Precise enzyme digestion method for biological cells

Abstract

The invention provides a precise enzyme digestion method for biological cells, and relates to the technical field of biology. According to the method, various biological cells such as animal and plant cells and food crop cells are subjected to efficient and accurate enzyme digestion through the synergistic effect of an ultrasonic microfluid device and an intelligent enzyme combination system. The ultrasonic microfluid device utilizes mechanical oscillation to increase cell membrane permeability, and the intelligent enzyme combination system screens specific enzymes according to cell types to realize efficient cutting. Meanwhile, the online monitoring system monitors, feeds back and controls the enzyme digestion process in real time, so that the optimal reaction condition is ensured. The method is mild and accurate in operation, can efficiently extract high-purity products such as small molecule peptides, is suitable for multiple fields, can remarkably improve the extraction efficiency and the product quality, reduces damage to cells, and provides a new way for biological product production.

Description

Technical Field

[0001] The present invention relates to the technical field of biological cell technology, and specifically provides a precise enzymatic digestion method for biological cells. Background Art

[0002] In the field of biotechnology, extracting specific components from biological cells is a key technology, which has wide applications in industries such as food, medicine, and cosmetics. In the prior art, for the treatment of animal and plant cells, food crop cells, etc., traditional enzymatic digestion methods are usually adopted, or the cell structure is destroyed by physical and chemical means to release the target components. Although these traditional methods can achieve the extraction of cell contents to a certain extent, there are still many defects and deficiencies.

[0003] In the prior art, common extraction methods include physical disruption methods (such as grinding, ultrasonic disruption, etc.), chemical solvent extraction methods, and traditional enzymatic digestion methods. Although physical disruption methods can effectively destroy the cell wall and cell membrane structures, they often introduce large mechanical damages, resulting in the destruction and loss of useful components inside the cells, and at the same time, a large amount of impurities are introduced, increasing the difficulty of subsequent purification. Chemical solvent extraction methods have the problem of solvent residue, which may not only affect the purity and safety of the extract, but also cause environmental pollution. Traditional enzymatic digestion methods, although having certain specificity and mildness, when dealing with cells with cell walls, usually need to remove the cell wall first to obtain protoplasts and then perform enzymatic digestion, which not only increases the operation steps and costs, but also may lead to the loss and contamination of cell contents. In addition, traditional enzymatic digestion methods are difficult to achieve precise control of the enzymatic digestion process, and it is easy to occur incomplete enzymatic digestion or over-enzymatic digestion, thus affecting the yield and quality of the target product.

[0004] In summary, the prior art has problems such as complex operation, low extraction efficiency, low product purity, and large cell damage when extracting specific components from biological cells. Therefore, a method that can efficiently, precisely, and mildly extract specific components from biological cells is needed to meet the requirements of various industries for high-quality biological products. The present invention aims to provide a precise enzymatic digestion method for biological cells, and solve the above problems in the prior art through innovative technical means, realizing efficient and precise enzymatic digestion of biological cells and improving the extraction efficiency and quality of the target product. Summary of the Invention

[0005] (1) Technical Problems to be Solved

[0006] In view of the deficiencies of the prior art, the present invention provides a precise enzymatic digestion method for biological cells, which solves the problems raised in the above background art.

[0007] (2) Technical Solutions

[0008] To achieve the above object, the present invention is realized by the following technical solutions: A precise enzymatic digestion method for biological cells, comprising the following steps:

[0009] S1. Suspend the biological cells to be processed in an appropriate buffer solution, remove the supernatant by centrifugation, and then repeatedly wash the cells several times with sterile physiological saline or a specific buffer solution to remove dust, microorganisms, serum components, and other impurities that may interfere with the enzymatic digestion reaction on the cell surface, and re-suspend the washed cells in a suitable buffer solution, adjusting the cell density to a suitable range to prepare for the subsequent enzymatic digestion reaction;

[0010] S2. Mix the washed biological cells with a specific biological enzyme to form an enzymatic digestion reaction system;

[0011] S3. Introduce the enzymatic digestion reaction system into an ultrasonic microfluidic device, use the mechanical oscillation of ultrasonic waves to increase the cell membrane permeability, and at the same time precisely control the reaction fluid flow rate and path through the microfluidic device;

[0012] S4. According to the cell type and target product, screen specific enzymes from the enzyme database through an intelligent enzyme combination system, and predict the synergistic effect through an AI model to determine the optimal enzyme combination and dosage;

[0013] S5. Use an on-line monitoring system to monitor the enzymatic digestion process in real time, and feed the data back to the control system to automatically adjust the enzyme concentration, temperature, and time parameters;

[0014] S6. Separate and purify the digested mixture to obtain the target product, including but not limited to small molecule peptides.

[0015] Preferably, in S3, the ultrasonic frequency of the ultrasonic microfluidic device is 5 - 10 MHz, the power is 20 - 50 W, the size of the microfluidic channel is 100 - 1000 μm, and the flow rate is 1 - 10 μL / min.

[0016] Preferably, in S4, the intelligent enzyme combination system includes an enzyme database and an AI model. The enzyme database covers the characteristics and action mechanisms of various biological enzymes, and the AI model is based on machine learning algorithms and is used to predict the synergistic effect of enzymes and determine the optimal combination and dosage.

[0017] Preferably, in S5, the on-line monitoring system includes a fluorescent probe and a mass spectrometer. The fluorescent probe is used to label cell structures and components, and the mass spectrometer is used to analyze the types and quantities of the released digested products in real time.

[0018] Preferably, the biological cells include animal cells and plant cells, and the target products include intracellular essence components such as small molecule peptides, polysaccharides, and alkaloids.

[0019] Preferably, the temperature of the enzymatic digestion reaction is 25 - 37 °C, and the time is 2 min - 18 h. The specific conditions depend on the cell type and the selected enzyme.

[0020] Preferably, the separation and purification method is one or more of centrifugation, filtration, and ultrafiltration.

[0021] Preferably, the obtained small molecule peptides can be used in the fields of food, medicine, and cosmetics.

[0022] (III) Beneficial Effects

[0023] The present invention provides a precise enzymatic digestion method for biological cells, having the following beneficial effects:

[0024] 1. Through the synergistic effect of the ultrasonic microfluidic device and the intelligent enzyme combination system, the present invention realizes the efficient and precise enzymatic digestion of biological cells. The mechanical oscillation of ultrasonic waves can increase the permeability of the cell membrane, promoting the rapid penetration of enzymes into the cell interior, while the intelligent enzyme combination system selects the best specific enzyme combination based on the cell type and the target product, ensuring that the enzymatic digestion reaction has high specificity and efficiency. Compared with traditional methods, the enzymatic digestion efficiency of the present invention can be increased by several times, significantly shortening the extraction time, thereby greatly improving the production efficiency and meeting the requirements of large-scale industrial production.

[0025] 2. With the help of the online monitoring system and the real-time feedback control mechanism, the present invention can carry out refined control of the enzymatic digestion process. The combined use of fluorescence probes and mass spectrometers can monitor the changes in cell structure and product generation in real time. After the data is fed back to the control system, the key parameters such as enzyme concentration, temperature, and time can be automatically and precisely adjusted to ensure that the enzymatic digestion reaction is always in the optimal state, avoiding the generation of impurities caused by over-enzymatic digestion or incomplete enzymatic digestion.

[0026] 3. The present invention is applicable to various biological cell types such as animal and plant cells and food crop cells, having wide applicability. At the same time, the entire enzymatic digestion process is carried out under relatively mild conditions, with the temperature controlled at 25 - 37 °C, without the need for extreme conditions such as high temperature, high pressure, or strong acids and bases. It can not only minimize the damage to the useful components of cells to the greatest extent but also reduce energy consumption and cost investment, realizing a green and efficient biological cell extraction process and providing strong technical support for the sustainable development of biological products. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a process schematic diagram of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts belong to the scope of protection of the present invention.

[0029] Embodiment 1:

[0030] As Figure 1 shown, an embodiment of the present invention provides a precise enzymatic digestion method for biological cells, including the following steps:

[0031] S1. Preparation work: Take mouse fibroblasts, suspend them in phosphate buffer solution (PBS), centrifuge (1000 rpm, 5 min) to remove the supernatant, wash repeatedly with sterile PBS 3 times to remove serum and impurities, and then resuspend the cells in the reaction buffer, adjusting the cell density to 1×10 6 cells / mL.

[0032] S2. Enzyme combination selection: Select a combination of trypsin and peptidase. Trypsin preliminarily hydrolyzes large protein molecules, and peptidase further cuts them into small peptides.

[0033] S3. Mix the cells with the enzyme, place them in a 37°C water bath shaker, and incubate with shaking at 150 rpm. After 30 min, introduce the mixture into an ultrasonic microfluidic device, set the ultrasonic frequency to 5 MHz, the power to 30 W, the microfluidic channel size to 200 μm, and the flow rate to 5 μL / min, and continuously process for 1 h.

[0034] S4. Termination and purification: Add a trypsin inhibitor to terminate the reaction, then centrifuge (1500 rpm, 10 min) to collect the supernatant, and then ultrafilter (cut-off molecular weight 3 kDa) to remove unreacted enzymes and small molecule impurities to obtain small peptides.

[0035] S5. After enzymatic digestion, use high performance liquid chromatography (HPLC) to detect the content of small peptides, and find that the content reaches 2.3 mg / mL and the purity reaches 96%. The cell viability assay shows that after treatment with the ultrasonic microfluidic device, the loss of cell activity is less than 5%, proving that this method causes little damage to cells.

[0036] Embodiment 2:

[0037] An embodiment of the present invention provides a precise enzymatic digestion method for biological cells, including the following steps:

[0038] S1. Preparation work: Pick Arabidopsis thaliana leaves and cut them into 0.5 cm 2Small pieces were soaked and washed with PBS to remove surface dust and microorganisms. The leaf tissues were placed in a mortar, a small amount of quartz sand and PBS were added, and gently ground to release cells. The ground slurry was filtered, and the filtrate was centrifuged (1500 rpm, 10 min) to collect cells. After washing twice with PBS, the cells were resuspended in the reaction buffer, and the cell density was adjusted to 2×10 6 cells / mL.

[0039] S2. Use a combination of cellulase and pectinase. These two enzymes can effectively decompose the plant cell wall and release the intracellular contents.

[0040] S3. Enzymatic digestion reaction. The pretreated cells were incubated with the enzyme mixture at 25°C and 100 rpm to initiate the enzymatic digestion reaction. After 1 hour of reaction, the mixture was transferred to an ultrasonic microfluidic device. The ultrasonic frequency was set to 8 MHz, the power was 40 W, the size of the microfluidic channel was 300 μm, and the flow rate was 8 μL / min, and the treatment was continued for 2 hours.

[0041] S4. Termination and purification. After the enzymatic digestion reaction was completed, an appropriate amount of enzyme inhibitor was added to terminate the reaction process. Subsequently, the reaction mixture was centrifuged (3000 rpm, 15 min), the supernatant was taken, and the product was further purified by ultrafiltration technology (cut-off molecular weight 5 kDa) to obtain the required small molecule peptides.

[0042] S5. After detection, the yield of small molecule peptides after enzymatic digestion was 1.8 mg / mL, and the purity was as high as 94%. At the same time, it was observed that the decomposition of the cell wall was relatively complete and the contents were released sufficiently, indicating that this enzymatic digestion method can effectively extract the essence components in plant cells.

[0043] Example 3:

[0044] The embodiment of the present invention provides a precise enzymatic digestion method for biological cells, including the following steps:

[0045] S1. Preparation work. Wheat grains were selected, and the endosperm part was crushed after dehulling. The crushed endosperm powder was soaked in PBS solution for 2 hours to allow the cells to fully absorb water and swell. The soaking solution was filtered, the cells were collected, washed 3 times with PBS to remove large particle impurities such as starch. The washed cells were suspended in the reaction buffer, and the cell density was adjusted to 3×10 6 cells / mL.

[0046] S2. Enzyme combination selection. A combination of hemicellulase and protease was selected. Hemicellulase can decompose the hemicellulose component in the cell wall, while protease is used to decompose proteins to facilitate the subsequent extraction of small molecule peptides.

[0047] S3. Enzymatic digestion reaction: The cell and enzyme mixture is incubated in a water bath shaker at 30 °C and 80 rpm to initiate enzymatic digestion. After the reaction proceeds for 2 hours, the mixture is introduced into an ultrasonic microfluidic device. Set the ultrasonic frequency at 6 MHz, the power at 35 W, the microfluidic channel size at 250 μm, and the flow rate at 6 μL / min, and continuously process for 1.5 hours.

[0048] S4. Termination and purification: Add the corresponding enzyme inhibitor to terminate the reaction. Centrifuge (2500 rpm, 20 min) to collect the supernatant, and use ultrafiltration (cut-off molecular weight 4 kDa) to remove impurities to obtain a product rich in small molecule peptides.

[0049] The content of small molecule peptides measured after enzymatic digestion is 2.7 mg / mL, and the purity reaches 95%. The results show that the method of the present invention can be effectively applied to the extraction of small molecule peptides from food cells, can fully decompose the cell wall, release the nutrients inside the cells, and has high extraction efficiency and good product purity, providing a new technical means for the deep processing of food.

[0050] In order to more intuitively display the application effects of the precise enzymatic digestion method for biological cells of the present invention in different cell types, the key experimental data in three examples are summarized and compared as shown in the following table:

[0051]

[0052] The above three examples fully demonstrate the high efficiency, precision and wide applicability of the method of the present invention in the enzymatic digestion treatment of animal cells, plant cells and food cells. By reasonably selecting the enzyme combination and optimizing the parameters of the ultrasonic microfluidic device, target products such as small molecule peptides with high purity are successfully extracted. The experimental data show that the present invention can not only effectively improve the enzymatic digestion efficiency and product purity, but also minimize the damage to cells to the greatest extent, reduce the extraction cost, and create higher economic benefits for enterprises. At the same time, the low-temperature and mild reaction conditions of this method are also beneficial to maintaining the biological activity of the extract, further expanding its application prospects in the fields of food, medicine, cosmetics, etc.

[0053] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A precise enzymatic cleavage method for biological cells, characterized in that, It includes the following steps: S1. Suspend the biological cells to be processed in an appropriate buffer solution, remove the supernatant by centrifugation, then wash the cells repeatedly several times with sterile physiological saline or a specific buffer solution, and re-suspend the washed cells in a suitable buffer solution, adjusting the cell density to a suitable range; S2. Mix the washed biological cells with a specific biological enzyme to form an enzymatic digestion reaction system; S3. Introduce the enzymatic digestion reaction system into an ultrasonic microfluidic device, use the mechanical oscillation of ultrasonic waves to increase the cell membrane permeability, and at the same time precisely control the reaction fluid flow rate and path through the microfluidic device; S4. According to the cell type and target product, screen specific enzymes from the enzyme database through an intelligent enzyme combination system, and predict the synergistic effect through an AI model to determine the optimal enzyme combination and dosage; S5. Use an on-line monitoring system to monitor the enzymatic digestion process in real time, and feed the data back to the control system to automatically adjust parameters such as enzyme concentration, temperature and time; S6. Separate and purify the mixture after enzymatic digestion to obtain the target product, including but not limited to small molecule peptides.

2. The precise enzymatic cleavage method for biological cells according to claim 1, wherein: In S3, the ultrasonic frequency of the ultrasonic microfluidic device is 5 - 10 MHz, the power is 20 - 50 W, the size of the microfluidic channel is 100 - 1000 μm, and the flow rate is 1 - 10 μL / min.

3. The precise enzymatic cleavage method for biological cells according to claim 1, characterized in that: In S4, the intelligent enzyme combination system includes an enzyme database and an AI model. The enzyme database covers the characteristics and action mechanisms of various biological enzymes, and the AI model is based on machine learning algorithms and is used to predict the synergistic effect of enzymes and determine the optimal combination and dosage.

4. The precise enzymatic cleavage method for biological cells according to claim 1, wherein: In S5, the on-line monitoring system includes a fluorescence probe and a mass spectrometer. The fluorescence probe is used to label cell structures and components, and the mass spectrometer is used to analyze the types and quantities of the released enzymatic digestion products in real time.

5. A precise enzymatic cleavage method for biological cells according to claim 1, wherein: The biological cells include animal cells and plant cells, and the target products include but not limited to small molecule peptides, polysaccharides, and alkaloids.

6. The precise enzymatic cleavage method for biological cells according to claim 1, characterized in that: The temperature of the enzymatic digestion reaction is 25 - 37 °C, and the time is 2 min - 18 h.

7. A precise enzymatic cleavage method for biological cells according to claim 1, characterized in that: The separation and purification method is one or more of centrifugation, filtration, and ultrafiltration.

8. A precise enzymatic cleavage method for biological cells according to claim 1, characterized in that: The obtained small molecule peptides can be used in the fields of food, medicine, and cosmetics.

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