A method for disrupting microalgal cells

Abstract

This invention discloses a method for disrupting microalgal cells. The method involves first grinding the algal solution containing microalgae with liquid nitrogen for 9-12 minutes, followed by 4-6 freeze-thaw cycles to obtain disrupted microalgal cells. The specific steps are as follows: (1) After centrifuging the algal solution containing microalgae, the supernatant is removed, and 0.01 M PBS phosphate buffer solution is added for resuscitation, then transferred to a mortar; (2) Liquid nitrogen is added to the mortar to freeze the algal solution, and then grinding is performed for 9-12 minutes using a grinding rod cooled with liquid nitrogen; (3) The ground algal solution is transferred to a centrifuge tube, and after being brought to a final volume with PBS phosphate buffer solution, it undergoes 4-6 freeze-thaw cycles. Finally, the frozen and thawed algal solution is stored in a -20°C freezer and refrigerated in the dark for 24 hours. After being taken out, it is allowed to cool naturally to obtain disrupted microalgal cells. This disruption method can efficiently disrupt microalgal cells without affecting the active substances within the cells.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and specifically relates to a method for disrupting microalgal cells. Background Technology

[0002] Microalgae are a type of microorganism widely distributed on Earth that can efficiently perform photosynthesis. They are characterized by their small size, rapid reproduction, strong environmental adaptability, and high biomass yield. Microalgae contain abundant high-value-added substances such as proteins, unsaturated fatty acids, natural pigments, and polysaccharides, and have broad development prospects in the fields of food, cosmetics, bioenergy, and medicine. However, microalgae usually have a robust cell wall, mainly composed of cellulose, pectin, xylan, and other substances, which is difficult to break down. As a protective layer for the cell, the cell wall effectively prevents the release of intracellular substances, thus hindering the extraction and research of intracellular active substances from microalgae. Therefore, effectively disrupting the cell is a key aspect of microalgae research.

[0003] Currently, methods for disrupting microbial cell walls include physical, chemical, and biological methods. Physical methods include bead milling, ultrasonic disruption, pulsed electric field, repeated freeze-thaw cycles, and liquid nitrogen grinding. These methods can generally achieve a disruption rate of over 70% for large algae particles, but are often less effective for small-sized microalgae with thick cell walls. Chemical methods include acid-heat methods and ionic liquid disruption methods, achieving a disruption rate of approximately 60%. However, chemical methods generate heat during cell disruption, which can affect the activity of intracellular substances. Furthermore, chemical reagents can contaminate intracellular components, affecting their application. Biological methods include enzymatic dissolution, which is gentle but time-consuming and inefficient. Summary of the Invention

[0004] In view of the above-mentioned shortcomings of the existing technology, the purpose of this invention is to provide a method for breaking microalgae cells, which can efficiently break microalgae cells without affecting the active substances inside the cells.

[0005] The technical solution of this invention is implemented as follows:

[0006] A method for disrupting microalgal cells involves first grinding the algal solution containing microalgae with liquid nitrogen for 9–12 minutes, followed by 4–6 freeze-thaw cycles to obtain the disrupted microalgal cells.

[0007] Furthermore, the specific steps are as follows:

[0008] (1) After centrifuging the algal solution containing microalgae, remove the supernatant, add 0.01M PBS phosphate buffer solution to suspend it, and then transfer it to a mortar.

[0009] (2) Add liquid nitrogen to the mortar to freeze the algal solution, and then grind it for 9 to 12 minutes using a grinding rod cooled by liquid nitrogen;

[0010] (3) Transfer the ground algal solution to a centrifuge tube, and after adjusting the volume with PBS phosphate buffer solution, perform 4 to 6 freeze-thaw cycles. Finally, store the frozen and thawed algal solution in a -20°C freezer and refrigerate in the dark for 24 hours. After taking it out, let it cool naturally to obtain the broken microalgal cells.

[0011] Furthermore, the microalgal cells in the algal solution had a particle size of 3–7 μm and a cell density of approximately 45.1 × 10⁻⁶. 5 cells / mL.

[0012] Furthermore, the algal solution was Microcystis aeruginosa cultured in BG11 medium.

[0013] Furthermore, the grinding time in step (2) is 10 minutes.

[0014] Furthermore, step (3) involves 5 freeze-thaw cycles.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] 1. Microalgae cell walls are relatively thick. Although liquid nitrogen grinding can break the cell walls, prolonged grinding time can damage intracellular substances. This invention first uses liquid nitrogen grinding to break the cell walls and controls the grinding time to 9-12 minutes to ensure the breakage rate while effectively avoiding damage to the intracellular substances of microalgae. Then, repeated freeze-thaw cycles are used to further lyse the cells, allowing the intracellular substances to dissolve automatically, thereby effectively ensuring the integrity of the intracellular structure.

[0017] 2. This invention can effectively improve the cell breakage rate of microalgae cells, which can reach 60%, and can effectively shorten the breakage time, improve efficiency, and will not affect the active substances in the cells. Attached Figure Description

[0018] Figure 1 -Comparison of cell structure of Example 1 and microalgal cells under a microscope

[0019] Figure 2 - Comparative images of cell structures under a microscope for Examples 2-8 and Comparative Examples 1-6.

[0020] Figure 3 - Results of phycobiliprotein content determination in Example 1 and Comparative Examples 1-6. Detailed Implementation

[0021] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0022] The following examples and comparative examples all used *Microcystis aeruginosa* (FACHB-905), which was purchased from the Freshwater Algae Culture Bank of the Institute of Hydrobiology, Chinese Academy of Sciences. BG11 culture medium was purchased from Haibo Biotechnology Co., Ltd. The algae were inoculated into BG11 culture medium according to the specified ratio and cultured in an intelligent incubator (Ningbo Southeast Instrument Co., Ltd., GXZ+PGX) at 25℃. The light conditions were 2500 lx, and the light-dark ratio was 12 h:12 h. Experiments were conducted when the algae reached the logarithmic growth phase.

[0023] Example 1

[0024] A method for disrupting microalgal cells, specifically including the following steps:

[0025] (1) Take 4 mL of algal solution in the logarithmic growth phase into a 10 mL centrifuge tube, centrifuge at 4℃ and 10000 rpm for 10 min, remove the supernatant, and add 1 mL of 0.01 M PBS phosphate buffer solution for resuspending.

[0026] (2) Then transfer to a mortar and grind with liquid nitrogen for 10 min;

[0027] (3) The ground algal solution was brought to a final volume of 4 mL with PBS phosphate buffer solution and then placed in liquid nitrogen for freeze-thaw. After about 30 seconds, the algal solution was taken out and thawed in running water. This process was repeated 5 times.

[0028] (4) Place the freeze-thawed algal solution in a -20℃ refrigerator in the dark for 24 hours;

[0029] (5) The cell disruption rate was determined by placing 0.1 mL of algal solution in a phytoplankton counting frame and observing it under a 20×10x electron microscope (BX53F, OLYMPLUS). Images were taken using the Wanshen AlgaeC plankton identification and counting software, and intact cells were counted. Cell disruption rate. Use the following formula:

[0030]

[0031] Where: N i —Number of cells before fragmentation;

[0032] N f —Number of cells after breakage.

[0033] The experiment was conducted with three parallel samples, and the results were averaged.

[0034] Example 2

[0035] Same as Example 1, except that the grinding time in this example is 5 minutes.

[0036] Example 3

[0037] Same as Example 1, except that the grinding time in this example is 8 minutes.

[0038] Example 4

[0039] Same as Example 1, except that the grinding time in this example is 9 minutes.

[0040] Example 5

[0041] Same as Example 1, except that the grinding time in this example is 11 minutes.

[0042] Example 6

[0043] Similar to Example 1, except that the freeze-thaw cycle in this example is repeated 3 times.

[0044] Example 7

[0045] Similar to Example 1, except that the freeze-thaw cycle in this example is repeated 4 times.

[0046] Example 8

[0047] Similar to Example 1, except that the freeze-thaw cycle in this example is repeated 6 times.

[0048] Comparative Example 1

[0049] The following are the specific steps involved in disrupting microalgal cells using an ultrasonic disruptor:

[0050] (1) Take 4 mL of algal solution during the logarithmic growth phase into a 10 mL centrifuge tube and place the centrifuge tube into a beaker containing ice for an ice bath.

[0051] (2) Place the algal liquid into an ultrasonic disruptor for disruption. The disruption conditions are: 200W, ultrasonic for 3 seconds, intermittent for 2 seconds, for a total of 20 minutes.

[0052] (3) The obtained cells are subjected to a cell breakage rate test, and the steps are the same as step (5) in Example 1.

[0053] Comparative Example 2

[0054] The microalgal cells were disrupted using a repeated freeze-thaw cycle. The specific steps included the following:

[0055] (1) Take 4 mL of algal solution during the logarithmic growth phase into a 10 mL centrifuge tube, centrifuge at 4℃ and 10000 rpm for 10 min, and remove the supernatant.

[0056] (2) The centrifuged algal solution was brought to a final volume of 4 mL with PBS phosphate buffer solution and then placed in liquid nitrogen for freeze-thaw. After about 30 seconds, the algal solution was taken out and thawed in running water. This process was repeated 5 times.

[0057] (3) The obtained cells are subjected to a cell breakage rate test, and the steps are the same as step (5) in Example 1.

[0058] Comparative Example 3

[0059] Microalgal cells were disrupted using a combination of repeated freeze-thaw cycles and ultrasonic disruption. The specific steps included the following:

[0060] (1) Take 4 mL of algal solution during the logarithmic growth phase into a 10 mL centrifuge tube, centrifuge at 4℃ and 10000 rpm for 10 min, and remove the supernatant.

[0061] (2) The centrifuged algal solution was brought to a final volume of 4 mL with PBS phosphate buffer solution and then placed in liquid nitrogen for freeze-thaw. After about 30 seconds, the algal solution was taken out and thawed in running water. This process was repeated 5 times.

[0062] (3) The algal solution was then subjected to ultrasonic disruption under the following conditions: 200W, 3s of ultrasonic stimulation, 2s of intermittent stimulation, for a total of 20min.

[0063] (4) The obtained cells are subjected to a cell breakage rate test, and the steps are the same as those in step (5) of Example 1.

[0064] Comparative Example 4

[0065] The microalgal cells were disrupted using liquid nitrogen grinding, and the specific steps included the following:

[0066] (1) Take 4 mL of algal solution during the logarithmic growth phase into a 10 mL centrifuge tube, centrifuge at 4℃ and 10000 rpm for 10 min, and remove the supernatant.

[0067] (2) After centrifugation, the algal solution was brought to a volume of 1 mL with PBS phosphate buffer solution to resuspend the cells. Then, it was placed in a mortar and ground. Before grinding, an appropriate amount of liquid nitrogen was added to the algal solution to prevent the algal cell material from being damaged by excessive temperature. The grinding time was 10 min. After grinding, the solution was transferred to a centrifuge tube. Finally, the container was washed with PBS phosphate buffer solution and the volume of algal solution was brought to 4 mL.

[0068] (3) The obtained cells are subjected to a cell breakage rate test, and the steps are the same as step (5) in Example 1.

[0069] Comparative Example 5

[0070] Microalgae were broken down using a combination of repeated freeze-thaw cycles and grinding. The specific steps included the following:

[0071] (1) Take 4 mL of algal solution during the logarithmic growth phase into a 10 mL centrifuge tube, centrifuge at 4℃ and 10000 rpm for 10 min, and remove the supernatant.

[0072] (2) The centrifuged algal solution was brought to a final volume of 4 mL with PBS phosphate buffer solution and then placed in liquid nitrogen for freeze-thaw. After about 30 seconds, the algal solution was taken out and thawed in running water. This process was repeated 5 times.

[0073] (3) After thawing the algal solution in (2), put it into a mortar and grind it for 10 minutes. After grinding, transfer it to a centrifuge tube, and finally wash the container with PBS phosphate buffer and bring the volume of algal solution to 4 mL.

[0074] (4) The obtained cells are subjected to a cell breakage rate test, and the steps are the same as those in step (5) of Example 1.

[0075] Comparative Example 6

[0076] The microalgal cells were disrupted using a combination of grinding and ultrasonic disruption methods. The specific steps included the following:

[0077] (1) Take 4 mL of algal solution during the logarithmic growth phase into a 10 mL centrifuge tube, centrifuge at 4℃ and 10000 rpm for 10 min, and remove the supernatant.

[0078] (2) After centrifugation, the algal solution was brought to a volume of 1 mL with PBS phosphate buffer solution to resuspend the cells. Then, it was placed in a mortar and ground. Before grinding, an appropriate amount of liquid nitrogen was added to the algal solution to prevent the algal cell material from being damaged by excessive temperature. The grinding time was 10 min. After grinding, the solution was transferred to a centrifuge tube. Finally, the container was washed with PBS phosphate buffer solution and the volume of algal solution was brought to 4 mL.

[0079] (3) The algal solution was then subjected to ultrasonic disruption under the following conditions: 200W, 3s of ultrasonic stimulation, 2s of intermittent stimulation, for a total of 20min.

[0080] (4) The obtained cells are subjected to a cell breakage rate test, and the steps are the same as those in step (5) of Example 1.

[0081] 1. Comparison of cell structure between Example 1 and microalgal cells under a microscope is shown in the figure below. Figure 1 As shown, Figure 1 (a) shows the cell structure before it breaks down. Figure 1 (b) is a diagram of the cell structure after disruption in Example 1. Comparative microscopic images of Examples 2-8 and Comparative Examples 1-6 are shown below. Figure 2 As shown,; Figure 2 (c) to (o) are cell structure diagrams after disruption in Examples 2 to 8 and Comparative Examples 1 to 6, respectively.

[0082] 2. The detection results of microalgal cell fragmentation rate in Examples 1-8 and Comparative Examples 1-6 are shown in Table 1.

[0083] Table 1 Microalgal cell fragmentation rate

[0084]

[0085] As shown in Table 1: (1) The breakage rates of ultrasonic breakage, repeated freeze-thaw, and liquid nitrogen grinding were 37.0±3.1, 27.8±2.2, and 18.6±2.8%, respectively. The breakage rate of the combination of liquid nitrogen grinding and repeated freeze-thaw could reach 60.5±1.1%, which was 23.5%, 32.7%, and 41.9% higher than that of ultrasonic breakage, repeated freeze-thaw, and liquid nitrogen grinding, respectively. The combination of ultrasonic and repeated freeze-thaw could increase the breakage rate to 48.1±3.1%, but the breakage rate was still lower than that of grinding and repeated freeze-thaw.

[0086] (2) As can be seen from Examples 1 to 5, as the grinding time increases, the increase in the breakage rate begins to increase slowly. When the grinding time is 10 min, the increase is larger, and then it increases slowly again. When the grinding time is 10 min, a high breakage rate and working efficiency can be guaranteed.

[0087] (3) As can be seen from Examples 1 and 6-8, as the number of repeated freeze-thaw cycles increases, the increase in breakage rate begins to increase slowly. When the number of repeated freeze-thaw cycles is 5, the increase is larger, and then it increases slowly again. When the number of repeated freeze-thaw cycles is 5, a high breakage rate and working efficiency can be guaranteed.

[0088] 3. Phycobiliproteins are important photosynthetic pigments in microalgal cells, composed of phycobilins and apoproteins. As natural pigments, they can be used in cosmetics, dyes, and medical industries. The extraction amount of phycobiliproteins can indirectly reflect the cell disruption rate; therefore, the phycobiliprotein content of microalgal cells disrupted by the above methods was determined. The specific steps included: 4 mL of disrupted microalgal cells from Example 1 and Comparative Examples 1 to 6 were transferred to 10 mL centrifuge tubes and centrifuged at 10,000 rpm for 10 min at 4°C. The absorbance of the supernatant at 562 nm, 620 nm, and 652 nm was measured using a UV-Vis spectrophotometer (TU-1950, Beijing Purkinje). The concentrations of phycoerythrin (PE), phycocyanin (PC), and allophycocyanin (APC) were calculated using the following formula:

[0089]

[0090]

[0091]

[0092] The results of phycobiliprotein content determination after algal cell disruption using different disruption methods are as follows: Figure 3 The results of the ultrasound group (Comparative Example 1), freeze-thaw + ultrasound group (Comparative Example 3), and ultrasound + grinding group (Comparative Example 6) were the lowest, followed by the freeze-thaw + grinding group (Comparative Example 5). The grinding + freeze-thaw group (Example 1) had the highest content, with PE, PC, and APC concentrations of 14.9±0.3, 23.1±0.5, and 27±0.6 μg / L, respectively. Compared with the ultrasound group (Comparative Example 1), freeze-thaw group (Comparative Example 2), freeze-thaw + ultrasound group (Comparative Example 3), grinding group (Comparative Example 4), and freeze-thaw + grinding group (Comparative Example 5), the total phycobiliprotein content extracted by the grinding + freeze-thaw group used in this invention was increased by 39.7, 13.8, 39.7, 18.1, and 20.1 μg / L, respectively, significantly improving the extraction yield of phycobiliproteins.

[0093] In summary, the results of the algal cell disruption rate and the determination of phycobiliprotein content after disruption showed that grinding and repeated freeze-thaw cycles alone resulted in a low disruption rate of microalgal cells, thus affecting the extraction of intracellular substances. While ultrasound and the combination of ultrasound and freeze-thaw cycles achieved higher disruption rates, the large amount of energy generated during ultrasound disruption damaged the intracellular structure, affecting the extraction and analysis of substances. Although repeated freeze-thaw cycles followed by grinding also resulted in a relatively high disruption rate, the extraction of phycobiliproteins was relatively low. This may be because repeated freeze-thaw cycles had already partially lysed the cells, and further grinding would damage intracellular substances, thus affecting the extraction of intracellular substances. The grinding + repeated freeze-thaw cycle method of this invention can achieve a high disruption rate of microalgal cells and has a higher extraction rate of intracellular substances than other traditional methods.

[0094] Finally, it should be noted that the above embodiments of the present invention are merely illustrative examples and not intended to limit the implementation of the invention. Those skilled in the art can make other variations and modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A method for disrupting microalgal cells, characterized in that, First, the algal solution containing microalgae is ground with liquid nitrogen for 10-12 minutes, followed by 5-6 freeze-thaw cycles to obtain fragmented microalgal cells. The microalgae is Microcystis aeruginosa. The specific steps are as follows: (1) After centrifuging the algal solution containing microalgae, remove the supernatant, add 0.01 M PBS phosphate buffer solution to suspend it, and then transfer it to a mortar; (2) Add liquid nitrogen to the mortar to freeze the algal solution, and then grind it for 10-12 minutes with a grinding rod cooled by liquid nitrogen to break the cell wall of the microalgae, while avoiding damage to the intracellular substances of the microalgae. (3) Transfer the ground algal solution to a centrifuge tube and make up the volume with PBS phosphate buffer solution. Then, freeze and thaw 5 to 6 times to allow the intracellular substances of the microalgae to dissolve automatically. Finally, store the frozen and thawed algal solution in a refrigerator at -20 ℃ and refrigerate in the dark for 24 h. After taking it out, let it cool naturally to obtain the broken microalgae cells.

2. The method for disrupting microalgal cells according to claim 1, characterized in that, The microalgal cells in the algal solution had a particle size of 3–7 μm and a cell density of 45.1 × 10⁻⁶. 5 cells / mL.

3. The method for disrupting microalgal cells according to claim 1, characterized in that, The grinding time in step (2) is 10 min.

4. The method for disrupting microalgal cells according to claim 1, characterized in that, Step (3) involves 5 freeze-thaw cycles.

Images