Wall breaking process of chlamydomonas reinhardtii

Abstract

The invention relates to the technical field of biology, in particular to a wall breaking process of chlamydomonas reinhardtii. The invention provides a chlamydomonas reinhardtii wall breaking process combining ultrasonic waves and repeated freezing and thawing at 37 DEG C. Tests prove that the protein extraction rate of the method is obviously higher than that of other four wall breaking methods, and the process has an unexpected synergistic effect in protein extraction according to a probability addition method. The whole process is simple to operate, low in cost and higher in extraction efficiency, and has higher industrial application value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the cell wall disruption process of Chlamydomonas reinhardtii. Background Technology

[0002] The rich nutrients and unique bioactive components of microalgae, along with their health benefits and pharmacological properties, have become an active element in new product development, earning them the titles of "superfood" and "food of the future." They have become a hot topic and focus of research in the health-conscious food industry. Currently, microalgae are widely used in baked goods, functional foods, snacks, beverages, yogurt, smoothies, pasta, and vegetable soups.

[0003] Chlamydomonas reinhardtii possesses advantages such as high photosynthetic efficiency, rapid growth, short reproductive cycle, and simple cultivation conditions; it can be cultivated not only through photoautotrophic cultivation but also in fermenters. Chlamydomonas reinhardtii is rich in protein, crude polysaccharides, dietary fiber, and various vitamins and minerals. It is one of the most nutritionally valuable natural plant proteins that have been developed and utilized in recent years.

[0004] Most of the nutrients in microalgae are located inside the cells, while the rigid cell walls on the outer layer of microalgae hinder the extraction of intracellular substances. Various methods are typically used for cell wall disruption pretreatment to promote the release of intracellular compounds. Compared to the cell walls of ordinary microorganisms, the cell walls of microalgae are more tightly structured, making disruption more difficult, and it is challenging to ensure that the substances inside the algae are not damaged during disruption.

[0005] However, existing Chlamydomonas reinhardtii cell wall breaking processes still face technical problems such as complex operation, high cost, and insufficient extraction efficiency. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a cell wall breaking process for Chlamydomonas reinhardtii that is simple to operate, low in cost, and has higher extraction efficiency.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a cell wall disruption process for Chlamydomonas reinhardtii, which is based on repeated freeze-thaw cycles and ultrasonic lysis to disrupt the cell wall of Chlamydomonas reinhardtii algal solution.

[0009] The freeze-thaw cycle includes freezing temperatures of -30℃, -29℃, -28℃, -27℃, -26℃, -25℃, -24℃, -23℃, -22℃, -20℃, -19℃, -18℃, -17℃, -16℃, -15℃, -14℃, -13℃, -12℃, -11℃, or -10℃, and melting temperatures of 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, or 40℃.

[0010] In some specific embodiments of the present invention, the number of repeated freeze-thaw cycles in the above-mentioned cell wall breaking process is 2, 3, 4 or 5.

[0011] In some specific embodiments of the present invention, the ultrasonic pyrolysis power of the above-mentioned cell wall breaking process is 180 W, 190 W, 200 W, 210 W, 220 W, 230 W, 240 W, 250 W, 260 W or 270 W.

[0012] In some specific embodiments of the present invention, the ultrasonic lysis time of the above-mentioned cell wall breaking process is 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min or 40 min.

[0013] In some specific embodiments of the present invention, the material-to-liquid ratio of the Chlamydomonas rhinestones liquid in the above-mentioned cell wall breaking process is 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14 or 1:15.

[0014] In some specific embodiments of the present invention, the above-mentioned cell-wall breaking process includes:

[0015] Chlamydomonas reinhardtii was mixed with a solvent to obtain an algal solution;

[0016] The algal solution is frozen and thawed 2, 3, 4 or 5 times to obtain a freeze-thawed algal solution;

[0017] The frozen-thawed algal solution was lysed by ultrasound to break down the cell walls.

[0018] In some specific embodiments of the present invention, the above-mentioned cell-wall breaking process includes:

[0019] Chlamydomonas reinhardtii was mixed with a solvent to obtain an algal solution;

[0020] The frozen-thawed algal solution was ultrasonically lysed.

[0021] The ultrasonically lysed algal solution is freeze-thawed 2, 3, 4, or 5 times to complete the cell wall disruption.

[0022] In some specific embodiments of the present invention, the freeze-thaw process described above in the cell wall breaking process includes: freezing the algal solution at -30℃, -29℃, -28℃, -27℃, -26℃, -25℃, -24℃, -23℃, -22℃, -20℃, -19℃, -18℃, -17℃, -16℃, -15℃, -14℃, -13℃, -12℃, -11℃ or -10℃, and then thawing the algal solution at 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃ or 40℃.

[0023] In some specific embodiments of the present invention, the above-mentioned cell disruption process further includes a centrifugation step after ultrasonic lysis and before cell disruption is completed.

[0024] In some specific embodiments of the present invention, the ultrasonic pyrolysis power of the above-mentioned cell wall breaking process is 180 W, 190 W, 200 W, 210 W, 220 W, 230 W, 240 W, 250 W, 260 W or 270 W.

[0025] In some specific embodiments of the present invention, the ultrasonic lysis time of the above-mentioned cell wall breaking process is 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min or 40 min.

[0026] In some specific embodiments of the present invention, the material-to-liquid ratio of the Chlamydomonas rhinestones liquid in the above-mentioned cell wall breaking process is 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14 or 1:15.

[0027] This application re-tested the cell wall disruption effects of five different methods on *Chlamydomonas reinhardtii*. The results showed that the combination of ultrasound and repeated freeze-thaw cycles at 37°C yielded the best cell wall disruption effect, with a protein extraction rate of 69.80%. The combination of ultrasound and repeated freeze-thaw cycles at 4°C resulted in a protein extraction rate of 54.09%. Ultrasound alone yielded a protein extraction rate of 41.79%, repeated freeze-thaw cycles at 4°C alone yielded a rate of 26.46%, and repeated freeze-thaw cycles at 37°C alone yielded a rate of 34.31%. Clearly, the combination of ultrasound and repeated freeze-thaw cycles at 37°C resulted in a higher protein extraction rate than the other four methods. Furthermore, based on the probability addition method, it can be determined that the combination of ultrasound and repeated freeze-thaw cycles at 37°C produced an unexpected synergistic effect, making the entire process simpler, lower in cost, and more efficient in extraction, thus possessing high industrial application value. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0029] Figure 1 Show the BSA protein standard curve;

[0030] Figure 2 This demonstrates the effect of ultrasonic power on protein extraction rate.

[0031] Figure 3 This demonstrates the effect of ultrasound time on protein extraction rate.

[0032] Figure 4 The effect of the feed-to-liquid ratio on protein extraction rate;

[0033] Figure 5 This study demonstrates the effect of different cell wall disruption methods on protein extraction rate. Detailed Implementation

[0034] This invention discloses a cell wall disruption process for *Chlamydomonas reinhardtii*. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0035] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0036] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0037] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0038] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and does not constitute a limitation on the scope of the invention. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0039] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0040] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0041] The present invention will be further illustrated below with reference to the embodiments.

[0042] Example

[0043] 1. Materials and Methods

[0044] 1.1 Raw material pretreatment

[0045] The prepared Chlamydomonas rhinestones mud is freeze-dried, and then the freeze-dried mud is ground into algae powder. After thorough grinding, it is placed in a sealed bag and stored in a dry and ventilated place to prevent mold and damage.

[0046] 1.2 Preparation of BCA working solution

[0047] BCA working solution preparation: Mix BCA reagents A and B at a volume ratio of 1:50 to prepare the BCA working solution. Prepare and use immediately.

[0048] 1.3 Preparation of protein standard solutions and plotting of protein standard curves

[0049] 1) Preparation of protein standards: Dissolve the protein standards completely at room temperature. Take 150 μL of 5 mg / mL BSA protein standard solution and add 600 μL of PBS solution to dilute to 750 μL, so that the final concentration is 1.0 mg / mL.

[0050] 2) Take 9 stoppered test tubes. Use a pipette to place the standard protein solution into each stoppered test tube. Then, add an appropriate amount of pure water to each test tube, followed by 2 mL of the prepared BCA working solution. Shake the test tubes to mix thoroughly, and let them stand at 37°C for 30 minutes. Finally, place them in cuvettes and put them in a UV spectrophotometer to measure the absorbance at 562 nm. Record the absorbance values ​​for constructing a standard protein curve (as shown in Table 1). Use the protein curve to calculate the protein content of the samples in the later stages. The protein standard curve is shown in Table 1. Figure 1 .

[0051] Table 1: Determination of protein standard curve

[0052]

[0053] 1.4 Repeated freeze-thaw cell disruption process flow

[0054] Weigh 10 g of *Chlamydomonas reinhardtii* powder using an analytical balance, place it in a beaker, add 100 mL of sterile water, mix the algal solution thoroughly, shake well, and then pour it into a centrifuge tube. Freeze the tube at -20°C for 90 min, then place the centrifuge tube in a preheated water bath, shaking appropriately during the reaction to ensure a more complete reaction. Repeat the process three times. Based on the method used in this experiment to measure the protein content of *Chlamydomonas reinhardtii* dissolved in its internal components, determine the protein content in the algal solution after the reaction.

[0055] 1.5 Single-factor experiment on the extraction rate of proteins from Chlamydomonas reinhardtii by ultrasound

[0056] 1.5.1 Effect of ultrasonic power on the protein extraction rate of Chlamydomonas reinhardtii

[0057] Weigh 10 g of Chlamydomonas reinhardtii algal powder using an analytical balance, place it in a beaker, and add 100 mL of sterile water. Based on the experimental design, use appropriate pulverizing power. After the reaction, centrifuge the algal solution, collect the supernatant, and determine its protein content using the BCA method. Record the data. As shown in Table 2, the experimental power was selected at 180, 210, 240, 270, and 300 W, and the reaction time was 30 min.

[0058] Table 2: Ultrasonic Experiment

[0059]

[0060] 1.5.2 Effect of sonication time on the protein extraction rate of Chlamydomonas reinhardtii

[0061] Weigh 10 g of Chlamydomonas reinhardtii algal powder using an analytical balance, place it in a beaker, and add 100 mL of sterile water. The grinding power was adjusted according to the experimental design. After the reaction, the algal solution was centrifuged, and the supernatant was used to determine the protein content of its contents using the BCA method. The data were recorded. As shown in Table 3, the experimental power was 240 W, and the reaction times were 20, 25, 30, 35, and 40 min.

[0062] Table 3: Ultrasonic Experiment

[0063]

[0064] 1.5.3 Effect of solid-liquid ratio on protein extraction rate from Chlamydomonas reinhardtii

[0065] The reaction was carried out according to Table 4 with material-to-liquid ratios of 1:5, 1:10, 1:15, 1:20, and 1:25 (g / mL). After the reaction was completed, the algal solution was placed in a centrifuge tube and centrifuged at low speed for 5 minutes. After centrifugation, an appropriate amount of supernatant was taken out and the absorbance of protein in the algal solution was determined by BCA method. The data were recorded and the protein content in the algal solution was calculated.

[0066] Table 4: Experiment on the ratio of feed to liquid

[0067]

[0068] 1.6 Effect of combined ultrasound and repeated freeze-thaw cycles on protein extraction rate from Chlamydomonas reinhardtii

[0069] Weigh 10 g of Chlamydomonas reinhardtii algal powder using an analytical balance and place it in a beaker. Add 100 mL of sterile water. Perform the algal fragmentation process according to the designed power and time: reaction power of 210, 240, and 270 W; reaction time of 25, 30, and 35 min; and material-to-liquid ratio of 1:5, 1:10, and 1:15. After thoroughly mixing and shaking the algal solution, freeze it at -20℃ for 90 min. Then, place the centrifuge tube in a preheated water bath, shaking it appropriately during the reaction to ensure complete reaction. Repeat the process three times. After the reaction, centrifuge the algal solution, collect the supernatant, and determine its protein content using the BCA method. Record the data and calculate the protein content after the reaction.

[0070] 1.7 Determination of protein content in samples

[0071] After the reaction, the algal solution with cell wall disruption was centrifuged at 4000 rpm for 5 min using a low-speed centrifuge. The supernatant was collected, and its absorbance was measured using BCA working solution to calculate the protein content. Alternatively, 100 μL of the supernatant was added to 2 mL of BCA solution, mixed well, and incubated at 37℃ for 30 min. The mixture was then poured into a cuvette, and its absorbance was measured at 562 nm using a UV spectrophotometer. The absorbance was recorded to prepare a protein standard curve. This process was repeated three times, using a blank reagent as a control. The absorbance was measured according to the prepared standard curve, and the protein content of the sample was calculated.

[0072] The formula for calculating protein content is as follows:

[0073] Sample protein content (μg / mL) = M × dilution factor

[0074] in:

[0075] A1 represents the absorbance of the protein sample at 562 nm;

[0076] A0 is the absorbance of the blank sample at 562 nm;

[0077] M represents the value of the standard curve of the introduced protein after (A1-A0).

[0078] 2. Results and Analysis

[0079] 2.1 Effect of ultrasonic disruption on the cell wall disruption of Synechococcus cells

[0080] Cell wall disruption of *Chlamydomonas reinhardtii* was performed under different ultrasonic powers. The disruption was observed under a microscope, and the protein content in the contents after the reaction was determined using the BCA method. The results of ultrasonic cell disruption are shown below. Figure 2 As shown, when the cell-wall breaking power is 240 W, the protein content of the contents measured after cell-wall breaking is significantly increased. When the power is increased to 270 W, the protein content decreases. Therefore, the optimal cell-wall breaking condition for ultrasound is 240 W.

[0081] 2.2 Effect of ultrasound time on the protein extraction rate of Chlamydomonas reinhardtii

[0082] Cell wall disruption was performed at different ultrasonic times, and the effect on the degree of disruption of *Chlamydomonas reinhardtii* cell walls was observed under a microscope. The protein content in the post-reaction contents was determined using the BCA method. The reaction time was set to 20–40 min, with samples taken every 5 minutes. The experimental results are as follows: Figure 3As shown, the protein content of the contents measured at 20-30 min increases with the increase of ultrasonic time, but the protein content of the contents measured at more than 30 min decreases. This is because the ultrasonic time is too long and the temperature is too high, which damages the protein. Therefore, the optimal cell disruption condition is determined to be 30 min.

[0083] 2.3 Effect of solid-liquid ratio on protein extraction rate from Chlamydomonas reinhardtii

[0084] The effect of different feed-to-liquid ratios on cell wall disruption of *Chlamydomonas reinhardtii* was observed. After the reaction, the protein content in the algal solution was determined using the BCA method, and the cell wall disruption before and after the reaction was observed using an optical microscope. The results are as follows: Figure 4 As shown, when the reaction mixture ratio was greater than 1:20 (g / mL), the algal cells were too closely spaced and difficult to count, as observed under an optical microscope. At a mixture ratio of 1:10 (g / mL), the algal cells were appropriately spaced, facilitating counting and yielding the best results, with a protein extraction rate of 45.9449%. At a mixture ratio of 1:15 (g / mL), the algal cells were more dispersed, and the protein extraction rate decreased significantly. Therefore, a mixture ratio of 1:10 (g / mL) was determined to be the optimal ratio for cell disruption.

[0085] 2.4 Effect of combined ultrasound and repeated freeze-thaw cycles on the protein extraction rate of Chlamydomonas reinhardtii

[0086] The cell disruption results of 5 different cell disruption methods are as follows: Figure 5 As shown, the combination of ultrasound and repeated freeze-thaw cycles at 37℃ yielded the best cell wall disruption effect, with a protein extraction rate of 69.80%. The combination of ultrasound and repeated freeze-thaw cycles at 4℃ achieved a protein extraction rate of 54.09%. Ultrasound alone yielded a protein extraction rate of 41.79%, repeated freeze-thaw cycles at 4℃ alone yielded a rate of 26.46%, and repeated freeze-thaw cycles at 37℃ alone yielded a rate of 34.31%. The experiment revealed that a single method cannot completely disrupt the cell wall of *Chlamydomonas reinhardtii*. The combination of ultrasound and repeated freeze-thaw cycles at 37℃ achieved a higher protein extraction rate than the other four methods, further disrupting cells that failed to be broken down after ultrasound treatment through freeze-thaw cycles, thus achieving the desired cell wall disruption effect. Therefore, it is determined that the combination of ultrasound and repeated freeze-thaw cycles at 37℃ yields a higher protein extraction rate than the other four methods.

[0087] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The cell wall breaking process of Chlamydomonas reinhardtii, characterized in that, Cell wall disruption of Chlamydomonas reinhardtii algal solution was achieved through repeated freeze-thaw cycles and ultrasonic lysis. The freezing temperature for the freeze-thaw process is -22 to -18°C, and the thawing temperature is 35 to 39°C.

2. The cell-wall breaking process as described in claim 1, characterized in that, The number of freeze-thaw cycles is 2 to 4.

3. The cell-wall breaking process as described in claim 1, characterized in that, The power of the ultrasonic pyrolysis is 180~270 W.

4. The cell-wall breaking process as described in claim 1, characterized in that, The ultrasonic lysis time is 25~40 min.

5. The cell-wall breaking process as described in claim 1, characterized in that, The ratio of the Chlamydomonas rhinestones solution to the substrate is 1:(5~15).

6. The cell-wall breaking process as described in claim 1, characterized in that, include: Chlamydomonas reinhardtii was mixed with a solvent to obtain an algal solution; The algal solution is freeze-thawed 2-4 times to obtain a freeze-thawed algal solution; The frozen-thawed algal solution was lysed using ultrasound to break down the cell walls. The single freeze-thaw cycle includes freezing the algal solution at -22 to -18°C and then thawing the algal solution at 35 to 39°C.

7. The cell-wall breaking process as described in claim 6, characterized in that, After ultrasonic lysis but before cell wall disruption, a centrifugation step is also included.

8. The cell-wall breaking process as described in claim 6, characterized in that, The power of the ultrasonic pyrolysis is 180~270 W.

9. The cell-wall breaking process as described in claim 6, characterized in that, The ultrasonic lysis time is 25~40 min.

10. The cell-wall breaking process as described in claim 6, characterized in that, The ratio of the Chlamydomonas rhinestones solution to the substrate is 1:(5~15).

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