CN102702308B - Expansion solvent system extraction device and process for separating toad maggot proteins by using same - Google Patents
Expansion solvent system extraction device and process for separating toad maggot proteins by using same Download PDFInfo
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- CN102702308B CN102702308B CN201210179076.6A CN201210179076A CN102702308B CN 102702308 B CN102702308 B CN 102702308B CN 201210179076 A CN201210179076 A CN 201210179076A CN 102702308 B CN102702308 B CN 102702308B
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Abstract
The invention discloses an expansion solvent system extraction device and a process for separating toad maggot proteins by using the same, which relates to an expansion solvent system extraction device and a protein separating process. The aims of the invention are fulfilled through a technical scheme comprising the following steps of: (1) loading toad maggots into a normal-temperature swelling device for swelling; (2) adding swelled toad maggot powder into the expansion solvent system extraction device for extracting to obtain crude toad maggot proteins; (3) performing micro-filtration and ultrafiltration treatment; (4) pumping into an ultrasonic standing wave liquid phase preparation chromatogram, and continuously purifying and separating toad maggot proteins out; and (5) performing freeze drying in vacuum to obtain a finished product. During separation of proteins, processes such as normal-temperature swelling and smashing, water-free low-temperature expansion solvent system extraction, tangential flow micro-filtration and ultrafiltration, solid-free fixed phase ultrasonic standing wave liquid phase preparation chromatography purification and the like are adopted, and the content of extracted, separated and purified toad maggot proteins is up to 91.8-93.5 percent. The process has the advantages of definite extraction components, high consistence and controllable quality.
Description
Technical field
The present invention relates to a kind of expanded solvents system extraction element and protein separation process, particularly a kind of expanded solvents system extraction element and utilize it to separate the technique of toad maggot protein.
Background technology
Protein is the most important nitrogen-containing organic compound that forms organism, is the basic substance of life.Protein is the biopolymer being combined into by 20 seed amino acids, generally by 100, with upper amino acid, form, amino acid links together with putting in order according to different ratios, formed various kinds of protein, various protein is composition difference not only, and three-dimensional arrangement, physicochemical property and the biochemical functions of molecule all present different characteristics.Isolated protein carries out according to above-mentioned difference just.
CN102091318A(201110007300.9) disclose toad maggot and separated toad peptide antibiotic and antimicrobial preparation method thereof.Take toad maggot as raw material, adopt water solvent extraction process or dilute alkaline soln extraction process or dilute alcohol solution extraction process, one or more technique purifying in the techniques such as coupled ion exchange resin absorption method, ultrafiltration and micro-filtration, gel chromatography, ultrasonic standing wave preparative liquid chromatogram, then vacuum lyophilization is prepared into toad peptide antibiotic.
CN101991608A (201010551771.1) discloses fly maggot extract and its production and use, and its main component is water soluble protein, and molecular weight is 2～16Kpa.
CN101288436(200810111136.4) a kind of method and application with prepairing insect albumen powder from sterile maggot disclosed, as aseptic fly-maggot, through press filtration, decomposition, filtration, acid adjustment degree, precipitation, washing, dry and make insect protein.
The people such as Zhang Aijun have studied the edible fly maggot protein of alkaline extraction, and temperature is got in its liquid ratio, lixiviate, alkali concn has remarkably influenced to extracting yield.
ZL200410013742.4 discloses the method that adopts Chinese medicine toad maggot, can mass industrialized production toad maggot.
The conventional isolated protein technique of extracting has buffered soln extraction method, normal saline extraction method, dilute alkaline soln extraction method, after extracting, recycle the differential liberation purifying of solubleness, utilize the differential liberation purifying of molecular size and shape and utilize the differential liberation purifying of charge property.Because toad maggot protein is a kind of protein of preciousness, guarantee that maggot proteins extraction efficiency and output all need special processing condition just can reach best effect.
Summary of the invention
The problem existing for prior art, the invention provides a kind of expanded solvents system extraction element and utilizes it to separate the technique of toad maggot protein, and the toad maggot protein content of extraction separation and purification reaches 91.8～93.5%, and extraction efficiency is high.
Expanded solvents system extraction element of the present invention is by spherical expanded solvents system extractor, head tank, solid-liquid separator, gas-solid separator, finished pot, external source carbonic acid cylinder, carbon dioxide storage tank, carbonic acid gas circulating pressure pump and solvent tank composition, spherical expanded solvents system extractor by pipeline respectively with head tank, solid-liquid separator, carbonic acid gas circulating pressure pump and solvent tank are connected, solid-liquid separator is connected with gas-solid separator, gas-solid separator is connected with finished pot and carbon dioxide storage tank respectively by pipeline, carbon dioxide storage tank is connected with external source carbonic acid cylinder and carbonic acid gas circulating pressure pump respectively by pipeline.
The present invention separates toad maggot protein according to following technical scheme:
(1) 1000g toad maggot is packed in normal temperature expanding apparatus, be forced into 1～8Mpa, time 10～60min, then relief pressure is to normal pressure rapidly, and toad maggot is by expanded;
(2) the toad maggot powder after expanded joins in expanded solvents system extraction element, add expanded solvents, be forced into 2～10Mpa and form expanded solvents system, at 10～25 ℃, extract 10～60min, toad maggot protein dissolves, then be depressurized to normal pressure, obtain toad maggot protein crude product;
(3) after adding 10～50Kg pure water to stir 1000g toad maggot protein crude product, pump into the Microfilter micro-filtration processing of membrane pore size 0.5～10 μ m, after micro-filtration, solution pumps into the ultrafilter uf processing of membrane pore size 5～120nm;
(4) by the material dissolution after micro-filtration and uf processing in solvent system, pump in ultrasonic standing wave preparative liquid chromatogram, ultrasonic standing wave power 0.1～5.0KW, frequency 500～1000kHz, flow velocity 50～1000mL/min, UV-detector wavelength 280nm, continuous purification is isolated toad maggot protein;
(5), by the toad maggot protein of separation and purification vacuum lyophilization 2～24h under the condition of temperature-30～30 ℃, vacuum tightness 10～500Kpa, during moisture content of material 5～8%, obtain finished product.
The present invention is when isolated protein, adopt the expanded pulverizing of normal temperature, anhydrous low-temperature expansion solvent system extracts, tangential flow micro-filtration or ultrafiltration, without techniques such as stationary solid phase ultrasonic standing wave preparative liquid chromatogram purifying, the toad maggot protein content of extraction separation and purification reaches 91.8～93.5%.It is clear and definite that this technique is extracted composition, high conformity, and quality controllable, technological process is simple, and extraction efficiency is high, is easy to suitability for industrialized production, and energy-conservation, there is no environmental pollution.
Accompanying drawing explanation
Fig. 1 is the structural representation of expanded solvents system extraction element.
Embodiment
Embodiment one: present embodiment separates toad maggot albumen by the following method:
(1) dry 1000g toad maggot is packed in normal temperature expanding apparatus (200520108339.8) and to being filled with nitrogen in installing and add 1～5Mpa to pressing, time 10～60min, then relief pressure is to normal pressure rapidly, and toad maggot is by expanded;
(2) the toad maggot powder after expanded joins in expanded solvents system extraction element, add 1～10Kg liquid carbon dioxide, 0.1～2Kg ethanol and 0.3～1.0Kg ethyl acetate, be forced into 2～10Mpa and form expanded solvents system, keep 10～20 ℃ of temperature, time 10～30min, toad maggot protein dissolves, and is then depressurized to normal pressure, the protein in formation toad maggot powder and the mixed solution of ethanol and ethyl acetate;
(3) after adding 10～50Kg pure water to stir 1000g mixed solution, pump into the Microfilter micro-filtration processing of membrane pore size 0.5～10 μ m, after micro-filtration, solution pumps into the ultrafilter uf processing of membrane pore size 10～120nm;
(4) in the solvent system material dissolution after micro-filtration and uf processing being formed according to 4:1:5 volume ratio at methyl tertiary butyl ether one acetonitrile one water, pump in ultrasonic standing wave preparative liquid chromatogram (200920274485.8), ultrasonic standing wave power 0.1～5.0KW, frequency 500～1000kHz, flow velocity 50～1000mL/min, UV-detector wavelength 280nm, continuous purification is isolated toad maggot protein;
(5), by the toad maggot protein of separation and purification vacuum lyophilization 2～24h under the condition of temperature-30～30 ℃, vacuum tightness 10～500Kpa, during moisture content of material 5～8%, obtain finished product.
As shown in Figure 1, the system of expanded solvents described in present embodiment extraction element is by spherical expanded solvents system extractor 1, head tank 2, solid-liquid separator 3, gas-solid separator 4, finished pot 5, external source carbonic acid cylinder 6, carbon dioxide storage tank 7, carbonic acid gas circulating pressure pump 8 and solvent tank 9 form, spherical expanded solvents system extractor 1 by pipeline respectively with head tank 2, solid-liquid separator 3, carbonic acid gas circulating pressure pump 8 and solvent tank 9 are connected, solid-liquid separator 3 is connected with gas-solid separator 4, gas-solid separator 4 is connected with finished pot 5 and carbon dioxide storage tank 7 respectively by pipeline, carbon dioxide storage tank 7 is connected with external source carbonic acid cylinder 6 and carbonic acid gas circulating pressure pump 8 respectively by pipeline.
Embodiment two: present embodiment separates toad maggot albumen by the following method:
(1) 1000g toad maggot is packed in normal temperature expanding apparatus (200520108339.x), to being filled with carbon dioxide to pressure 1～8Mpa in device, the rapid relief pressure of time 10～60min is to normal pressure, and toad maggot is by expanded;
(2) the toad maggot after expanded is put into expanded solvents system extraction element, add 100～200mL methyl alcohol and 1.0～2.0kg carbonic acid gas and 50～100mL ammoniacal liquor, be forced into 3～10Mpa and form expanded solvents system, at 10～25 ℃, extract 10～60min, then reduce pressure to normal pressure, the toad maggot protein that is dissolved in expanded solvents system is separation with other solid substances;
(3) after isolated toad maggot protein crude product adds the pure water of 10～50 times to stir, pump into membrane pore size 0.5～10 μ m Microfilter micro-filtration and pump into again membrane pore size 5～120nm ultrafilter uf processing;
(4) material dissolution after micro-filtration and uf processing is by mass concentration 12.5%K
2hPO
4in the solvent system of mass ratio 12.5%PEG1000 composition, after stirring, pump in ultrasonic standing wave merit liquid phantom preparing chromatogram (200920274485.8), ultrasonic standing wave power 0.5～5KW, frequency 750～1000kHz, flow velocity 50～1000mL/min, UV-detector wavelength 280nm, continuous purification separates toad maggot protein;
(5) the purifying toad maggot protein of collecting is dry with vacuum freeze drier, service temperature-30～30 ℃, vacuum tightness 10～500Kpa, time 2～24h, is dried to moisture 5～8% finished products.
Claims (3)
1. a separating technology for toad maggot protein, is characterized in that described method steps is as follows:
(1) 1000g toad maggot is packed in normal temperature expanding apparatus, be forced into 1～8Mpa, time 10～60min, then relief pressure is to normal pressure rapidly, and toad maggot is by expanded;
(2) the toad maggot powder after expanded joins in expanded solvents system extraction element, add expanded solvents, be forced into 2～10Mpa and form expanded solvents system, at 10～25 ℃, extract 10～60min, toad maggot protein dissolves, then be depressurized to normal pressure, obtain toad maggot protein crude product, described expanded solvents system is by 1～10Kg liquid carbon dioxide, 0.1～2Kg ethanol and 0.3～1.0Kg ethyl acetate are also forced into 2～10Mpa composition, described expanded solvents system extraction element is by spherical expanded solvents system extractor (1), head tank (2), solid-liquid separator (3), gas-solid separator (4), finished pot (5), external source carbonic acid cylinder (6), carbon dioxide storage tank (7), carbonic acid gas circulating pressure pump (8) and solvent tank (9) composition, spherical expanded solvents system extractor (1) by pipeline respectively with head tank (2), solid-liquid separator (3), carbonic acid gas circulating pressure pump (8) and solvent tank (9) are connected, solid-liquid separator (3) is connected with gas-solid separator (4), gas-solid separator (4) is connected with finished pot (5) and carbon dioxide storage tank (7) respectively by pipeline, carbon dioxide storage tank (7) is connected with external source carbonic acid cylinder (6) and carbonic acid gas circulating pressure pump (8) respectively by pipeline,
(3) after adding 10～50Kg pure water to stir 1000g toad maggot protein crude product, pump into the Microfilter micro-filtration processing of membrane pore size 0.5～10 μ m, after micro-filtration, solution pumps into the ultrafilter uf processing of membrane pore size 5～120nm;
(4) by the material dissolution after micro-filtration and uf processing in solvent system, pump in ultrasonic standing wave preparative liquid chromatogram, ultrasonic standing wave power 0.1～5.0KW, frequency 500～1000kHz, flow velocity 50～1000mL/min, UV-detector wavelength 280nm, continuous purification is isolated toad maggot protein, and described solvent system is comprised of according to 4:1:5 volume ratio methyl tertiary butyl ether one acetonitrile one water;
(5), by the toad maggot protein of separation and purification vacuum lyophilization 2～24h under the condition of temperature-30～30 ℃, vacuum tightness 10～500Kpa, during moisture content of material 5～8%, obtain finished product.
2. the separating technology of toad maggot protein according to claim 1, is characterized in that described expanded solvents system replaces with by 100～200mL methyl alcohol, 1.0～2.0kg carbonic acid gas and 50～100mL ammoniacal liquor and is forced into the solvent that 2～10Mpa forms.
3. the separating technology of toad maggot protein according to claim 1, is characterized in that described solvent system replaces with by mass concentration 12.5%K
2hPO
4solvent with mass ratio 12.5%PEG1000 composition.
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| CN104740594A (en) * | 2015-03-02 | 2015-07-01 | 哈尔滨工业大学 | Application of gecko maggot protein in preparing anti-lymphoma drugs |
| CN104623621A (en) * | 2015-03-02 | 2015-05-20 | 哈尔滨工业大学 | Application of gecko maggot protein to preparation of drug for treating nasopharyngeal cancer |
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| CN102091318B (en) * | 2011-01-14 | 2012-11-28 | 哈尔滨工业大学 | Toad peptide antibiotics separated from toad maggots and preparation method of antibacterial drugs thereof |
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