CN103848883B - A kind of method of aqueous two-phase extraction abalone ovum protein polypeptide concentrated solution - Google Patents
A kind of method of aqueous two-phase extraction abalone ovum protein polypeptide concentrated solution Download PDFInfo
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Abstract
The present invention relates to biologically active substance separating and purifying technology, specifically a kind of method of aqueous two-phase extraction abalone ovum protein polypeptide concentrated solution.The method of being specially is: prefabricated abalone ovum homogenate, and separation and purification after double water-phase continuous extraction, wherein double-aqueous phase system overall consistency is 0.06 ~ 0.16kg/m
3, viscosity is 4 ~ 16mpa/s, pH value 6 ~ 8, extraction time 30 ~ 50min, and total amount and the abalone ovum homogenate ingredient proportion of two phase aqueous extraction system are 1:0.4 ~ 0.5.The method phase-splitting time that the present invention adopts is short, saves extraction time, and save energy consumption, after phase-splitting, aglucon liquid is reused, and there is not organic solvent residual, and abalone ovum protein polypeptide concentrated solution and nutritive food have broad incorporation, applicability.
Description
Technical field:
The present invention relates to biologically active substance separating and purifying technology, specifically a kind of method of aqueous two-phase extraction abalone ovum protein polypeptide concentrated solution.
Background technology:
Be rich in protein polypeptide, polysaccharide, nucleic acid, gonadin in abalone ovum, have anti-oxidant, remove too much free radical, be delay senility, the biologically active substance of alleviating fatigue.The nutrition of abalone and being divided into of functional mass part science: " an ovum; I.e. abalone ovum; Two fourths; I.e. abalone meat; Three limit; I.e. abalone shirt rim." the abalone ovum that ranks the first as seen is the important integral part of abalone body nutritive substance.But traditional abalone working method but by abalone ovum as waste disallowable fall, cause the waste of abalone nutritional resource.Active effective ingredient in abalone ovum after deliberation, how in processing abalone process, retaining abalone ovum, and effectively can extract active abalone proteins peptide material and obtain application, is also the target that scientific worker and marine organisms processing enterprise are pursued.
But abalone ovum protein peptide matters namely will be made to be separated by double water-phase, activeconstituents will be made again simultaneously to be retained, make wherein small molecular protein peptide be purified simultaneously, improve the function value of abalone ovum, edibleness is a large technical barrier.
Summary of the invention:
The present invention, for solving the problems of the technologies described above, provides a kind of method of aqueous two-phase extraction abalone ovum protein polypeptide concentrated solution
For achieving the above object, the technical solution used in the present invention is;
Prefabricated abalone ovum homogenate, separation and purification after double water-phase three continuous extractions, wherein double-aqueous phase system overall consistency is 0.06 ~ 0.16kg/m
3, viscosity is 4 ~ 16mpa.s, pH value 6 ~ 8, extraction time 30 ~ 50min.
Total amount and the abalone ovum homogenate ingredient proportion of two phase aqueous extraction system are 1:0.4 ~ 0.5.
Described two phase aqueous extraction system is divided into 3 assembly bases, and the content often organized is specially:
A group one-level extraction aglucon
Upper phase aglucon is the polyoxyethylene glycol of mass concentration 12 ~ 18% or the aqueous solution of dextran;
Lower phase aglucon is the sodium-chlor of mass concentration 10 ~ 16% or the phosphatic aqueous solution;
B group secondary extraction aglucon;
Upper phase aglucon is the polyoxyethylene glycol of mass concentration 17 ~ 23% or the aqueous solution of dextran;
Lower phase aglucon is mass concentration 16 ~ 22% sodium-chlor or the phosphatic aqueous solution;
C group three grades extraction aglucon;
Upper phase aglucon is the aqueous solution of mass concentration 10 ~ 12% polyoxyethylene glycol or dextran;
Lower phase aglucon is mass concentration 9 ~ 11% sodium-chlor or the phosphatic aqueous solution.
Described double water-phase continuous extraction concrete steps are:
The A first step extracts:
Getting aqueous two-phase extraction A assembly base is input in No. 1 stainless steel cauldron, add prefabricated amount abalone ovum homogenate, control stirring velocity 500r/min, pH value 8,10 ~ 20min is extracted under double water-phase, obtain aqueous two-phase extraction liquid, again by aqueous two-phase extraction liquid input disc separator, under 8400 ~ 12000r/min condition, carry out being separated 5 ~ 10min, in collection, phase liquid enters second stage extraction, upper phase liquid is the liquid phase containing abalone ovum homogenate, and wherein the mass ratio of abalone ovum homogenate and A assembly base is 1:0.8-1;
The B second stage extracts:
Upper phase liquid after first step extracting and separating is input in No. 2 stainless steel cauldrons, again B assembly base is input in No. 2 stainless steel cauldrons, control stirring velocity 400r/min, pH value 7, after carrying out aqueous two-phase extraction 10 ~ 20min, second time extraction liquid is inputted in disc separator, under 8400 ~ 9700r/min condition, carries out being separated 6 ~ 8min, in collection, phase liquid enters third stage extraction
Upper phase liquid wherein after first step extracting and separating and the mass ratio of B assembly base are 1:0.4-0.45;
The C third stage extracts:
Upper phase liquid after the extracting and separating of the second stage is input in No. 3 stainless steel cauldrons, again C assembly base is input in No. 3 stainless steel cauldrons, control stirring velocity is 300r/min, pH value 6, after carrying out aqueous two-phase extraction 10 ~ 20min, will input in disc separator by extraction liquid for the third time, carry out being separated 7-9min at 8400 ~ 9300r/min, in collection, phase liquid is through 0.12 ~ 0.24 μm of filtration, obtains aqueous two-phase extraction abalone ovum protein polypeptide active liquid
Wherein, Upper phase liquid after the extracting and separating of the second stage and the mass ratio of C assembly base are 1:0.3-0.35
Described A, B, C, 3 assembly bases all mix in the ratio of upper and lower phase mass ratio 1:1-1.2, and by first going up the order input inputting mutually and to be input to mutually under after stainless steel cauldron in stainless steel cauldron.
Separation and purification after described double water-phase continuous extraction, separation and purification concrete steps are: by aqueous two-phase extraction abalone ovum protein polypeptide active liquid, carry out ultra micro membrane separation purification, successively through the separation and purification group of ultra micro film 10000 dalton ultra micro film; The separation and purification group of 5000 dalton's ultra micro films; The group of 2000 dalton's ultra micro films; Collect by the outer composition of the film of ultra micro film; The last nanofiltration separation again through 300 dalton's ultra micro films is concentrated, obtains aqueous two-phase extraction abalone ovum protein polypeptide concentrated solution.
The quality index of described aqueous two-phase extraction abalone ovum protein polypeptide concentrated solution is molecular weight≤2000 dalton, protein content >=50%, saltiness≤6ml/L, the abalone ovum protein polypeptide concentrated solution of moisture≤40%, abalone ovum protein total recovery >=80% of the present invention.
Tool of the present invention has the following advantages
1. water content 80% in aqueous two-phase extraction abalone ovum protein polypeptide concentrated solution process aglucon system, abalone ovum protein polypeptide not volatility in extraction, partition equilibrium, keeps biological activity.
2. the aqueous two-phase extraction abalone ovum protein polypeptide concentrated solution phase-splitting time is short, saves extraction time, and save energy consumption, after phase-splitting, aglucon liquid is reused, and there is not organic solvent residual.
3. aqueous two-phase extraction abalone ovum protein polypeptide concentrated solution, extraction environment is gentle, and in normality static state, carry out phase-splitting without high temperature, high pressure, biocompatibility is high.
4. aqueous two-phase extraction abalone ovum protein polypeptide concentrated solution, polymer poly ethylene glycol or dextran (PEG6000-8000; DX.D37-48) stable and provide protection is had to the structure of the multistage protein of abalone ovum.
5. aqueous two-phase extraction abalone ovum protein polypeptide concentrated solution, extraction process is uncomplicated, is easy to industrialization and continuity operation.
6. aqueous two-phase extraction abalone ovum protein polypeptide concentrated solution, associativity is strong, applied range, is applicable to the interpolation of all kinds of formulation nutrition, protective foods.
Accompanying drawing explanation
Fig. 1 is aqueous two-phase extraction abalone ovum protein polypeptide process flow sheet of the present invention;
Fig. 2 is embodiment 1 double water-phase abalone ovum protein polypeptide extraction liquid (2,000 membrane concentration liquid) molecular weight determination;
Fig. 3 is embodiment 3 double water-phase abalone ovum protein polypeptide extraction liquid (nanofiltration membrane concentrated solution) molecular weight determination.
Embodiment; , below in conjunction with enforcement power, the present invention is described in further details.
Embodiment 1
Get after fresh abalone oophyte 150kg drinkable water cleans up, adopt stainless steel colloidal mill, abalone oophyte is broken, be input in high-shear emulsion machine (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.), add 150kg drinkable water, stir and make 300kg abalone ovum homogenate through the high-shear emulsifying of 3000r/min, filter through 100 orders, the abalone ovum homogenate 300kg obtaining concentration 18% is for subsequent use.Select polyoxyethylene glycol (PEG8000) 21.6kg, drinkable water (H
2o) 98.4kg, is uniformly mixed makes { the upper phase 120kg of A} first assembly base, solute (massfraction) concentration 18% with the composite tank of 600L stainless steel (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.).Select NaC1,19.2kg. again, H
2o100.8kg.Make { phase 120kg under A} first assembly base, solute (massfraction) concentration 16%.Start underflow pump (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.) under 150pa pressure by { on A} first group, phase aglucon 120kg adds in 1200L stainless steel cauldron (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.), start agitator degree of control 500r/min, add { phase aglucon 120kg under A} first group while stirring, after stopping stirring, recycle pump is adopted to input in 1200L stainless steel cauldron by the abalone ovum homogenate 300kg of concentration 18%, after extraction 10min, obtain aqueous two-phase extraction abalone ovum protein polypeptide active liquid.Its A group aqueous two phase extraction technique Con trolling index is; System partition ratio 1.83, density 0.16kg/m
3viscosity 16mpa.s, pH value 6, adopt underflow pump (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.) under 150pa pressure, 540kg extraction liquid is input in the disc separator (manufacture of oasis, Nanjing Machinery Co., Ltd.) of 12000r/min, be separated 5min, collecting phase liquid 432kg yield on separating machine is 80%, and lower phase liquid 108kg preserves in separating tank.By liquid that 432kg is separated, be input in No. 2 1200L stainless steel cauldrons with recycle pump, select PEG6000,19.8kg, H
2o, 70.2kg, adopt in the composite tank of 300L stainless steel (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.) and be mixed { phase 90kg on B} second assembly base, solute (massfraction) concentration 23% underflow pump (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.) joins in No. 2 1200L stainless steel cauldrons (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.) under 150pa pressure, and startup agitator speed is 400r/min.Prepare NaC1(80% again), 14.4kg.; H2O, 75.6kg; Adopt in the composite tank of 300L stainless steel (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.) and be mixed { phase 90kg under B} second assembly base, solute (massfraction) concentration 16% joins in stainless steel cauldron (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.), after carrying out double water-phase { B } second group extraction 10min, its B group aqueous two phase extraction technique Con trolling index is; System divides disposition, and 3.33, density 0.10kg/m
3viscosity 11mpa.s, pH value 7, underflow pump (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.) is adopted to be input to 9700r/min under 150pa pressure, carry out being separated 6min in disc separator (manufacture of oasis, Nanjing Machinery Co., Ltd.), obtain phase 550.8kg on { B } second group of aqueous two-phase extraction, yield is 90%, and lower phase liquid 61.2kg returns in No. 2 stainless steel cauldrons (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.).By phase liquid on 550.8kg, with in recycle pump (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.) input No. 3 1200L stainless steel cauldrons (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.), select PEG6000:10.8kg; H
2o:79.2kg, add in the composite tank of 300L stainless steel (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.), mix, make phase aglucon 90kg on { C } the 3rd group, solute (massfraction) concentration 12%, start underflow pump is input in No. 3 1200L stainless steel cauldrons (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.) under 150Pa pressure, start agitator, speed control at 300r/min, then prepares NaC1(80%), 9.9kg, H
2o:80.1kg, { C } the 3rd group of subordinate base 90kg is made with the composite tank of 300L stainless steel (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.), solute (massfraction) concentration 11%, enter in No. 3 1200L stainless steel cauldrons (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.), after carrying out double water-phase { C } the 3rd group extraction 10min, its C group aqueous two phase extraction technique Con trolling index is; System divides disposition, and 2.33, density 0.06kg/m
3, viscosity 6mpa.s, pH value 8, adopts underflow pump (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.) to be input under 150pa pressure in disc separator (manufacture of oasis, Nanjing Machinery Co., Ltd.), in 9300r/min, is separated 7min.Obtain phase liquid 657.72kg saltiness 196ml/L on { C } the 3rd group of aqueous two-phase extraction, yield is 90%.Adopt recycle pump (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.) by 657.72kg aqueous two-phase extraction abalone ovum protein polypeptide active liquid; in input ultra micro membrane sepn (purifying) unit (Dalian eight reaches space science Institute for Applied Technology and manufactures); (purifying) group (adopting molecular weight cut-off 10000 dalton ultra micro film) is separated through first; collect by the outer composition of the film of ultra micro film; percent of pass is 80%, obtains 526.2kg saltiness 27ml/L; Be separated (purifying) group (adopting molecular weight cut-off 5000 dalton ultra micro film) through second again, collect by the outer composition of the film of ultra micro film, percent of pass is 85%, obtains 449.27kg saltiness 21ml/L; (purifying) group (adopting molecular weight cut-off 2000 dalton ultra micro film) is separated by the 3rd, collect by the outer composition of the film of ultra micro film, percent of pass be 90% 404.3kg retain concentrated by the daltonian nanofiltration membrane of molecular weight cut-off 300 again, in separation and purification liquid more than 60% moisture and salinity mixed solution 242.58kg; Composition in film under collection nanofiltration membrane retains, obtain aqueous two-phase extraction abalone ovum protein polypeptide concentrated solution finished product 134.72kg, its saltiness 5.7ml/L, molecular weight is < 2000 dalton, protein content 23.6%, moisture 58.6%.
Embodiment 1 application examples;
A. by 134, the aqueous two-phase extraction abalone ovum protein polypeptide concentrated solution of 72kg, adopt oral liquid auto-filling unit (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.) through composite, even liquid, filtration, filling one-tenth 50min abalone ovum protein polypeptide oral liquor 2694 bottles, obtain 2694 bottles of finished products through sterilising packaging.
B. by the aqueous two-phase extraction abalone ovum protein polypeptide concentrated solution of 134.72kg, adopt vacuum-freeze-dry unit (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.) to take up thickness 20mm weight 3600g with each lyophilized plate and fill 38 dishes altogether,-35 DEG C, warp, 2h are frozen into solid-state, vacuumize 180Pa, heating sublimation to 50 DEG C, 23h, desorb 1h in 60 DEG C.Obtain 37.72kg protein content 81.6%, moisture 4, the abalone ovum protein polypeptides freeze-dry powder of 3%, yield is 28%, and the capsule being processed into 300mg, through packaging, can obtain 125733 abalone ovum protein polypeptides freeze-dry powder capsule finished products.
C. the aqueous two-phase extraction abalone ovum protein polypeptide concentrated solution of 134.72kg is added 1212.48kg fresh milk, through composite (10% abalone ovum protein polypeptide concentrated solution), emulsification, is spray dried to abalone proteins polypeptide milk powder 145.5kg finished product, flour extraction rate 12%.
Embodiment 2
Embodiment 2 difference from Example 1 is:
1.. abalone ovum homogenate;
Prefabricated abalone ovum homogenate, select freezing abalone oophyte 100kg moisture 73.6%, protein content 23.3% makes raw material.Broken after cleaning, 100kg pure water is added by abalone ovum mass ratio, 200kg homogenate is made through 3000r/min high-shear emulsion machine (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.), (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.) percent of pass 85.8% is filtered with 80 orders, require homogenate concentration (massfraction) 22%, obtain abalone ovum homogenate 171.6kg for subsequent use.
2. double water-phase aglucon;
Abalone ovum protein polypeptide extraction aglucon summation is selected to be that 429kg is allocated as follows;
A group extraction aglucon 171.6kg accounts for 40% of aglucon summation;
Upper phase aglucon: dextran (DX48) 10.3kg pure water (H
2o) 75.5kg.Upper middle solute (massfraction) concentration 12%, 85.8kg mutually.
Lower phase aglucon;
Phosphoric acid salt { K
2pO4}, 8.58kg
Pure water H
2o, 77.22kg;
Lower phase solute (massfraction) concentration 10%, 85.8kg.
B group extraction aglucon 128.7kg accounts for 30% of aglucon summation;
Upper phase aglucon: DX10.94kg;
Pure water H
2o; 65.63kg;
Upper middle solute (massfraction) concentration 17%, 65.35kg mutually.
Lower phase aglucon
Phosphoric acid salt K
2pO4,7.72kg;
Pure water H
2o; 56.63kg;
Lower middle solute (massfraction) concentration 6%, 64.35kg mutually.
C group extraction aglucon 128.7kg accounts for 30% of aglucon summation;
Upper phase aglucon; DX48; 6.435kg;
Pure water H
2o; 57.915kg;
Upper middle solute concentration 10%64.35kg mutually.
Lower phase aglucon;
Phosphoric acid salt K
2pO4:5.79kg;
Pure water H
2o:58.56kg;
Lower middle solute (massfraction) concentration 9%, 64.35kg mutually.
2. aqueous two-phase extraction flow process
Aqueous two-phase extraction abalone ovum protein polypeptide is use continuous extraction flow process.The mixing of double water-phase adopts stainless steel cauldron (Wenzhou Hai Chuan Machinery Co., Ltd. manufactures), stirring velocity 500r/min with dispersion; Be separated and adopt disc centrifuge (manufacture of oasis, Nanjing Machinery Co., Ltd.), velocity of separation 8400r/min; Abalone ovum protein peptide purification adopts 300-10000 Dalton molecular weight ultra micro membrane separation purification unit (Dalian eight reaches space science Institute for Applied Technology and manufactures).Controlling double-aqueous phase system overall consistency is 0.16kg/m
2, viscosity is 16mpa.s, pH value 6, extracting and separating time 30min, and two phase aqueous extraction system and abalone ovum homogenate ingredient proportion are that 1:0.4 obtains 600.6kg, abalone ovum protein polypeptide total recovery more than 80%.
The A first step extracts:
Get aqueous two-phase extraction A assembly base 171.6kg underflow pump is input in 1200L stainless steel cauldron (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.) under 200pa pressure, add abalone ovum homogenate 171.6kg, control stirring velocity 500r/min, extraction 10min, controlling double-aqueous phase system overall consistency is 0.12kg/m
2viscosity is 13mpa.s, pH value 6, A group two phase aqueous extraction system partition ratio 1.93, adopting underflow pump (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.) under 200Pa pressure, starch the full liquid 343.2kg of aqueous two-phase extraction inputs in disc separator, in 8400r/min, carry out being separated 20min, in collection, phase liquid 329.94kg enters second stage extraction.
The B second stage extracts:
Upper phase liquid 329.94kg after adopting recycle pump (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.) first step to be separated is input in 1200L stainless steel cauldron (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.), again with inputting B assembly base 128.7kg under underflow pump 200pa pressure, control stirring velocity 400r/min, after extracting 10min mutually, controlling double-aqueous phase system overall consistency is 0.14kg/m
2viscosity is 15mpa.s, pH value 7, B group two phase aqueous extraction system partition ratio 3.75, adopting underflow pump will extract full liquid 458.64kg under 200pa pressure inputs in disc separator (manufacture of oasis, Nanjing Machinery Co., Ltd.), carry out being separated 8min at 9700r/min, in collection, phase liquid 412.78kg enters third stage extraction.
The C third stage extracts:
Recycle pump is adopted to be input to by the upper phase liquid 412.78kg after the extracting and separating of the second stage in 1200L stainless steel cauldron (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.), use underflow pump (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.) under 200pa pressure, input C assembly base 128.7kg again, control stirring velocity is 300r/min, after extracting 20/min mutually, controlling double-aqueous phase system overall consistency is 0.16kg/m
2viscosity is 16mpa.s, pH value 8, C group two phase aqueous extraction system partition ratio 3.6, adopt underflow pump under 200pa pressure, inputting extracting full liquid 541.48kg mutually in disc-type separator (manufacture of oasis, Nanjing Machinery Co., Ltd.), dividing 9min in 9300r/min phase, in collection, phase liquid 487.33kg is through 0.12-0.24 μm of filtration, obtains aqueous two-phase extraction abalone ovum protein polypeptide active liquid 480kg.
D purifying:
Adopt recycle pump by aqueous two-phase extraction abalone ovum protein polypeptide active liquid 480kg saltiness 125ml/L, separation and purification is carried out in input ultra micro membrane sepn (purifying) unit (Dalian eight reaches space science Institute for Applied Technology and manufactures), through ultra micro film 10000 dalton 384kg saltiness 17ml/L, percent of pass 80%; 5000 dalton 362.4kg saltiness 12ml/L, percent of pass 85%; 2000 dalton 293.76kg percent of pass 90%; 300 dalton's nanofiltrations retain concentrated 117kg; Obtain aqueous two-phase extraction abalone ovum protein polypeptide concentrated solution 117kg, saltiness 5ml/L, protein content 23%, molecular weight≤2000 dalton, the total recovery of abalone ovum protein is 81%.Wherein the separation (purifying) of first three groups is organized dalton and is left composition by film, and the composition namely outside film, leaves becoming within film during 300 dalton's nanofiltration, be the abalone ovum protein polypeptide of high-content.
Embodiment 2 application examples:
1. get aqueous two-phase extraction 117kg abalone ovum protein polypeptide concentrated solution, adopt oral liquid auto-filling unit (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.) through composite, even liquid, filter, filling, 20mg scavenging free radicals is made in sterilizing, beautifying face and moistering lotion abalone ovum protein polypeptide oral liquor 5850 bottles of finished products.
2. get aqueous two-phase extraction 117kg abalone ovum protein polypeptide concentrated solution, through frozen vacuum dryer group (manufacture of Wenzhou Hai Chuan Machinery Co., Ltd.), control liquid thickness 30mm, quick-frozen-35 DEG C; Be frozen into solid-state in 2h; Distil in 50 DEG C 17h; Desorb 2h in 60 DEG C, vacuum degree control, at 120pa, obtains 32.76kg protein content 82.1%, the abalone ovum protein polypeptides freeze-dry powder of moisture 4.6%, and flour extraction rate 28%, can be processed into 200mg strengthening immunity, antifatigue abalone ovum protein polypeptide ball 163800 ball finished product.
3. get aqueous two-phase extraction 117kg abalone ovum protein polypeptide concentrated solution, add concentrated Succus Mali pumilae 1053kg,
Through composite, emulsification, centrifugal spray drying 140.4kg, flour extraction rate 12%, quantitative 50g is distributed into
Nutrition abalone ovum protein polypeptide apple powder 2808 bags of finished products.
Effective constituent
1. aqueous two-phase extraction abalone ovum protein polypeptide raw material effective substance:
1. abalone ovum (fresh goods) moisture content detects, and adopts Ao Haosi MB-23 moisture content Quick Measuring instrument to detect: 100mg abalone ovum (fresh goods) water content is 73.6%.(the abalone ovum raw material adopted in embodiment 1 and 2).
2. abalone ovum (fresh goods) protein detection, adopts Kjeldahl determination to detect: 100mg abalone ovum (fresh goods) protein content is 23.3%.The abalone ovum raw material adopted in embodiment 1 and 2).
2. aqueous two-phase extraction abalone ovum protein polypeptide finished product effective substance:
1. abalone ovum protein polypeptides freeze-dry powder (embodiment 2 application examples 2. in) protein detection, adopts Kjeldahl determination to detect: 100mg abalone ovum protein polypeptides freeze-dry powder protein content is 82.1%.
2. abalone ovum protein polypeptide concentrated solution (in embodiment 2, D) protein detection, adopts Kjeldahl determination to detect: 100mg abalone ovum protein polypeptide concentrated solution protein content is 23%.
3. aqueous two-phase extraction abalone ovum protein peptide purification liquid (the separation and purification liquid in embodiment 1) saltiness detects:
1) adopt JL-26 salinity tester, detect saltiness: 196ml/L. in aqueous two-phase extraction abalone ovum protein polypeptide active liquid liquid 657.72 ㎏
2) aqueous two-phase extraction abalone ovum protein peptide purification liquid saltiness: 27mg/L in 10000D ultra micro film 526.2 ㎏
3) aqueous two-phase extraction abalone ovum protein peptide purification liquid retains saltiness: 5.7ml/L in concentrated solution 134.72 ㎏ through 300D nanofiltration membrane
4) aqueous two-phase extraction abalone ovum protein peptide purification concentrated solution saltiness: 21mg/L in 5000D ultra micro film 449.27 ㎏
3. aqueous two-phase extraction abalone ovum protein polypeptide phase base Concentration Testing: the JC08-HYD-III type numeral tester of liquid concentration adopting Beijing North letter future electronic science and technology, test phase base concentration is as follows;
A group extraction phase base: upper phase concentration 12-18%; Lower phase concentration 10-16%.
B group extraction phase base: upper phase concentration 17-23%; Lower phase concentration 6-8%.
C group extraction phase base: upper phase concentration 12-16%; Lower phase concentration 9-11%.
4. aqueous two-phase extraction abalone ovum protein polypeptide concentrated solution middle-molecular-weihydroxyethyl of the present invention detects: (entrusting Changchun Inst. of Applied Chemistry, Chinese Academy of Sciences)
Embodiment 1 double water-phase abalone ovum protein polypeptide extraction concentrated solution (2,000 membrane concentration liquid) molecular weight determination (as Fig. 2)
ASTRA4.90.08summaryReportforNo1-080105-s
File:K:\MAJIA\GPC\badakongjian\No1-080105-s.adf
Illustrate:
MolarMassMoments(molecular weight) (g/mol)
Mn(number-average molecular weight): 1.189e+03
Mw(weight-average molecular weight): 2.338e+03
Polydispersity (Mw/Mn) (molecular weight distribution): 1.967
COLLECTIONINFORMATION
Collectiontime:SatJan05,201109:06PMPacificStandardTime
Instrumenttype:DAWNEOS
Celltype:K5
Laserwavelength:690.0nm
Solventname:water
SolventRI:1.330
Calibrationconstants
DAWN:9.0950e-06
AUX1:2.3095e-05
Flowrate:0.500mL/min
QELSInformation
Instrument:WyattQELS
Solventviscosity:8.9450e-0330.0°C(gmcm
2/sec)
PROCESSINGINFORMATION
Processingtime:MonJan07,201109:06PMPacificStandardTime
DAWN/AUX1delay:0.183mL
Fitmethod/model:Debye
Calculationmethod:dn/dc+AUXConstant
QELSdelaytimerange:1.10e-06-1.00e+00sec
Detectorsused:67891011121415
RESULTS
Time(min.):10.317-24.333
Slices:842
Fitdegree:1
InjectedMass(g):2.5000e-03
Calc.Mass(g):2.5048e-03
dn/dc(mL/g):0.151
Polydispersity(Mw/Mn):1.967
MolarMassMoments(g/mol)
Mn:1.189e+03
Mw:2.338e+03
Embodiment 2 double water-phase abalone ovum protein polypeptide extraction concentrated solution (nanofiltration membrane concentrated solution) molecular weight determination
ASTRA4.90.08summaryReportfor2-080105-7mg-s
File:K:\MAJIA\GPC\badakongjian\2-080105-7mg-s.adf
Illustrate:
MolarMassMoments(molecular weight) (g/mol)
Mn(number-average molecular weight): 7.631e+02
Mw(weight-average molecular weight): 1.023e+03
Polydispersity (Mw/Mn) (molecular weight distribution): 1.340
COLLECTIONINFORMATION
Collectiontime:SatJan05,201109:05PMPacificStandardTime
Instrumenttype:DAWNEOS
Celltype:K5
Laserwavelength:690.0nm
Solventname:water
SolventRI:1.330
Calibrationconstants
DAWN:9.0950e-06
>>AUX1:2.3095e-05
Flowrate:0.500mL/min
QELSInformation
Instrument:WyattQELS
Solventviscosity:8.9450e-0330.0°C(gmcm
2/sec)
PROCESSINGINFORMATION
Processingtime:SatJan05,201109.05PMPacificStandardTime
DAWN/AUX1delay:0.183mL
Fitmethod/model:Debye
Calculationmethod:dn/dc+AUXConstant
QELSdelaytimerange:1.10e-06-1.00e+00sec
Detectorsused:91011121415
RESULTS
Time(min.):17.167-25.317
Slices:490
Fitdegree:1
InjectedMass(g):3.5000e-03
Calc.Mass(g):3.4617e-03
dn/dc(mL/g):0.162
Polydispersity(Mw/Mn):1.340
MolarMassMoments(g/mol)
Mn:7.631e+02
Mw:1.023e+03。
Claims (5)
1. a method for aqueous two-phase extraction abalone ovum protein polypeptide concentrated solution, is characterized in that: prefabricated abalone ovum homogenate, and separation and purification after double water-phase three continuous extractions, wherein double-aqueous phase system overall consistency is 0.06 ~ 0.16kg/m
3, viscosity is 4 ~ 16mpa.s, pH value 6 ~ 8, extraction time 30 ~ 50min;
Separation and purification after described double water-phase continuous extraction, separation and purification concrete steps are: by aqueous two-phase extraction abalone ovum protein polypeptide active liquid, carry out ultra micro membrane separation purification, successively through the separation and purification group of ultra micro film 10000 dalton ultra micro film; The separation and purification group of 5000 dalton's ultra micro films; The group of 2000 dalton's ultra micro films; Collect by the outer composition of the film of ultra micro film; The last nanofiltration separation again through 300 dalton's ultra micro films is concentrated, obtains aqueous two-phase extraction abalone ovum protein polypeptide concentrated solution.
2. the method for aqueous two-phase extraction abalone ovum protein polypeptide concentrated solution according to claim 1, is characterized in that:
Total amount and the abalone ovum homogenate ingredient proportion of two phase aqueous extraction system are 1:0.4 ~ 0.5.
3. the method for aqueous two-phase extraction abalone ovum protein polypeptide concentrated solution according to claim 1 or 2, is characterized in that: described two phase aqueous extraction system is divided into 3 assembly bases, and the content often organized is specially:
A group one-level extraction aglucon
Upper phase aglucon is the polyoxyethylene glycol of mass concentration 12 ~ 18% or the aqueous solution of dextran;
Lower phase aglucon is the sodium-chlor of mass concentration 10 ~ 16% or the phosphatic aqueous solution;
B group secondary extraction aglucon;
Upper phase aglucon is the polyoxyethylene glycol of mass concentration 17 ~ 23% or the aqueous solution of dextran;
Lower phase aglucon is mass concentration 16 ~ 22% sodium-chlor or the phosphatic aqueous solution;
C group three grades extraction aglucon;
Upper phase aglucon is the aqueous solution of mass concentration 10 ~ 12% polyoxyethylene glycol or dextran;
Lower phase aglucon is mass concentration 9 ~ 11% sodium-chlor or the phosphatic aqueous solution.
4. the method for aqueous two-phase extraction abalone ovum protein polypeptide concentrated solution according to claim 3, is characterized in that: described double water-phase continuous extraction concrete steps are:
The A first step extracts:
Getting aqueous two-phase extraction A assembly base is input in No. 1 stainless steel cauldron, add prefabricated amount abalone ovum homogenate, control stirring velocity 500r/min, pH value 8,10 ~ 20min is extracted under double water-phase, obtain aqueous two-phase extraction liquid, again by aqueous two-phase extraction liquid input disc separator, under 8400 ~ 12000r/min condition, carry out being separated 5 ~ 10min, in collection, phase liquid enters second stage extraction, upper phase liquid is the liquid phase containing abalone ovum homogenate, and wherein the mass ratio of abalone ovum homogenate and A assembly base is 1:0.8-1;
The B second stage extracts:
Upper phase liquid after first step extracting and separating is input in No. 2 stainless steel cauldrons, again B assembly base is input in No. 2 stainless steel cauldrons, control stirring velocity 400r/min, pH value 7, after carrying out aqueous two-phase extraction 10 ~ 20min, second time extraction liquid is inputted in disc separator, under 8400 ~ 9700r/min condition, carries out being separated 6 ~ 8min, in collection, phase liquid enters third stage extraction
Upper phase liquid wherein after first step extracting and separating and the mass ratio of B assembly base are 1:0.4-0.45;
The C third stage extracts:
Upper phase liquid after the extracting and separating of the second stage is input in No. 3 stainless steel cauldrons, again C assembly base is input in No. 3 stainless steel cauldrons, control stirring velocity is 300r/min, pH value 6, after carrying out aqueous two-phase extraction 10 ~ 20min, will input in disc separator by extraction liquid for the third time, carry out being separated 7-9min at 8400 ~ 9300r/min, in collection, phase liquid is through 0.12 ~ 0.24 μm of filtration, obtains aqueous two-phase extraction abalone ovum protein polypeptide active liquid
Wherein, Upper phase liquid after the extracting and separating of the second stage and the mass ratio of C assembly base are 1:0.3-0.35
Described A, B, C, 3 assembly bases all mix in the ratio of upper and lower phase mass ratio 1:1-1.2, and by first going up the order input inputting mutually and to be input to mutually under after stainless steel cauldron in stainless steel cauldron.
5. the method for aqueous two-phase extraction abalone ovum protein polypeptide concentrated solution according to claim 1, it is characterized in that: the quality index of described aqueous two-phase extraction abalone ovum protein polypeptide concentrated solution is molecular weight≤2000 dalton, protein content >=50%, saltiness≤6ml/L, the abalone ovum protein polypeptide concentrated solution of moisture≤40%, abalone ovum protein total recovery >=80%.
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