CN117210333A - Microorganism wall breaking method based on combination of ultrasonic and grinding - Google Patents
Microorganism wall breaking method based on combination of ultrasonic and grinding Download PDFInfo
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Abstract
The invention discloses a microorganism wall breaking method based on combination of ultrasound and grinding, which comprises the following steps: (1) Preparing a microbial sample to be broken into a solution, and then adding the solution into a sample tank filled with glass beads, so that the glass beads and the microbial sample solution to be broken are fully mixed; the diameter of the glass beads is 150-500um; the total volume of the glass beads accounts for 10-70% of the whole sample tank volume; (2) Applying ultrasonic waves to a to-be-broken microorganism sample solution in a sample tank by adopting an ultrasonic transducer, and breaking the wall of microorganisms under the combined action of the ultrasonic waves and glass beads; the voltage at the two ends of the ultrasonic transducer is 10-200V; the ultrasonic time is 1-60min; and (3) separating out glass beads after wall breaking is completed. The microbial wall breaking method has the characteristics of high efficiency, low loss, low pollution, low cost, easy operation and the like.
Description
Technical Field
The invention relates to the technical field of microbial wall breaking, in particular to a microbial wall breaking method based on combination of ultrasound and grinding.
Background
Microorganisms can release their intracellular active ingredients by physically or chemically destroying their cell wall and cell membrane structures. The wall-broken microorganism has high bioavailability and nutritive value, and can be used for preparing health food, medicine, feed, etc.
The method can be used for extracting important biomolecules such as nucleic acid, protein, enzyme and the like by destroying the cell wall and cell membrane structures of microorganisms, and provides raw materials and technical support for the fields such as genetic engineering, medical detection, vaccine preparation and the like.
Common wall breaking techniques are physical and chemical wall breaking techniques. The physical wall breaking technology mainly uses mechanical equipment such as a high-pressure homogenizer, a bead impact crusher and the like to perform mechanical actions such as shearing, impact or friction on microbial cells. The method has the advantages of high efficiency and strong controllability, and can be used for efficiently breaking the wall of the thick-wall microorganism. But has complex operation, high cost and easy pollution or heat generation. The chemical wall breaking technology refers to the chemical reaction of dissolving or hydrolyzing microbial cells by using acid-base solution, enzyme preparation, surfactant and other chemical reagents. Its advantages are simple operation, low cost and high suitability for mass production. The disadvantages are low efficiency and the susceptibility to stability or purity of the active substance. And chemical solvents have problems such as long-term storage failure. The ultrasonic wall breaking technology is also a physical wall breaking technology, and the wall breaking is carried out on microbial cells through the cavitation of ultrasonic waves, so that the power of the thick-wall microorganisms is generally increased, but the higher the power is, the lower the energy efficiency ratio is, and the high-power input is difficult to ensure the high-efficiency wall breaking effect.
The Chinese patent with publication number CN 114507601A discloses a wall breaking method for preparing nano-scale selenium-enriched yeast, which comprises the following steps: (1) Mixing selenium-enriched yeast with water to obtain selenium-enriched yeast suspension; (2) Adding a photocatalyst into the selenium-enriched yeast suspension prepared in the step (1), uniformly mixing, placing in a dark box, stirring, performing light treatment, and performing centrifugal separation to obtain a photocatalytic selenium-enriched yeast suspension; (3) Ball milling is carried out on the prepared photocatalysis selenium-enriched yeast suspension to obtain ball milling selenium-enriched yeast suspension; (4) Ultrasonically crushing the prepared ball-milling selenium-enriched yeast suspension; (5) Repeating the operations of the steps (3) and (4) to obtain the nano-scale selenium-enriched yeast, wherein the nano-scale selenium-enriched yeast is crushed by the combination of photocatalysis, ball milling and ultrasound. The process firstly uses ball milling and then ultrasonic crushing, and the time efficiency is low.
The Chinese patent with publication number of CN 115074250A discloses a mild and efficient chlorella cell wall breaking method, which comprises the following steps: centrifuging and concentrating chlorella, treating the algae liquid with cellulase, thoroughly freezing the mixture liquid after enzyme treatment under the conditions of liquid nitrogen or minus 80 ℃, melting in water bath at 37 ℃, repeatedly freezing and thawing for 2-5 times, and breaking the wall by using ultrasonic with power of 300-500 w. The process belongs to a method combining ultrasonic and chemical wall breaking, and has the defects of long-term storage failure of chemical reagents and overhigh ultrasonic power, and the process is complex.
Disclosure of Invention
Aiming at the problems that the prior microorganism wall breaking method needs large volume of devices, is difficult to store reagents and the like, the invention provides the microorganism wall breaking method based on the combination of ultrasonic and grinding, which has simple and rapid extraction process and does not need to be additionally matched with chemical reagents such as lysate, protease and the like for use.
The technical scheme of the invention is as follows:
a microorganism wall breaking method based on combination of ultrasonic and grinding comprises the following steps:
(1) Preparing a microbial sample to be broken into a solution, and then adding the solution into a sample tank filled with glass beads, so that the glass beads and the microbial sample solution to be broken are fully mixed; the diameter of the glass beads is 150-500um; the total volume of the glass beads accounts for 10-70% of the whole sample tank volume;
(2) Applying ultrasonic waves to a to-be-broken microorganism sample solution in a sample tank by adopting an ultrasonic transducer, and breaking the wall of microorganisms under the combined action of the ultrasonic waves and glass beads; the voltage at the two ends of the ultrasonic transducer is 10-200V; the ultrasonic time is 1-60min;
(3) And separating out glass beads after wall breaking is completed.
In the method for breaking the wall of the microorganism, the microorganism damages the cell wall and cell membrane structures of the microorganism in a sample under the actions of cavitation effect, vibration effect and thermal effect of ultrasonic waves, and meanwhile, the microorganism is physically ground through glass beads. The grinding actions of ultrasonic waves on the glass beads are mutually cooperated, so that the cell wall or cell membrane structure of microorganisms can be quickly destroyed under the condition that chemical lysate and protease are not required to be matched, the quick wall breaking effect is achieved, and compared with the conventional microorganism wall breaking method, the wall breaking time is greatly shortened, and the wall breaking efficiency is improved.
The microbial sample to be broken is saliva, blood or tissue fluid containing bacteria, cells and/or viruses.
Further preferably, the bacterium is a gram positive bacterium. The cell wall of gram-positive bacteria is difficult to break, and cannot be broken by simple osmotic shock, and generally requires lysozyme treatment to digest the higher peptidoglycan in its cell wall. Under the action of lysozyme, the method can break the cell wall of gram-positive bacteria based on a microorganism cell wall breaking method combining ultrasonic treatment and grinding.
Preferably, the glass beads have a diameter of 150-250um.
The diameter of the glass beads is related to the mass of the individual glass beads. Since the grinding action of the glass beads is mainly generated by the high-frequency vibration action of the ultrasonic transducer, the weight of the glass beads is directly related to the grinding action. If the diameter is too large, the vibration effect of the glass beads is poor, and the corresponding grinding effect is smaller. Too small a diameter can affect the subsequent solid-liquid separation process. When the diameter of the glass beads is 150-250um, the vibration grinding effect of the glass beads can be mutually cooperated with the ultrasonic effect, so that a better microbial wall breaking effect is achieved.
Preferably, the total volume of the glass beads is 30-50% of the total sample cell volume.
The percentage of the total volume of the glass beads to the whole sample tank volume is related to the ratio of the glass beads to the sample amount, and the total volume of the glass beads is too small, so that the collision probability among the glass beads is relatively low, and the grinding effect is relatively poor; too much total volume of the glass beads results in a relatively large overall weight and poor grinding due to vibration effects. When the total volume of the glass beads accounts for 30-50% of the whole sample tank volume, the whole grinding effect is good.
Preferably, the glass beads are acid-washed glass beads.
The acid-washed glass beads are sufficiently washed by concentrated sulfuric acid, so that the surface impurities are less, and the pollution to a sample is avoided.
The ultrasonic transducer is contacted with the bottom wall of the sample tank to apply ultrasonic waves to the microbial sample solution to be broken. The size of the ultrasonic transducer can cover or be slightly smaller than the bottom wall of the sample tank.
Preferably, the ultrasonic transducer is a monolithic piezoelectric ceramic.
Preferably, the thickness of the ultrasonic transducer is 5-30mm.
The invention adopts the ultrasonic transducer with smaller volume (the thickness is not more than 30 mm) to realize the rapid wall breaking of microorganisms and can be integrated in a smaller space.
Preferably, the voltage across the ultrasound transducer is 100-200V.
The wall breaking effect is mainly influenced by the cavitation of ultrasound and the grinding of glass beads. The higher the voltage of the ultrasonic transducer is, the higher the sound pressure is, and the stronger the cavitation effect is, but after a certain power is reached, the stronger cavitation effect cannot be brought by continuously increasing the power. The grinding action of the glass beads is mainly affected by the displacement of the ultrasonic transducer, and the displacement d=d33×v, so the higher the voltage is, the larger the displacement is, and the stronger the grinding action is. However, an excessively high ultrasonic voltage may cause cleavage of nucleic acid, which adversely affects subsequent detection results, and the piezoelectric ceramic has a certain withstand voltage value, exceeding which may cause damage to the piezoelectric ceramic. When the voltage at the two ends of the ultrasonic transducer is 100-200V, the cavitation effect of the ultrasonic and the grinding effect of the glass beads are better.
Preferably, in the step (2), the frequency of the ultrasonic wave is 20-200kHz; further preferably 20-40kHz.
The ultrasonic frequency mainly affects cavitation effect. The best cavitation frequency is typically 20-40kHz.
Preferably, in the step (2), the ultrasonic time is 1-30min.
When the microbial sample to be broken is gram positive bacteria which are difficult to break, the ultrasonic time is 5-30min, and when the microbial sample to be broken is a cell, the extraction time can be shortened to 1-5min. If the ultrasonic wave is too long, the nucleic acid is broken, which is unfavorable for the subsequent detection. The wall breaking method has higher wall breaking efficiency.
In step (3), the glass beads may be separated by centrifugation, filtration, or the like.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention is based on the microorganism wall breaking method combining ultrasound and grinding, adopts the grinding action of ultrasound matched with glass beads to act on microorganism samples at the same time, comprehensively utilizes cavitation effect, vibration effect and thermal effect of ultrasound and mechanical grinding action of glass beads, and can quickly destroy the cell wall structure of microorganisms without matching with chemical lysate and protease. When the sample is gram positive bacteria which are difficult to break, the wall breaking time is required to be 5-30min, and when the sample is cells, the wall breaking time can be shortened to be 1-5min, and compared with the conventional wall breaking method, the wall breaking time is greatly shortened, and the wall breaking efficiency is greatly improved.
(2) In the microorganism wall breaking method based on the combination of ultrasonic and grinding, the volume of the ultrasonic transducer is smaller, so that the ultrasonic transducer can be integrated in a smaller space.
The microorganism wall breaking method based on the combination of the ultrasonic and the grinding comprehensively utilizes three effects of the ultrasonic and the mechanical grinding effect of the glass beads, can rapidly realize the wall breaking of a microorganism sample in a limited space, and has the characteristics of high efficiency, low loss, low pollution, low cost, easy operation and the like.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are intended to facilitate the understanding of the present invention without any limitation thereto.
Judging whether the wall breaking is realized or not through the nucleic acid in the solution.
Because staphylococcus aureus is a gram-positive bacterium that is more difficult to extract nucleic acids, the bacterium was used for testing. After extraction was completed, the clear solution was subjected to PCR testing and compared with a sample of the supernatant obtained by direct centrifugation. The extraction effect was observed by the results of Ct values.
Examples 1 to 4, comparative example 1
Pure bacterial solutions of staphylococcus aureus were propagated through a shaker one day prior to the experiment, and staphylococcus aureus samples were formulated as solutions at the time of the experiment.
Examples 1-4 glass beads of about 200 μm or 400 μm in diameter were respectively loaded into the sample cell, the total volume of the glass beads was 10%,30%,50% of the volume of the sample cell, respectively, and then the sample cell was packaged with a pressure sensitive film. The staphylococcus aureus sample solution is placed in a sample tank with glass beads, an ultrasonic module with an ultrasonic transducer is contacted with the bottom of the sample tank, and ultrasonic is applied for 10min at the resonance frequency of the ultrasonic transducer, and the voltage is 100V. Under the cavitation effect, vibration effect and thermal effect of the ultrasound, releasing nucleic acid in bacteria in the sample solution to be broken into the solution. The solution was separated from the glass beads by centrifugation and the solution containing the nucleic acid was subjected to a PCR test.
Comparative example 1A sample solution of Staphylococcus aureus was centrifuged at 12000rpm and 4℃for 5 minutes in a centrifuge to obtain a precipitate and a supernatant, and the supernatant was taken out to directly carry out PCR amplification.
Finally, comparing the ultrasonic group with comparative example 1 of the supernatant of the direct degerming liquid, and the result shows that the nucleic acid content of the ultrasonic group is far higher than that of the comparative group, which shows that the wall breaking is realized.
The test results of examples 1 to 4 and comparative example 1 are shown in Table 1.
TABLE 1
| Diameter of glass bead | Glass bead ratio | Ct value | |
| Example 1 | 200μm | 10％ | 27.42 |
| Example 2 | 200μm | 30％ | 25.5 |
| Example 3 | 200μm | 50％ | 23.15 |
| Example 4 | 400μm | 50％ | 26.4 |
| Comparative example 1 | 30.85 |
Examples 4 to 6, comparative example 2, comparative example 3
Pure bacterial solutions of staphylococcus aureus were propagated through a shaker one day prior to the experiment, and staphylococcus aureus samples were formulated as solutions at the time of the experiment.
Comparative example 2 and examples 4 to 6 each had a glass bead of about 200um diameter filled into the sample cell, and the total volume of the glass beads was 0%,10%,30%,50% of the volume of the sample cell, respectively, and then the sample cell was packaged with a pressure sensitive film. The staphylococcus aureus sample solution is placed in a sample tank with glass beads, an ultrasonic module with an ultrasonic transducer is contacted with the bottom of the sample tank, and ultrasonic is applied for 10min at the resonance frequency of the ultrasonic transducer, and the voltage is 100V. Under the cavitation effect, vibration effect and thermal effect of the ultrasound, releasing nucleic acid in bacteria in the sample solution to be broken into the solution. The solution was separated from the glass beads by centrifugation and the solution containing the nucleic acid was subjected to a PCR test.
Comparative example 3 staphylococcus aureus sample solution was centrifuged at 12000rpm,4 ℃ for 5 minutes in a centrifuge to obtain precipitate and supernatant, and the supernatant was taken out to directly perform PCR amplification.
Finally, comparing the ultrasonic group with comparative example 1 of the supernatant of the direct degerming liquid, and the result shows that the nucleic acid content of the ultrasonic group is far higher than that of the comparative group, which shows that the wall breaking is realized.
The test results of examples 4 to 6, comparative example 2, comparative example 3 are shown in Table 2.
TABLE 2
| Glass bead ratio | Ct value | |
| Comparative example 2 | 0％ | 32.3 |
| Examples4 | 10％ | 32.1 |
| Example 5 | 30％ | 27.2 |
| Example 6 | 50％ | 21.48 |
| Comparative example 3 | 32.5 |
As can be seen from Table 2, the wall breaking effect of the ultrasonic on staphylococcus aureus is very little, after the glass beads are added, the ultrasonic is matched with the grinding effect of the glass beads to act on the microorganism sample, the cavitation effect, the vibration effect and the thermal effect of the ultrasonic and the mechanical grinding effect of the glass beads are comprehensively utilized, the cell wall structure of the microorganism can be quickly broken without matching with chemical lysate and protease, and the wall breaking efficiency is greatly improved.
Examples 7 to 15
Pure bacterial solutions of staphylococcus aureus were propagated through a shaker one day prior to the experiment, and staphylococcus aureus samples were formulated as solutions at the time of the experiment.
Glass beads with diameters of about 200um, which account for 50% of the volume of the sample cell, are respectively filled into the sample cell, and then the sample cell is packaged by a pressure sensitive film.
The staphylococcus aureus sample solution is put into a sample tank with glass beads, an ultrasonic module with an ultrasonic transducer is contacted with the bottom of the sample tank, and ultrasonic different voltages and times are respectively applied at the resonance frequency of the ultrasonic transducer.
The solution was separated from the glass beads by centrifugation. The solution containing the nucleic acid was subjected to a PCR test.
Comparative example 4A sample solution of Staphylococcus aureus was centrifuged at 12000rpm and 4℃for 5 minutes in a centrifuge to obtain a precipitate and a supernatant, and the supernatant was taken out to directly perform PCR amplification.
Finally, the ultrasound group is compared with a control group of the directly sterilized liquid supernatant. The test results of examples 7 to 15 and comparative example 4 are shown in Table 3.
TABLE 3 Table 3
| Voltage (V) | Time | Ct value | |
| Example 7 | 100V | 10min | 25.64 |
| Example 8 | 150V | 10min | 24.19 |
| Example 9 | 200V | 10min | 22.27 |
| Implementation of the embodimentsExample 10 | 100V | 20min | 25.2 |
| Example 11 | 150V | 20min | 22.72 |
| Example 12 | 200V | 20min | 24.47 |
| Example 13 | 100V | 30min | 23.4 |
| Example 14 | 150V | 30min | 25.48 |
| Example 15 | 200V | 30min | 26.7 |
| Comparative example 4 | 27.54 |
As can be seen from the data in table 3, the Ct values of the control group were improved differently for different voltages and ultrasound times, indicating that the bacteria were ruptured. The ultrasonic voltage is increased or the ultrasonic time is prolonged within a certain range, so that the microbial wall breaking effect can be improved, but after the ultrasonic voltage or the ultrasonic time exceeds a certain value, the Ct value is increased instead, and the Ct value is judged by nucleic acid amplification, so that the too high ultrasonic voltage or the too long ultrasonic time can cause the breaking of nucleic acid.
The foregoing embodiments have described the technical solutions and advantages of the present invention in detail, and it should be understood that the foregoing embodiments are merely illustrative of the present invention and are not intended to limit the invention, and any modifications, additions, substitutions and the like that fall within the principles of the present invention should be included in the scope of the invention.
Claims (10)
1. The microbial wall breaking method based on the combination of ultrasonic and grinding is characterized by comprising the following steps of:
(1) Preparing a microbial sample to be broken into a solution, and then adding the solution into a sample tank filled with glass beads, so that the glass beads and the microbial sample solution to be broken are fully mixed; the diameter of the glass beads is 150-500um; the total volume of the glass beads accounts for 10-70% of the whole sample tank volume;
(2) Applying ultrasonic waves to a to-be-broken microorganism sample solution in a sample tank by adopting an ultrasonic transducer, and breaking the wall of microorganisms under the combined action of the ultrasonic waves and glass beads; the voltage at the two ends of the ultrasonic transducer is 10-200V; the ultrasonic time is 1-60min;
(3) And separating out glass beads after wall breaking is completed.
2. The method for breaking cellular wall of microorganism based on combination of ultrasound and grinding according to claim 1, wherein the microbial sample to be broken is saliva, blood or tissue fluid containing bacteria, cells and/or viruses.
3. The method for breaking cellular wall by microorganism based on combination of ultrasonic and grinding according to claim 1, wherein the diameter of the glass beads is 150-250um.
4. A method of breaking cellular walls by micro-organisms based on a combination of ultrasound and grinding according to claim 1 or 3, characterized in that the total volume of the glass beads is 30-50% of the whole sample cell volume.
5. The method for breaking cellular walls by microorganisms based on the combination of ultrasound and grinding according to claim 1 or 3, wherein the glass beads are acid-washed glass beads.
6. The method for breaking cellular walls of microorganisms based on the combination of ultrasound and grinding according to claim 1, characterized in that an ultrasonic transducer is in contact with the bottom wall of the sample cell to apply ultrasonic waves to the solution of the microbial sample to be broken; the size of the ultrasonic transducer is not smaller than the bottom wall of the sample tank.
7. The method for breaking cellular wall of microorganism based on the combination of ultrasound and grinding according to claim 1 or 6, wherein the ultrasonic transducer is a monolithic piezoelectric ceramic; the thickness of the ultrasonic transducer is 5-30mm.
8. The method for breaking cellular wall of microorganism based on combination of ultrasound and grinding according to claim 1 or 6, wherein the voltage across the ultrasound transducer is 100-200V.
9. The method for breaking cellular wall of microorganism based on the combination of ultrasonic and grinding according to claim 1, wherein in the step (2), the frequency of ultrasonic wave is 20-200kHz.
10. The method for breaking cellular wall of microorganism based on combination of ultrasonic and grinding according to claim 1, wherein in the step (2), the ultrasonic time is 1-30min.
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