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Preparation method of sutorius brunneissimus agglutinin with relatively high agglutination activity

A lectin and activity technology, which is applied in the field of the preparation of the lectin of Boletus taeniae, and achieves the effect of helping development and utilization

Pending Publication Date: 2019-12-13
KUNMING INST OF EDIBLE FUNGI CHINA NAT SUPPLY & MARKETING GENERAL COOP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Boletus tea brown is rich in nutrients such as protein, amino acid, dietary fiber, trace elements, etc. At the same time, studies have also found that it is rich in physiologically active components such as ergosterol, monomethyl fumarate, and nicotinamide. But there is no report about the lectin of Boletus theophaeus

Method used

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preparation example Construction

[0017] The preparation method of the boletus tea brown lectin with high agglutination activity according to the present invention comprises pretreatment, leaching, first centrifugation, static precipitation, second centrifugation and post-processing steps, specifically including:

[0018] A. Pre-treatment: select fresh fruiting body of Boletus tea brown, cut into pieces, add Tris-HCl buffer solution and pound in a tissue grinder to obtain material a;

[0019] B. Leaching: add Tris-HCl buffer solution 10-15 times the volume of material a to material a to extract to obtain extract b;

[0020] C. Centrifugation for the first time: centrifuge the extract b and discard the precipitate, collect the supernatant to obtain material c;

[0021] D. Static precipitation: Add solid ammonium sulfate to the material c to 30~40% saturation, and static precipitation to obtain material d;

[0022] E, the second centrifugation: the material d is centrifuged and the precipitate is collected to o...

Embodiment 1

[0038] (1) Extraction of crude lectin: Weigh and cut the fresh fruiting body of Boletus tea brown, add a small amount of Tris-Hcl buffer (0.01mol / L, pH7.4), mash with a tissue grinder, and then follow the Add Tris-Hcl buffer with a solid-liquid ratio of 1:10, extract at 4°C for 24-48h, filter the filtrate and centrifuge at 4°C, 12000rmp / min for 15min, discard the precipitate, and collect the supernatant. Add solid ammonium sulfate to 35% saturation, dialyze at 4°C for 12 hours, centrifuge at 4°C and 12000rmp / min for 15min, and collect the precipitate. Add 5 mL of phosphate buffered saline (PBS buffer, 0.01mol / L, pH7.4), fully dialyze to remove salt, detect agglutination activity, and obtain crude lectin after freeze-drying.

[0039] (2) DAEA-Cellulose ion-exchange column chromatography: Take the crude lectin from step (1), dissolve it in phosphate buffer, and load it on a DAEA-Cellulose ion-exchange column that has been pre-balanced with 3 column volumes in phosphate buffer T...

Embodiment 2

[0042] (1) Extraction of crude lectin: Weigh and cut the fresh fruiting body of Boletus tea brown, add a small amount of Tris-Hcl buffer (0.01mol / L, pH7.4), mash with a tissue grinder, and then follow the Add Tris-Hcl buffer at a solid-liquid ratio of 1:12, extract at 4°C for 36 hours, filter the filtrate and centrifuge at 10,000 rpm for 15 minutes at 4°C, discard the precipitate, and collect the supernatant. Add solid ammonium sulfate to 40% saturation, dialyze at 4°C for 12 hours, centrifuge at 10,000rmp / min for 15min at 4°C, and collect the precipitate. Add 5 mL of phosphate buffered saline (PBS buffer, 0.01mol / L, pH7.4), fully dialyze to remove salt, detect agglutination activity, and obtain crude lectin after freeze-drying.

[0043] (2) DAEA-Cellulose ion-exchange column chromatography: Take the crude lectin from step (1), dissolve it in phosphate buffer, and load it on a DAEA-Cellulose ion-exchange column that has been pre-balanced with 3 column volumes in phosphate buff...

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Abstract

The invention discloses a preparation method of sutorius brunneissimus agglutinin with relatively high agglutination activity. The preparation method comprises the following steps: crushing fresh sutorius brunneissimus sporocarp, and performing extraction, precipitation, centrifuging, dialysis desalination, freeze-drying and the like to obtain a lectin crude product; and carrying out DAEA ion exchange column chromatography, sephadex G-100 molecular gel filtration chromatography and the like to obtain the sutorius brunneissimus agglutinin. The sutorius brunneissimus agglutinin with high agglutination activity is obtained through the processes of extraction, precipitation, dialysis desalination, ion exchange, gel chromatography and the like on fresh sutorius brunneissimus, and multidirectional development and utilization of active ingredients of the sutorius brunneissimus are facilitated.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a preparation method of boletus theopherus agglutinin with high agglutination activity. Background technique [0002] Tea brown boletus (Sutorius brunneissimus), also known as sheep liver, black sheep liver, black boletus, etc., belongs to Basidiomycota, Agaricomycetes, Boletes, Boletaceae, Heterochromic Boletus , is a medicinal and edible fungus widely distributed in southwest China such as Yunnan, Guizhou, and Sichuan. Studies have shown that Boletus tea brown has functions such as anti-tumor, lowering blood sugar, protecting liver and gallbladder, improving human immunity, and regulating endocrine. Due to the special nutritional mode, the fungus has not yet been artificially cultivated, and its development and utilization is becoming a research hotspot of scholars at home and abroad. [0003] Lectin is a glycoprotein or carbohydrate-binding protein of non-imm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/375C07K1/36C07K1/34C07K1/30C07K1/18C07K1/16C07K1/14
CPCC07K14/375
Inventor 严明邓雅元杨璐敏游金坤王婷婷张月娇
Owner KUNMING INST OF EDIBLE FUNGI CHINA NAT SUPPLY & MARKETING GENERAL COOP
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