A selenium Spirulina mutant strain and its application

Abstract

The present invention relates to a selenium Spirulina mutant strain, with the classification number: Spirulina platensis, the preservation number: CCTCC NO: M 20221180, the strain name: FACHB-Se1500, and the preservation location: Wuchang District, Bayi Road, Luojia Mountain, Wuhan City, Hubei Province, China Center for Type Culture Collection. In the present invention, Spirulina is mutated under the condition of ultraviolet mutagenesis to obtain a Spirulina mutant strain with a high content of selenomethionine, and the content can be increased by 64.8%. Using this as a material, a Spirulina mutant strain with high selenium tolerance is further screened. Among them, the algal strain that can tolerate 800 mg / L Na2SeO3 has the best salt tolerance effect and can tolerate 1000 mM NaCl.

Description

Technical Field

[0001] The present invention relates to the breeding and screening of selenium-rich Spirulina, in particular to a selenium Spirulina mutant strain and its application. Background Art

[0002] The optimal growth temperature of Spirulina is 35 - 37 °C, and it has good heat resistance. The optimal growth pH range is 8.3 - 11.0. When the pH is greater than 11.0, it will be unfavorable for growth. Under normal nutrition and temperature conditions, light becomes an important factor affecting the growth of Spirulina. When culturing outdoors, the light source is mainly the sun. The management during the large-pond cultivation is an important guarantee for stable and high yields. The main contents of the management are to regularly measure and record the air temperature, water temperature, pH, OD value, remove debris, and regularly switch on and off the stirrer. Generally, when culturing in the algal species pond and large pond, attention should be paid to controlling the pH around 10. The methods are generally to increase NaHCO3, add or replace fresh culture medium, increase the supply of CO2, etc. The optimal temperature control is 25 - 35 °C. Summary of the Invention

[0003] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a selenium Spirulina mutant strain with strong salt tolerance and high organic selenium content and its application. The present invention has screened 1 strain of high selenium-rich Spirulina through ultraviolet mutagenesis, which can efficiently convert inorganic selenium into organic selenium and is resistant to Na2SeO3 up to 800 mg / L.

[0004] To achieve the above-mentioned one purpose of the present invention, the technical solution adopted by the present invention is as follows:

[0005] A selenium Spirulina mutant strain, with the classification number: Spirulina platensis, deposit number: CCTCC NO: M20221180, strain name: FACHB-Se1500, deposit location: China Center for Type Culture Collection, Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0006] Moreover, the culture conditions of the mutant strain are: the temperature is 20 °C, pH = 9.5, and the light intensity is 40 μE·M -2 ·S -1 and culture is carried out under this condition.

[0007] Moreover, the yield of ergothioneine of the selenium Spirulina mutant strain is 22 - 33 μg / g.

[0008] Moreover, the selenium Spirulina mutant strain is resistant to a Na2SeO3 concentration of 800 mg / L.

[0009] The beneficial effects of the present invention are as follows:

[0010] Selenoneine is a selenium-containing small molecule with antioxidant function. In the present invention, Spirulina was mutated under ultraviolet mutagenesis conditions to obtain a Spirulina mutant strain with a high content of selenoneine, and the content can be increased by 64.8%. Using this as the material, Spirulina mutant strains with high selenium tolerance were further screened. Among them, the algal strain that can tolerate 800 mg / L Na2SeO3 has the best salt tolerance effect and can tolerate 1000 mM NaCl.

[0011] In the present invention, strains with relatively high salt tolerance were obtained through salt tolerance screening. During the comparison process, the applicant found that there is a positive relationship between salt tolerance and the conversion of high Na2SeO3 directly. Description of the Drawings

[0012] Figure 1 It is a microscope photograph of Spirulina in Example 1 of the present invention. Detailed Embodiments

[0013] The present invention will be further described in detail below in conjunction with the embodiments and the drawings. This embodiment is implemented on the premise of the technology of the present invention. Now, the detailed implementation methods and specific operation processes are given to illustrate the creativity of the present invention. However, the protection scope of the present invention is not limited to the following embodiments.

[0014] Example 1

[0015] The applicant detected selenoneine in the fermentation Spirulina products through analysis. In order to obtain a transformed algal strain with a high concentration of selenoneine, the method of obtaining mutants by ultraviolet mutagenesis was used to obtain mutant Spirulina algal strains. The specific method is as follows:

[0016] The original strain is Spirulina platensis (FACHB-882, from the Freshwater Algae Species Bank of the Chinese Academy of Sciences). The steps are as follows.

[0017] 1. Mutagenesis: The Spirulina was broken into single cells by ultrasonic fragmentation method. The culture dish containing the algal solution was placed in an ultraviolet mutagenesis instrument, and the ultraviolet irradiation time was 40 s, using a 20 W ultraviolet lamp tube.

[0018] 2. Screening: After ultraviolet mutagenesis, the algal strain was placed in a dark environment to recover overnight. It was transferred to the normal Zarrouk medium to grow for one week. 192 "single species" of Spirulina were separated by the micro-pipette method and expanded for culture. After culturing in the Zarrouk medium containing 100 mg / L Na2SeO3 for 2 weeks, the cells were lysed and selenoneine was extracted, and its content was detected by the HPLC-ICP-MS method.

[0019] 3. A total of 3 Spirulina algal strains with the content of selenoneine increased by more than 15% were screened, namely SEN-16#, SEN-73#, and SEN-136#.

[0020] Algal strain number Ergothioneine selenium content Wild type 19.9 μg / g SEN-16# 32.8 μg / g SEN-73# 23.1 μg / g SEN-136# 30.2 μg / g

[0021] Zarrouk Medium Formula (1L)*

[0022]

[0023] *CaCl2·2H2O (1000X): Weigh 8 g of CaCl2·2H2O, dissolve it in distilled water, and make up the volume to 200 mL; FeSO4·7H2O (1000X): Weigh 2 g of FeSO4·7H2O, dissolve it in distilled water, and make up the volume to 200 mL.

[0024] A5 Medium Formula (1L)

[0025]

[0026] In order to obtain transformed strains with high selenium content, using the SEN-136# algal strain as the parent, screening was carried out by the ultraviolet mutation method to obtain high-selenium spirulina algal strains. The specific method is as follows:

[0027] 1. Mutation: Break the obtained spirulina into single cells, place the culture dish containing the algal solution into an ultraviolet mutagenesis instrument, the ultraviolet irradiation time is 40 s, use a 20 W ultraviolet lamp tube, and place it in the dark condition for overnight recovery.

[0028] 2. Screening: Screen using Zarrouk medium with different Na2SeO3 concentration gradients, and the gradient ranges are: 400 mg / L, 600 mg / L, 800 mg / L, 1000 mg / L, 1200 mg / L.

[0029] Cultivation conditions: 25 - 30 °C, pH = 9.5, light intensity is 40 μE·M -2 ·S -1 and cultivate under this condition, and the medium is Zarrouk medium containing Na2SeO3.

[0030] Detect the selenium content of the algal strains obtained from the above 5 rounds of screening respectively

[0031] Mutant algal strain number <![CDATA[Addition amount of Na2SeO3]]> Total selenium content of algal strain FACHB-Se500 400 mg / L 350.5 μg / g FACHB-Se1000 600 mg / L 819.21 μg / g FACHB-Se1500 800 mg / L 1286.9 μg / g FACHB-Se2000 1000 mg / L 1328.3 μg / g FACHB-Se2500 1200 mg / L 1632.7 μg / g

[0032] According to the change of the current spirulina cultivation environment, the land salinization is becoming more and more serious, and the salt tolerance of the algal strains is becoming increasingly important. Considering the salt tolerance, the applicant conducts salt tolerance selection on the above strains, and the steps are as follows:

[0033] The strains screened in the above 5 rounds were subjected to salt tolerance screening using Zarrouk medium with different NaCl concentration gradients. The gradient ranges were 400 mmol / L, 600 mmol / L, 800 mmol / L, 1000 mmol / L, and 1200 mmol / L respectively.

[0034] Cultivation conditions: 25 - 30 °C, pH = 9.5, light intensity of 40 μE·M -2 ·S -1 The cultivation results under these conditions showed that the salt tolerance of the FACHB - Se1500 strain was significantly improved, and it could tolerate 1000 mmol / L of NaCl. However, there was no significant difference in the salt tolerance of FACHB - Se500, FACHB - Se1000, and FACHB - Se2000 compared with the wild type, and the highest tolerance was 800 mmol / L of NaCl.

[0035] Based on the comprehensive consideration of salt tolerance and selenium - enrichment characteristics, the FACHB - Se1500 algal beads were used as the strain for subsequent production in this application and were preserved. The preservation number: CCTCC NO: M 20221180, strain name: FACHB - Se1500, preservation location: China Center for Type Culture Collection (CCTCC), Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0036] Application Example 1

[0037] Strain cultivation conditions: 25 - 30 °C, pH = 9.5, 1000 mmol / L of NaCl, 800 mg / L of Na2SeO3, light intensity of 40 μE·M -2 ·S -1 Cultivation was carried out under these conditions. Medium: Zarrouk medium, for 10 days.

[0038] Protein purification method: Weigh the broken algal powder and dissolve it in 200 mL of 0.1 M NaOH solution; stir at 80 revolutions per minute for 2 h; centrifuge at 6000 rpm for 15 min at 10 °C to obtain a precipitate and supernatant;

[0039] Take the supernatant, add acetic acid to adjust the pH to 5, and let it stand for 1 h to fully precipitate the protein; centrifuge at 6000 rpm for 15 min at 10 °C to obtain a protein precipitate and supernatant; the protein precipitate is the selenium - containing protein.

[0040] Through detection, the contents of some selenium compounds in the extracted selenium - containing protein are as follows

[0041]

[0042] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A selenium Spirulina mutant strain, characterized in that: Classification number: Spirulina platensis , deposit number: CCTCC NO: M20221180, strain name: FACHB-Se1500; The selenium Spirulina mutant strain is resistant to a Na2SeO3 concentration of 800 mg / L and can tolerate 1000 mmol / L of NaCl.

2. The selenium spirulina mutant strain according to claim 1, wherein: The culture conditions of the mutant strain are as follows: the medium is Zarrouk medium, and the strain culture conditions are: 25 - 30 °C, pH = 9.5, 1000 mmol / L of NaCl, 800 mg / L of Na2SeO3, and the light intensity is 40 μE·M -2 ·S -1 Under these conditions, the culture is carried out with Zarrouk medium for 10 days.

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