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Method for purifying papain through double-water phase extraction-ultrasonic wave combined separation

A papain, two-phase technology, applied in the directions of hydrolase, biochemical equipment and methods, enzymes, etc., can solve the problems of increasing the distribution coefficient and yield of the target product, accelerating the system phase separation, increasing the difficulty of operation and economic cost, etc. , to achieve the effect of conventional equipment, fast phase separation and easy operation

Inactive Publication Date: 2016-09-07
瑞安市智造科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing two-phase extraction technology needs to be supplemented by centrifugation and other operations after the phase separation of the system, thereby accelerating the phase separation of the system, and adding neutral salt KCl to the system to increase the distribution coefficient and yield of the target product increases the operational difficulty and economic cost

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] (1) Preparation of crude extract

[0020] Add 10g of papaya milk to 500g of deionized water and soak for 6 hours, freeze at -20°C for 3 hours, then thaw at 25°C, repeat the freeze-thaw process 3 times to break the wall, then centrifuge the obtained solution, and collect the supernatant Obtained 17g of crude extract containing papain, the purity was 55.3%, and the yield was 94.2%.

[0021] (2) Aqueous two-phase extraction

[0022] Take 6.5g of the crude extract containing papain, 2g of sodium hydroxymethylcellulose and 1.5g of ammonium sulfate and add them to the Erlenmeyer flask. Upper phase, the upper phase is placed in a dialysis membrane with a molecular weight cut-off of 5000 and dialyzed for 20 hours to obtain an aqueous solution of papain, then freeze-dried at -15°C for 12 hours to obtain 6g of papain powder, wherein the purity of papain is 95.6, The yield was 88.9%, and the total yield was .

Embodiment 2

[0024] (1) Preparation of crude extract

[0025] Add 10g of papaya milk to 600g of deionized water and soak for 4 hours, freeze at -20°C for 5 hours, then thaw at 25°C, repeat the freeze-thaw process 3 times to break the wall, then centrifuge the obtained solution, and collect the supernatant 18.1 g of crude extract containing papain was obtained with a purity of 51.5% and a yield of 93.0%.

[0026] (2) Aqueous two-phase extraction

[0027] Take 6.6g of the crude extract containing papain, 2g of sodium hydroxymethylcellulose and 1.5g of ammonium sulfate and add them to the Erlenmeyer flask. For the upper phase, place the upper phase in a dialysis membrane with a molecular weight cut-off of 5000 and dialyze for 12 hours to obtain an aqueous solution of papain, then freeze-dry at -15°C for 15 hours to obtain 6.3g of papain powder, wherein the purity of papain is 95.2 %, the yield is 90.6%, and the total yield is 84.3%.

Embodiment 3

[0029] (1) Preparation of crude extract

[0030] Add 10g of papaya milk to 700g of deionized water and soak for 5 hours, freeze at -20°C for 8 hours, and then thaw at 25°C, repeat the freeze-thaw process for 3 times to break the wall, then centrifuge the obtained solution, and collect the supernatant 16.6 g of crude extract containing papain was obtained, the purity was 55.0%, and the yield was 91.2%.

[0031] (2) Aqueous two-phase extraction

[0032] Take 5.9g of papain-containing crude extract, 2.5g of sodium hydroxymethylcellulose and 1.6g of magnesium sulfate and add them to the Erlenmeyer flask. After shaking and mixing, put them in an ultrasonic wave for 16 minutes, and then separate the phases after standing for 13 minutes. Collect the upper phase, place the upper phase in a dialysis membrane with a molecular weight cut-off of 5000 and dialyze for 18 hours to obtain an aqueous solution of papain, then freeze-dry at -15°C for 15 hours to obtain 5.6g of papain powder, wh...

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Abstract

The invention provides a method for purifying papain through double-water phase extraction-ultrasonic wave combined separation. The method has the concrete operation steps of performing wall breaking on papaya juice by a repeated freeze thawing method; then, collecting a supernatant to obtain papain coarse extraction liquid; using sodium carboxmethylcellulose-inorganic salt double water phases for extraction; putting the materials into ultrasonic waves for ultrasonic wave treatment for 15 to 20 minutes; then, performing still standing for 8 to 15 minutes; taking an upper phase to perform dialysis for 12 to 20 hours in a dialysis film with the interception relative molecular weight being 5000; performing freeze drying to obtain papain powder. The double-water phase extraction-ultrasonic wave combined method is used for purifying the papain; the advantages of high selectivity and high system phase separating speed are realized; in addition, the operation is simple and convenient; the equipment is conventional; the method is suitable for industrial production. The purity of the papain in the papain powder prepared by the method is as high as 97.1 percent; the total yield reaches 84.2 percent.

Description

technical field [0001] The invention belongs to the technical field of biochemical separation, and relates to a method for separating and purifying papain, in particular to a method for separating and purifying papain in combination with two-phase extraction and ultrasonic waves. Background technique [0002] Papain is widely found in the milk of immature papaya fruits. It is a protein single chain composed of 212 amino acids. It has the characteristics of high temperature resistance, strong activity, good stability, and strong protein hydrolysis ability. It is a pure natural product that can act on various proteins. It is not sensitive to pH changes, metal ions and detergents. It has many uses and has been widely used in various industries such as food, chemical industry, light textile industry, medicine and health. In meat processing, papain is the best meat tenderizer because of its higher thermal stability compared with other proteases. Papain is used to treat beer dur...

Claims

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Application Information

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IPC IPC(8): C12N9/50
CPCC12N9/63C12Y304/22002
Inventor 任新年
Owner 瑞安市智造科技有限公司
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