) of the selective medium of the present invention, respectively, and incubated in a water bath at 37℃for 3 hours, during which time it was observed whether air bubbles appeared in the pouring tube and whether the color was changed.
The experimental results are shown in Table 5 and FIG. 3. FIG. 3 shows a selective medium of the present invention in the left tube and BGLB in the right tube.
As can be seen from the results of Table 5 and FIG. 3, since only E.coli was inoculated, the color of the indicator of the selective medium of the present invention was changed and the results of the gas production, gas production and color observation were the same as the theoretical results, whereas the indicator of BGLB medium was not discolored and no bubbles were produced, showing that viable bacteria were present, but gas production was not yet occurring, and a long time was required for gas production, and according to the description of the prior art, detection of E.coli using BGLB medium required cultivation for 18-48 hours, and failed to meet the requirements for rapid evaluation in situ evaluation, whereas the method of the present invention required only 3 hours under the same cultivation conditions, indicating that the use of the selective medium of the present invention, has a higher specificity, a faster timeliness, and can meet the requirements for rapid evaluation in situ in production practice.
Example 5: indicator for screening auxiliary observation
The preparation method of the escherichia coli culture solution, the antibacterial peptide diluent and the selective culture medium is the same as that of example 1;
According to the characteristics of lactose growth metabolism acid production in the escherichia coli, three different color developers are added into a selective culture medium, and the names and the addition amounts are as follows:
indicator 1: bromocresol purple, 0.01g/L;
indicator 2: TTC (chlorotrityl tetrazole), 0.05g/L;
indicator 3: resazurin, 0.001g/L;
The selective medium added with the indicator is divided into 8ml of 18 x 180mm glass test tubes, each test tube is added with a Du Shixiao tube (with the tube diameter of 6 x 30 mm), then a silica gel plug is added for sealing, and the sealing film is wound tightly by a rubber band and a sealing film for sealing. Taking 1mL of escherichia coli culture solution and 1mL of antibacterial peptide diluent, lightly adding the escherichia coli culture solution and the antibacterial peptide diluent into a selective culture medium test tube, and culturing the escherichia coli culture solution in a 37 ℃ water bath for 3 hours; meanwhile, a control group is prepared, 1mL of escherichia coli culture solution and 1mL of sterile water are gently added into a selective culture medium test tube, and water bath culture is carried out for 3 hours at 37 ℃. During which it was observed whether bubbles appeared in the pouring tube in the test tube and whether the color was changed. At this time, the concentration of the antimicrobial peptide used was 1/100×1/10=0.1%.
The experimental results are shown in table 6, fig. 4 and fig. 5:
As can be seen from the results in Table 6, 1) the experimental group and the control group using the selective medium, the experimental results are normal, and are consistent with the theoretical results, and the antibacterial peptide inhibits the growth and the gas production of Escherichia coli; 2) Different indicators are adopted, and the influence on the experimental result is different, wherein after bromocresol purple is added, the color change contrast is strong (indicator bacteria such as escherichia coli grow, and the reaction system turns yellow; the reaction system is purple when the growth is not carried out) and the gas production phenomenon of the escherichia coli in the system are kept consistent (the indicator bacteria escherichia coli grows and produces bubbles; no bubbles are produced without growth). After TTC and resazurin are added as indicators, the antibacterial effect of the antibacterial peptide is changed in color, but the change contrast is not strong, so that bromocresol purple is selected as the indicator of the method.
The method of the invention can be finally optimized as follows:
And (3) inoculating 1mL of quantitative indicator strain escherichia coli into a prepared escherichia coli selective liquid culture medium test tube (sterilized and containing a pouring tube), adding 1mL of samples to be tested with various concentrations, standing, incubating in a 37 ℃ incubator for 3 hours, and observing the color change of the culture medium in the test tube and the generation condition of bubbles in the pouring tube. The visual result judgment can be carried out on whether the growth condition of the indicator bacteria (escherichia coli ATCC 25922) is inhibited or not by observing the color change and the bubble generation condition of the test tube culture medium inoculated with the indicator bacteria, so that the rapid evaluation of the sample to be tested is realized.
The preparation method of the escherichia coli selective medium comprises the following steps: 20.0g of tryptone, 5.0g of sodium chloride, 5.0g of lactose, 2.75g of dipotassium hydrogen phosphate, 2.75g of potassium dihydrogen phosphate, 0.1g of sodium lauryl sulfate and 0.01g of bromocresol purple per liter, and adjusting the pH value to be 7.0+/-0.2;
The method has the advantages that: 1) The method does not need to carry out multiple cell density measurement (spectrophotometry) on experimental indicator bacteria escherichia coli, so that the exposure risk is reduced; 2) The method has the advantages that the sterilization requirements on experimental evaluation samples (antibacterial peptides, bacteriocins and the like) are reduced, the difficulty of sample treatment is reduced, 3) the transparency requirements on the samples are reduced, the observation of experimental results is simple and convenient, the experimental results can be judged through visual experimental phenomena such as color change (yellow and purple color change are obvious in distinction), whether bubbles are generated or not, and the like, and the rapid and effective evaluation on the samples can be realized in 3 hours.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.
Claims (8)
1. A method for rapidly assessing the inhibitory capacity of an antimicrobial component against e.coli comprising the steps of:
S1, preparing an escherichia coli selective medium: dissolving 20.0g of tryptone, 5.0g of sodium chloride, 5.0g of lactose, 2.75g of dipotassium hydrogen phosphate, 2.75g of monopotassium phosphate and 0.1g of sodium lauryl sulfate in distilled water, adjusting pH=7.0+/-0.2, and fixing the volume to 1L by using distilled water;
s2, split charging and sterilization: subpackaging the E.coli selective medium prepared in the step S1 into glass test tubes, adding a pouring tube of a Du' S small tube into each test tube, adding a silica gel plug for sealing, tightly winding and sealing by using a rubber band or a sealing film, and maintaining the pressure at 121 ℃ for 20min for sterilization;
s3, culturing and observing: 1ml of escherichia coli culture solution and 1ml of antibacterial component diluent are gently added into a test tube prepared by the method S2, water bath culture is carried out for 3 hours at 37 ℃, whether bubbles appear in a Du Shixiao tube in the test tube or not is observed, and the antibacterial component without bubbles is judged to have inhibition capability on escherichia coli;
The selective culture medium of the escherichia coli prepared in the step S1 also contains 0.01g of bromocresol purple;
The preparation method of the escherichia coli culture solution comprises the following steps: culturing Escherichia coli indicator bacteria in LB liquid medium at 37deg.C and 180rpm for 20 hr, and adjusting the concentration of Escherichia coli culture solution with LB liquid medium to make it consistent with 0.5 turbidity of the Mirabilitum.
2. The method of rapidly assessing the inhibitory capacity of an antimicrobial component against e.coli according to claim 1, wherein the antimicrobial component is an antimicrobial peptide, an essential oil or a probiotic preparation.
3. The method of rapidly assessing the inhibitory capacity of an antimicrobial component against e.coli according to claim 2, wherein the probiotic preparation comprises viable bacteria.
4. The method for rapidly evaluating the inhibitory capacity of an antimicrobial component against escherichia coli according to claim 1, wherein the preparation method of the antimicrobial component diluent comprises the following steps: 1g of the antibacterial component to be measured is weighed, dissolved in 100mL of sterile water, centrifuged at 4000rpm for 5min, and the supernatant is taken to prepare the antibacterial component diluent.
5. The method for rapidly evaluating the inhibitory capacity of an antibacterial component against escherichia coli according to claim 1, wherein the escherichia coli indicator is prepared by the following steps: thawing the escherichia coli frozen stock solution at 4 ℃, dipping the escherichia coli frozen stock solution in an ultra-clean bench by using a sterile inoculating loop, streaking on an LB flat plate medium, culturing overnight at 37 ℃, selecting 2-3 single colonies, inoculating the single colonies into 100ml of LB liquid medium, culturing for 12-16 h by shaking at 37 ℃ and a shaking table at 100rpm after inoculating, and obtaining the escherichia coli frozen stock solution after culturing.
6. The method for rapidly evaluating the inhibitory capacity of an antibacterial component against escherichia coli according to claim 1, wherein the LB liquid medium is prepared by the following steps: 10g of tryptone, 5g of yeast extract powder and 10g of sodium chloride are dissolved in distilled water, the pH value is regulated to be 7.0+/-0.2 at 25 ℃, distilled water is used for fixing the volume to be 1L, and the pressure is maintained for 20min at 121 ℃ for sterilization.
7. The method for rapidly evaluating the inhibitory ability of an antibacterial component against E.coli according to claim 5, wherein the LB plate medium is prepared by dissolving 10g of tryptone, 5g of yeast extract and 10g of sodium chloride in distilled water, adjusting pH to be 7.0.+ -. 0.2 at 25 ℃, adding 17g of agar, stirring uniformly and sterilizing at 121 ℃ for 20min after constant volume to 1L with distilled water.
8. Use of the method for rapidly assessing the inhibitory capacity of an antibacterial component against e.coli according to any one of claims 1 to 7 in the detection of a feed component.
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| JP4338889B2 (en) * | 2000-12-20 | 2009-10-07 | 森永乳業株式会社 | Rapid detection method for coliform bacteria and / or E. coli and rapid detection kit used in this method |
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| CN106811404A (en) * | 2017-01-22 | 2017-06-09 | 贵州勤邦食品安全科学技术有限公司 | A kind of test piece of quick detection coliform and preparation method thereof, detection method |
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