CN103289969A - Method for extracting superoxide dismutase from plant stem leaves - Google Patents

Method for extracting superoxide dismutase from plant stem leaves Download PDF

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Publication number
CN103289969A
CN103289969A CN2013102285472A CN201310228547A CN103289969A CN 103289969 A CN103289969 A CN 103289969A CN 2013102285472 A CN2013102285472 A CN 2013102285472A CN 201310228547 A CN201310228547 A CN 201310228547A CN 103289969 A CN103289969 A CN 103289969A
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China
Prior art keywords
dismutase
superoxide
juice
molecular weight
weight cut
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Pending
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CN2013102285472A
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Chinese (zh)
Inventor
衣悦涛
靳志明
冯大伟
刘胜一
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Yantai Institute of Coastal Zone Research of CAS
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Yantai Institute of Coastal Zone Research of CAS
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Priority to CN2013102285472A priority Critical patent/CN103289969A/en
Publication of CN103289969A publication Critical patent/CN103289969A/en
Pending legal-status Critical Current

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Abstract

The invention discloses a method for extracting superoxide dismutase from plant stem leaves. The method comprises the following steps: adding a phosphate buffer and pulping to slurry by a feed liquid ratio (W/V) of 1:1-1:2; squeezing the slurry obtained and collecting juice; coarsely filtering by 100-300-mesh filter cloth; filtering the filtered juice first through an ultrafiltration membrane with molecular weight cut-off of 40000-60000 and then through an ultrafiltration membrane with molecular weight cut-off of 6000-20000 to obtain a superoxide dismutase coarse extract; and further purifying and spraying and drying to obtain the superoxide dismutase. The method disclosed by the invention is wide in source of raw materials, low in cost, harmless and free from viruses to human beings and animals, free from any side effects and high in product activity.

Description

A kind of method of from plant stem-leaf, extracting superoxide-dismutase
Technical field
The present invention relates to a kind of preparation method of enzyme, be specifically related to a kind of method of from plant, extracting superoxide-dismutase.
Background technology
Superoxide-dismutase (Superoxide Dismutase), be called for short: SOD is a kind of antioxidase that extensively is present in the organism, can catalysis superoxide anion generation disproportionation reaction, and now confirmed, the disease that is caused by oxygen anion is kind more than 60 nearly.Because modern life pressure, environmental pollution, various radiation and excess motion all can cause oxyradical to form in a large number, and superoxide-dismutase can resist and block the infringement that cell is caused because of oxyradical, and in time repair damaged cell, recovery because of free radical cause to cell damage, thereby resist the toxic action of ultra-oxygen anion free radical, in radioprotective, antitumor, degeneration-resistant border and delay aspects such as body aging and play an important role.Superoxide-dismutase is existing the application in medicine, food, agricultural, and some products also command a ready sale in the market accordingly.
Superoxide-dismutase is wide at the distributed pole of organic sphere, almost from the animal to the plant, even from people to the unicellular organism, its existence is arranged.The superoxide-dismutase product overwhelming majority on the domestic and international market is extracted from animal blood now, but from animal blood, extract superoxide-dismutase and have very big defective, the exclusiveness of animality superoxide-dismutase, be difficult for cross infection and other potentially dangerous of blood diseases such as normal temperature preservation, acquired immune deficiency syndrome (AIDS), the use of the superoxide-dismutase product that extracts has been prohibited in many countries and regions from animal; Relatively shortage of animal blood source in addition, the mask work amount is big again, caused production cost huge, through a series of technological process, the active conservation rate of resulting superoxide-dismutase is also lower, so the extraction raw material of superoxide-dismutase turns to plant from animal gradually now, and the traditional extraction technique isolation technique mainly adopts substep to saltout to separate SOD, this can cause corresponding chemical pollution, the raising and the market requirement that the product safety performance are required along with people are so corresponding extraction superoxide-dismutase processing method is also changing with it.
The method that has now had some from plant, to extract superoxide-dismutase, but also have some defectives.Tradition is generally used ammonium sulfate precipitation, and this can cause certain chemical pollution; Extract with the organic reagent precipitator method, and just there is certain toxicity in organic reagent itself, probably human body is worked the mischief in the leaching process, and is easy in product residual; Use the high temperature thermal change, extracting method such as centrifugation extract, and temperature and centrifugal rotational speed are had relatively high expectations, and can improve preparation cost accordingly, are difficult to realize suitability for industrialized production.
Number of patent application is 201010151796.2, adopt ultra-filtration membrane ultrafiltration behind centrifugation, the chromatography in the patent Herba Houttuyniae of applicant Zhang Weiguo, trifoliate orange seed, Rosa roxburghii, phyllanthus emblica, calabash melon, the fruit of Christophine SOD extraction process, but this arts demand is successively through three kinds of affinity columns, process is than complicated and time consumption, the pre-treatment centrifugal condition is too high, is not suitable for producing in enormous quantities.Number of patent application is 201110004935.3, the patent of applicant Gong Senmiao adopts subzero fractionation just to separate the back second ultrafiltration from the processing method of root, stem and the leaf extraction superoxide-dismutase of burdock, use 60,000 and 10,000 ultra-filtration membrane respectively, but the film of selecting for use is fixed, the scope of application is less, and use membrane sepn separately, not in conjunction with additive method, corresponding purity is lower.
Summary of the invention
The objective of the invention is to solve above-mentioned the deficiencies in the prior art, provide a kind of and extract the novel method of superoxide-dismutase from plant stem-leaf, the inventive method raw material sources are wide, cost is low and harmless, virus-free to humans and animals, without any side effects.
In order to realize above-mentioned purpose of the present invention, the present invention takes following technical scheme:
A kind of method of extracting superoxide-dismutase from plant comprises following concrete steps:
1) gets the fresh cauline leaf of plant, clear water flush away surface irregularities.
2) pressing solid-liquid ratio (W/V) is 1:1~1:2, and the pH that adds 0.1M is 7~8 phosphoric acid buffer, breaks into slurries with hollander.
3) slurries that obtain are squeezed, collect juice.
4) filter residue adding deionized water carries out twice pressing, and juice and previous step merge, and discard waste residue.
5) juice after merging is with 100 orders~300 purpose filter cloth coarse filtrations.
6) juice after the filtration is earlier 40000~60000 ultra-filtration membrane through molecular weight cut-off, is 6000~20000 ultra-filtration membrane again through molecular weight cut-off, obtains the superoxide-dismutase crude extract.
7) crude extract is further purified through sephadex column, obtains the superoxide-dismutase refined liquid.
8) obtain the superoxide-dismutase finished fluid, can obtain solid superoxide-dismutase powder finished product through spray-drying process.
Preferable, step 2) phosphoric acid buffer is formulated with SODIUM PHOSPHATE, MONOBASIC and Sodium phosphate dibasic in.
Preferable, step 2) in, during making beating, Controllable Temperature is built in 30~50 ℃.
Preferable, adopting the aperture in the step 3) is that the belt filter press of 140-390 micron squeezes, and pressure condition is 0.4~0.8MPa/cm 2
Preferable, sephadex column is SephadexG-50 in the step 7).
Preferable, in the step 8), the spraying drying inlet temperature is between 120~135 ℃.
Beneficial effect of the present invention:
The present invention is intended to propose a kind of processing method of extracting superoxide-dismutase from plant stem-leaf, the extraction raw material of this processing method is the fresh plant cauline leaf, raw material sources are wide, cost is low and harmless, virus-free to humans and animals, without any side effects, can be used for medicine, food, beverage and healthcare products etc.; This processing method is handled through twice squeezing after making beating, and crushing juice rate can reach more than 80%, the extraction yield height, and physical condition is easier to reach; The mode separation and purification superoxide-dismutase that adopts membrane separation technique to combine with chromatography column, with two kinds of technology separately use compare, better effects if can improve the purity of superoxide-dismutase product greatly; The waste residue that produces in this processing method can be used as biological fodder, and is that 40000~60000 ultra-filtration membrane is held back liquid down and also contained abundant leaf protein through molecular weight cut-off, both can handle obtaining other protein products again, also can directly drink to animal.
Description of drawings
Fig. 1 is the present invention extracts superoxide-dismutase from plant stem-leaf explained hereafter schema.
Embodiment
Below in conjunction with concrete embodiment the present invention is described in further detail.
Embodiment 1 extracts superoxide-dismutase from fresh Rumex
The Production Flow Chart of present embodiment as shown in Figure 1, take by weighing the fresh Rumex cauline leaf that 50kg cleans up, the pH that adds 50kg0.1mol/L is 7.4 phosphate buffer solution (formulated with SODIUM PHOSPHATE, MONOBASIC and Sodium phosphate dibasic), smashes homogenate with hollander, and holding temperature is at 35 ℃ during making beating.Slurries are entered belt filter press squeeze, 140 microns in strainer aperture, pressure is 0.8MPa/cm 2, collect juice 90kg, filter residue is added the 12kg deionized water adopt identical condition to squeeze, discard waste residue, twice filtrate is merged 100kg altogether, filter with 300 purpose filter clothes.Filtrate is earlier 40000 ultra-filtration membrane through molecular weight cut-off, obtains the liquid 25kg greater than 40000 molecular weight; Be 10000 ultra-filtration membrane again through molecular weight cut-off less than the liquid of 40000 molecular weight, obtain superoxide-dismutase crude extract 4.5kg; Crude extract is used the 4%(weight percent through sephadex column (SephadexG-50)) low ethanol carries out wash-out, obtains the superoxide-dismutase refined liquid; Behind the concentrating under reduced pressure, the ratio of survey superoxide-dismutase is lived and is 830.2u/ milligram albumen; Use spray-drying process, in inlet temperature spraying drying under 130 ℃ of conditions, obtain solid superoxide-dismutase powder-product 113.84g.
Embodiment 2 extracts superoxide-dismutase from fresh soda is fluffy
Take by weighing the fluffy cauline leaf of fresh soda that 50kg cleans up, the pH that adds 75kg0.1mol/L is 7.0 phosphate buffer solution (formulated with SODIUM PHOSPHATE, MONOBASIC and Sodium phosphate dibasic), smashes homogenate with hollander, and holding temperature is at 45 ℃ during making beating.Slurries are entered belt filter press squeeze, 190 microns in strainer aperture, pressure is 0.4MPa/cm 2, collect juice 115kg, filter residue is added the 11kg deionized water adopt identical condition to squeeze, discard waste residue, twice filtrate is merged 126kg altogether, filter with 100 purpose filter clothes.Filtrate is earlier 50000 ultra-filtration membrane through molecular weight cut-off, obtains the liquid 36kg greater than 50000 molecular weight; Be 20000 ultra-filtration membrane again through molecular weight cut-off less than the liquid of 50000 molecular weight, obtain superoxide-dismutase crude extract 5.7kg; Crude extract is used the 5%(weight percent through sephadex column (SephadexG-50)) low ethanol carries out wash-out, obtains the superoxide-dismutase refined liquid; Behind the concentrating under reduced pressure, the ratio of survey superoxide-dismutase is lived and is 657.2u/ milligram albumen; Use spray-drying process, in inlet temperature spraying drying under 120 ℃ of conditions, obtain solid superoxide-dismutase powder-product 106.92g.
Embodiment 3 extracts superoxide-dismutase from fresh spinach
Take by weighing the fresh spinach cauline leaf that 50kg cleans up, the pH that adds 100kg0.1mol/L is 7.8 phosphate buffer solution (formulated with SODIUM PHOSPHATE, MONOBASIC and Sodium phosphate dibasic), smashes homogenate with hollander, and holding temperature is at 30 ℃ during making beating.Slurries are entered belt filter press squeeze, 170 microns in strainer aperture, pressure is 0.6MPa/cm 2, collect juice 126kg, filter residue is added the 28kg deionized water adopt identical condition to squeeze, discard waste residue, twice filtrate is merged 155kg altogether, filter with 200 purpose filter clothes.Filtrate is earlier 60000 ultra-filtration membrane through molecular weight cut-off, obtains the liquid 47kg greater than 60000 molecular weight; Be 6000 ultra-filtration membrane again through molecular weight cut-off less than the liquid of 60000 molecular weight, obtain superoxide-dismutase crude extract 6.4kg; Crude extract is used the 4%(weight percent through sephadex column (SephadexG-50)) low ethanol carries out wash-out, obtains the superoxide-dismutase refined liquid; Behind the concentrating under reduced pressure, the ratio of survey superoxide-dismutase is lived and is 1002.8u/ milligram albumen; Use spray-drying process, in inlet temperature spraying drying under 135 ℃ of conditions, obtain solid superoxide-dismutase powder-product 117.34g.

Claims (6)

1. method of extracting superoxide-dismutase from plant comprises following concrete steps:
1) gets the fresh cauline leaf of plant, clear water flush away surface irregularities;
2) pressing solid-liquid ratio (W/V) is 1:1~1:2, and the pH that adds 0.1M is 7~8 phosphoric acid buffer, breaks into slurries with hollander;
3) slurries that obtain are squeezed, collect juice;
4) filter residue adding deionized water carries out twice pressing, and juice and previous step merge, and discard waste residue;
5) juice after merging is with 100 orders~300 purpose filter cloth coarse filtrations;
6) juice after the filtration is earlier 40000~60000 ultra-filtration membrane through molecular weight cut-off, is 6000~20000 ultra-filtration membrane again through molecular weight cut-off, obtains the superoxide-dismutase crude extract;
7) crude extract is further purified through sephadex column, obtains the superoxide-dismutase refined liquid;
8) obtain the superoxide-dismutase finished fluid, can obtain solid superoxide-dismutase powder finished product through spray-drying process.
2. phosphoric acid buffer is formulated with SODIUM PHOSPHATE, MONOBASIC and Sodium phosphate dibasic the method for claim 1, wherein step 2).
3. the method for claim 1, wherein step 2), during making beating, Controllable Temperature is built in 30~50 ℃.
4. the method for claim 1, wherein adopting the aperture in the step 3) is that the belt filter press of 140-390 micron squeezes, and pressure condition is 0.4~0.8MPa/cm 2
5. the method for claim 1, wherein sephadex column SephadexG-50 in the step 7).
6. the method for claim 1, wherein in the step 8), the spraying drying inlet temperature is between 120~135 ℃.
CN2013102285472A 2013-06-08 2013-06-08 Method for extracting superoxide dismutase from plant stem leaves Pending CN103289969A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103509097A (en) * 2013-10-11 2014-01-15 中国科学院烟台海岸带研究所 Extraction process method for plant leaf protein
CN103509763A (en) * 2013-10-11 2014-01-15 中国科学院烟台海岸带研究所 Process method for extracting leaf protein and superoxide dismutase from plants
CN106279387A (en) * 2016-08-09 2017-01-04 越西县绿色原野现代生态农业发展有限公司 A kind of grass leaf protein extraction process

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102010852A (en) * 2010-10-21 2011-04-13 湛江师范学院 Method for extracting sisal hemp superoxide dismutase (SOD)
CN102586200A (en) * 2011-01-12 2012-07-18 龚森淼 Process method for extracting superoxide dismutase from root, stem and leaf of burdock

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102010852A (en) * 2010-10-21 2011-04-13 湛江师范学院 Method for extracting sisal hemp superoxide dismutase (SOD)
CN102586200A (en) * 2011-01-12 2012-07-18 龚森淼 Process method for extracting superoxide dismutase from root, stem and leaf of burdock

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JINGBING WANG ET AL.: "Isolation and purification of superoxide dismutase from garlic using two-stage ultrafiltration", 《JOURNAL OF MEMBRANE SCIENCE》 *
柳建平,罗安才: "青梅果超氧化物歧化酶粗酶的提取及活性研究", 《安徽农业科学》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103509097A (en) * 2013-10-11 2014-01-15 中国科学院烟台海岸带研究所 Extraction process method for plant leaf protein
CN103509763A (en) * 2013-10-11 2014-01-15 中国科学院烟台海岸带研究所 Process method for extracting leaf protein and superoxide dismutase from plants
CN106279387A (en) * 2016-08-09 2017-01-04 越西县绿色原野现代生态农业发展有限公司 A kind of grass leaf protein extraction process

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Application publication date: 20130911