High-efficiency extraction method of macadamia nut leaf total proteins

An extraction method and protein technology, applied in the field of protein extraction, can solve the problems of easy oxidative browning and low protein purity, and achieve the effect of avoiding protein degradation, wide distribution of protein size and good quality

Active Publication Date: 2020-05-19
SOUTH SUBTROPICAL CROPS RES INST CHINESE ACAD OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art, and provide a high-efficiency extraction method for the total protein of macadamia nut leaves. The total protein of macadamia nut leaves extracted by this method has high purity, and the SDS-PAGE electrophoresis band is neat and clear, and is suitable for In Western Blot analysis, it effectively solved the problem that the total protein of macadamia nut leaves was easily oxidized and browned during the traditional extraction process, and the extracted protein was not high in purity.

Method used

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  • High-efficiency extraction method of macadamia nut leaf total proteins
  • High-efficiency extraction method of macadamia nut leaf total proteins
  • High-efficiency extraction method of macadamia nut leaf total proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Lysis Solution PW1: Prepared with acetone, in which TCA content is 10%, PVP is 0.01%, and DTT is 20mM;

[0038] Extract PW2: prepared with sterile distilled water, in which Tris saturated phenol content is 55%, sucrose is 10%, SDS is 1%, Tris-HCl is 0.05mol / L pH 8.0, DTT is 20mM, PMSF is 1mM;

[0039] Solution PW3: Prepared with sterile distilled water, in which SDS content is 0.2%, urea is 7mol / L, thiourea is 2mol / L, Tris-HCl is 25mmol / L pH 6.8, DTT is 10mM.

[0040] A method for efficiently extracting total protein from leaves of macadamia nuts, comprising the following steps:

[0041]1) Take 1.0 g of fresh leaves of Nanya No. 1 macadamia nuts, put them in a 10mL sampling cup pre-cooled with liquid nitrogen, add steel balls, and use a tissue grinder to grind at 50Hz for 1min, then take out the sampling cup and freeze again in liquid nitrogen, and repeat the grinding for two times. The first time, grind the sample into powder; take the powder into a centrifuge tube, a...

Embodiment 2

[0046] Lysis Solution PW1: Prepared with acetone, in which TCA content is 11%, PVP is 0.05%, and DTT is 50mM;

[0047] Extract PW2: prepared with sterile distilled water, in which Tris saturated phenol content is 60%, sucrose is 11%, SDS is 0.8%, Tris-HCl is 0.03mol / L pH 7.8, DTT is 30mM, PMSF is 0.5mM;

[0048] Solution PW3: prepared with sterile distilled water, in which the SDS content is 0.1%, urea is 7mol / L, thiourea is 1mol / L, Tris-HCl is 20mmol / L pH 6.6, and DTT is 10mM.

[0049] A method for efficiently extracting total protein from leaves of macadamia nuts, comprising the following steps:

[0050] 1) Take 1.5g of fresh leaves of Nanya No. 1 macadamia nuts, put them in a 10mL sampling cup pre-cooled with liquid nitrogen, add steel balls, use a tissue grinder to grind at 55Hz for 1min, then take out the sampling cup and freeze again in liquid nitrogen, and repeat the grinding for two times. The second time, grind the sample into powder; take the powder into a centrifug...

Embodiment 3

[0055] Lysis Solution PW1: Prepared with acetone, in which TCA content is 12%, PVP is 0.03%, and DTT is 35mM;

[0056] Extract PW2: prepared with sterile distilled water, in which Tris saturated phenol content is 65%, sucrose is 15%, SDS is 1.2%, Tris-HCl is 0.06mol / L pH 8.2, DTT is 50mM, PMSF is 1.5mM;

[0057] Solution PW3: Prepared with sterile distilled water, in which SDS content is 0.3%, urea is 8mol / L, thiourea is 3mol / L, Tris-HCl is 30mmol / L pH 7.0, DTT is 50mM.

[0058] A method for efficiently extracting total protein from leaves of macadamia nuts, comprising the following steps:

[0059] 1) Take 2.0g of fresh leaves of Nanya No. 1 macadamia nut, put them in a 10mL sampling cup pre-cooled with liquid nitrogen, add steel balls, and use a tissue grinder to grind at 60Hz for 1min, then take out the sampling cup and freeze it again in liquid nitrogen, and repeat the grinding for two times. The second time, grind the sample into powder; take the powder into a centrifuge ...

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Abstract

The invention discloses a high-efficiency extraction method of macadamia nut leaf total proteins. The method comprises the following steps: grinding and crushing fresh macadamia nut leaves by using liquid nitrogen; adding a pre-cooling cracking liquid PW1 for primary extraction; performing solid-liquid separation to obtain a first precipitate; cleaning and airing the first precipitate; adding an extracting solution PW2 for secondary extraction; performing solid-liquid separation to obtain a second supernatant; adding a pre-cooling methanol solution; performing centrifuging to obtain a second precipitate; cleaning and drying the second precipitate; adding a dissolving solution PW3 for tertiary extraction; and performing solid-liquid separation to obtain a third supernatant, namely the macadamia nut leaf total proteins. According to the invention, the fresh macadamia nut leaves are taken as materials, and the three extracting solutions are added into macadamia nut leaf powder, so that oxidation of raw materials can be effectively avoided, degradation of proteins can be avoided, the yield of the total proteins is high, purity of the extracted protein is high, the SDS-PAGE electrophoresis bands are neat and clear, and the macadamia nut leaf powder is suitable for Western Blot analysis.

Description

technical field [0001] The invention belongs to the technical field of protein extraction. More specifically, it relates to a high-efficiency extraction method for total protein in leaves of macadamia nuts. Background technique [0002] Macadamia ternifolia F.Muell., also known as Queensland chestnut, macadamia nut, and macadamia walnut, belongs to the genus Macadamia of the family Longanaceae. It is an evergreen tree native to southeastern Queensland and northern New South Wales, Australia. Coastal subtropical rainforest at 25°-31° south latitude. Macadamia nuts are rich in nutrients, with an oil content of 70% to 79%, especially rich in unsaturated fatty acids, rich in protein, and rich in phosphorus, calcium, iron, vitamins, amino acids, etc., known as the "Queen of Dried Fruits" and "No. King". At present, research on macadamia at home and abroad is mainly focused on the promotion of cultivation techniques, germplasm identification, resource utilization, etc. There ar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14
CPCC07K1/145
Inventor 井敏敏陈倪刘恒曾辉
Owner SOUTH SUBTROPICAL CROPS RES INST CHINESE ACAD OF TROPICAL AGRI SCI
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