A method for improving the swimming ability and proliferation ability of green swimming cells of Haematococcus pluvialis

Abstract

The invention relates to a method for improving the swimming ability and proliferation ability of green swimming cells of Haematococcus pluvialis, and belongs to the technical field of Haematococcus pluvialis cultivation. The invention abandons the limitation of improving the proliferation ability of Haematococcus pluvialis to improve the astaxanthin yield, and starts from improving the movement ability of Haematococcus pluvialis to obtain a method for improving the movement ability and proliferation ability of green swimming cells. The invention significantly improves the movement ability and proliferation ability of green swimming cells by adding sodium percarbonate (2Na2CO3·3H2O2) in the green algae cultivation stage, and finally improves the dry weight and astaxanthin yield of Haematococcus pluvialis. In the case of adding sodium percarbonate, buffer Tris and playing white noise at the same time, the swimming speed of green swimming cells is increased to 4.16 times of the original swimming speed, the proliferation ability is increased to 2.478 times, the dry weight after cultivation is increased by 89.74% at most, and the astaxanthin yield can be increased by 37.17% at most.

Description

Technical Field

[0001] The invention belongs to the technical field of Haematococcus pluvialis cultivation, and specifically relates to a method for improving the swimming ability and proliferation ability of green swimming cells of Haematococcus pluvialis. Background Art

[0002] The astaxanthin content in Haematococcus pluvialis can reach 10-40mg, making it the organism with the highest natural astaxanthin content in nature. In addition, the astaxanthin in Haematococcus pluvialis is 100% left-handed, with a stable structure that is consistent with the astaxanthin structure required by the human body and animals, making it easy to absorb and able to maximize its various biological effects.

[0003] Haematococcus pluvialis is a biflagellate unicellular algae, and its cultivation is divided into the green algae (green swimming cells) cultivation stage and the red algae (red spore cells) cultivation stage. The green algae cultivation stage is an important stage for algae cell proliferation. At present, the research on the green algae cultivation stage mainly focuses on the proliferation ability of green algae, and the astaxanthin production is increased by improving the proliferation ability of Haematococcus pluvialis. It has been reported that adding organic carbon sources to the culture medium can promote cell proliferation in the green swimming cell stage. However, the addition of organic carbon sources may cause the bacterial growth rate in the culture medium to be greater than the proliferation rate of green swimming cells, making bacteria the dominant population in the culture medium, and inhibiting the green swimming cell population to a certain extent. In addition, bacteria secrete extracellular polymers, that is, high molecular polymers secreted outside the body. It has been reported that the extracellular polymers secreted by bacteria, such as chitosan and polysaccharides, can flocculate algae cells through charge neutralization or physical encapsulation, resulting in a large amount of precipitation in the green swimming cell cultivation stage, a large reduction in the number of swimming cells, and a large increase in the number of immotile spore cells. In addition, the addition of organic carbon sources will increase the risk of eutrophication pollution of aquaculture wastewater. Summary of the invention

[0004] In response to the problems raised by the background technology, the present invention can effectively improve the motility of green swimming cells of Haematococcus pluvialis by adding sodium percarbonate during the green algae cultivation stage, breaking through the current limitation of increasing astaxanthin production by only improving the proliferation ability of Haematococcus pluvialis. It not only improves the motility of green swimming cells, but also improves the proliferation ability of green swimming cells, and ultimately increases the dry weight and astaxanthin yield of Haematococcus pluvialis.

[0005] Haematococcus pluvialis is a biflagellate unicellular algae. The energy generated by the beating of flagella can propel it to move in the water, making it mobile. It can obtain light energy, absorb nutrients, and escape from enemies through vertical, horizontal, and instantaneous movements. The present invention significantly improves the movement and proliferation capacity of green swimming cells by adding sodium percarbonate (2Na2CO3·3H2O2) during the green algae cultivation stage, and ultimately increases the dry weight and astaxanthin content of Haematococcus pluvialis.

[0006] To achieve the above object, the present invention is implemented through the following technical solutions:

[0007] The method for improving the swimming ability and proliferation ability of green swimming cells of Haematococcus pluvialis is achieved by adding sodium percarbonate 2Na2CO3·3H2O2 to the culture medium, wherein the addition amount of sodium percarbonate 2Na2CO3·3H2O2 is 10-50 μg / ml.

[0008] Furthermore, the added amount of sodium percarbonate 2Na2CO3·3H2O2 is 20-50 μg / ml.

[0009] Furthermore, during the green algae cultivation stage, a pH buffer Tris is used, wherein the amount of Tris added is 5-50 mM.

[0010] Furthermore, the amount of Tris added is 10-50 mM.

[0011] Furthermore, white noise is played during the green algae cultivation stage; the white noise is 20-100 decibels of white noise.

[0012] Furthermore, white noise of 20-80 decibels was played during the green algae cultivation stage.

[0013] Beneficial effects of the present invention:

[0014] The invention can obviously improve the movement ability and proliferation ability of green swimming cells by adding sodium percarbonate in the green algae cultivation stage of Haematococcus pluvialis, and finally improve the dry weight and astaxanthin yield of Haematococcus pluvialis.

[0015] The invention uses a Tris buffer while adding sodium percarbonate, which can further improve the movement ability and proliferation ability of green swimming cells and significantly improve the dry weight and astaxanthin yield of Haematococcus pluvialis.

[0016] The present invention can also choose to play white noise at the same time. By playing white noise, the motility of swimming cells of Haematococcus pluvialis can be further improved, the proliferation of green swimming cells can be promoted, and the dry weight and astaxanthin content of Haematococcus pluvialis can be increased. The present invention can increase the swimming speed of green swimming cells to a maximum of 4.16 times, the proliferation capacity to 2.478 times, the dry weight after cultivation can be increased by a maximum of 89.74%, and the astaxanthin yield can be increased by a maximum of 37.17%.

[0017] The present invention does not require the addition of organic carbon and can not only promote the proliferation of green swimming cells, but also improve the movement ability of the green swimming cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a comparison chart of the effects of different sodium percarbonate addition amounts on the cultivation of Haematococcus pluvialis in Example 1 of the present invention;

[0019] Figure 2 This is a comparison of the swimming ability of green motile cells in different culture media in Example 2 of the present invention;

[0020] Figure 3 is a comparison chart of the number of motile cells of green motile cells under different culture media in Example 2 of the present invention;

[0021] Figure 4 This is a comparison chart of the dry weight of algae powder after culture in different culture media in Example 2 of the present invention;

[0022] Figure 5 This is a comparison chart of astaxanthin content of algae powder after culture in different culture media in Example 2 of the present invention;

[0023] Figure 6 This is the pH value change of different culture media in Example 4 of the present invention. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0025] The method for improving the swimming ability and proliferation ability of green swimming cells of Haematococcus pluvialis comprises adding sodium percarbonate 2Na2CO3·3H2O2 to the culture medium, wherein the addition amount of sodium percarbonate 2Na2CO3·3H2O2 is 10-50 μg / ml.

[0026] For flagellar microalgae, the redox signals in the environment and cells are related to the coordinated movement of flagella, and the addition of sodium percarbonate can promote the motility of flagella by regulating the redox state in flagella. Appropriate sodium percarbonate concentration can promote the motility of green motile cells, increase the movement speed of green motile cells, and ultimately increase the dry weight and astaxanthin content of Haematococcus pluvialis. In addition, the sodium percarbonate added in the present invention can be used as a food additive, which is more safe.

[0027] The method of the present invention can also use pH buffer Tris during the green algae cultivation stage, wherein the amount of Tris added is 5-50mM.

[0028] During the green algae cultivation stage of Haematococcus pluvialis, as nutrients are consumed and cell metabolites increase, the pH value of the culture fluid will continue to rise. Haematococcus pluvialis, which has the characteristic of rapid sedimentation in a highly alkaline environment, will have its swimming cells' flagella fall off under high pH conditions, transform into amoenospores, and settle to the bottom of the culture container. In addition, during the cultivation of green swimming cells, the concentration of inorganic carbon may increase, which will cause water acidification. By adding the pH buffer Tris, the pH value of the cell culture fluid can be stabilized under the optimal conditions. In addition, Tris can also neutralize the alkaline effect caused by the addition of 2Na2CO3·3H2O.

[0029] The method of the present invention can simultaneously play 20-100 decibels of white noise during the green algae cultivation stage. Studies have shown that music can cause cells in plants to resonate, strengthen the plant's ability to resist pests and diseases, accelerate growth and development, and increase crop yields. In the late 20th century, the United States invented music specifically for promoting plant growth, and continued to promote it to dozens of countries in the fields of edible fungus cultivation and fermentation, as well as horticultural crop cultivation. However, the above studies are all based on higher plants, especially crops. There is no research on the effect of music on microalgae. The present invention can significantly improve the motility of green swimming cells and their movement speed by playing white noise during the green algae cultivation stage, and has a good proliferation effect on green algae.

[0030] In order to more clearly illustrate the present invention, it is described in detail through the following examples. Example 1

[0031] A method for improving the swimming ability and proliferation ability of green swimming cells of Haematococcus pluvialis, which is as follows:

[0032] During the indoor green algae cultivation stage of Haematococcus pluvialis, conventional BG11 culture medium was used and different concentrations of sodium percarbonate were added for cultivation.

[0033] The green algae culture stage starts from the initial inoculation day as day 0. On the 8th day of culture, samples are taken to detect the swimming speed and number of swimming cells. The culture results with different sodium percarbonate addition amounts are shown in the following table.

[0034] Table 1 Green motile cells at different sodium percarbonate concentrations

[0035]

[0036] After the green algae stage cultivation is completed, the culture is transferred outdoors to the columnar reactor for red algae stage cultivation. After 15 days of cultivation, the spores of Haematococcus pluvialis are harvested and dried into algae powder. The cultivation results of culture media with different sodium percarbonate concentrations are tested. The test results are shown in the following table.

[0037] Table 2 Dry weight of algae powder and astaxanthin content after culture at different sodium percarbonate concentrations

[0038]

[0039] By comparison, it can be seen that the addition of sodium percarbonate can significantly increase the swimming speed of swimming cells. Compared with the absence of sodium percarbonate, the swimming speed of swimming cells increased from 14.49±1.18μm / s to 27.11±0.50μm / s after adding 20μg / ml sodium percarbonate, and the swimming speed increased by 87.1%, which shows that the addition of sodium percarbonate can significantly increase the swimming speed of green algae cells. In addition, the experimental results also show that the addition of sodium percarbonate can significantly increase the dry weight of algae powder and the astaxanthin content. Example 2

[0040] A method for improving the swimming ability and proliferation ability of green swimming cells of Haematococcus pluvialis

[0041] During the indoor green algae cultivation stage of Haematococcus pluvialis, taking the 250ml triangular flask cultivation stage as an example, conventional BG11 culture medium was used for cultivation without any additional treatment, conventional BG11 culture medium was added with 10mM buffer Tris for cultivation, conventional BG11 culture medium was added with 20μg / ml sodium percarbonate for cultivation, conventional BG11 culture medium was added with 10mM buffer Tris and 20μg / ml sodium percarbonate for cultivation, and conventional BG11 culture medium was added with 10mM buffer Tris and 20μg / ml sodium percarbonate and 60dB white noise treatment for cultivation.

[0042] The green algae culture stage starts from the initial inoculation day as day 0. On the 8th day of culture, samples were taken to detect the swimming speed and number of swimming cells. The results are shown in the following table.

[0043] Table 3 Green motile cells cultured in different culture media

[0044]

[0045] After the green algae stage culture is completed, the column reactor is transferred outdoors for red algae stage culture. After 15 days of culture, the spores of Haematococcus pluvialis are harvested and dried into algae powder. The results are shown in the following table.

[0046] Table 4 Effects of different culture media on dry weight of algae powder and astaxanthin content

[0047]

[0048] The test results showed that the swimming speed and number of swimming cells in the green algae stage in the conventional BG11 culture medium without any additional treatment were the lowest, and the dry weight of algae powder and astaxanthin content after drying were also the lowest; the swimming speed and number of swimming cells in the green algae stage in the conventional BG11 culture medium with 10mM buffer Tris and 20μg / ml sodium percarbonate and 60dB white noise treatment were the highest, and the dry weight of algae powder and astaxanthin content after drying were also the highest. When sodium percarbonate, buffer Tris and white noise were added at the same time, the swimming speed of green swimming cells increased to 4.16 times the original swimming speed, the proliferation ability increased to 2.478 times, the dry weight after cultivation increased by up to 89.74%, and the astaxanthin yield increased by up to 37.17%. Example 3

[0049] A method for improving the swimming ability and proliferation ability of green swimming cells of Haematococcus pluvialis

[0050] During the indoor green algae cultivation stage of Haematococcus pluvialis, conventional BG11 culture medium was used, 10mM buffer Tris and 20μg / ml sodium percarbonate were added for cultivation, and white noise treatments of 20dB, 60dB, 80dB and 100dB were applied respectively during the entire cultivation stage.

[0051] The green algae culture stage starts from the initial inoculation day as day 0. On the 8th day of culture, samples were taken to test the swimming speed and number of swimming cells. The test results are shown in the following table.

[0052] Table 5 Comparison of the effects of white noise decibels on green swimming cells

[0053]

[0054] After the green algae stage cultivation is completed, the culture is transferred outdoors to the columnar reactor for the red algae stage cultivation. After 15 days of cultivation, the Haematococcus pluvialis spores are harvested and dried into algae powder. The dry weight and astaxanthin content of the algae powder are tested. The test results are shown in the following table.

[0055] Table 6 Effect of white noise decibel on dry weight of algae powder and astaxanthin content

[0056]

[0057] The results showed that compared with the 20dB, 80dB and 100dB groups, the 60dB white noise group had the highest swimming speed and the largest number of swimming cells during the green algae cultivation stage. The dry weight and astaxanthin content of the red algae after harvest were significantly higher than those of the other groups. Example 4

[0058] A method for improving the swimming ability and proliferation ability of green swimming cells of Haematococcus pluvialis

[0059] During the indoor green algae cultivation stage of Haematococcus pluvialis, conventional BG11 culture medium was added with 20 μg / ml sodium percarbonate and 60 dB white noise was applied for cultivation. In addition, 5 mM, 10 mM, and 50 mM buffer Tris were added to the culture medium, respectively.

[0060] The green algae culture stage starts from the initial inoculation day as day 0. On the 8th day of culture, samples were taken to test the swimming speed and number of swimming cells. The test results are shown in the following table.

[0061] Table 7 Comparison of the effects of buffer addition on green motile cells

[0062]

[0063] At the same time, the pH value changes of the algae solution in each experimental group from the 0th to the 8th day of culture were detected. The results showed that the pH value of the algae solution in the group with 5mM Tris buffer increased to above 8.5 in the last 2 days. The addition of 10mM Tris buffer could effectively stabilize the pH value of the algae solution between 7.5-8.5, while there was no significant difference between the group with 50mM Tris buffer and the group with 10mM Tris buffer (see Appendix). Figure 6 ).

[0064] After the green algae stage cultivation is completed, the culture is transferred outdoors to the columnar reactor for red algae stage cultivation. After 15 days of cultivation, the spores of Haematococcus pluvialis are harvested and dried into algae powder. The test results are shown in the following table.

[0065] Table 8 Effect of buffer addition on algae powder dry weight and astaxanthin content

[0066]

[0067] The results showed that compared with the 5mM group and the 50mM group, the group with 10mM buffer addition had the highest swimming speed and the largest number of swimming cells during the green algae cultivation stage, and the dry weight and astaxanthin content of the red algae after harvest were significantly higher than those of the other groups. Example 5

[0068] A method for improving the swimming ability and proliferation ability of green swimming cells of Haematococcus pluvialis

[0069] During the indoor green algae cultivation stage of Haematococcus pluvialis, conventional BG11 culture medium was added with 10mM Tris buffer and 60dB white noise was applied for cultivation. In addition, 10μg / ml, 20μg / ml and 50μg / ml sodium percarbonate were added to the culture medium respectively.

[0070] The green algae culture stage starts from the initial inoculation day as day 0. On the 8th day of culture, samples were taken to test the swimming speed and number of swimming cells. The test results are shown in the following table.

[0071] Table 9 Comparison of the effects of sodium percarbonate concentration on green swimming cells

[0072]

[0073] After the green algae stage cultivation is completed, the column reactor is transferred outdoors for red algae stage cultivation. After 15 days of cultivation, the spores of Haematococcus pluvialis are harvested and dried into algae powder. The test results are shown in the following table.

[0074] Table 10 Effect of sodium percarbonate concentration on dry weight of algae powder and astaxanthin content

[0075]

[0076] The results showed that the swimming speed of swimming cells in the green algae cultivation stage was the highest in the group adding 50μg / ml sodium percarbonate, but the number of swimming cells in the group adding 20μg / ml sodium percarbonate was the highest. After the red algae were harvested, the dry weight of the group adding 20μg / ml sodium percarbonate was the highest, and the astaxanthin content of the group adding 50μg / ml sodium percarbonate was the highest.

[0077] Description of the test method:

[0078] The number of green swimming cells was calculated by counting under an optical microscope using a commonly used plankton counting plate.

[0079] Motile cell swimming speed (green motile cell swimming speed) detection method description:

[0080] A Pasteur pipette was used to pick algae from a single swimming cell under a 100x optical microscope. 100 μl of culture solution was added to the counting area of ​​the plankton counting plate in advance, and the randomly picked single swimming cells were transferred to the droplets on the counting plate. Then, a coverslip was placed on the plate. After finding a single swimming cell under a 40x optical microscope, a PCO-sCMOS high-speed camera was used to record a 3-minute video. The video was a ND format file with 1 frame per second. After the 3-minute video recording was completed, the video was imported into MATLAB software for image processing and analysis. The regionprops function was used to obtain the movement trajectory of the swimming cell. The movement trajectory information of the 1st and 3rd minutes of the 3-minute video was deleted, and only the movement trajectory information of the middle 1 minute was retained. Logger pro was used to analyze the total distance of the movement trajectory. The movement speed was calculated by the ratio of the movement distance to the movement time.

[0081] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for improving the swimming ability and proliferation ability of green swimming cells of Haematococcus pluvialis, characterized in that: During the green algae cultivation stage, sodium percarbonate 2Na2CO3·3H2O2 is added to the culture medium, wherein the added amount of sodium percarbonate 2Na2CO3·3H2O2 is 20-50 μg / ml.

2. The method for improving the swimming ability and proliferation ability of green swimming cells of Haematococcus pluvialis according to claim 1, characterized in that: During the green algae cultivation stage, a pH buffer Tris is used, wherein the amount of Tris added is 10-50 mM.

3. The method for improving the swimming ability and proliferation ability of green swimming cells of Haematococcus pluvialis according to claim 1 or 2, characterized in that: During the green algae cultivation stage, white noise is played; the white noise is 60-80 decibels.

Images