CN102796163A - Method for extracting and separating proteins from cake by using ionic liquid and enzyme process - Google Patents
Method for extracting and separating proteins from cake by using ionic liquid and enzyme process Download PDFInfo
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- CN102796163A CN102796163A CN2012103287856A CN201210328785A CN102796163A CN 102796163 A CN102796163 A CN 102796163A CN 2012103287856 A CN2012103287856 A CN 2012103287856A CN 201210328785 A CN201210328785 A CN 201210328785A CN 102796163 A CN102796163 A CN 102796163A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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Abstract
The invention relates to a field of separation and purification of proteins, in particular to a novel method for extracting and separating proteins from cake by using an ionic liquid and an enzyme process. The method for extracting and separating proteins from cake by using an ionic liquid and an enzyme process disclosed by the invention comprises the following steps of: (1) smashing and sieving cake serving as a raw material, and degreasing to obtain degreased cake powder; (2) performing enzymolysis on the degreased cake powder under the condition that the pH is 7-10, the temperature is 40-65 DEG C and the material to liquid ratio is 1:7-1:15 in a state that the total enzyme activity is 90-250 U.g<-1> for 1-5 hours, and centrifugally separating; (3) performing alkali-solution and acid-isolation on the obtained precipitate and hydrolysate, and performing freeze drying to obtain crude proteins; (4) adding the crude proteins obtained in the step (3) into an ionic liquid/salt dual-water-phase system, and separating and purifying proteins at the temperature of 40-70 DEG C and at the pH of 4-9; and (5) separating proteins from an ionic liquid phase obtained in the step (4) with a membrane separation method, recovering an ionic liquid, and performing freeze drying to obtain high-purity protein powder. The method is easy to operate, is environment-friendly, and is a novel environment-friendly, energy-saving and efficient protein separating and purifying method; and the protein purity is high, and the bioactivity is kept.
Description
Technical field
The present invention relates to the protein separation field, be specifically related to a kind of ionic liquid and Enzymatic Extraction utilized and separate method of protein in the grouts.
Background technology
Oil meal generally contains the composition that albumen, polyphenol, phytic acid, polysaccharide etc. have higher utility value.Grouts are the very high plant protein resources of a kind of potential nutritive value, and its protein content is 35% ~ 40%, and this is the highest composition of utility value in the grouts.Containing the multiple necessary amino acid of a large amount of needed by human body in this albumen, also is very important food protein source therefore.We are example at this with the rape seed protein in the rapeseed meal; The dregs of rapeseed cake total amino acid content accounts for 83.8% of its total protein; Its lysine content and soybean are approaching, and methionine content is also higher than soybean, are applicable to the beans that is used for replenishing cereal and many these amino acids of shortage.Compare with other protein, the advantage of vegetable seed protein is: it is very approaching that 1. its amino acid compositional model and FAO and WHO recommend pattern, more better than Sunlover 10; 2. sulfur-containing amino acid---methionine(Met), Gelucystine and histidine content are high than soybean protein concentrate.Sulfur-containing amino acid is one type of most important amino acid in the food, also belongs to necessary amino acid, and effect is great aspect food sense organ function, trophic function and reason regulatory function.Sulfur-containing amino acid and basic group that rape seed protein is rich in the cereal and is lacked are sour, can remedy cereal amino acid defective.China's diet formula is main with cereal, and it is not enough that rape seed protein can remedy China people diet formula, satisfies production such as food better and lives to the protein needs with compatriots.
Ionic liquid is the green solvent of a kind of alternative traditional volatile organic matter of developing rapidly over past ten years, not volatile, not flammable, advantages such as stability is high, dissolving power is strong, function adjustable that ionic liquid has.Ionic liquid can form double-aqueous phase system with multiple material such as salt, sugar, tensio-active agent etc., and it is showing excellent performance aspect extracting and separating biological material, and this can open up a kind of new green stripping technique undoubtedly.
Traditional method for extracting and separating protein exists still that extraction yield is low, cost is high and shortcoming such as complicated operation, therefore seeks the emphasis that a kind of efficient, green and economic method for extracting and separating protein has become technical study.Thereby; The inventor according to ionic liquid-salt double-aqueous phase system than the technological not available plurality of advantages of traditional double water; As simple to operate, pollution-free, separation efficiency is high, may command emulsification, ionic liquid etc. can be recycled; In conjunction with the proteinic technology of enzymolysis and extraction, researched and developed a kind of more efficient and economic green sub proteinic new technology in grouts.Present method has kept proteinic biological activity not to be damaged in extraction separation to the utmost simultaneously, has improved proteinic quality.
Summary of the invention
The object of the present invention is to provide a kind of ionic liquid and Enzymatic Extraction utilized to separate method of protein in the grouts.
The object of the invention is realized through following technical scheme:
A kind of ionic liquid and Enzymatic Extraction utilized separated method of protein in the grouts, may further comprise the steps:
(1) with using organic solvent degreasing behind the raw material grouts crushing screening, obtains degreasing grouts powder;
(2) be that 7 ~ 10 buffered soln mixes the back and adds prozyme and process mixed solution with the described degreasing grouts of step (1) powder and pH value, behind enzymolysis 2 ~ 5 h under 40 ~ 65 ℃ of conditions, spinning obtains enzymolysis solution and deposition with mixed solution; The feed liquid mass ratio of said degreasing grouts powder and buffered soln is 1:7 ~ 1:15, and the total enzyme work in the said mixed solution is 90 ~ 250 Ug
-1
(3) then the described deposition alkali of step (2) is dissolved the back spinning, after the supernatant that separation is obtained merges with the described enzymolysis solution of step (2), carry out the heavy also spinning of acid, will precipitate and wash postlyophilization, obtain crude protein;
(4) crude protein that step (3) is obtained is added in ionic liquid/salt double-aqueous phase system, leaving standstill after the concussion under the condition of 40 ~ 70 ℃ of temperature, pH4 ~ 9, gets upper strata ionic liquid phase again;
(5) at last the ionic liquid of step (4) is used the membrane separation process separating protein mutually, and reclaim ionic liquid, the protein lyophilize is obtained the high purity protein powder.
Above-mentioned utilize ionic liquid and Enzymatic Extraction are separated method of protein in the grouts; It is; Enzyme in the said prozyme is AMS and cellulase, perhaps AMS, cellulase and polygalacturonase, and the enzyme activity of AMS and cellulase is than being 5:1 ~ 2:3.
Above-mentioned utilize ionic liquid and Enzymatic Extraction are separated method of protein in the grouts, and it is that in the mixed solution, the enzyme activity of AMS and cellulase is respectively 60 ~ 150 Ug
-1With 30 ~ 90 Ug
-1
Above-mentioned utilize ionic liquid and Enzymatic Extraction are separated method of protein in the grouts, and it is that described buffered soln is phosphoric acid buffer or borate buffer.
Above-mentioned utilize ionic liquid and Enzymatic Extraction are separated method of protein in the grouts, and it is that the concentration of crude protein is 20 ~ 100 mgL described in the described step (4)
-1, said ion liquid concentration is 200 ~ 600 mgmL
-1, the concentration of said salt is 90 ~ 220 mgmL
-1Be preferably: the concentration of said crude protein is 50 ~ 80 mgL
-1, said ion liquid concentration is 300 ~ 400 mgmL
-1, the concentration of said salt is 130 ~ 180 mgmL
-1The concentration that further is preferably crude protein is 70 ~ 80 mgL
-1, ion liquid concentration is 350 ~ 380 mgmL
-1, the concentration of salt is 150 ~ 180 mgmL
-1
Described ionic liquid/salt double-aqueous phase system is hydrophilic ionic-liquid/salt double-aqueous phase system, and described ionic liquid is wetting ability glyoxaline ion liquid, pyridines ionic liquid or guanidine class ionic liquid, and described salt is phosphoric acid salt, carbonate or vitriol.
Described wetting ability glyoxaline ion liquid is [Amim] Cl, [Bmim] Cl or [Bmim] BF
4, described pyridines ionic liquid is [Epy] Br or [BPy] BF
4, described phosphoric acid salt is K
2HPO
4, KH
2PO
4Or K
3PO
4, described carbonate is NaHCO
3, Na
2CO
3Or (NH
4)
2SO
4Preferred described ionic liquid/salt double-aqueous phase system is [Bmim] Cl/K
2HPO
4, [Amim] Cl/K
2HPO
4, [Epy] Br/K
2HPO
4, [Bmim] BF
4/ K
2HPO
4Further be preferably [Bmim] Cl/K
2HPO
4
The centrifugal speed of spinning is 3000 ~ 5000 rmin described in step (2) and the step (3)
-1
The number of times of deposition washing is 3 ~ 6 times in the step (3); In the step (4), the time of said concussion is 20 ~ 50min, and concussion speed is 100 ~ 300 rmin
-1, the said time of leaving standstill is 30 ~ 60min.
Organic solvent described in the step (1) is sherwood oil, cyclohexane, normal hexane or No. four solvents.
Obtain crude protein with the alkali extraction and acid precipitation method in the technical scheme steps of the present invention (3), wherein " alkali extraction and acid precipitation " is known in those skilled in the art.Utilize the principle of protein " alkali extraction and acid precipitation "; Be that protein is under alkaline condition, in relatively dissolving easily in water (extraction), when under acidic conditions, arriving iso-electric point (protein solubleness when iso-electric point is minimum) then; The principle of the easy aggregate and precipitate of protein is come out protein extraction.
Raw material grouts of the present invention are the grouts that squeezed of low temperature or high temperature preferably.
The enzyme that uses among the present invention is the mixture of AMS and cellulase, perhaps the mixture of AMS, cellulase and polygalacturonase.Once useful other several methods of applicant extract protein or other enzyme carries out enzymolysis, but the result is not very desirable, and the problem of existence is more, and its key is how to solve efficiently, keeps proteinic higher structure and keeps bioactive problem.If use traditional alkali extraction and acid precipitation method to extract protein, extraction yield is lower, and the time is longer.If use proteolytic enzyme to carry out enzyme digestion reaction, it is improper that proteolytic enzyme or enzymatic hydrolysis condition are selected, and protein can be hydrolyzed into polypeptide, loses higher structure.The applicant discovers that at pH be 7 ~ 10; 40 ~ 65 ℃ of temperature; Only add in the grouts reaction solution of solid-liquid ratio 1:7 ~ 1:15 a small amount of AMS and cellulase mixture or AMS, cellulase and or the mixture of polygalacturonase; Extraction yield is improved, successfully accomplished the present invention.
AMS, cellulase, polygalacturonase can be distinguished hydrolyzed starch, Mierocrystalline cellulose and pectin, with cellulose skeleton, the collapse plant cell wall of effective degrading plant cell walls, thereby protein are discharged and don't destroy proteinic structure.When AMS and cellulase mixing effect, perhaps can embody above-mentioned function preferably when AMS, cellulase and polygalacturonase mixing effect, at this moment, the enzyme work of AMS and cellulase is respectively 60 ~ 150 Ug
-1With 30 ~ 90 Ug
-1The present invention discovers if enzyme dosage is too much, and it influences little to proteinic extraction yield but the waste resource, and proteinic composition and mensuration are produced certain influence.Therefore, preferably be controlled at 90 ~ 250 Ug to total enzyme work among the present invention
-1, AMS compares at 5:1 ~ 2:3 with the enzyme activity of cellulase.In addition, the raw material of the AMS that uses among the present invention, cellulase and polygalacturonase should be first-selected the enzyme high kind of living, possibly save cost like this and increase the benefit, more than several kinds of enzymes all can buy from market.
With degreasing grouts powder and pH value is that 7 ~ 10 buffered soln mixes the back and adds prozyme, carries out enzyme digestion reaction and extracts protein.Carry out heated and stirred in the common container, its condition is 40 ~ 65 ℃, 2 ~ 5 h, feed liquid mass ratio 1:7 ~ 1:15, general stirring velocity 50 ~ 100 rmin
-1Then in rotating speed 3000 ~ 5000 rmin
-1After centrifugal, will precipitate alkali and dissolve the back spinning, alkali will be dissolved supernatant and the heavy and spinning of enzymolysis solution merging back acid, will precipitate and wash postlyophilization, obtain crude protein.Proteinic extraction yield can reach more than 96%, and raw protein content is more than 80%.
Proteinic main points are in the extraction separation grouts of the present invention: enzymolysis process extracts the protein in the grouts, and the proteinic technology of coupled ion liquid double water-phase method separation and purification, and wherein the selection of ionic liquid and salt and consumption are crucial.
The ionic liquid double-aqueous phase system that uses among the present invention is preferably 1-butyl-3-Methylimidazole villaumite ([Bmim] Cl)/K
2HPO
4, 1-allyl group-3-Methylimidazole villaumite ([Amim] Cl)/K
2HPO
4System, [Epy] Br/K
2HPO
4System or [Bmim] BF
4/ K
2HPO
4System.Once useful several kinds of ionic liquids and salt formation double water-phase carry out separation and purification, but not really desirable, have more problem, and its key is how to solve the problem that forms double water-phase and improve separation and purification efficient.Discover that phosphoric acid salt can form double water-phase with ionic liquid preferably, and [Bmim] Cl, [Amim] Cl, [Epy] Br or [Bmim] BF
4With K
2HPO
4The double water-phase that forms is higher to proteinic separation and purification efficient, has successfully accomplished the present invention.
The ionic liquid double-aqueous phase system that adopts among the present invention is ionic liquid-inorganic salt double-aqueous phase system.Ionic liquid molecules can be regarded as by hydrophobic and hydrophilic two portions and form.Hydrophobic part is assembled nucleation in the aqueous solution, hydrophilic segment outwards opens the formation micella.Add auxiliary inorganic salt and possibly play non-distinctive salting out, promote the formation of double water-phase with the osmotic pressure effect.After protein gets into double-aqueous phase system; Because surface properties, charge effect and the effect of various power (like hydrophobic key, hydrogen bond and ionic linkage etc.) and the influence of solution environmental; Make them different in upper and lower concentration in mutually; Be that each composition distributes in two alternate selectivity, thereby reach the purpose of extracting and separating.
In with ionic liquid-inorganic salt double-aqueous phase system separation and purification crude protein, the concentration of crude protein is 20 ~ 100 mgL
-1, ionic liquid concentration is 200 ~ 600 mgmL
-1, inorganic salt concentration is 90 ~ 220 mgmL
-1Through repeatedly testing to such an extent that optimal conditions is: the concentration of crude protein is 50 ~ 80 mgL
-1, ionic liquid concentration is 300 ~ 400 mgmL
-1, inorganic salt concentration is 130 ~ 180 mgmL
-1And when the inorganic salt concentration of system is lower than 90 mgmL
-1Or ionic liquid concentration is lower than 200 mgmL
-1The time system can't form double water-phase, along with the increase of inorganic salt concentration, phase volume ratio reduces gradually up and down, but protein increases in two alternate partition ratios gradually, when inorganic salt concentration reaches 150 mgmL
-1The time, rape seed protein is maximum in two alternate partition ratios, and this moment is the highest to the percentage extraction of rape seed protein; At 150 ~ 180 mgmL
-1The time, partition ratio will tend towards stability; Inorganic salt concentration is greater than 220 mgmL
-1The time, partition ratio will reduce to some extent.When ionic liquid concentration is 300 ~ 400 mgmL
-1The time vegetable seed proteic percentage extraction increase gradually, when ionic liquid concentration greater than 400 mgmL
-1The time, after reducing gradually, the percentage extraction of rape seed protein is tending towards certain.Therefore, preferred: the concentration of grouts crude protein of the present invention is 50 ~ 80 mgL
-1, ionic liquid concentration is 300 ~ 400 mgmL
-1, inorganic salt concentration is 130 ~ 180 mgmL
-1In addition, the used ionic liquid purity of the present invention needs greater than 99%.
After above raw materials mix, carry out the aqueous two-phase extraction separating protein.In constant temperature oscillator, add thermal shocking, add the thermal shocking condition and be generally 40 ~ 70 ℃ of Heating temperatures, times 20 ~ 50 min, hunting speed 100 ~ 300 rmin
-1, pH 4 ~ 9 o'clock system is higher to proteinic percentage extraction, most preferably is pH 6 ~ 9.Phase-splitting is complete after leaving standstill 30 ~ 60 min, and ionic liquid can be isolated protein through membrane separation process mutually, after the protein lyophilize high-purity protein, ionic liquid can be able to reclaim after rotary evaporation, vacuum-drying and use.Proteinic percentage extraction of the present invention is up to 98%, and the ionic liquid recovery is more than 90%, and the ionic liquid that reclaims to proteinic percentage extraction still up to more than 94%, this method is efficiently energy-conservation again.
The method for extracting and separating protein key that the present invention adopted is that water enzymolysis process and the acting in conjunction of ionic liquid double water-phase method to obtain highly purified protein, so as to improving the utility value of grouts, obtain nutritious protein.
Ionic liquid is a kind of novel green solvent that occurs in recent years.In room temperature or near it is made up of ion with organic melting salt that liquid state exists fully under the room temperature.Ionic liquid not only has the advantage of conventional organic solvents, and compares with organic solvent and to have shown many excellent properties, and is wide like the liquid state TR, can be from stable existence in 15 ℃ ~ 300 ℃ scopes; Has good physical and chemical stability; Almost do not have vp, eliminated the pollution problem of volatile organic solvent environment; Do not have combustibility, with organism good dissolving ability is arranged all inorganic in a large number; Double effects with solvent and catalyzer can be used as reaction solvent and catalyst active carrier; Specific conductivity is high.Simultaneously, ion liquid character can be optimized through regulating zwitterion.This has just determined ionic liquid will become the ideal substitute of volatile organic solvent.Therefore, the people is arranged ionic liquid, supercritical CO
2With double water-phase and be called big green solvent of 21 centurys three, have broad application prospects.
Ionic liquid double-aqueous phase system generally is the double-aqueous phase system that is formed by hydrophilic ionic-liquid, inorganic salt (like phosphoric acid salt, carbonate, vitriol, oxyhydroxide etc.) and water, and it combines the advantage of ionic liquid and double-aqueous phase system.Ionic liquid aqueous two-phase extraction separating protein belongs to liquid-liquid extraction; Its principle is similar with the principle of water-organic extractant phase; But it is as a kind of separation system of novel green efficiently; Compare with the traditional polymer double-aqueous phase system, the ionic liquid double water-phase has that viscosity is low, phase-splitting is fast, be difficult for emulsification, percentage extraction is high, ionic liquid can recycle etc. advantage, therefore more and more receive the attention of academia and industrial community.
Technical scheme proposed by the invention is more energy-efficient, meets modern's low-carbon environment-friendly requirement.
Protein collection of the present invention mainly is meant with ionic liquid recovery technology isolates protein with last ionic liquid mutually with ultra-filtration membrane, gets protein powder after the lyophilize; And ionic liquid can be able to reclaim with rotary evaporation and vacuum-drying.
Protein has following characteristics in the enzyme process coupled ion liquid double water-phase extraction separation grouts of the present invention:
1. the efficient of extraction separation is high, lipidated protein is high: the present invention adopts the water enzymolysis process to combine the protein in the extraction separation grouts with ionic liquid double water-phase method; Protein has been carried out multiple extraction separation and operated simple and easyly, and unicity and complicated operation that has solved most methods etc. makes the unfavorable problem of lipidated protein.
2. economical, environment friendly and pollution-free: the ionic liquid that the present invention adopts is a kind of green solvent of alternative traditional volatile organic matter, and can repeatedly recycle.This product has catered to the requirement of modern low-carbon energy-saving, and market outlook are wide.
Embodiment
Embodiment 1
(1) with high temperature rapeseed meal crushing screening, petroleum ether degreasing gets degreasing rapeseed meal powder;
(2) degreasing rapeseed meal powder is mixed with the phosphoric acid buffer of pH=8.0 the back adds AMS and cellulase is processed mixed solution, with mixed solution behind enzymolysis 4h under 60 ℃ of conditions, 4000 rmin
-1Centrifugal 20min obtains enzymolysis solution and deposition.
The feed liquid mass ratio of said degreasing rapeseed meal powder and phosphoric acid buffer is 1:15;
The enzyme activity of AMS and cellulase is respectively 110 Ug in the said mixed solution
-1With 60 Ug
-1, total enzyme activity is 170 Ug
-1
(3) step (2) deposition is added in the water of 10 times of amounts, use 0.5 molL
-1NaOH transfers to pH=10 and carries out the molten reaction of alkali, behind the 2h, and 4000 rmin
-1Centrifugal 20min, supernatant and step (2) enzymolysis solution that separation is obtained merge, and add 0.5 molL
-1It is heavy that hydrochloric acid transfers pH to 4.2 ~ 4.8 to carry out acid, 4000 rmin
-1Centrifugal 20min, after distilled water wash deposition 5 times, lyophilize obtains bulky vegetable seed albumen;
(4) bulky vegetable seed albumen is added to [Bmim] Cl/K
2HPO
4In the double-aqueous phase system, 65 ℃ of temperature, pH 7.0, hunting speed 200 rmin
-1Constant-temperature shaking 30 min leave standstill 40 min again, get upper strata ionic liquid phase;
Bulky vegetable seed protein concentration is 70 mgL
-1, [Bmim] Cl concentration is 350 mgmL
-1, K
2HPO
4Concentration is 150 mgmL
-1
(5) ionic liquid is used the membrane separation process separating protein mutually, and reclaims ionic liquid with removing moisture after rotary evaporation and the vacuum-drying, and the protein lyophilize is obtained high purity rape seed protein powder.
Embodiment 2
(1) with high temperature rapeseed meal crushing screening, the cyclohexane degreasing gets degreasing rapeseed meal powder;
(2) degreasing rapeseed meal powder is mixed with the phosphoric acid buffer of pH=8.0 the back adds AMS and cellulase is processed mixed solution, with mixed solution behind enzymolysis 3 h under 55 ℃ of conditions, 4000 rmin
-1Centrifugal 20min obtains enzymolysis solution and deposition.
The feed liquid mass ratio of said degreasing rapeseed meal powder and phosphoric acid buffer is 1:10;
The enzyme activity of AMS and cellulase is respectively 90 Ug in the said mixed solution
-1With 50 Ug
-1, total enzyme activity is 140 Ug
-1
(3) step (2) deposition is added in the water of 10 times of amounts, use 0.5 molL
-1NaOH transfers to pH=10 and carries out the molten reaction of alkali, behind the 2h, and 4000 rmin
-1Centrifugal 20 min, supernatant and step (2) enzymolysis solution that separation is obtained merge, and add 0.5 molL
-1It is heavy that hydrochloric acid transfers pH 4.2 ~ 4.8 to carry out acid, 4000 rmin
-1Centrifugal 20 min, after distilled water wash precipitated 5 times, lyophilize obtained bulky vegetable seed albumen;
(4) bulky vegetable seed albumen is added to [Amim] Cl/K
2HPO
4In the double-aqueous phase system, 65 ℃ of temperature, pH 7.0,200 rmin
-1Isothermal vibration 40 min leave standstill 40 min again, get upper strata ionic liquid phase;
Bulky vegetable seed protein concentration is 80 mgL
-1, [Amim] Cl concentration is 400 mgmL
-1, K
2HPO
4Concentration is 180 mgmL
-1
(5) ionic liquid is used the membrane separation process separating protein mutually, and reclaims ionic liquid with removing moisture after rotary evaporation and the vacuum-drying, and the protein lyophilize is obtained high purity rape seed protein powder.The ionic liquid recovery is more than 90%.
Embodiment 3
(1) with low temperature rapeseed meal crushing screening, the normal hexane degreasing gets degreasing rapeseed meal powder;
(2) degreasing rapeseed meal powder is mixed the back with the borate buffer of pH=9.0 and adds AMS, cellulase and polygalacturonase and process mixed solution, with mixed solution behind enzymolysis 5 h under 60 ℃ of conditions, 4000 rmin
-1Centrifugal 20min obtains enzymolysis solution and deposition.
The feed liquid mass ratio of said degreasing rapeseed meal powder and borate buffer is 1:15;
The enzyme activity of AMS, cellulase and polygalacturonase is respectively 70 Ug in the said mixed solution
-1, 40 Ug
-1With 50 Ug
-1, total enzyme activity is 160 Ug
-1
(3) step (2) deposition is added in the water of 10 times of amounts, use 0.5 molL
-1NaOH transfers to pH=10 and carries out the molten reaction of alkali, behind the 2h, and 4000 rmin
-1Centrifugal 20min, supernatant and step (2) enzymolysis solution that separation is obtained merge, and add 0.5 molL
-1It is heavy that hydrochloric acid transfers pH 4.2 ~ 4.8 to carry out acid, 4000 rmin
-1Centrifugal 20min, after distilled water wash precipitated 5 times, lyophilize obtained bulky vegetable seed albumen;
(4) bulky vegetable seed albumen is added to [Epy] Br/K
2HPO
4In the double-aqueous phase system, 65 ℃ of temperature, pH 6.5,200 rmin
-1Isothermal vibration 30 ~ 45 min leave standstill 30 min again, get upper strata ionic liquid phase;
Bulky vegetable seed protein concentration is 60 mgL
-1, [Epy] Br concentration is 300 mgmL
-1, K
2HPO
4Concentration is 130 mgmL
-1
(5) ionic liquid is used the membrane separation process separating protein mutually, and reclaims ionic liquid with removing moisture after rotary evaporation and the vacuum-drying, and the protein lyophilize is obtained high purity rape seed protein powder.
Embodiment 4
(1) with low temperature leached tea oil slag crushing screening, No. four solvent degreasings get degreasing leached tea oil slag powder;
(2) degreasing leached tea oil slag powder is mixed the back with the borate buffer of pH=9 and adds AMS, cellulase and polygalacturonase and process mixed solution, with mixed solution behind enzymolysis 4 h under 60 ℃ of conditions, 4000 rmin
-1Centrifugal 25 min obtain enzymolysis solution and deposition.
The feed liquid mass ratio of said degreasing leached tea oil slag powder and borate buffer is 1:10;
The enzyme activity of AMS, cellulase and polygalacturonase is respectively 100 Ug in the said mixed solution
-1, 65 Ug
-1With 75 Ug
-1, total enzyme activity is 240 Ug
-1
(3) step (2) deposition is added in the water of 10 times of amounts, use 0.5 molL
-1NaOH transfers to pH=9.5 ~ 10.5 and carries out the molten reaction of alkali, behind 2 h, and 4000 rmin
-1Centrifugal 20min, supernatant and step (2) enzymolysis solution that separation is obtained merge, and add 0.5 molL
-1It is heavy that hydrochloric acid transfers pH 3 ~ 4 to carry out acid, 4000 rmin
-1Centrifugal 20 min, after distilled water wash precipitated 5 times, lyophilize obtained thick tea seed albumen;
(4) thick tea seed albumen is added to [Bmim] BF
4/ K
2HPO
4In the double-aqueous phase system, 70 ℃ of temperature, pH 6.8,200 rmin
-1Isothermal vibration 30 min leave standstill 30 min again, get upper strata ionic liquid phase;
Thick tea seed protein concentration is 70 mgL
-1, [Bmim] BF
4Concentration is 380 mgmL
-1, K
2HPO
4Concentration is 160 mgmL
-1
(5) ionic liquid is used the membrane separation process separating protein mutually, and reclaims ionic liquid with removing moisture after rotary evaporation and the vacuum-drying, and the protein lyophilize is obtained high-purity tea seed protein powder.
Adopt the influence of different ionic liquid/salt double-aqueous phase systems and reaction conditions among the table 1 embodiment 1-4 to the protein extraction rate
| ? | Unit | Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 |
| Vegetable seed/tea seed protein concentration | mg·L -1 | 70 | 80 | 60 | 70 |
| Ionic liquid | — | [Bmim]Cl | [Amim]Cl | [Epy]Br | [Bmim]BF 4 |
| Ionic liquid concentration | mg·mL -1 | 350 | 400 | 300 | 380 |
| K 2HPO 4Concentration | mg·mL -1 | 150 | 180 | 130 | 160 |
| Temperature | ℃ | 65 | 65 | 65 | 70 |
| pH | — | 7.0 | 7.0 | 6.5 | 6.8 |
| Percentage extraction | % | 98.24 | 92.35 | 88.94 | 94.87 |
Embodiment 5
(1) with low temperature rapeseed meal crushing screening, petroleum ether degreasing gets degreasing rapeseed meal powder;
(2) degreasing rapeseed meal powder is mixed the back with the pH=9.0 borate buffer and adds AMS, cellulase and polygalacturonase and process mixed solution, with mixed solution behind enzymolysis 3 ~ 5h under 50 ~ 60 ℃ of conditions, 4000 rmin
-1Centrifugal 20 min obtain enzymolysis solution and deposition.
The feed liquid mass ratio of said degreasing rapeseed meal powder and borate buffer is 1:10;
The enzyme activity of AMS, cellulase and polygalacturonase is respectively 100 Ug in the said mixed solution
-1, 70Ug
-1And 50Ug
-1, total enzyme activity is 220Ug
-1
(3) step (2) deposition is added in the water of 10 times of amounts, use 0.5 molL
-1NaOH transfers to pH=10 and carries out the molten reaction of alkali, behind the 2h, and 4000 rmin
-1Centrifugal 20min, supernatant and step (2) enzymolysis solution that separation is obtained merge, and add 0.5 molL
-1It is heavy that hydrochloric acid transfers pH 4.2 ~ 4.8 to carry out acid, 4000 rmin
-1Centrifugal 20min, after distilled water wash precipitated 5 times, lyophilize obtained bulky vegetable seed albumen;
(4) bulky vegetable seed albumen is added to [Bmim] Cl/K
2HPO
4In the double-aqueous phase system, 200 rmin under certain temperature and pH condition
-1Isothermal vibration 30 min leave standstill 30min again, get upper strata ionic liquid phase; Rape seed protein concentration, [Bmim] Cl, K
2HPO
4Concentration and extraction temperature get different values with extraction pH, see table 2.
(5) ionic liquid is used the membrane separation process separating protein mutually, and reclaims ionic liquid with removing moisture after rotary evaporation and the vacuum-drying, and the protein lyophilize is obtained high purity rape seed protein powder, and percentage extraction is seen table 2.
Table 2 protein concentration, [Bmim] Cl concentration, K
2HPO
4Concentration, temperature and pH are to the influence of protein extraction rate
The present invention has investigated different protein concentrations, [Bmim] Cl concentration, K through embodiment 5
2HPO
4Concentration, extraction temperature and extraction pH separate the influence to the protein extraction rate to the ionic liquid aqueous two-phase extraction, and the result is as shown in table 2.The result proves: the consumption of vegetable seed crude protein is 70 ~ 80 mgL
-1, [Bmim] Cl consumption is 350 ~ 380 mgmL
-1, K
2HPO
4Consumption is 150 ~ 180 mgmL
-1, temperature is 65 ~ 70 ℃, pH 6.8 ~ 8.0 o'clock [Bmim] Cl/K
2HPO
4Double-aqueous phase system reaches more than 97% the percentage extraction of rape seed protein.
Claims (10)
1. one kind is utilized ionic liquid and Enzymatic Extraction to separate method of protein in the grouts, it is characterized in that may further comprise the steps:
(1) with using organic solvent degreasing behind the raw material grouts crushing screening, obtains degreasing grouts powder;
(2) be that 7 ~ 10 buffered soln mixes the back and adds prozyme and process mixed solution with the described degreasing grouts of step (1) powder and pH value, behind enzymolysis 2 ~ 5 h under 40 ~ 65 ℃ of conditions, spinning obtains enzymolysis solution and deposition with mixed solution; The feed liquid mass ratio of said degreasing grouts powder and buffered soln is 1:7 ~ 1:15, and the total enzyme work in the said mixed solution is 90 ~ 250 Ug
-1
(3) then the described deposition alkali of step (2) is dissolved the back spinning, after the supernatant that separation is obtained merges with the described enzymolysis solution of step (2), carry out the heavy also spinning of acid, will precipitate and wash postlyophilization, obtain crude protein;
(4) crude protein that step (3) is obtained is added in ionic liquid/salt double-aqueous phase system, leaving standstill after the concussion under the condition of 40 ~ 70 ℃ of temperature, pH4 ~ 9, gets upper strata ionic liquid phase again;
(5) at last the ionic liquid of step (4) is used the membrane separation process separating protein mutually, and reclaim ionic liquid, the protein lyophilize is obtained the high purity protein powder.
2. ionic liquid and the Enzymatic Extraction utilized according to claim 1 separated method of protein in the grouts; It is characterized in that; Enzyme in the said prozyme is AMS and cellulase; Perhaps AMS, cellulase and polygalacturonase, and the enzyme activity of AMS and cellulase is than being 5:1 ~ 2:3.
3. ionic liquid and the Enzymatic Extraction utilized according to claim 2 separated method of protein in the grouts, it is characterized in that in the mixed solution, the enzyme activity of AMS and cellulase is respectively 60 ~ 150 Ug
-1With 30 ~ 90 Ug
-1
4. ionic liquid and the Enzymatic Extraction utilized according to claim 1 separated method of protein in the grouts, it is characterized in that described buffered soln is phosphoric acid buffer or borate buffer.
5. ionic liquid and the Enzymatic Extraction utilized according to claim 1 separated method of protein in the grouts, it is characterized in that the concentration of crude protein is 20 ~ 100 mgL described in the described step (4)
-1, said ion liquid concentration is 200 ~ 600 mgmL
-1, the concentration of said salt is 90 ~ 220 mgmL
-1Be preferably: the concentration of said crude protein is 50 ~ 80 mgL
-1, said ion liquid concentration is 300 ~ 400 mgmL
-1, the concentration of said salt is 130 ~ 180 mgmL
-1
6. separate method of protein in the grouts according to claim 1 or 5 described ionic liquid and the Enzymatic Extraction utilized; It is characterized in that; Described ionic liquid/salt double-aqueous phase system is hydrophilic ionic-liquid/salt double-aqueous phase system; Described ionic liquid is wetting ability glyoxaline ion liquid, pyridines ionic liquid or guanidine class ionic liquid, and described salt is phosphoric acid salt, carbonate or vitriol.
7. ionic liquid and the Enzymatic Extraction utilized according to claim 6 separated method of protein in the grouts, it is characterized in that described wetting ability glyoxaline ion liquid is [Amim] Cl, [Bmim] Cl or [Bmim] BF
4, described pyridines ionic liquid is [Epy] Br or [BPy] BF
4, described phosphoric acid salt is K
2HPO
4, KH
2PO
4Or K
3PO
4, described carbonate is NaHCO
3, Na
2CO
3, described vitriol is (NH
4)
2SO
4Preferred described ionic liquid/salt double-aqueous phase system is [Bmim] Cl/K
2HPO
4, [Amim] Cl/K
2HPO
4, [Epy] Br/K
2HPO
4, [Bmim] BF
4/ K
2HPO
4
8. ionic liquid and the Enzymatic Extraction utilized according to claim 1 separated method of protein in the grouts, it is characterized in that the centrifugal speed of spinning is 3000 ~ 5000 rmin described in step (2) and the step (3)
-1
9. ionic liquid and the Enzymatic Extraction utilized according to claim 1 separated method of protein in the grouts, it is characterized in that the number of times of deposition washing is 3 ~ 6 times in the step (3); In the step (4), the time of said concussion is 20 ~ 50min, and concussion speed is 100 ~ 300 rmin
-1, the said time of leaving standstill is 30 ~ 60min.
10. ionic liquid and the Enzymatic Extraction utilized according to claim 1 separated method of protein in the grouts, it is characterized in that the organic solvent described in the step (1) is sherwood oil, cyclohexane, normal hexane or No. four solvents.
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