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Extraction method for chlorella vulgaris metallothioneins with whitening functions

A metallothionein and extraction method technology, which is applied in the field of chlorella metallothionein extraction, can solve the problems of difficulty in microbial culture and protein separation and extraction, high production cost, and non-compliance with animal protection requirements.

Inactive Publication Date: 2012-09-19
JIMEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cost of metallothionein obtained by the second method is lower than that of animal induction (that is, the first method), but due to the complexity of the medium material, the operation is difficult
[0004] To sum up, the current extraction of metallothionein either uses animals as raw materials, which has high production costs and does not meet the international requirements for animal protection; or uses microorganisms as raw materials, which make it difficult to culture microorganisms and separate and extract proteins.

Method used

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  • Extraction method for chlorella vulgaris metallothioneins with whitening functions
  • Extraction method for chlorella vulgaris metallothioneins with whitening functions
  • Extraction method for chlorella vulgaris metallothioneins with whitening functions

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 1) Add 0.05 mol / L zinc chloride to the algae liquid of Chlorella in the logarithmic growth phase, control the final concentration of zinc in the algae liquid within the range of 5-100 μmol / L, and continue to cultivate Chlorella 24- 120 h, during the cultivation process, the flasks were manually shaken 5 times a day, each time for about 3 min. Take 100 mL of each chlorella liquid in the culture group, centrifuge at 10000 r / min, collect the algal cells, wash the algal cells twice with 5 mmol / L EDTA-2Na and ultrapure water respectively, add 10 mmol / L Tris-HCl With 5 mL of buffer solution, the algal cells were disrupted by ultrasonic wave in ice bath for 5 min, centrifuged at 12000 r / min for 10 min, and the supernatant was collected.

[0026] 2) Chromatographic separation was performed using a TSK-gel column (G3000PWxl, 7.8 mm i.d.×300 mm, 6 μm), with 10 mmol / L Tris-HCl buffer as the mobile phase, and the eluate was passed through inductively coupled plasma mass spectrometr...

Embodiment 2

[0032] 1) Add ZnCl to Chlorella in logarithmic growth phase 2 , so that the final concentration of zinc in the algae liquid reached 50 μmol / L, and aerated for 72 hours.

[0033] 2) Algae cells were collected by centrifugation. After repeated freezing and thawing for 5 times, the cells were disrupted by ultrasonic waves, and the supernatant was collected by centrifugation at 12000 r / min for 10 min.

[0034] 3) Same as steps (4) and (5) in Example 1

[0035]4) Through the test of inhibition of tyrosinase activity, it was proved that when the concentration of chlorella metallothionein was 1.2 mg / mL, the activity of monophenolase could not be displayed within 2300 s, and the activity of diphenolase could be reduced by 87%. , indicating that the protein has a strong whitening effect.

Embodiment 3

[0037] 1) Add ZnCl to Chlorella in logarithmic growth phase 2 , so that the final concentration of zinc in the algal fluid reached 5 μmol / L, and aerated for 120 hours.

[0038] 2) Algae cells were collected by centrifugation, and the cells were disrupted by ultrasonic waves in an ice bath (540 w, 1.2 s on and 3 s off each time), and observed with a microscope until the cells were completely disrupted. The supernatant was collected by centrifugation at 12000 r / min for 10 min.

[0039] 3) Same as steps (4) and (5) in Example 1

[0040] 4) Through the test of inhibition of tyrosinase activity, the half-inhibition concentration of metallothionein on monophenolase activity is 0.5 mg / mL, and the half-inhibition concentration of diphenolase activity is 0.7 mg / mL, indicating that the protein Has whitening function.

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Abstract

The invention discloses an extraction method for chlorella vulgaris metallothioneins with whitening functions. According to the method, zinc chloride is adopted for stress culture of common marine chlorella vulgaris, the optimal induction condition of the metallothioneins can be obtained through investigation of different zinc stress concentrations and stress times, and the metallothionein products are obtained through the processes of extraction, separation and purification. Tyrosinase activity inhibiting tests prove that when the concentration of the chlorella vulgaris metallothioneins is 1.2 mg / ml, the activity of diphenolase can be reduced by 87 percent and monophenolase doesn't show activity within 2300 s, which shows that the chlorella vulgaris metallothioneins have favorable whitening functions and a wide application prospect in the fields of cosmetics and health products.

Description

technical field [0001] The invention relates to the preparation of chlorella metallothionein and a test method for its whitening function, in particular to an extraction method of chlorella metallothionein with whitening function. Background technique [0002] Metallothioneins (MTs) are a class of proteins with low molecular weight, high sulfhydryl content, and the ability to bind a large number of metal ions. Due to the special molecular structure and amino acid composition of MTs, this type of protein has physiological functions such as anti-oxidation, anti-radiation, and excretion of heavy metal ions in the body. However, at present, MTs are mostly induced and extracted from mammals, and the production cost and technical difficulty of the products are high, which makes MTs expensive. With the deepening of research, it is found that MTs exist in plants, prokaryotic algae and eukaryotic microorganisms. Although their content is usually very low in normal organism environme...

Claims

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Application Information

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IPC IPC(8): C07K14/405C07K1/16
Inventor 黄志勇杨洪
Owner JIMEI UNIV
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