CN104478987A - Special extracting solution for oil palm leaf total proteins and extracting method thereof - Google Patents

Special extracting solution for oil palm leaf total proteins and extracting method thereof Download PDF

Info

Publication number
CN104478987A
CN104478987A CN201410657130.2A CN201410657130A CN104478987A CN 104478987 A CN104478987 A CN 104478987A CN 201410657130 A CN201410657130 A CN 201410657130A CN 104478987 A CN104478987 A CN 104478987A
Authority
CN
China
Prior art keywords
oil palm
total protein
protein matter
leaves total
special extract
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201410657130.2A
Other languages
Chinese (zh)
Other versions
CN104478987B (en
Inventor
杨耀东
吴翼
肖勇
李静
周立霞
夏薇
沈雁
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Coconut Research Institute of Chinese Academy of Tropical Agricultural Sciences
Original Assignee
Coconut Research Institute of Chinese Academy of Tropical Agricultural Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Coconut Research Institute of Chinese Academy of Tropical Agricultural Sciences filed Critical Coconut Research Institute of Chinese Academy of Tropical Agricultural Sciences
Priority to CN201410657130.2A priority Critical patent/CN104478987B/en
Publication of CN104478987A publication Critical patent/CN104478987A/en
Application granted granted Critical
Publication of CN104478987B publication Critical patent/CN104478987B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Landscapes

  • Peptides Or Proteins (AREA)

Abstract

The invention relates to the field of proteomics research and discloses a special extracting solution for oil palm leaf total proteins and an extracting method thereof. The extracting method comprises the following steps: adding the oil palm special extracting solution into oil palm leaves which are ground into powder, performing total protein extraction of the oil palm leaves, wherein the extracted liquid supernatant amount and the concentration of the extracted proteins are obviously increased according to quantitative measurement. Over 3mg of total proteins can be obtained from every 2g of oil palm leaves; and the extracted protein solution is clear in color, not or slightly thick and greatly improved in purity, so that the follow-up study is greatly convenient.

Description

A kind of special extract of oil palm leaves total protein matter and extracting method
Technical field
The present invention relates to proteomics research field, refer to a kind of special extract and extracting method of oil palm leaves total protein matter especially.
Background technology
In the research of proteomics, in plant, the extraction of protein is the basis of all research, also directly affects the result of subsequent experimental.At present, method conventional in the extraction of plant protein has trichoroacetic acid(TCA)-acetone precipitation (TCA) method, E-TCA method and phenol (Phe) to take out method.Although above method can remove the impurity of the interferencing protein analytical resultss such as polysaccharide, ester class and phenols plant tissue from different aspect, unsatisfactory at the extraction effect of tropical crops protein.Chinese invention patent CN1908007A discloses extracting method and the special extract thereof of a kind of plant total protein, proposes the extracting method of a kind of plant total protein, is applicable to majority of plant, the especially extraction of gross protein in halophytes.Due to three kinds of important chemical composition sodium tetraborate (Borax), polyvinylpolypyrrolidone (PVPP) and phenol (Phenol) required in leaching process, so called after BPP method.Use BPP method to extract tropical crops, as coconut, betel nut, banana, rubber, wood work waits the extraction of plant leaf gross protein, also achieves good effect.
Oil palm (Elaeis guineensis) belongs to perennial monocotyledons, rich in oil in the pulp of oil palm, kernel, in various oil crops, has the title of " world oil king ".Utilize BPP method to extract its leaves total protein, in leaching process, supernatant liquor comparatively thickness, very difficult with pipettor Aspirate supernatant, and cause oil palm blade extraction efficiency to reduce, total protein purity is not high, has had a strong impact on the carrying out of subsequent experimental.This phenomenon summer temperature higher be particularly serious, often cause total protein extraction failure.
Summary of the invention
The present invention proposes a kind of special extract and extracting method of oil palm leaves total protein matter, effectively can remove the thick substances in supernatant liquor, improve total protein extraction productive rate and purity.
Technical scheme of the present invention is achieved in that
A kind of special extract of oil palm leaves total protein matter, with the cumulative volume of described oil palm leaves total protein matter special extract note, comprise the component of following mass concentration: complexing agent 35-40g/L, sodium tetraborate 15-20g/L, reductive agent 20-40g/L, sucrose 200-400g/L; The special extract pH value of described oil palm gross protein is 4-7.
Further, described complexing agent is ENTA or EDTA-2Na.
Further, described reductive agent is the mixture of vitamins C and beta-mercaptoethanol.
Further, ascorbic mass concentration is 5-10g/L, and the mass concentration of beta-mercaptoethanol is 15-28g/L.
Further, the special extract pH value of described oil palm leaves total protein matter is 4-5.
Further, described pH value regulates reagent used to be hydrochloric acid.
A kind of extracting method of oil palm leaves total protein matter, comprise the following steps: oil palm blade is pulverized last, add described oil palm leaves total protein matter special extract, room temperature whirlpool concussion 4-6min, remove supernatant liquor after centrifugal, then carry out the extraction of gross protein according to BPP method.
Further, with leaf quality note, the amount that described oil palm leaves total protein matter special extract adds is 4-6 (V/W) times amount.
Further, in described centrifugal process, temperature is 3-5 DEG C, centrifugal force 10000-17000RCF, and centrifugation time is 8-12min.
Beneficial effect of the present invention:
Oil palm special extract of the present invention, join after oil palm blade grinds in powder, effectively can remove the dope in supernatant liquor, greatly reduce the difficulty of experimental implementation, and leaching process is not by the impact of temperature, summer or winter all can carry out experimental implementation, easy and simple to handle, for oil palm leaf protein Quality Research provides the foundation.Extract the protein soln color clarification obtained, not or only have minute quantity thick substances, purity does not improve greatly.Carry out detection by quantitative through Bradford method to the protein soln extracted, the total protein of the oil palm blade adopting BPP method to extract, gained extracting solution of protein concentration is generally 5-7 μ g μ L -1, and adopting extracting method of the present invention, gained total protein concentration generally can reach 8 μ g μ L -1above, most concentration is at 10 μ g μ L -1left and right.Meanwhile, after testing, carry out the extraction of oil palm leaves total protein by adding oil palm special extract of the present invention, gross protein extraction yield improves greatly.
Embodiment
Below in conjunction with the embodiment of the present invention, be clearly and completely described the technical scheme in the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment 1
A special extract for oil palm leaves total protein matter, with the cumulative volume of described oil palm leaves total protein matter special extract note, comprises the component of following mass concentration: EDTA 35g/L, sodium tetraborate 15g/L, vitamins C 5g/L, beta-mercaptoethanol 15g/L, sucrose 200g/L; Add concentrated hydrochloric acid and regulate pH value to 4.
An extracting method for the special extract that oil palm leaves total protein matter is extracted, comprises the following steps: oil palm blade is pulverized last, adds oil palm leaves total protein matter special extract of the present invention; With leaf quality note, the amount that described oil palm leaves total protein matter special extract adds is 4 (V/W) times amount, and room temperature whirlpool concussion 4min, removes supernatant liquor after centrifugal, then carry out the extraction of leaves total protein matter according to BPP method.Concrete operation step is as follows:
(1) get oil palm blade 2g, liquid nitrogen grinding powdered, proceed to 50mL centrifuge tube;
(2) add 8mL oil palm leaves total protein matter special extract, room temperature whirlpool concussion 4min, removes supernatant after centrifugal 8min, repeats this step 1 time under temperature 3 DEG C, centrifugal force 10000RCF condition;
(3) 10mL precooling extracting solution, 1mL 10%SDS is added, room temperature whirlpool concussion 5min;
(4) equal-volume Tris balance phenol (pH7.8) is added, room temperature whirlpool concussion 10min;
(5) 4 DEG C, 17000RCF, centrifugal 15min, upper-layer phenol goes to 10mL centrifuge tube mutually;
(6) equal-volume precooling extracting solution is added, room temperature whirlpool concussion 10min;
(7) 4 DEG C, 17000RCF, centrifugal 15min, upper-layer phenol goes to new pipe mutually;
(8) add the pre-cold methanol of 5 times of volumes containing supersaturated ammonium sulfate, mixing, puts-20 DEG C of more than precipitating proteins 6h or spends the night.
(9) supernatant is removed, resuspended 2 times of protein precipitation precooling methanol solution after centrifugal (4 DEG C, 17000RCF, 10min);
(10) remove supernatant after centrifugal (4 DEG C, 17000RCF, 10min), with precooling acetone soln resuspended for 2 times;
(11) centrifugal (4 DEG C, 17000RCF, 10min), precipitate air-dry after add appropriate lysate, more than room-temperature dissolution 2h.
(12) centrifugal (4 DEG C, 17000RCF, 30min), draws supernatant for subsequent use to 1.5mL centrifuge tube.
Embodiment 2
The special extract that oil palm leaves total protein matter is extracted, with the cumulative volume of described oil palm leaves total protein matter special extract note, comprises the component of following mass concentration: EDTA-2Na 37g/L, sodium tetraborate 18g/L, vitamins C 8g/L, beta-mercaptoethanol 22g/L, sucrose 300g/L; Add acetic acid and regulate pH value to 5.5.
An extracting method for the special extract that oil palm leaves total protein matter is extracted, comprises the following steps: oil palm blade is pulverized last, adds oil palm leaves total protein matter special extract of the present invention; With leaf quality note, the amount that described oil palm leaves total protein matter special extract adds is 5 (V/W) times amount, and room temperature whirlpool concussion 5min, removes supernatant liquor after centrifugal, then carry out the extraction of leaves total protein matter according to BPP method.
Its concrete operation step difference from Example 1 is, the add-on of step (2) oil palm leaves total protein matter special extract is 10mL, room temperature whirlpool concussion 5min; Under temperature 4 DEG C, centrifugal force 13000RCF condition, remove supernatant liquor after centrifugal 10min, all the other operations are identical with embodiment 1.
Embodiment 3
The special extract that oil palm leaves total protein matter is extracted, with the cumulative volume of described oil palm leaves total protein matter special extract note, comprises the component of following mass concentration: EDTA 40g/L, sodium tetraborate 20g/L, vitamins C 10g/L, beta-mercaptoethanol 28g/L, sucrose 400g/L; Add hydrochloric acid and regulate pH value to 7.
An extracting method for oil palm leaves total protein matter, comprises the following steps: oil palm blade is pulverized last, adds oil palm leaves total protein matter special extract of the present invention; With leaf quality note, the amount that described oil palm leaves total protein matter special extract adds is 8 (V/W) times amount, and room temperature whirlpool concussion 6min, removes supernatant liquor after centrifugal, then carry out the extraction of leaves total protein matter according to BPP method.
Its concrete operation step difference from Example 1 is, step (2) adds 16mL oil palm leaves total protein matter special extract, room temperature whirlpool concussion 5min, removes supernatant liquor after centrifugal 12min, repeats this step 1 time under temperature 5 DEG C, centrifugal force 17000RCF condition; All the other operations are identical with embodiment 1.
Embodiment 4
The special extract that oil palm leaves total protein matter is extracted, with the cumulative volume of described oil palm leaves total protein matter special extract note, comprises the component of following mass concentration: EDTA 35g/L, sodium tetraborate 18g/L, vitamins C 5g/L, beta-mercaptoethanol 28g/L, sucrose 300g/L; Add hydrochloric acid and regulate pH value to 5.
An extracting method for oil palm leaves total protein matter, comprises the following steps: oil palm blade is pulverized last, adds oil palm leaves total protein matter special extract of the present invention; With leaf quality note, the amount that described oil palm leaves total protein matter special extract adds is 6 (V/W) times amount, and room temperature whirlpool concussion 6min, removes supernatant liquor after centrifugal, then carry out the extraction of leaves total protein matter according to BPP method.
Its concrete operation step difference from Example 1 is, step (2) adds 12mL oil palm leaves total protein matter special extract, room temperature whirlpool concussion 6min, removes supernatant liquor after centrifugal 12min, repeats this step 1 time under temperature 5 DEG C, centrifugal force 10000RCF condition; All the other operations are identical with embodiment 1.
Embodiment 5
The special extract that oil palm leaves total protein matter is extracted, with the cumulative volume of described oil palm leaves total protein matter special extract note, comprises the component of following mass concentration: EDTA 37g/L, sodium tetraborate 19g/L, vitamins C 10g/L, beta-mercaptoethanol 22g/L, sucrose 200g/L; Add hydrochloric acid and regulate pH value to 4.5.
An extracting method for oil palm leaves total protein matter, comprises the following steps: oil palm blade is pulverized last, adds oil palm leaves total protein matter special extract of the present invention; With leaf quality note, the amount that described oil palm leaves total protein matter special extract adds is 5 (V/W) times amount, and room temperature whirlpool concussion 5min, removes supernatant liquor after centrifugal, then carry out the extraction of leaves total protein matter according to BPP method.
Its concrete operation step difference from Example 1 is, step (2) adds 10mL oil palm leaves total protein matter special extract, and room temperature whirlpool concussion 5min, removes supernatant liquor after centrifugal 10min under temperature 3 DEG C, centrifugal force 17000RCF condition; All the other operations are identical with embodiment 1.
In above embodiment, if thick substances is more in supernatant liquor, step (2) can be repeated 1-2 time.The special extract of oil palm leaves total protein of the present invention and extracting method effect expedition
Get 2g oil palm blade respectively, respectively according to extracting method and the conventional BPP method extraction leaves total protein matter of the present invention's three embodiments, each process repeats 10 operations.Obtaining extracting solution of protein to extraction adopts Bradford method to carry out quantitative assay, and calculate its mean value, result is as shown in table 1.
Table 1 extracting method extraction effect of the present invention compares
As shown in Table 1, the method that three embodiments of the present invention provide, the supernatant liquid measure that proteins extraction obtains, protein concn are all significantly increased.The ultimate capacity of 2g oil palm leaves total protein matter is all at more than 3mg; And adopting BPP method, the protein ultimate capacity of gained is at about 2mg.
Adopt BPP method, the supernatant fluid color extracting gained is faint yellow, and obvious thickness, foreign matter content is high, brings very large impact to follow-up research.And the treatment process described in the present invention's three embodiments, do not extract the supernatant liquor that obtains not or only have the thick substances of minute quantity, color is clarified, and foreign matter content reduces greatly, facilitates the carrying out of subsequent experimental research.In addition, extracting method provided by the present invention, extract operation not by the restriction of envrionment temperature, temperature high (summer), temperature low (winter) all can carry out the extraction of oil palm leaves total protein matter, greatly improve the efficiency that oil palm leaves total protein extracts, the correlative study also for carrying out oil palm leaf protein under Various Seasonal and weather condition creates condition.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.

Claims (9)

1. a special extract for oil palm leaves total protein matter, is characterized in that, remembers with the cumulative volume of described oil palm leaves total protein matter special extract, comprise the component of following mass concentration: complexing agent 35-40g/L, sodium tetraborate 15-20g/L, reductive agent 20-40g/L, sucrose 200-400g/L; The special extract pH value of described oil palm gross protein is 4-7.
2. the special extract of a kind of oil palm leaves total protein matter extraction according to claim 1, is characterized in that: described complexing agent is ENTA or EDTA-2Na.
3. the special extract of a kind of oil palm leaves total protein matter extraction according to claim 1, is characterized in that: described reductive agent is the mixture of vitamins C and beta-mercaptoethanol.
4. the special extract of a kind of oil palm leaves total protein matter extraction according to claim 3, it is characterized in that: ascorbic mass concentration is 5-10g/L, the mass concentration of beta-mercaptoethanol is 15-28g/L.
5. the special extract of a kind of oil palm leaves total protein matter according to claim 1, is characterized in that: the special extract pH value of described oil palm gross protein is 4-5.
6. the special extract of a kind of oil palm leaves total protein matter according to claim 1, is characterized in that: the reagent regulating pH value used is hydrochloric acid.
7. the extracting method of an oil palm leaves total protein matter, comprise the following steps: oil palm blade is pulverized last, add oil palm leaves total protein matter special extract described in the arbitrary claim of claim 1-6, room temperature whirlpool concussion 4-6min, remove supernatant liquor after centrifugal, then carry out the extraction of leaves total protein matter according to BPP method.
8. the extracting method of a kind of oil palm gross protein according to claim 7, is characterized in that: the volume added of described oil palm leaves total protein matter special extract is oil palm leaf quality 4-6 times (V/W).
9. a kind of oil palm leaves total protein matter extracting method according to claim 7, it is characterized in that: in described centrifugal process, temperature is 3-5 DEG C, centrifugal force 10000-17000RCF, and centrifugation time is 8-12min.
CN201410657130.2A 2014-11-18 2014-11-18 A kind of special extract and extracting method of oil palm leaves total protein matter Expired - Fee Related CN104478987B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201410657130.2A CN104478987B (en) 2014-11-18 2014-11-18 A kind of special extract and extracting method of oil palm leaves total protein matter

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201410657130.2A CN104478987B (en) 2014-11-18 2014-11-18 A kind of special extract and extracting method of oil palm leaves total protein matter

Publications (2)

Publication Number Publication Date
CN104478987A true CN104478987A (en) 2015-04-01
CN104478987B CN104478987B (en) 2018-06-19

Family

ID=52753608

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201410657130.2A Expired - Fee Related CN104478987B (en) 2014-11-18 2014-11-18 A kind of special extract and extracting method of oil palm leaves total protein matter

Country Status (1)

Country Link
CN (1) CN104478987B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105859831A (en) * 2016-04-18 2016-08-17 中国农业科学院果树研究所 Apple protein extraction method and related protein extracting solution
CN106939032A (en) * 2017-03-24 2017-07-11 艾比玛特医药科技(上海)有限公司 A kind of extract solution and extracting method for rapidly and efficiently extracting plant total protein

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1908007A (en) * 2006-08-07 2007-02-07 中国科学院植物研究所 Extraction method of plant total protein and special extract for the same
WO2009064404A2 (en) * 2007-11-15 2009-05-22 Univation Technologies, Llc Polymeriazation catalysts, methods of making; methods of using, and polyolefinproducts made therefrom
CN102532258A (en) * 2012-03-15 2012-07-04 中国热带农业科学院热带生物技术研究所 Method for extracting protein from component with low protein content

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1908007A (en) * 2006-08-07 2007-02-07 中国科学院植物研究所 Extraction method of plant total protein and special extract for the same
WO2009064404A2 (en) * 2007-11-15 2009-05-22 Univation Technologies, Llc Polymeriazation catalysts, methods of making; methods of using, and polyolefinproducts made therefrom
CN102532258A (en) * 2012-03-15 2012-07-04 中国热带农业科学院热带生物技术研究所 Method for extracting protein from component with low protein content

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
王海燕等: "巴西橡胶树幼叶和成熟叶比较蛋白质组学初步研究", 《中国农学通报》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105859831A (en) * 2016-04-18 2016-08-17 中国农业科学院果树研究所 Apple protein extraction method and related protein extracting solution
CN106939032A (en) * 2017-03-24 2017-07-11 艾比玛特医药科技(上海)有限公司 A kind of extract solution and extracting method for rapidly and efficiently extracting plant total protein
CN106939032B (en) * 2017-03-24 2021-06-22 艾比玛特医药科技(上海)有限公司 Extracting solution for rapidly and efficiently extracting total plant protein and extracting method

Also Published As

Publication number Publication date
CN104478987B (en) 2018-06-19

Similar Documents

Publication Publication Date Title
CN102210803B (en) Extraction and enrichment method of fritillaria total alkaloids
CN102618721A (en) Method for extracting germanium, indium and zinc from high iron, silicon and manganese materials containing germanium, indium and zinc
CN107586959B (en) Utilize the method for heavy metal in intensified by ultrasonic wave functional ionic liquids extraction water
CN102579612A (en) Method for extracting total alkaloid of aconitum soongaricum
CN104478987A (en) Special extracting solution for oil palm leaf total proteins and extracting method thereof
CN102210732A (en) Method for extracting panax notoginseng saponins from fresh panax notoginseng by enzyme treatment
CN105795095B (en) Preparation method of cardamine violifolia selenoprotein with low heavy metal content
CN103397184A (en) Method for separating uranium and iron from tertiary amine organic phase by back extraction
CN103543059A (en) Method for extracting inorganic arsenic in rice
CN106734142B (en) Eluting agent for eluting and repairing copper-nickel composite polluted site and using method
CN107585820A (en) A kind of extractant and extracting process to polyhydric phenols in phenol wastewater with high distribution coefficient
CN104713964A (en) Pretreatment method of polybrominated diphenyl ethers in environment solid matrix
CN103823003A (en) High performance liquid chromatography (HPLC) determination method of total formaldehyde content in man-made board
CN106539848A (en) A kind of preparation method of sweet persimmon flavone
CN101491618A (en) Method for separating tobacco total alkaloid from extract liquid of tobacco leftovers
CN102899318B (en) Method for extracting nanmu RNA (Ribonucleic Acid)
CN101491589A (en) Method for separating greater celandine, eomecon chionantha hance or toddalia asiatica total alkaloids
CN112898258B (en) Separation preparation method of high-purity hyoscyamine
CN101491647A (en) Method for separating ergot total alkaloids in ergot stem extract
CN103156897A (en) Method for removing pesticide residue dichloro-diphenyl trichloroethane (DDT) and benzene hexachloride from American ginseng extract
CN105477029A (en) Preparation method of evening primrose seed flavone
CN101029019A (en) Process for extracting separating arecoline in scale
CN101491617A (en) Method for separating Anisodus acutangulus total alkaloid from extract liquid of Anisodus acutangulus
CN103044410B (en) A kind of production technique extracting isorientin from bamboo product processing waste material
CN1709916A (en) Method for extracting high purity polysaccharide of membranous milk vetch and saponin of same

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
TR01 Transfer of patent right
TR01 Transfer of patent right

Effective date of registration: 20200724

Address after: Office 407, coconut research institute office building, 496 Wenqing Avenue, Wenchang City, Hainan Province 570100

Patentee after: Hainan rainforest coconut Technology Development Co.,Ltd.

Address before: 571339 Wen Qing Avenue, Wen Town, Wenchang, Hainan

Patentee before: COCONUT Research Institute OF CHINESE ACADEMY OF TROPICAL AGRICULTURAL SCIENCES

TR01 Transfer of patent right
TR01 Transfer of patent right

Effective date of registration: 20220926

Address after: 570100 next to Wenqing Avenue, Wencheng Town, Wenchang City, Hainan Province

Patentee after: COCONUT Research Institute OF CHINESE ACADEMY OF TROPICAL AGRICULTURAL SCIENCES

Address before: Office 407, Office Building, Coconut Research Institute, No. 496, Wenqing Avenue, Wenchang City, Hainan Province, 570100

Patentee before: Hainan rainforest coconut Technology Development Co.,Ltd.

CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20180619