Haematococcus pluvialis breed conservation culture solution and application of haematococcus pluvialis breed conservation culture solution in inhibition of kettles

Abstract

The invention relates to a haematococcus pluvialis breed conservation culture solution and application of the haematococcus pluvialis breed conservation culture solution in inhibition of kettles, and belongs to the technical field of haematococcus pluvialis culture. According to the haematococcus pluvialis breed conservation culture solution, on the basis of a basic culture medium, chitinase, potassium sorbate, sodium pyrosulfite and sodium dehydroacetate are added, so that the kettles can be effectively inhibited; the diameter, the area and the dry weight of the algae colony cultured by the culture solution are obviously increased; the cultured haematococcus pluvialis is continuously subjected to subsequent outdoor culture of algae species, the risk of kettle fungus infection can be eradicated or reduced from the source, the risk of kettle fungus infection is avoided or reduced in the later outdoor culture stage, and the loss caused by kettle fungus infection is avoided or reduced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of Haematococcus pluvialis cultivation, in particular to a Haematococcus pluvialis preservation culture solution and application thereof in inhibiting Chytrid. BACKGROUND

[0002] The large-scale cultivation of Haematococcus pluvialis mainly includes two stages: indoor expansion and outdoor cultivation. The indoor cultivation is mainly to culture the algae from the culture dish to the liquid algae, and the algae is transferred to the outdoor cultivation after about 60 days. The green algae stage of the outdoor cultivation is to proliferate the motile cells of Haematococcus pluvialis to expand the culture system and the culture amount, and the red algae stage is to accumulate astaxanthin by stress.

[0003] During the indoor cultivation of Haematococcus pluvialis, the preservation culture dish is used as the starting source, and agar culture solution is poured into the culture dish to separate and purify the algae cells. The single algae colony cultured by the streaking separation and purification of the preservation culture dish is free of bacterial and fungal contamination and is grown from single cells, and the single algae colony can completely retain the genetic characteristics of the single cells, has the characteristics of good separation effect and purity, and is a good method for separating, culturing and preserving the single-cell algae.

[0004] During the large-scale cultivation of Haematococcus pluvialis, the preservation culture dish can effectively ensure the genetic characteristics of the algae to be stable, specific and free of contamination, so the work at the source plays a crucial role. During the preservation culture dish process, a large number of single algae colonies can be obtained in one culture dish, and each algae colony has a large diameter, which will be very beneficial to the subsequent culture of the algae.

[0005] However, during the cultivation of Haematococcus pluvialis, there is a kind of endophytic pathogen in the algae cells, Chytrid (Paraphysoderma sedebokerense), which adheres to the surface of the Haematococcus pluvialis cells through zoospores, penetrates the cell wall through hyphae, absorbs intracellular nutrients, and inhibits the synthesis of astaxanthin, eventually leading to the death of the host cells, which poses a serious threat to the large-scale cultivation. Chytrid has a close symbiotic relationship with algae cells, and traditional chemical control is easy to harm the algae cells.

[0006] Recent research has proposed methods for eradicating chytrid fungi, including: First, using surfactants (such as SDBS) to disrupt the cell membranes of zoospores (which lack cell walls), inhibiting infection and harming algal cells with cell walls. This method is cost-effective (only 0.07 yuan per 1000 L of algal fluid is needed), but it can cause foaming and lead to cell loss. Second, maintaining a low temperature (10–22°C) can slow the growth of the fungus, but this method is energy-intensive. Third, using a combination of inhibitors (caspofungin + β-mannanase) at a pH of 8.0–9.5 can inhibit cell wall synthesis and control the infection rate to less than 2%, but this method is costly. Fourth, using antioxidants (such as butylated hydroxyanisole (BHA)) to neutralize reactive oxygen species, reduce oxidative damage, and block fungal infection pathways (effective at a concentration of 7 ppm), but overall effectiveness is low during chytrid outbreaks. The above control methods rely on targeted antimicrobial agents (such as surfactants and enzyme complexes) or environmental manipulation (such as low temperature and antioxidants). These methods not only require a balance between effectiveness, safety, and cost, but are also crucially applicable to outdoor aquaculture operations where chytrid infection has already occurred. However, when the infection rate exceeds 50%, cell mortality in the algal slurry can reach over 90%, leading to complete aquaculture failure. Once chytrid infection occurs during outdoor aquaculture, it can trigger catastrophic cell death within 3-5 days, ultimately resulting in a complete loss of initial human and material investment and a significant impact on production. Furthermore, chytrid outbreaks during outdoor aquaculture are sporadic and highly unpredictable. In outdoor aquaculture, waiting until infection is detected to intervene is highly ineffective. Furthermore, there are no reports of successful application of these methods in large-scale aquaculture systems to effectively suppress chytrid infection during outdoor aquaculture. Summary of the Invention

[0007] To overcome the problems existing in the background technology, the present invention provides a culture solution for preserving Haematococcus pluvialis seeds and its use in inhibiting chytrid fungus. The culture solution of the present invention, when used in the purification and preservation of Haematococcus pluvialis seeds in indoor culture dishes, can achieve source-end inhibition of chytrid fungus, a specific endophytic pathogenic microorganism of Haematococcus pluvialis. The cultivated and purified algal colonies have a larger diameter and area and are free of chytrid fungus infection. The algae species cultivated with the culture solution of the present invention are used as sterile, high-quality source algae species for production, avoiding or reducing the risk of chytrid fungus infection. This is of great significance for inhibiting chytrid fungus infection in the cultivation process of Haematococcus pluvialis, especially in large-scale cultivation processes.

[0008] To achieve the above objectives, a first object of the present invention is to provide a Haematococcus pluvialis seed preservation culture solution comprising chitinase, potassium sorbate, sodium metabisulfite and sodium dehydroacetate, wherein the culture solution can effectively inhibit chytrid infection.

[0009] Further, the culture solution further comprises chitinase 1-15 mg / L, potassium sorbate 5-40 mg / L, sodium pyrosulfite 0.1-3 mg / L and sodium dehydroacetate 3-20 mg / L.

[0010] Further, the culture solution further comprises a basic culture medium.

[0011] Further, the basic culture medium is any one of BG11 culture medium, BBM culture medium and SM culture medium.

[0012] The application further provides application of the culture solution in purification culture of Haematococcus pluvialis spores.

[0013] The application further provides application of the culture solution in inhibition of Chytrid.

[0014] The application further provides a purification culture method of Haematococcus pluvialis spores, which uses the culture solution.

[0015] The application further provides Haematococcus pluvialis spores obtained after purification of Haematococcus pluvialis by using the culture solution, which can effectively avoid Chytrid infection in outdoor culture.

[0016] The application has the following beneficial effects: The culture solution of the application is used as a culture solution for purification and preservation of Haematococcus pluvialis in an indoor culture dish, and the cultivated algae are obviously increased in diameter and area and are free of Chytrid infection. The algae cultivated by using the culture solution of the application are used as a source of algae spores for liquid algae spores, and the subsequent outdoor cultivation can eliminate or reduce the risk of Chytrid infection from the source, avoid or reduce the risk of Chytrid infection in the later outdoor breeding stage, and avoid or reduce the loss caused by Chytrid infection. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is an appearance comparison of the algae cultivated by using the culture solution of the application and the algae cultivated by using the culture solution of the comparative example 1 to the comparative example 3 and the example 1 in a culture dish; Figure 2 is a diameter comparison of the algae cultivated by using the culture solution of the application and the algae cultivated by using the culture solution of the comparative example 1 to the comparative example 3 and the example 1; Figure 3 is a diameter comparison bar chart of the algae cultivated by using the culture solution of the application and the algae cultivated by using the culture solution of the comparative example 1 to the comparative example 3 and the example 1; Figure 4 is an area comparison bar chart of the algae cultivated by using the culture solution of the application and the algae cultivated by using the culture solution of the comparative example 1 to the comparative example 3 and the example 1; Figure 5 is a dry weight comparison bar chart of the algae cultivated by using the culture solution of the application and the algae cultivated by using the culture solution of the comparative example 1 to the comparative example 3 and the example 1; Figure 6is a fluorescent staining image of the algae Chytridium sp. cultivated by the present application Comparative Example 1 to Comparative Example 3 and Example 1. DETAILED DESCRIPTION

[0018] In order to make the objectives, technical solutions and beneficial effects of the present application clearer, the technical solutions of the present application will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, other embodiments obtained by a person skilled in the art without creative work fall within the protection scope of the present application.

[0019] In order to make the objectives, technical solutions and beneficial effects of the present application clearer, the technical solutions of the present application will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, other embodiments obtained by a person skilled in the art without creative work fall within the protection scope of the present application. Example 1

[0020] The culture solution is prepared by adding chitinase, potassium sorbate, sodium pyrosulfite and sodium dehydroacetate into the BG11 culture medium, wherein the concentration of chitinase is 5.4 mg / L, the concentration of potassium sorbate is 12.62 mg / L, the concentration of sodium pyrosulfite is 1.43 mg / L, and the concentration of sodium dehydroacetate is 7.8 mg / L.

[0021] After the purification and preservation culture medium is completed, in the super-clean workbench, healthy single algae are picked up by using a sterile operation and a loop to transfer to the culture medium, inoculated by a three-zone streaking method, and the culture dish is sealed by using a sealing film after inoculation. Then, the culture dish is placed on a light culture rack in the preservation interval in a clean area of 100,000 levels for culture. The light is set to 2000-3000 lux, and a light period of 12h light / 12h darkness is adopted, the light source is a white LED lamp, and the distance from the culture dish is 15-20 cm. The temperature is controlled at 18-22℃.

[0022] Comparative Example 1 The culture solution is BG11 basic culture medium.

[0023] The culture method is the same as that in Example 1.

[0024] Comparative Example 2 The culture solution is prepared by adding chitinase into the BG11 basic culture medium, and the concentration of chitinase is 5.4 mg / L.

[0025] The culture method is the same as that in Example 1.

[0026] Comparative Example 3 The culture solution is prepared by adding chitinase, potassium sorbate, sodium pyrosulfite and sodium acetate into the BG11 basic culture medium, wherein the concentration of chitinase is 5.4 mg / L, the concentration of potassium sorbate is 12.62 mg / L, the concentration of sodium pyrosulfite is 1.43 mg / L, and the concentration of sodium acetate is 7.8 mg / L. Example 2

[0027] Preparation of culture medium: chitinase, potassium sorbate, sodium metabisulfite and sodium dehydroacetate were added to BG11 basal medium, with the chitinase concentration being 10 mg / L, potassium sorbate concentration being 30 mg / L, sodium metabisulfite concentration being 1.2 mg / L, and sodium dehydroacetate concentration being 5 mg / L.

[0028] The culture method is the same as in Example 1. Example 3

[0029] Preparation of culture medium: chitinase, potassium sorbate, sodium metabisulfite and sodium dehydroacetate were added to BG11 basal medium, with the chitinase concentration being 2 mg / L, potassium sorbate concentration being 7 mg / L, sodium metabisulfite concentration being 2.0 mg / L, and sodium dehydroacetate concentration being 18 mg / L.

[0030] The culture method is the same as in Example 1. Example 4

[0031] Preparation of culture medium: chitinase, potassium sorbate, sodium metabisulfite and sodium dehydroacetate were added to SM basal medium, with the chitinase concentration being 9 mg / L, potassium sorbate concentration being 12 mg / L, sodium metabisulfite concentration being 1.6 mg / L, and sodium dehydroacetate concentration being 10 mg / L.

[0032] The culture method is the same as in Example 1.

[0033] After culturing for 20 days, the algae colonies in Example 1 and Comparative Examples 1 to 3 were observed. Figures 1 to 4 As shown, attached Figure 1 For the appearance of algal colonies in culture dishes, Figure 1 It can be seen that the single algae colonies in the culture dish of Example 1 are discretely distributed with clear boundaries, and the diameters of the single algae species are relatively large. Three dishes were randomly selected from each group, and 5 single algae colonies were randomly selected from each dish. The algae colony images were taken under a 40x optical microscope, and the diameters of the algae colonies were measured. Figure 2 The images of algae colonies taken for demonstration purposes show that the average diameter of the algae colonies in Example 1 was approximately 5.72±0.19 mm, the average diameter of the algae colonies in Comparative Example 1 was approximately 2.41±0.20 mm, the average diameter of the algae colonies in Comparative Example 2 was approximately 2.38±0.21 mm, and the average diameter of the algae colonies in Comparative Example 3 was approximately 3.56±0.11 mm. The diameter and area of ​​the algae colonies in Example 1 were significantly larger than those in Comparative Examples 1 to 3, while the diameter and area of ​​the algae colonies in Comparative Example 3 were larger than those in Comparative Example 1, but still significantly lower than those in Example 1.

[0034] The dry weight of the algae colonies of Example 1 and Comparative Examples 1 to 3 was compared by randomly taking 5 dishes. The average dry weight of the algae colonies of Example 1 was 2.65 ± 0.12 g / dish, the average dry weight of the algae colonies of Comparative Example 1 was 0.55 ± 0.05 g / dish, the average dry weight of the algae colonies of Comparative Example 2 was 0.70 ± 0.04 g / dish, and the average dry weight of the algae colonies of Comparative Example 3 was 1.02 ± 0.05 g / dish. The dry weight of the algae colonies of Example 1 was significantly greater than that of the other groups.

[0035] The specific dry weight detection method is as follows: 5 ml of sterile water was taken, and all the algae colonies in the culture dish were taken out with a inoculation ring and put into 5 ml of sterile water. Then 1 ml of sterile water was injected into the culture dish to wash the algae cells on the agar surface. The sterile water containing the washed algae cells was collected into the 5 ml of sterile water containing the taken algae colonies. The washing was repeated 5 times, and finally 10 ml of cell suspension containing the culture dish algae colonies was prepared. The algae liquid was filtered through a filter membrane using a vacuum filtration device, and the algae cells were collected. The filter membrane was gently washed with distilled water for 3 times to remove residual medium salts. The algae-loaded filter membrane was placed in a culture dish and dried in an 80℃ oven for 4-6 hours until the weight was constant. After cooling, the weight was measured with an electronic balance (recorded as W1). The dry weight calculation formula is: dry weight (g / L) = (W1-W0) / V x 1000. Three parallel experiments were performed to take the average value.

[0036] The algae cell suspensions prepared from 3 dishes of each group of Example 1, Comparative Examples 1 to 3 were subjected to chitin fluorescence staining. The specific fluorescence staining method is as follows: According to the dry weight detection method, 10 ml of cell suspension containing the culture dish algae colonies was prepared, shaken and mixed, and 1 mL was taken. After centrifugation at 800 rpm for 5 minutes, the supernatant was discarded and the cells were resuspended and washed twice with sterile PBS buffer. The final concentration of the fluorescent dye Calcofluor White M2R was 10 μg / mL, and the dye was added and stained at room temperature for 15 minutes. The dye can specifically bind to the chitin of fungal cell wall. After staining, centrifugation was performed at 800 rpm for 5 minutes, and the dye was discarded. The cells were washed with PBS for 3 times to remove the unbound dye. 5 μL of sample was taken and dropped onto a glass slide, and a cover glass was added to avoid air bubbles. A fluorescence microscope was used with an excitation wavelength of 350-400 nm and an emission wavelength of 420-460 nm. The chitinous hyphae and spores appeared blue fluorescence.

[0037] The staining results are shown in the accompanying Figure 6 The results show that no chitinous fungi are present in Example 1, and chitinous fungi are present in all other groups. The number of chitinous fungi in Comparative Example 1 is the largest.

[0038] The algae colonies obtained in Examples 2 to 4 were further cultured outdoors, and no chitinous fungi were found, indicating that the combination of chitinase, potassium sorbate, sodium metabisulfite and dehydroacetic acid sodium has good effect on inhibiting chitinous fungi.

[0039] The main components of the BG11 basic medium used in the above examples are: agar 10.5 g / L, NaNO3 (sodium nitrate) 1.5 g / L, K2HPO4 (dibasic potassium phosphate) 0.04 g / L, MgSO4·7H2O (magnesium sulfate heptahydrate) 0.075 g / L, CaCl2·2H2O (calcium chloride dihydrate) 0.036 g / L, Na2CO3 (sodium carbonate) 0.02 g / L, citric acid 6.0 mg / L, ferric ammonium citrate 6.0 mg / L, EDTA disodium salt 1.0 mg / L, trace element solution A5 1.0 ml / L, and each liter of trace element solution A5 includes H3BO3 (boric acid) 2.86 g / L, MnCl2·4H2O (manganese chloride tetrahydrate) 1.81 g / L, ZnSO4·7H2O (zinc sulfate heptahydrate) 0.222 g / L, Na2MoO4·2H2O (sodium molybdate dihydrate) 0.39 g / L, CuSO4·5H2O (copper sulfate pentahydrate) 0.079 g / L, and Co(NO3)2·6H2O (cobalt nitrate hexahydrate) 0.0494 g / L.

[0040] It should be noted that the method of the present application is not limited to BG11, and adding chitinase, potassium sorbate, sodium pyrosulfite, and sodium dehydroacetate to BG11, BBM medium, and SM medium can also achieve good chlamydomonas inhibition effect.

[0041] Meanwhile, the basic medium suitable for the present application is not limited to BG11, BBM medium, SM medium, and other existing basic media, and any formula for adding chitinase, potassium sorbate, sodium pyrosulfite, and sodium dehydroacetate to the basic medium of Haematococcus pluvialis can inhibit chlamydomonas.

[0042] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application 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 detail without departing from the scope defined by the claims of the present application.

Claims

1. A Haematococcus pluvialis seed preservation culture solution, characterized in that: The culture solution contains chitinase, potassium sorbate, sodium pyrosulfite and sodium dehydroacetate.

2. The culture solution according to claim 1, characterized in that The culture solution contains 1-15 mg / L chitinase, 5-40 mg / L potassium sorbate, 0.1-3 mg / L sodium pyrosulfite and 3-20 mg / L sodium dehydroacetate.

3. The culture solution according to claim 1 or 2, characterized in that The culture solution also includes a basal culture medium.

4. The culture solution according to claim 3, characterized in that The basal culture medium is any one of BG11 culture medium, BBM culture medium and SM culture medium.

5. Use of the culture solution according to any one of claims 1 to 4 in the purification and cultivation of Haematococcus pluvialis algae. Use of the culture solution according to any one of claims 1 to 4 in inhibiting chytrid fungi.

7. A method for purifying and cultivating Haematococcus pluvialis species, characterized in that: The culture solution according to any one of claims 1 to 4 is used.

8. A Haematococcus pluvialis species obtained by breeding and purifying Haematococcus pluvialis using the culture solution according to any one of claims 1 to 4.

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