Culture method and application of beta-1, 3-glucan producing microalgae

Abstract

The invention discloses a culture method and application of beta-1, 3-glucan-producing microalgae, and realizes high-efficiency synergy from microalgae culture to separation. According to the enrichment culture medium, by optimizing the ratio of carbon and nitrogen sources and nutritional ingredients, efficient accumulation of beta-1, 3-glucan can be remarkably improved, meanwhile, the microalgae biomass is promoted, and the problem of low product content in conventional culture is solved. The matched microalgae culture method breaks through the limitation that traditional plate streak bacterial colonies are mixed, water drop separation consumes time and is prone to pollution, the high-purity single strain is accurately extracted from a complex water sample, the operation standardization degree is high, and the manual randomness influence is reduced. Through the synergistic application of the two, the microalgae with high biomass and high glucan content can be efficiently obtained, the separation purity can be guaranteed, high-quality resources are provided for scientific research and industrial production, and the application of the microalgae in the fields of food, medicine and the like is promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of algal separation, and in particular to a culture method of a beta-1,3-glucan-producing microalgae and application thereof. BACKGROUND

[0002] Microalgae are widely distributed in aquatic environments, and their economic value in functional foods, biomedicines and other fields is increasingly prominent, which is largely due to the rich beta-1,3-glucan in cells. However, there are many difficulties in the culture of microalgae and the acquisition of beta-1,3-glucan. In terms of culture, the traditional photosynthetic autotrophic method is inefficient, and the content of beta-1,3-glucan is low. The types of carbon and nitrogen sources used and the culture conditions are not much different from those reported in early literature, making it difficult to significantly improve production efficiency. Microalgae have significant value in the fields of biological energy, food, medicine and other fields, but the overall separation technology still needs to be improved: the traditional separation method is low in efficiency, and the precision is insufficient when obtaining a single high-purity microalgae strain from complex water samples. For example, when plate streaking culture is used, the colonies are often dense and mixed, and the target microalgae are difficult to distinguish from other impurities; and due to the influence of operation proficiency and randomness, it is difficult to avoid the mixing of impurities, and it is difficult to obtain high-purity microalgae, which cannot meet the requirements of scientific research and production for high-quality microalgae. Therefore, it is urgent to develop a rich culture medium that can significantly improve the content of beta-1,3-glucan in microalgae and to improve the method for efficient and stable separation, which is of great significance for promoting the development of microalgae industry and fully tapping the application value of beta-1,3-glucan. SUMMARY

[0003] Therefore, the present application aims to provide a culture method of a beta-1,3-glucan-producing microalgae which is reliable in implementation and flexible in application.

[0004] In order to achieve the above technical purposes, the technical scheme adopted by the present application is as follows:

[0005] A high-efficiency high-density culture medium for producing beta-1,3-glucan microalgae, comprising the following components at the following concentrations: 4.5-5.5 g / L glucose, 14.0-16.0 g / L agar, 0.4-0.6 g / L MgSO4·7H2O, 0.2-0.4 g / L KH2PO4, 0.070-0.080 g / L CaCl2·2H2O, 3.1-3.3 mg / L FeCl3·6H2O, 3.5-4.5 mg / L EDTA·2Na, 1.1-1.3 mg / L H3BO3, 0.17-0.19 mg / L MnCl2·4H2O, 0.015-0.025 mg / L ZnSO4·7H2O, 0.011-0.013 mg / L Co(NO3)2·6H2O, 0.005-0.007 mg / L NaMoO4·2H2O, 1.5-2.5 g / L C2H5OH, 95-105 mg / L vitamin B1, 0.4-0.6 mg / L vitamin B 12 , and 0.04-0.06 g sodium citrate.

[0006] As a preferred embodiment, preferably, the medium comprises the following components at the following concentrations: 5.0 g / L glucose, 15.0 g / L agar, 0.5 g / L MgSO4·7H2O, 0.3 g / L KH2PO4, 0.075 g / L CaCl2·2H2O, 3.2 mg / L FeCl3·6H2O, 4.0 mg / L EDTA·2Na, 1.2 mg / L H3BO3, 0.18 mg / L MnCl2·4H2O, 0.02 mg / L ZnSO4·7H2O, 0.012 mg / L Co(NO3)2·6H2O, 0.006 mg / L NaMoO4·2H2O, 2 g / L C2H5OH, 100 mg / L vitamin B1, 0.5 mg / L vitamin B 12 , and 0.05 g sodium citrate.

[0007] The above-mentioned high-density culture medium is close to the composition of the basic medium for microalgae culture, and can ensure that the microalgae grow well in the high-efficiency high-density culture medium.

[0008] In terms of biomass growth, the microalgae biomass can be greatly improved compared to traditional culture media, achieving efficient proliferation.

[0009] In terms of beta-1,3-glucan content, the proportion of beta-1,3-glucan in cell dry weight can be significantly increased, greatly enriching the target product.

[0010] In addition, the culture medium is low in cost, uses common and affordable materials as raw materials, reduces the use of expensive nutrients, reduces the culture cost, and ensures stable medium composition and easy access, providing feasibility for large-scale industrial cultivation of microalgae and beta-1,3-glucan production, and showing application potential in the fields of biological energy, food, medicine and other microalgae resource-dependent fields.

[0011] Based on the above, the present application also provides a microalgae culture medium, which comprises the above-mentioned high-efficiency enrichment culture medium for beta-1,3-glucan-producing microalgae.

[0012] Based on the above, the present application also provides a microalgae culture method, which comprises the following steps:

[0013] S1, the microalgae mother liquor is subjected to vacuum filtration, the filter paper is placed in the above-mentioned high-efficiency enrichment culture medium for beta-1,3-glucan-producing microalgae, and then weak light culture is carried out;

[0014] S2, 1 mL of the algal liquid after step S1 culture is subjected to centrifugal treatment, then the supernatant is discarded, and the algal cells are collected by washing with physiological saline or ddH2O containing 100 ug / mL or 50 ug / mL ampicillin for 8-10 times and then washing with physiological saline or ddH2O without ampicillin for 2 times and centrifuging to remove the supernatant;

[0015] S3, the collected algal cells are subjected to gradient dilution in the above-mentioned high-efficiency enrichment culture medium for beta-1,3-glucan-producing microalgae;

[0016] S4, 10% of the dilution liquid is coated, and the coated plate is inverted in the culture room and subjected to separation culture under light;

[0017] S5, after the completion of S4 culture, single algal colonies are picked up by using a sterile inoculation needle and inoculated on a plate with the above-mentioned high-efficiency enrichment culture medium for beta-1,3-glucan-producing microalgae for purification culture, and the separation and purification culture of the target microalgae are completed.

[0018] As a preferred implementation option, preferably, in step S1 of the present application, the microalgae culture environment parameters are: culture temperature 26℃, light intensity 2500Lx, and culture duration 2-3 days.

[0019] As a preferred implementation option, preferably, in step S2 of the present application, the algal liquid centrifugation parameters are: rotation speed 5000r / min, and centrifugation time 10min.

[0020] As a preferred implementation option, preferably, in step S4 of the present application, the microalgae separation culture environment parameters are: culture temperature 26℃, light intensity 6000-8000Lx, and culture duration 3-4 days.

[0021] As a preferred implementation option, preferably, in step S5 of the present scheme, the microalgae culture environment parameters are: culture temperature 26℃, light intensity 6000-8000Lx, light and dark cycle 12h:12h.

[0022] Based on the above, the present scheme also proposes a culture method for producing beta-1,3-glucan microalgae, which applies the microalgae culture method described above.

[0023] The microalgae culture method of the present scheme has remarkable results and outstanding advantages compared with traditional methods. It can efficiently extract single microalgae strains from complex water samples, effectively solve the problem of mixed and difficult to distinguish colonies in traditional plate streak culture, and significantly improve the separation accuracy. In terms of operation, the tedious and time-consuming multiple suction operation of the water droplet separation method is abandoned, the risk of impurity mixing is reduced, the demand for microalgae purity in scientific research and production is met, and the application of microalgae in the fields of biological energy, food, medicine and other fields is provided with solid protection. And the subsequent step of transferring the single colony after culture to the rich culture medium plate for purification culture and other steps are coordinated to achieve the purpose of accurately separating the specific algae species single colony of microalgae, making the whole separation process more scientific and efficient.

[0024] By adopting the technical scheme, the present application has the beneficial effects compared with the prior art: the present scheme proposes a high-efficiency rich culture medium for beta-1,3-glucan in microalgae and a microalgae culture method, which realizes efficient cooperation from microalgae culture to separation. The rich culture medium can significantly improve the efficient accumulation of beta-1,3-glucan by optimizing the ratio of carbon and nitrogen sources and nutrients, and can promote the biomass of microalgae, solving the problem of low product content in conventional culture. The matching microalgae culture method breaks through the limitations of traditional plate streaking colony mixing, water droplet separation time-consuming and easy pollution, accurately extracts high-purity single strain from complex water samples, and has high standardization degree of operation, reducing the influence of artificial randomness. The synergistic application of the two can not only efficiently obtain microalgae with high biomass and high glucan content, but also can guarantee the separation purity, provide high-quality resources for scientific research and industrial production, and promote the application in the fields of food, medicine and other fields. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without creative labor.

[0026] Figure 1 The figure shows the cell morphology of chlamydomonas cultured by the conventional medium and the rich culture medium;

[0027] Figure 2 Figure of β-1,3-glucan content in Leishnannia sp. cultured with different carbon sources, wherein A is glucose, B is sodium acetate, C is glycerol, and D is ethanol. DETAILED DESCRIPTION

[0028] The application will be further described below in conjunction with the accompanying drawings and examples. It is particularly pointed out that the following examples are only for illustration of the application, but do not limit the scope of the application. Similarly, the following examples are only part of the examples of the application, but not all examples. All other examples obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the application.

[0029] The embodiment provides a high-efficiency rich culture medium for β-1,3-glucan-producing microalgae, which comprises the following components at the following concentrations: 5.0 g / L glucose, 15.0 g / L agar, 0.5 g / L MgSO4·7H2O, 0.3 g / L KH2PO4, 0.075 g / L CaCl2·2H2O, 3.2 mg / L FeCl3·6H2O, 4.0 mg / L EDTA·2Na, 1.2 mg / L H3BO3, 0.18 mg / L MnCl2·4H2O, 0.02 mg / L ZnSO4·7H2O, 0.012 mg / L Co(NO3)2·6H2O, 0.006 mg / L NaMoO4·2H2O, 2 g / L C2H5OH, 100 mg / L vitamin B1, 0.5 mg / L vitamin B 12 , and 0.05 g sodium citrate.

[0030] In addition to the above, the embodiment further provides a microalgae culture method, which comprises the following steps.

[0031] S1, subjecting a microalgae mother liquor (Leishnannia sp. mother liquor) to vacuum filtration, placing filter paper in the high-efficiency rich culture medium for β-1,3-glucan in microalgae described above, and then placing in weak light for culture; wherein the microalgae culture environment parameters are as follows: culture temperature is 26°C, light intensity is 2500 Lx, and culture duration is 2-3 days;

[0032] S2, centrifuging 1 mL of the algal liquid after the culture in step S1 under the treatment conditions of a rotation speed of 5000 r / min and a centrifugation time of 10 min, then discarding the supernatant, washing with physiological saline or ddH2O containing 100 ug / mL ampicillin for 9 times, washing with physiological saline or ddH2O without ampicillin for 2 times, centrifuging to remove the supernatant, and collecting the algal cells;

[0033] S3, gradient dilution is carried out on the microalgae in the β-1, 3-glucan high-efficiency rich culture medium of the microalgae;

[0034] S4, 10% dilution is coated, and the coated plate is inverted in the culture room and separated and cultured under illumination; wherein, the microalgae separation and culture environment parameters are: culture temperature 26℃, illumination intensity 6000-8000Lx, and culture duration 3-4 days;

[0035] S5, after S4 culture is completed, single algae fall is picked by using a sterile inoculation needle, and is streaked and inoculated on a plate with the β-1, 3-glucan high-efficiency rich culture medium of the microalgae to carry out purification culture (the microalgae culture environment parameters are: culture temperature 26℃, illumination intensity 6000-8000Lx, and light-dark cycle 12h:12h), and the separation and purification culture of the target microalgae (Euglena gracilis) is completed.

[0036] In the embodiment scheme, the role of step S5 is that: after the previous microalgae separation and culture, the colony purity is still not ideal. At this time, the single colony is picked out and moved to the rich culture medium plate for microalgae to carry out purification culture, so that the single microalgae cell can grow into a single pure colony, and a pure culture with the same genetic background and consistent physiological characteristics is obtained. In this way, the subsequent research on the biological characteristics, physiological metabolism and genetic characteristics of the microalgae can be more accurate, and will not be disturbed by the mixed algae or microorganisms, so as to ensure that the experimental results are true and reliable. In addition, the purified microalgae can be stored on the rich culture medium plate for a long time, which is not only convenient to take, but also can maintain its genetic stability and physiological characteristics, reduce the possibility of strain degradation or variation, and thus the microalgae separation work is completed.

[0037] After 7 days of cultivation, the microalgae morphology is observed by using a bright field microscope, Figure 1 (1), Figure 1 (2) are respectively the naked algae cell morphology before and after magnification.

[0038] By replacing the carbon source in the β-1, 3-glucan high-efficiency rich culture medium of the microalgae, glucose is replaced by sodium acetate, glycerol and ethanol in turn, and then microalgae culture is carried out, and the results are as shown in Figure 2 , Figure 2 is the content diagram of β-1, 3-glucan in the microalgae cultured under different carbon sources, wherein A is glucose, B is sodium acetate, C is glycerol, and D is ethanol.

[0039] The above only describes some embodiments of the present application, and does not limit the protection scope of the present application, and any equivalent device or equivalent process conversion using the content of the present application specification and drawings, or direct or indirect application in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. A highly efficient enrichment culture medium for microalgae producing β-1,3-glucan, characterized in that, It comprises the following components at the following concentrations: 4.5–5.5 g / L glucose, 14.0–16.0 g / L agar, 0.4–0.6 g / L MgSO4·7H2O, 0.2–0.4 g / L KH2PO4, 0.070–0.080 g / L CaCl2·2H2O, 3.1–3.3 mg / L FeCl3·6H2O, 3.5–4.5 mg / L EDTA·2Na, 1.1–1.3 mg / L H3BO3, 0.17–0.19 mg / L MnCl2·4H2O, 0.015–0.025 mg / L ZnSO4·7H2O, 0.011–0.013 mg / L Co(NO3)2·6H2O, and 0.005–0.007 mg / L. NaMoO4·2H2O, 1.5–2.5 g / L; C2H5OH, 95–105 mg / L; Vitamin B1, 0.4–0.6 mg / L 12 0.04–0.06 g sodium citrate.

2. The highly efficient enrichment culture medium for β-1,3-glucan-producing microalgae as described in claim 1, characterized in that, It includes components at the following concentrations: 5.0 g / L glucose, 15.0 g / L agar, 0.5 g / L MgSO4·7H2O, 0.3 g / L KH2PO4, 0.075 g / L CaCl2·2H2O, 3.2 mg / L FeCl3·6H2O, 4.0 mg / L EDTA·2Na, 1.2 mg / L H3BO3, 0.18 mg / L MnCl2·4H2O, 0.02 mg / L ZnSO4·7H2O, 0.012 mg / L Co(NO3)2·6H2O, 0.006 mg / L NaMoO4·2H2O, 2 g / L C2H5OH, 100 mg / L vitamin B1, 0.5 mg / L vitamin B2 12 0.05g sodium citrate.

3. A microalgae culture medium, characterized in that, It includes the highly efficient enrichment culture medium for β-1,3-glucan-producing microalgae as described in claim 1 or 2.

4. A method for cultivating microalgae, characterized in that, It includes the following steps: S1. The microalgae mother liquor is filtered under reduced pressure, and the filter paper is placed in the high-efficiency enriched culture medium for β-1,3-glucan-producing microalgae as described in claim 1 or 2, and then incubated under weak light. S2. Take 1 mL of the algal culture after step S1 and centrifuge it. Then discard the supernatant and wash it with physiological saline or ddH2O containing 100 ug / mL or 50 ug / mL ampicillin. Repeat 8-10 times. Then wash it twice with physiological saline or ddH2O without ampicillin. Centrifuge to remove the supernatant and collect the algal cells. S3. Select the collected algal cells and perform gradient dilution in a high-efficiency enriched culture medium containing the β-1,3-glucan-producing microalgae as described in claim 1. S4. Take 10% dilution solution and spread it. Invert the plate after spreading in the culture room and carry out separation culture under light. After S5 and S4 cultures were completed, a single algal colony was picked up with a sterile inoculation needle and streaked onto a plate containing the highly efficient enriched medium for producing β-1,3-glucan microalgae as described in claim 1 for purification culture, thus completing the isolation, purification, and culture of the target microalgae.

5. The microalgae cultivation method as described in claim 4, characterized in that, In step S1, the microalgae culture environment parameters are: culture temperature 26℃, light intensity 2500Lx, and culture time 2-3 days.

6. The microalgae cultivation method as described in claim 4, characterized in that, In step S2, the centrifugation parameters for the algal solution are: rotation speed 5000 r / min, centrifugation time 10 min.

7. The microalgae cultivation method as described in claim 4, characterized in that, In step S4, the environmental parameters for microalgae isolation and culture are: culture temperature 26℃, light intensity 6000-8000Lx, and culture time 3-4 days.

8. The microalgae cultivation method as described in claim 4, characterized in that, In step S5, the microalgae culture environment parameters are: culture temperature 26℃, light intensity 6000-8000Lx, and light-dark cycle 12h:12h.

9. A method for cultivating microalgae that produce β-1,3-glucan, characterized in that, Its application includes the microalgae cultivation method as described in any one of claims 4 to 8.

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