A kind of enzymolysis preparation method of anticoagulant leech peptide

Abstract

The invention discloses a method for preparing an anticoagulant leech peptide by enzymolysis, which comprises: adding fresh hirudinaria manillensis which is refrigerated for not more than 24 hours into water, and homogenizing, wherein the weight ratio of water to the hirudinaria manillensis is (1-6):1; centrifuging the homogenate at a high speed of 10,000r / min for 10 minutes, filtering, settling, and mixing in a reactor according to a ratio of sediment to water of (3-15)g:100ml; keeping the pH value between 7.0 and 10.0, and adding an alkaline protease, wherein the weight ratio of the alkaline protease to the collagen in the bodies of the hirudinaria manillensis is (1-8):100; reacting at 40 to 70 DEG C for 1 to 5 hours with oscillating at a speed of 100 to 300rpm; and refreeze-drying and thus, obtaining the anticoagulant leech peptide. The method breaks through the limitation of source of natural hirudin anticoagulant active material, realizes the multiple purpose of the raw material and has the advantages of high product activity mild reaction conditions, environment friendliness, simple and controllable reaction process and the like.

Description

technical field

[0001] The invention relates to the field of enzymolysis preparation of bioactive peptides, in particular to an enzymolysis preparation method of anticoagulant leech peptide. Background technique

[0002] The Philippine leeches, commonly known as Phnom Penh leeches, leeches, and burdocks, are widely distributed in the south of the Yangtze River in my country, especially in Guangdong, Guangxi, and Hainan. For the treatment of lumps in the abdomen, blood stasis, amenorrhea, bruises and other diseases. Li Shizhen's "Compendium of Materia Medica" records: "Salty removes blood, bitterness beats blood; leech's salty and bitter, in order to remove blood accumulation, is the liver meridian blood and divides medicine, so it can clear the liver meridian and gather blood." The main component of phenanthrene is collagen, and it also contains a small amount of fatty acids and trace minerals.

[0003] Modern scientific research shows that the natural hirudin in the body i...

Examples

Embodiment 1

[0022] Take fresh Hirudo phenanthae that has been refrigerated for no more than 24 hours, add 4 times of water and mix it in a beater for homogenization, centrifuge the homogenate at 10000r / min for 10min, and refrigerate the supernatant for later use, weigh 9g of the precipitate and 100ml of water and mix it in a triangle In the bottle, adjust the pH to 8.0, add Alcalase 2.4L enzyme (alkaline protease, produced by Novozymes), shake in a water bath constant temperature oscillator at 55°C and 150 rpm, and react for 1.5h. Inactivate the enzyme for 10 minutes, adjust the pH to 7.0 after cooling, and freeze-dry to obtain anticoagulant leech peptide powder with anticoagulant activity greater than 500AT-U / g.

Embodiment 2

[0024] Take fresh Hirudo phenanthae that has been refrigerated for no more than 24 hours, add 3 times of water and mix it in a beater for homogenization, centrifuge the homogenate at 10,000r / min for 10min, and refrigerate the supernatant for later use, weigh 12g of the precipitate and mix it with 100ml of water in a triangular In the bottle, adjust the pH to 8.5, add Alcalase 2.4L enzyme, place in a water bath constant temperature oscillator at 55°C and 150 rpm, shake, react for 2 hours, inactivate the enzyme in a 90°C water bath for 10 minutes, and adjust the pH to 7.0 after cooling. After freeze-drying, the anticoagulant leech peptide powder with anticoagulant activity greater than 600AT-U / g can be obtained.

Embodiment 3

[0026] Take fresh Hirudo phenanthae that has been refrigerated for no more than 24 hours, add 2 times the water and mix it in a beater for homogenization, centrifuge the homogenate at 10,000r / min for 10min, and refrigerate the supernatant for later use, weigh 15g of the precipitate and 100ml of water and mix it in a triangle In the bottle, adjust the pH to 9.0, add Alcalase 2.4L enzyme, place in a water bath constant temperature oscillator at 55°C and 150 rpm, shake, react for 2.5 hours, inactivate the enzyme in a 90°C water bath for 10 minutes, and adjust the pH to 7.0 after cooling After freeze-drying, the anticoagulant hirudopeptide powder with anticoagulant activity greater than 600AT-U / g can be obtained.

technical field

[0001] The invention relates to the field of enzymolysis preparation of bioactive peptides, in particular to an enzymolysis preparation method of anticoagulant leech peptide. Background technique

[0002] The Philippine leeches, commonly known as Phnom Penh leeches, leeches, and burdocks, are widely distributed in the south of the Yangtze River in my country, especially in Guangdong, Guangxi, and Hainan. For the treatment of lumps in the abdomen, blood stasis, amenorrhea, bruises and other diseases. Li Shizhen's "Compendium of Materia Medica" records: "Salty removes blood, bitterness beats blood; leech's salty and bitter, in order to remove blood accumulation, is the liver meridian blood and divides medicine, so it can clear the liver meridian and gather blood." The main component of phenanthrene is collagen, and it also contains a small amount of fatty acids and trace minerals.

[0003] Modern scientific research shows that the natural hirudin in the body i...

Examples

Embodiment 1

[0022] Take fresh Hirudo phenanthae that has been refrigerated for no more than 24 hours, add 4 times of water and mix it in a beater for homogenization, centrifuge the homogenate at 10000r / min for 10min, and refrigerate the supernatant for later use, weigh 9g of the precipitate and 100ml of water and mix it in a triangle In the bottle, adjust the pH to 8.0, add Alcalase 2.4L enzyme (alkaline protease, produced by Novozymes), shake in a water bath constant temperature oscillator at 55°C and 150 rpm, and react for 1.5h. Inactivate the enzyme for 10 minutes, adjust the pH to 7.0 after cooling, and freeze-dry to obtain anticoagulant leech peptide powder with anticoagulant activity greater than 500AT-U / g.

Embodiment 2

[0024] Take fresh Hirudo phenanthae that has been refrigerated for no more than 24 hours, add 3 times of water and mix it in a beater for homogenization, centrifuge the homogenate at 10,000r / min for 10min, and refrigerate the supernatant for later use, weigh 12g of the precipitate and mix it with 100ml of water in a triangular In the bottle, adjust the pH to 8.5, add Alcalase 2.4L enzyme, place in a water bath constant temperature oscillator at 55°C and 150 rpm, shake, react for 2 hours, inactivate the enzyme in a 90°C water bath for 10 minutes, and adjust the pH to 7.0 after cooling. After freeze-drying, the anticoagulant leech peptide powder with anticoagulant activity greater than 600AT-U / g can be obtained.

Embodiment 3

[0026] Take fresh Hirudo phenanthae that has been refrigerated for no more than 24 hours, add 2 times the water and mix it in a beater for homogenization, centrifuge the homogenate at 10,000r / min for 10min, and refrigerate the supernatant for later use, weigh 15g of the precipitate and 100ml of water and mix it in a triangle In the bottle, adjust the pH to 9.0, add Alcalase 2.4L enzyme, place in a water bath constant temperature oscillator at 55°C and 150 rpm, shake, react for 2.5 hours, inactivate the enzyme in a 90°C water bath for 10 minutes, and adjust the pH to 7.0 after cooling After freeze-drying, the anticoagulant hirudopeptide powder with anticoagulant activity greater than 600AT-U / g can be obtained.