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Grub enzymolysis primary isolate high in specific activity and preparation method of grub enzymolysis primary isolate

A technology of grub enzymatic hydrolysis solution and grub enzyme, which is applied in the field of agricultural biology and can solve problems such as constraints and difficulties in peptide separation and purification

Active Publication Date: 2019-10-18
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the components with relatively concentrated molecular mass can be obtained through the optimization of the enzymatic hydrolysis process, the wide variety of peptides in the enzymatic hydrolysis products, their molecular weights are relatively close to each other, and the sensitivity of the active components, which largely causes The difficulty of peptide separation and purification has become a major factor restricting further research on the structure and function of active peptides

Method used

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  • Grub enzymolysis primary isolate high in specific activity and preparation method of grub enzymolysis primary isolate
  • Grub enzymolysis primary isolate high in specific activity and preparation method of grub enzymolysis primary isolate
  • Grub enzymolysis primary isolate high in specific activity and preparation method of grub enzymolysis primary isolate

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Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1 prepares grub enzymatic hydrolysis solution

[0033] Take a few washed grubs, put them in boiling water at 100°C for about 40 seconds, take them out immediately, dry the water, put them in a -20°C refrigerator, and store them for later use.

[0034] Accurately weigh 10 g of grubs, add a buffer system of pH 7.0 (or 6.8, 6.9, 7.1, 7.2) according to 5% substrate concentration (g / mL), and homogenize with a high-speed disperser at a speed of 8000 r / min to obtain insect slurry. Add lipase according to the enzyme-to-worm ratio (%) 0.6%, put it in a water bath constant temperature shaker, 150r / min (or 100r / min, 200r / min), 40°C (or 38°C, 39°C, 41°C, 42°C ) Enzymolysis for 3h. Put it in a water bath constant temperature shaker at 70°C for 30min, and let it stand overnight at 4°C. Centrifuge at 4000r / min for 15min at 4°C, take the supernatant, and freeze-dry to obtain the enzymatic hydrolyzate of grubs.

[0035] The determination of the total protein content of gru...

Embodiment 2

[0039] Example 2 Separation and Purification of Grub Antioxidant Peptides

[0040] Using HPLC preparative chromatography, the grub hydrolyzate was fractionated by SephadexG-15 (5μm, 300×21.2mm), and the grub antioxidant peptide extract was prepared Chromatographic conditions: mobile phase ultrapure water, flow rate 15mL / min (or 14mL / min, 16mL / min), the column temperature is 30°C (or 27°C, 28°C, 29°C, 31°C, 32°C, 33°C), and the wavelength is 220nm (or 215nm, 225nm). The gradient elution program was 16 min. Prepare grub enzymatic hydrolyzate solution with a concentration of 10mg / mL with ultrapure water, and the loading volume is 2mL. Collect a tube every 2 min according to the separation time of the grub extract in the preparative chromatography.

[0041] Such as figure 1 As shown, the grub extract obtained 8 fractions PB1 (Protaetia brevitarsis larvae), PB2, PB3, PB4, PB5, PB6, PB7 and PB8, and the collected samples were rotary evaporated at 45°C and 85r / min, and the primar...

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Abstract

The invention belongs to the field of agricultural biotechnology, and particularly relates to grub enzymolysis primary isolate high in specific activity and a preparation method of the grub enzymolysis primary isolate. The grub enzymolysis primary isolate is obtained through the following steps of separating grub enzymatic hydrolysate by adopting a sephadex column, and using ultra pure water as amobile phase; and collecting fraction at intervals. A high performance liquid chromatogram method is adopted for separating the grub lipase enzymolysis product, so that the obtained fraction has highoxidation resistance and specific activity.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a high-specific activity grub enzymolysis primary isolate and a preparation method thereof. Background technique [0002] Grubs are the collective name for the larvae of Scarabaeoedea, belonging to the order Coleoptera, also known as walnut worms, ground silkworms, and soil silkworms. Grubs contain a variety of amino acids and inorganic elements needed by humans. They have been clinically used to promote blood circulation for hundreds of years. Modern pharmacological studies have shown that grubs have anti-tumor, liver protection, treatment of mouth sores, children's asthma, stroke, cirrhosis of schistosomiasis, etc. It has a variety of medicinal activities and is a medicinal material with great research and development value. Studies have found that the crude extract of grub has anticoagulant activity, and several fibrin (ogen) dissolving agents have antith...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/06C07K1/16C07K1/20A61P39/06
CPCA61P39/06C07K1/16C07K1/20C07K7/06C12P21/06
Inventor 石冬冬李秀梅杨培龙张海英闻治国满晨
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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