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Enzymolysis preparation method of phosphatidylinositol

A technology of phosphatidylinositol and phospholipase, which is applied in the field of enzymatic hydrolysis and preparation of phosphatidylinositol, can solve the problems of large solvent consumption, high cost, difficulty in wide application, etc., and achieves short reaction time, simple method and low cost Effect

Inactive Publication Date: 2012-09-05
GUANGDONG INST OF MICROORGANISM +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is difficult to obtain a single phospholipid only by separation method, and the solvent consumption is large, the cost is high, and it is difficult to be widely used.

Method used

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  • Enzymolysis preparation method of phosphatidylinositol
  • Enzymolysis preparation method of phosphatidylinositol
  • Enzymolysis preparation method of phosphatidylinositol

Examples

Experimental program
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Effect test

preparation example Construction

[0019] 1. Preparation and determination of phosphatidylinositol products

[0020] Step 1, pretreatment of raw materials

[0021] Weigh 5g of powdered soybean lecithin, dissolve the sample with 50mL of cold chloroform (4°C), add 60mL of cold methanol after fully dissolved, and stir for 1h. Centrifuge at 2°C and 5000r / min for 10min, discard the upper layer solution, collect the solid and dissolve it with 25mL of cold chloroform (4°C), then add 60mL of alkaline methanol for leaching, stir well, and centrifuge for 10min under the same conditions to collect the solid. The above operation is repeated twice. The finally obtained insoluble solid is the pretreated soybean lecithin after drying.

[0022] Step two product purification

[0023] Dissolve every 50 mg of the enzymatic hydrolysis product with 10-100 mL of n-hexane solvent, fully stir at 200 r / min, 4 °C for 1 h, centrifuge at 8000 r / min, 4 °C, concentrate and dry the supernatant in a vacuum desiccator, The dried product is...

Embodiment 1

[0027] Refer to the method of raw material pretreatment in step 1 to process soybean lecithin to obtain soybean lecithin after pretreatment. Weigh 100 mg of soybean lecithin after pretreatment, dissolve it in 20 mL of ether, add 0.001 mol / LCa 2+ 10mL of Tris-HCl buffer solution, adjust the pH to 8, add 50units of phospholipase D, react in a constant temperature water bath at 37°C, at a speed of 800r / min for 4h, and obtain the enzymatic hydrolyzate, refer to step 2, use 100mL for every 50mg of enzymatically hydrolyzate Dissolve in n-hexane solvent, stir thoroughly at 200r / min, 4°C for 1h, centrifuge at 8000r / min, 4°C, place the supernatant in a vacuum desiccator to concentrate and dry, and use the dried product with a volume fraction of 5% The phosphatidylinositol product was obtained by rinsing three times with acidic ethanol (pH=6), and the purity of the phosphatidylinositol was 80.18%.

Embodiment 2

[0029] This example is basically the same as Example 1, except that the amount of phospholipase D added is 150 units to obtain a phosphatidylinositol product with a purity of 90.13%.

[0030] Second, the pH factor (7-9)

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Abstract

The invention discloses an enzymolysis preparation method of phosphatidylinositol. The enzymolysis preparation method comprises the following steps of: dissolving pretreated soybean phosphatide in solvent, and adding phospholipase D in the solvent, wherein the addition of the phospholipase D is 50 to 150 units per 100mg the pretreated soybean phosphatide; stirring for 3 to 12 hours at 36 DEG C to 40 DEG C and pH value ranging from 7.0 to 9.0, thus obtaining an enzymolysis product; and performing extraction on the enzymolysis product by virtue of normal hexane, concentrating and drying extraction liquid, washing by virtue of ethanol or ethanol aqueous solution, and drying to obtain the phosphatidylinositol product. The pretreated soybean phosphatide is prepared by the following steps of: dissolving soybean phosphatide in chloroform, settling by virtue of methanol, collecting sediments, repeating the steps for several times, and drying the final obtained sediment to obtain the pretreated soybean phosphatide. The enzymolysis preparation method is simple and relatively low in cost; and the purity of the prepared phosphatidylinositol is high, and thus the phosphatidylinositol can serve as a chromogenic substrate to be applied to a chromogenic medium for quickly detecting LM (listeria monocytogenes).

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to an enzymatic hydrolysis preparation method of phosphatidylinositol. Background technique: [0002] Listeria monocytogenes (LM) is an important foodborne pathogen. In recent years, the rapid detection method of designing chromogenic substrates for LM-specific inositol phospholipase C (Cphosphatidylinositol phospholipase C, PI-PLC) and β-D-glucosidase (β-D-glucosidase) has attracted much attention. PI-PLC enzyme can specifically act on the structure of phosphatidylinositol (PI) to decompose free aliphatic hydrocarbon chains. Due to the hydrophobic effect of macromolecular aliphatic hydrocarbon chains, when applied to chromogenic medium, LM colonies An opaque white halo forms around it to distinguish LM from other foodborne pathogens and non-Listeria monocytogenes. [0003] At present, there are few related studies on the separation and purification of PI in China, and th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P9/00
Inventor 吴清平李琳张菊梅蔡芷荷郭伟鹏
Owner GUANGDONG INST OF MICROORGANISM
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