Method for extracting selenium protein and ergothioneine from spirulina

Abstract

The application relates to a method for extracting ergothioneine and selenoprotein from spirulina, wherein after culturing FACHB-Se1500 strains under the above-mentioned nitrogen deficiency induction condition, a product is obtained, and after purification, selenoprotein containing ergothioneine is obtained; the FACHB-Se1500 strains are cultured in a Zarrouk+Se culture medium for 10 days, and the strain culture conditions are as follows: 25-30 DEG C, pH=9.5, and light intensity is 40 muE.m-2.s-1 ‑2 ·S ‑1 After reaching the plateau, the strains are respectively transferred to a Zarrouk-N, a Zarrouk-Fe and a Zarrouk-Mg culture medium for induction culture for 2 days, and after collecting the algal powder, selenoprotein containing ergothioneine is obtained after purification. The application further screens a spirulina mutant strain with high selenium tolerance, wherein the algal strain tolerating 800 mg / L Na2SeO3 has the best salt tolerance and can tolerate 1000 mM NaCl; and it is found again through the nitrogen deficiency induction that the ergothioneine content can reach 103.65 mu g / g.

Description

Technical Field

[0001] This invention belongs to the field of ergot selenoside production methods, and in particular a method for extracting ergot selenoside and selenoprotein from spirulina. Background Technology

[0002] The method disclosed in Japanese Patent No. 5669056 is a method for extracting ergot selenoside from the organs or blood of fish. However, the amount of ergot selenoside contained in the method is very small, and there is a problem that a large number of fish are needed to obtain a large amount of ergot selenoside.

[0003] The Japanese Ministry of Health, Labour and Welfare, in its report from the "Review Committee on the Formulation of Dietary Intake Standards for Japanese People (2015 Edition)" published on March 28, 2018, the enzyme Egt1, which reacts with selenocysteine, was used to synthesize ergot selenoside in vivo by overexpressing the gene SPBC1604.01 encoding this enzyme in transformed Schizophrenia marina. However, the problem of very low ergot selenoside yield from the transformation of Schizophrenia marina remains.

[0004] All of the above-mentioned methods for producing ergot selenoside have very low yields and cannot meet the needs. In order to overcome the problems of the prior art, the present invention provides a method for extracting ergot selenoside and selenoprotein from spirulina selenium, which can significantly increase the yield of ergot selenoside. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for extracting ergot selenoside and selenoprotein from Spirulina. This invention uses ultraviolet mutagenesis to screen a highly selenium-rich Spirulina strain, which, after induced culture, can increase the expression level of ergot selenoside, and further allows for the extraction of selenoprotein products containing ergot selenoside.

[0006] To achieve the aforementioned objective of this invention, the technical solution adopted by this invention is as follows:

[0007] A method for extracting ergot selenoside and selenoprotein from Spirulina involves culturing the FACHB-Se1500 algal strain under the aforementioned nitrogen-deficient induction conditions to obtain the product, followed by purification to obtain a protein powder containing ergot selenoside.

[0008] Furthermore, the FACHB-Se1500 algal strain was cultured in Zarrouk+Se medium for 10 days under the following conditions: 25-30℃, pH=9.5, and light intensity of 40 μE·M. -2 ·S -1 After reaching the plateau phase, they were transferred to Zarrouk-N, Zarrouk-Fe, or Zarrouk-Mg media for induction culture for 2 days.

[0009] Furthermore, the purification method is as follows:

[0010] (1) Take 13.3 g of Spirulina powder, add 200 mL of 0.1 M NaOH solution, and stir for 2 h;

[0011] (2) After stirring in the previous step, put the solution into an Eppendorf Centrifuge 5910 R centrifuge and centrifuge at 6000 rpm for 10 minutes. Take the supernatant and discard the precipitate.

[0012] (3) Add 8 mL of glacial acetic acid to the supernatant obtained in the previous step to adjust the pH value to 4.0, and stir for 2 h;

[0013] (4) After stirring in the previous step, put the solution into an Eppendorf Centrifuge 5910 R centrifuge and centrifuge at 6000 rpm for 10 minutes. Take the precipitate and discard the supernatant.

[0014] (5) Add 200 mL of anhydrous ethanol to resuspend the above and stir for 1 h;

[0015] (6) Centrifuge at 6000 rpm for 10 min, collect the precipitate, discard the supernatant, and dry to obtain protein powder;

[0016] (7) Take 1g of protein powder, dissolve it with 4 mL of PBS buffer and 30 mg of alkaline protease, break it up with an ultrasonic cell disruptor at P = 60% power for 30 min, and place it in a 60℃ water bath for 30 min.

[0017] (8) Centrifuge at 5000 rpm / min, 4℃ for 15 min, take the supernatant, and make up to 100 mL with Milli-Q water. After stirring for 10 min, decolorize using a C18 column to obtain ergot selenium.

[0018] Furthermore, the classification number of the aforementioned FACHB-Se1500 is: Spirulina platensis Accession number: CCTCC M20221180, strain name: FACHB-Se1500, deposit location: China Center for Type Culture Collection, Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0019] The beneficial effects of this invention are as follows:

[0020] The strain involved in this invention can achieve a maximum selenoneine yield of 30.2 μg / g. Selenone is a selenium-containing small molecule with antioxidant functions. After UV mutagenesis, the selenone content of the product increased by 64.8%. Using this material, Spirulina mutant strains with high selenium tolerance were further screened. Among them, the strain tolerant to 800 mg / L Na2SeO3 showed the best salt tolerance and could tolerate 1000 mM NaCl. Through induction under nitrogen-deficient conditions, the selenone content could reach 103.65 μg / g. Attached Figure Description

[0021] Figure 1 This is a microscope photograph of Spirulina from Embodiment 1 of the present invention. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings. These embodiments are implemented based on the technology of the present invention. Detailed implementation methods and specific operating procedures are provided to illustrate the inventiveness of the present invention, but the scope of protection of the present invention is not limited to the following embodiments.

[0023] Example 1

[0024] A method for extracting ergot selenoside from spirulina, comprising the following steps:

[0025] The Spirulina strain used in this embodiment is a high-yield ergot selenoside, named FACHB-Se1500, with accession number CCTCC NO. M20221180. The strain name is FACHB-Se1500, and the deposit location is China Center for Type Culture Collection (CCTCC), Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0026] Selenoproteins and ergot selenosides are two high-value selenium-containing products found in spirulina. To obtain these two products, the applicant first increased the expression level of ergot selenosides through induced culture. The specific method is as follows:

[0027] The FACHB-Se1500 algal strain was cultured in Zarrouk+Se medium for 10 days. Culture conditions: 25-30℃, pH 9.5, light intensity 40 μE·M. -2 ·S -1 After reaching the plateau phase, the algae were transferred to Zarrouk-N, Zarrouk-Fe, and Zarrouk-Mg media for induced culture for 2 days, and algal powder was collected. The results showed that under nitrogen-deficient culture conditions, the ergot selenium content in the algal powder increased by 1.93 times.

[0028]

[0029] Zarrouk+Se medium formulation (1 L)*

[0030]

[0031] * CaCl2·2H2O (1000 X): Weigh 8 g CaCl2·2H2O, dissolve in distilled water, and bring the volume to 200 mL; FeSO4·7H2O (1000 X): Weigh 2 g FeSO4·7H2O, dissolve in distilled water, and bring the volume to 200 mL.

[0032] A5 culture medium formula (1 L)

[0033]

[0034] Zarrouk-N medium formulation (1 L)*

[0035]

[0036] Zarrouk-Fe medium formulation (1 L)*

[0037]

[0038] Zarrouk-Mg medium formulation (1 L)*

[0039]

[0040] To obtain high-purity selenoprotein products containing ergot selenoside, the applicant cultured the FACHB-Se1500 algal strain under the aforementioned nitrogen-deficiency induction conditions, following the screening conditions described above. The FACHB-Se1500 strain was cultured in Zarrouk + Se medium for 10 days. The culture conditions were: 25-30℃, pH=9.5, and light intensity of 40 μE·M. -2 ·S -1 After reaching the plateau phase, the algae were transferred to Zarrouk-N medium for induction culture for 2 days. After collecting the algal powder, a bio-extraction and purification method was established, as follows:

[0041] 1. Take 13.3 g of Spirulina powder, add 200 mL of 0.1 M NaOH solution, and stir for 2 h;

[0042] 2. Place the solution stirred in the previous step into an Eppendorf Centrifuge 5910 R centrifuge at 6000 rpm for 10 minutes, collect the supernatant and discard the precipitate;

[0043] 3. Add 8 mL of glacial acetic acid to the supernatant obtained in the previous step to adjust the pH to 4.0, and stir for 2 hours;

[0044] 4. Place the solution stirred in the previous step into an Eppendorf Centrifuge 5910 R centrifuge at 6000 rpm for 10 minutes, collect the precipitate and discard the supernatant;

[0045] 5. Add 200 mL of anhydrous ethanol to resuspend the above mixture and stir for 1 hour;

[0046] 6. Centrifuge at 6000 rpm for 10 min, collect the precipitate, and discard the supernatant;

[0047] 7. The obtained precipitate is dried using a freeze vacuum dryer to obtain protein powder;

[0048] 8. Dissolve 1g of protein powder in 4 mL of PBS buffer and 30 mg of alkaline protease. Dissolve the protein powder using an ultrasonic cell disruptor at 60% power for 30 min. Place the disrupted protein powder in a 60℃ water bath for 30 min.

[0049] 9. Centrifuge at 5000 rpm / min, 4℃ for 15 min, collect the supernatant, and dilute to 100 mL with Milli-Q water. After stirring for 10 min, decolorize using a C18 column to obtain ergot selenium.

[0050] The content of ergot selenine was determined by high performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS; HPLC: Dionex Ultimate 3000; ICP-MS: Thermo X2). (Separation conditions: stationary phase, C18 column (Shiseido); mobile phase, 3% methanol, 97% aqueous phase containing 25 mM ammonium acetate and pH adjusted to 4.0 with 0.1% trifluoroacetic acid).

[0051] The content of some selenium compounds in the extracted selenoprotein was analyzed as follows:

[0052]

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for extracting ergoselenoside and selenoprotein from Spirulina, characterized in that: The FACHB-Se1500 algal strain was cultured in Zarrouk+Se medium for 10 days. After reaching the plateau phase, it was transferred to Zarrouk-N medium for induced culture. After collecting algal powder, it was further processed by alkaline extraction and acid precipitation, alkaline protease hydrolysis, and C18 column decolorization to release proteins and ergot selenin. The classification number of FACHB-Se1500 is: Spirulina platensis Accession 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.

2. The method for extracting ergoselenoside and selenoprotein from Spirulina according to claim 1, characterized in that: The purification method is as follows: (1) Take 13.3 g of Spirulina powder, add 200 mL of 0.1 M NaOH solution, and stir for 2 h; (2) After stirring in the previous step, put the solution into an Eppendorf Centrifuge 5910 R centrifuge and centrifuge at 6000 rpm for 10 minutes. Take the supernatant and discard the precipitate. (3) Add 8 mL of glacial acetic acid to the supernatant obtained in the previous step to adjust the pH value to 4.0, and stir for 2 h; (4) After stirring in the previous step, put the solution into an Eppendorf Centrifuge 5910 R centrifuge and centrifuge at 6000 rpm for 10 minutes. Take the precipitate and discard the supernatant. (5) Add 200 mL of anhydrous ethanol to resuspend the above and stir for 1 h; (6) Centrifuge at 6000 rpm for 10 min, collect the precipitate, discard the supernatant, and dry to obtain protein powder; (7) Take 1g of protein powder, dissolve it with 4 mL of PBS buffer and 30 mg of alkaline protease, break it up with an ultrasonic cell disruptor at P = 60% power for 30 min, and place it in a 60℃ water bath for 30 min. (8) Centrifuge at 5000 rpm and 4℃ for 15 min, take the supernatant, make up to 100 mL with Milli-Q water, stir for 10 min, and then decolorize with a C18 column to obtain ergot selenium.

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