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Method for separating and purifying bromelain by circulating aqueous two-phase system

A technology of bromelain and two-phase aqueous system, which is applied in the field of separation and purification of active ingredients of natural products, can solve the problems of complex bromelain separation technology, difficulty in scaling up, destruction of enzyme activity and structure, etc., to achieve maximum utilization, The effect of improving the purification factor and improving the recovery rate of enzyme activity

Active Publication Date: 2016-11-16
江西颐迪科技有限公司
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  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the defects of current bromelain separation technology, such as complex process, difficult scale expansion, and destruction of enzyme activity and structure, and provide a method for two-phase aqueous extraction of bromelain in pineapple peel based on block polymers. The process is simple, the cost is low, and the operating conditions are mild, which can protect the original structure and activity of the target enzyme, and the polymer can be recycled and reused, with little environmental pollution

Method used

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  • Method for separating and purifying bromelain by circulating aqueous two-phase system
  • Method for separating and purifying bromelain by circulating aqueous two-phase system
  • Method for separating and purifying bromelain by circulating aqueous two-phase system

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Embodiment 1

[0032] (1) Sample pretreatment: After squeezing the pineapple peel and filtering it, the obtained filtrate was centrifuged in a centrifuge at 4°C at 8000 rpm for 20 minutes to obtain a relatively clear pineapple peel juice, and the bromelain in it was determined by the Folin method The activity was 90, and the protein content was determined by the Coomassie brilliant blue method.

[0033] (2) Study on the stability of bromelain: the crude pineapple peel juice was stored at pH = 4, the concentration was 8% K 2 HPO 4 , (NH 4 ) 3 C 6 h 5 o 7 and K 3 C 6 h 5 o 7 In the solution, after storing at 15 °C for 0.5 h, the enzyme activity was measured, and the ratios of the obtained enzyme activity to the enzyme activity before storage were 65.3%, 61.4% and 60.2%, respectively. It can be seen that the enzyme activity decreased under this environment , Bromelain has a certain loss, but in K 2 HPO 4The least loss in the system.

[0034] (3) One-time extraction of bromelain by ...

Embodiment 2

[0037] (1) Sample pretreatment: After the pineapple peel is squeezed and filtered, the filtrate obtained is centrifuged in a centrifuge at 4 °C at 8000 rpm for 20 minutes to obtain a relatively clear pineapple peel juice, and the bromelain in it is determined by the Folin method The activity was 90, and the protein content was determined by the Coomassie brilliant blue method.

[0038] (2) Study on the stability of bromelain: the crude pineapple peel juice was stored at pH = 10, the concentration was 15% K 2 HPO 4 , (NH 4 ) 3 C 6 h 5 o 7 and K 3 C 6 h 5 o 7 In the solution, after storage at 70 ℃ for 12 h, the enzyme activity was measured, and the ratios of the obtained enzyme activity to the enzyme activity before storage were 2.5%, 2.1% and 1.8%. Therefore, long-term storage under high temperature and high pH environment will significantly reduce the enzyme activity and cause more loss of bromelain.

[0039] (3) One-time extraction of bromelain by aqueous two-phase...

Embodiment 3

[0042] (1) Sample pretreatment: After the pineapple peel is squeezed and filtered, the filtrate obtained is centrifuged in a centrifuge at 4 °C at 8000 rpm for 20 minutes to obtain a relatively clear pineapple peel juice, and the bromelain in it is determined by the Folin method The activity was 90, and the protein content was determined by the Coomassie brilliant blue method.

[0043] (2) Stability study of bromelain: the crude pineapple peel juice was stored in 12% K at pH = 7.5 2 HPO 4 , (NH 4 ) 3 C 6 h 5 o 7 and K 3 C 6 h 5 o 7 In the solution, after storage at 8 ℃ for 4 h, the enzyme activity was measured, and the ratios of the obtained enzyme activity to the enzyme activity before storage were 82.6%, 65.6% and 62.5%. Therefore, the enzyme activity in the three salt solutions stored in this environment is slightly reduced, and the loss of bromelain is less, and the pH, temperature and time in this environment are suitable storage environments; and at K 2 HPO 4...

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Abstract

The invention relates to a method for separating and purifying bromelain by circulating an aqueous two-phase system, and belongs to the technical field of separation and purification of effective ingredients of natural products. The method comprises the following steps: firstly, pretreating to obtain coarsely extracted pineapple juice; then, extracting bromelain for the first time by an aqueous two-phase system, detecting the activity and protein content of top-phase bromelain, and determining an extraction effect; thirdly, taking out polymer, and diluting by adding a proper amount of distilled water, regulating the pH value of the solution, and extracting bromelain for the second time by a cloud point method; and finally, taking the separated polymer for circulating extraction experiments in the aqueous next two-phase system. The method has a simple process flow, low cost and mild operation condition and can be used for protecting the structure and activity of target enzyme; and the polymer can be recycled, and has small environmental pollution.

Description

technical field [0001] The invention relates to a method for separating and purifying bromelain by a circulating two-phase system, in particular to a method for separating bromelain by polymer-salt two-phase extraction, and belongs to the technical field of separation and purification of effective components of natural products. Background technique [0002] Bromelain is derived from a tropical fruit, pineapple, in which bromelain is divided into stem bromelain and fruit bromelain according to the extraction site. In the food industry, bromelain is the best meat tenderizer. In the UK, around 95% of meat is processed with bromelain. The optimal reaction temperature of bromelain is low, which is very beneficial to its application in the food industry. [0003] At present, bromelain separation and purification methods mainly include precipitation method, ion exchange chromatography, immobilized metal ion affinity chromatography, etc. The common problems in these methods are: ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/50
CPCC12N9/63C12Y304/22033
Inventor 李媛媛韩娟陆春花王赟唐旭倪良高蒙蒙
Owner 江西颐迪科技有限公司
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