CN104513307A - Method for recovery of Kunitz and Bowman-Birk trypsin inhibitors from soybean whey - Google Patents
Method for recovery of Kunitz and Bowman-Birk trypsin inhibitors from soybean whey Download PDFInfo
- Publication number
- CN104513307A CN104513307A CN201410742435.3A CN201410742435A CN104513307A CN 104513307 A CN104513307 A CN 104513307A CN 201410742435 A CN201410742435 A CN 201410742435A CN 104513307 A CN104513307 A CN 104513307A
- Authority
- CN
- China
- Prior art keywords
- soybean whey
- kunitz
- soybean
- protein
- trypsin inhibitor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
- C07K14/8114—Kunitz type inhibitors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicines Containing Plant Substances (AREA)
- Peptides Or Proteins (AREA)
Abstract
Belonging to the field of agricultural product processing and by-product comprehensive utilization, the invention relates to a method for recovery of Kunitz and Bowman-Birk trypsin inhibitors from soybean whey. The method of the invention comprises the steps of: (1) pretreatment of soybean whey; (2) primary separation of whey soy protein; (3) complex coacervation of soybean whey protein and polyanionic polysaccharide; and (4) recovery of Kunitz and Bowman-Birk trypsin inhibitors. By subjecting the raw material to pretreatment, primary separation and complex coacervation, a final protein polysaccharide compound can be obtained, and after ultrafiltration for sugar removal, ultrafiltration for desalination, and twice centrifugation on the final protein polysaccharide compound, a protein solution containing purified Kunitz and Bowman-Birk trypsin inhibitors can be obtained, and through vacuum freeze drying of the protein solution, Kunitz and Bowman-Birk trypsin inhibitor high purity samples can be obtained. The method provided by the invention has the characteristics of comprehensive utilization of soybean whey, low equipment requirement, simple operation, and no environmental pollution. And the trypsin inhibitors have high recovery rate, high purity, and high protein activity.
Description
Technical field
The present invention relates to the recovery method of soybean whey protein, specifically refer to a kind of method utilizing polyanionic polysaccharide to reclaim Kunitz and the Bowman Birk type trypsin inhibitor in soybean whey, belong to processing of farm products and byproduct field of comprehensive utilization.
Background technology
Soybean whey protein remains in the protein that can not precipitate for acid in soybean whey, and the proportion in soybean whey protein shared by 2S component is larger; Except containing except sphaeroprotein, albumin in whey protein, also mainly contain: 1. Kunitz trypsin inhibitor (KTI, pH3.0 ~ 10.0,20kDa); 2. Bowman-Brik trypsin inhibitor (BBI, pH3.0 ~ 10.0,20KDa); 3. beta-amylase (61.7KDa); 4. lectin (pH2.2 ~ 10.8,120KDa); 5. the several physiological active substances such as lipoxygenase (LOX, 102KDa), accounts for the 9%-15.3% of soybean protein.
In medical science and biology, inhibitor has become the useful tool of proteolysis research.Because proteinase inhibitor relates to some diseases as in film adenositis, shock and emophysematous proteolytic enzyme in control, and as conditioning agent for regulating the treatment potentiality of Mammalian Fertilization effect aspect, they are especially concerned.Proteinase inhibitor is known at least to be suppressed trypsinase, Quimotrase and may suppress to regulate and control other crucial transmembrane protein enzyme multiple of some critical metabolic functions.The topical of proteolytic enzyme is useful for the illness of such as atopic dermatitis, and atopic dermatitis is a kind of common format of skin inflammation, its major part that can be confined to minority block or relate to health.The active Pigmented ability preventing ultraviolet from causing with them of fading of proteinase inhibitor is proved to be in vitro and in vivo all.Proteinase inhibitor is also in the news and is conducive to wound healing.Such as, SLPI is proved to be and has reversed disorganization when being applied topically and accelerated wound healing process.In addition, serpin can also contribute to reducing pain in lupus erythematosus patient.
At present, Trypsin inhibitor SBTI anti-oxidant action is mainly manifested in and suppresses trypsinase and chymotrypsin activity, reduction protein digestion absorb and cause pancreas enlargement two aspects.These are considered to affect, and human digestive absorbs has bioactive soybean whey protein, and have unusual effect to some disease of prophylactic treatment now, this makes soybean whey become a kind of effective raw material sources.Such as, the trypsin inhibitor of lower concentration is wide spectrum anticarcinogen, can prevent colon cancer, the generation of the kinds cancer such as liver cancer, oral carcinoma, lung cancer, also has the effect reducing cholesterol levels; The activity of pancreas growth and increase pancreas digestive ferment can be strengthened, and to animal endothelial cell growth factor (ECGF), there is activation; Some inflammatory development processes of glomerulonephritis or pyelonephritis can be controlled.In addition, micro-trypsin inhibitor, for treating diabetes, regulates Regular Insulin imbalance to have certain effect.Pharmaceutically extract trypsin inhibitor (Trypsin inhibitor,Trasylol) and treat acute pancreatitis.
Existing lot of documents reports the recovery method of soybean whey protein.The method of membrane sepn preparation and extraction soybean whey protein disclosed in CN1954689 and CN1364765.Such as CN1537865 is disclosed extracts the composition such as whey-protein, nucleic acid from yellow plasm of soybean; CN1027812 openly knows clearly from whey process flow, to reclaim and is separated the method for soybean whey protein and other component.A kind of preparation method with the soybean whey protein of antitumor action disclosed in CN102746375.Prior art all adopts the method such as membrane sepn or ion exchange chromatography, generally has complicated numerous and complicated flow process.The polyanionic polysaccharide that present invention employs edible natural, as salvage material, makes full use of the complex coacervation principle of albumen and polysaccharide, and its complex coacervation thing formed both directly can utilize as foodstuffs material, also completely can realize being separated of albumen and polysaccharide.From the angle of resource recycling, the present invention not only effectively can reduce the anti-nutrition composition of soybean whey, can also prepare two kinds of trypsin inhibitor components with high purity and physiologically active.
Summary of the invention
The laboratory scale that the object of this invention is to provide a kind of simple easy handling prepares the method reclaiming also purifying Kunitz (KTI) and Bowman Birk (BBI) type trypsin inhibitor, KTI and BBI purity prepared by this invention is about 90%-93%, still keeps natural trypsin inhibitor activity.
Technical scheme of the present invention: a kind of method reclaiming Kunitz and Bowman Birk type trypsin inhibitor from soybean whey, step is:
(1) pre-treatment of soybean whey: soybean whey is adjusted to pH 4.5-4.8, the centrifugal 30min of 9500rpm is except precipitation; Again be adjusted to pH 8.0-10.0, the centrifugal 30min of 9500rpm, collect supernatant liquor, obtain test soybean whey liquid; PH is regulated by pH adjusting agent;
(2) primary separation of soybean whey protein: the above-mentioned soybean whey liquid measuring certain volume, regulate pH to 4.5-5.0, at 4-30 DEG C, adding ammonium sulfate to whole saturation ratio is the centrifugal 30min of 30%-40%, 9500rpm, collecting precipitation, add deionized water dissolving, make protein mass final concentration be 1%-10%, molecular weight cut-off 3500 dialysis desalting 36-48h, trapped fluid vacuum lyophilization, obtains the soybean whey protein component containing Kunitz and Bowman Birk type two kinds of trypsin inhibitors;
(3) soybean whey protein and polyanionic polysaccharide complex coacervation: soybean whey protein component prepared by step (2), with the soybean whey protein solution that deionized water preparation w/v final concentration is 0.1%-0.4%, regulate pH to 7.0, and prepare the polyanionic polysaccharide solution of w/v 0.1%-0.4% simultaneously, regulate pH to 7.0; In above-mentioned soybean whey protein solution, slowly add the polyanionic polysaccharide solution of v/v 2.5%-10%, regulate the centrifugal 20min of pH to 5.0-3.0,4000rpm, collecting precipitation; To supernatant liquor again addition polymerization anionic polysaccharide soln repeat precipitation 1-2 time, merging throw out, obtain the complex coacervation thing of KSTI and polyanionic polysaccharide;
Merge above-mentioned go precipitate after supernatant liquor, continue the polyanionic polysaccharide solution slowly adding v/v 5%-10%, regulate the centrifugal 20min of pH to 4.5-3.8,4000rpm, collect supernatant liquor, obtain the complex solution containing Bowman Birk type trypsin inhibitor and polyanionic polysaccharide.
(4) Kunitz and Bowman Birk type trypsin inhibitor is reclaimed: be suspended in the deionized water of 2-3 times of volume by the complex coacervation thing of the KSTI/polyanionic polysaccharide obtained, regulate pH to 7.0-9.0, cross ultra-filtration membrane 100kD-300kD except sugar, collect filtered solution; Molecular weight cut-off 3000-4000 ultrafiltration desalination, trapped fluid vacuum lyophilization, obtains the KSTI of purifying;
By the complex solution of Bowman Birk type trypsin inhibitor/polyanionic polysaccharide obtained, regulate pH to 7.0-9.0, cross ultra-filtration membrane 100kD-300kD except sugar, collect filtered solution; Molecular weight cut-off 3000-4000 ultrafiltration desalination, trapped fluid vacuum lyophilization, obtains the Bowman Birk type trypsin inhibitor of purifying.
Described pH adjusting agent is: acid: organic acid: include but not limited to formic acid, acetic acid; Mineral acid: include but not limited to hydrochloric acid, sulfuric acid; Prioritizing selection mineral acid; Alkalescence: include but not limited to sodium hydroxide, potassium hydroxide, sodium bicarbonate, prioritizing selection sodium hydroxide;
Described polyanionic polysaccharide includes but not limited to: carrageenin, sulfated galactan and other controlling sulfate polyose, gum arabic, sodium alginate, pectin; Preferential carrageenin; Its average molecular weight range is greater than 300kD, but is no more than 2000kD.
The source of step (1) soybean whey comprises: conventional soybean protein isolate produces the by product formed, and laboratory utilizes defatted soybean meal, after alcohol wash, and the whey liquid after extraction and isolation albumen; The pre-treatment of soybean whey, its condition is: the soybean whey producing soybean protein isolate need through the static 1-2d of pH4.5, and centrifugal de-precipitation, to deviate from residual soybean protein isolate.
The primary separation of step (2) soybean whey protein, its condition is: before process of carrying out saltouing, and is preferentially readjusted by soybean whey liquid within the scope of pH to acid 4.5-4.8; Temperature range is the most suitable with low temperature, includes but not limited to 4-30 DEG C; Its ammonium sulfate saturation ratio should be not less than 30%, but not higher than 40%, prioritizing selection high saturation; Its corresponding ammonium sulfate saturation ratio need according to ammonium sulfate saturation table, according to actual temperature, and red-tape operati; Resolution of precipitate need add the deionized water of 2-4 times of volume, dissolves completely; Desalting treatment includes but not limited to dialysis, and ultrafiltration, nanofiltration, need ensure that desalination is complete.
Step (3) soybean whey protein and polyanionic polysaccharide complex coacervation, its condition is: the isocyatic protein solution of prioritizing selection mixes according to aforementioned proportion with polysaccharide soln; If select not isoconcentration solution to mix, need ensure that ultimate density is than consistent with above ratio of mixture.
Step (4) reclaims Kunitz and Bowman Birk type trypsin inhibitor, and its condition is: except sugared ultra-filtration membrane molecular weight cut-off scope is not less than 100kD, and be not more than 300kD; Desalting treatment includes but not limited to ultrafiltration, dialysis, and nanofiltration, need ensure that desalination is complete; The KSTI KTI reclaimed and Bowman Birk type trypsin inhibitor BBI detects sample purity through polyacrylamide gel electrophoresis SDS-PAGE and high performance liquid chromatography SEC-HPLC, protein content is measured through biuret method, sugar degree is measured through Phenol sulfuric acid procedure GB/T 15672-2009, moisture is measured, through calcination gravimetric determination ash content through desiccating method GB/T 5497-1985.
Beneficial effect of the present invention: the present invention fully utilizes soybean whey, equipment requirements is low, simple to operate, non-environmental-pollution.And the trypsin inhibitor rate of recovery is high, purity is high, and protein-active is high.
Accompanying drawing explanation
Fig. 1. the recovery process schema of KTI and BBI.
Fig. 2. the SEC-HPLC of the purity of KTI and BBI detects collection of illustrative plates.
Fig. 3. the purity SDS-PAGE of KTI and BBI detects collection of illustrative plates.
Embodiment
Present invention process flow process is as accompanying drawing
Embodiment 1:
Soybean whey is adjusted to pH 4.5, centrifugal (9500rpm, 30min) is except precipitation; Again be adjusted to pH 8.0, centrifugal (9500rpm, 30min), abandons precipitation, collects supernatant liquor.Above-mentioned supernatant liquor is regulated pH to 4.5, measure 1L solution, solid ammonium sulfate to 35% saturation ratio is added according to ammonium sulfate saturation table at 4-30 DEG C, centrifugal (9500rpm, 30min), KTI and the BBI albumen crude product obtaining containing is about 2.12g, adds 30mL deionized water dissolving, (molecular weight cut-off: 3500) desalination 36h, the preparation of trapped fluid vacuum lyophilization is containing KTI and BBI inhibitor sample in dialysis.
Accurately take KTI and BBI inhibitor sample and the 0.2g polyanionic polysaccharide powder of the above-mentioned primary separation of 0.2g respectively, join in 100mL deionized water respectively, be stirred to two kinds of materials to dissolve completely, being mixed with mass concentration w/v is respectively 0.2% homogeneous albumen and polysaccharide soln.Above-mentioned soybean whey protein and polyanionic polysaccharide solution are all adjusted to pH7.0.Slowly 6.67%(v/v is added in above-mentioned soybean whey protein solution) polyanionic polysaccharide solution, magnetic agitation, regulates pH to 4.3, centrifugal (4000rpm, 20min), collecting precipitation; To supernatant liquor again addition polymerization anionic polysaccharide soln repeat 1 time, merge throw out, obtain the complex coacervation thing of KTI and polyanionic polysaccharide.Merge twice supernatant liquor, continue the polyanionic polysaccharide solution slowly adding v/v 5%, regulate the centrifugal 20min of pH to 4.5,4000rpm, collect supernatant liquor, obtain the complex solution containing Bowman Birk and polyanionic polysaccharide.
The complex coacervation thing of the KTI obtained and polyanionic polysaccharide is suspended in the deionized water of 2-3 times of volume, regulate pH to 7.5, cross molecular weight cut-off be 100kD ultra-filtration membrane except sugar, collect filtered solution; (molecular weight cut-off: 3000) desalination, obtains the KTI of purifying in ultrafiltration.
By the solution containing BBI and polyanionic polysaccharide mixture obtained, regulate pH to 7.5, cross molecular weight cut-off be the ultra-filtration membrane of 100kD except sugar, collection filtered solution; (molecular weight cut-off: 3000) desalination, obtains the BBI of purifying in ultrafiltration.Take each 3.0mg of KTI and BBI of purifying, be dissolved in 1.0mL ultrapure water, be mixed with the solution that concentration is 3.0mg/mL, cross 0.45 μm of millipore filtration, SEC-HPLC detects purity of protein, and does SDS-PAGE electrophoretic analysis.
After measured, above example gained KTI, about containing albumen 95.5 %, total reducing sugar 1.2%, ash content 1.8% and moisture 1.5%; Above example gained BBI, about containing albumen 95.0 %, total reducing sugar 1.8%, ash content 1.2% and moisture 2.0%.
Embodiment 2:
Soybean whey is adjusted to pH 4.5, centrifugal (9500rpm, 30min), except precipitation; Again be adjusted to pH 8.5, centrifugal (9500rpm, 30min), abandons precipitation, collects supernatant liquor.Above-mentioned supernatant liquor is regulated pH to 4.7, measure 1.5L solution, solid ammonium sulfate to 40% saturation ratio is added according to ammonium sulfate saturation table at 4-30 DEG C, centrifugal (9500rpm, 30min), KTI and the BBI albumen crude product obtaining containing is about 3.35g, adds 40mL deionized water dissolving, dialysis (molecular weight cut-off: 3500) desalination 48 hours, vacuum lyophilization prepares sample.
Accurately take KTI and BBI inhibitor sample and the 0.4g polyanionic polysaccharide powder of the above-mentioned primary separation of 0.4g respectively, join in 100ml deionized water, be stirred to two kinds of materials and dissolve completely, being mixed with mass concentration is 0.4% homogeneous albumen and polysaccharide soln.Above-mentioned soybean whey protein and polyanionic polysaccharide solution are all adjusted to pH7.0.Slowly 5.0%(v/v is added in above-mentioned soybean whey protein solution) polyanionic polysaccharide solution, magnetic agitation, regulates pH to 4.0, centrifugal (4000rpm, 20min), collecting precipitation; 1 time is repeated again to supernatant liquor, merges throw out, obtain the mixture of KTI and polyanionic polysaccharide.Merge twice supernatant liquor, continue the polyanionic polysaccharide solution slowly adding v/v 7 %, regulate the centrifugal 20min of pH to 4.2,4000rpm, collect supernatant liquor, obtain the complex solution containing Bowman Birk and polyanionic polysaccharide.
The mixture of the KTI obtained and polyanionic polysaccharide is suspended in the deionized water of 2-3 times of volume, regulate pH to 8.0, cross molecular weight cut-off be 200kD ultra-filtration membrane except sugar, collect filtered solution; (molecular weight cut-off: 3500) desalination, obtains the KTI of purifying.By the complex solution of the BBI that obtains and polyanionic polysaccharide, regulate pH to 7.5, cross molecular weight cut-off be the ultra-filtration membrane of 200kD except sugar, collect filtered solution; (molecular weight cut-off: 3500) desalination, obtains the BBI of purifying in ultrafiltration.Take each 4.0mg of KTI and BBI of purifying, be dissolved in 1.0ml ultrapure water, be mixed with the solution that concentration is 4.0mg/mL, cross 0.45 μm of millipore filtration, SEC-HPLC detects purity of protein, and does SDS-PAGE electrophoretic analysis.
After measured, above example gained KTI, about containing albumen 96.3 %, total reducing sugar 1.0%, ash content 1.0% and moisture 1.7%; Above example gained BBI, about containing albumen 96.0 %, total reducing sugar 1.4%, ash content 1.1% and water 1.5%.
Embodiment 3:
Soybean whey is adjusted to pH 4.8, centrifugal (9500rpm, 30min), except precipitation; Again be adjusted to pH 9.0, centrifugal (9500rpm, 30min), abandons precipitation, collects supernatant liquor.Above-mentioned supernatant liquor is regulated pH to 4.8, measure 2L solution, 30% saturated ammonium sulfate is added according to ammonium sulfate saturation table at 4-30 DEG C, centrifugal, KTI and the BBI albumen crude product obtaining containing is about 0.356g, add 10mL deionized water dissolving, dialysis (molecular weight cut-off: 3500) desalination 48 hours, vacuum lyophilization prepares sample.
Accurately take KTI and BBI inhibitor sample and the 0.15g polyanionic polysaccharide powder of the above-mentioned primary separation of 0.15g respectively, join in 100mL deionized water, be stirred to two kinds of materials to dissolve completely, being mixed with mass concentration is 0.15% homogeneous albumen and polysaccharide soln.Above-mentioned soybean whey protein and polyanionic polysaccharide solution are all adjusted to pH7.0.Slowly 10%(v/v is added in above-mentioned soybean whey protein solution) polyanionic polysaccharide solution, magnetic agitation, regulates pH to 4.2, centrifugal (4000rpm, 20min), collecting precipitation; 1 time is repeated again to supernatant liquor, merges throw out, obtain the mixture of KTI and polyanionic polysaccharide.Merge twice supernatant liquor, continue the polyanionic polysaccharide solution slowly adding v/v 10%, regulate the centrifugal 20min of pH to 3.8,4000rpm, collect supernatant liquor, obtain the complex solution containing Bowman Birk and polyanionic polysaccharide
The mixture of the KTI obtained and polyanionic polysaccharide is suspended in the deionized water of 2-3 times of volume, regulate pH to 8.5, cross molecular weight cut-off be 300kD ultra-filtration membrane except sugar, collect filtered solution; (molecular weight cut-off: 4000) desalination, obtains the KTI of purifying in ultrafiltration.By the complex solution of the BBI that obtains and polyanionic polysaccharide, regulate pH to 8.5, cross molecular weight cut-off be the ultra-filtration membrane of 300kD except sugar, collect filtered solution; (molecular weight cut-off: 4000) desalination, obtains the BBI of purifying in ultrafiltration.Take each 2.0mg of KTI and BBI of purifying, be dissolved in 1.0ml ultrapure water, be mixed with the solution that concentration is 2.0mg/ml, cross 0.45 μm of millipore filtration, SEC-HPLC detects purity of protein, and does SDS-PAGE electrophoretic analysis.
After measured, above example gained KTI, about containing albumen 95.5 %, total reducing sugar 1.6%, ash content 1.1% and moisture 1.8%; Above example gained BBI, about containing albumen 97.0 %, total reducing sugar 1.1%, ash content 0.9% and moisture 1.0%.
Claims (6)
1. from soybean whey, reclaim a method for Kunitz and Bowman Birk type trypsin inhibitor, it is characterized in that step is:
(1) pre-treatment of soybean whey: soybean whey is adjusted to pH 4.5-4.8, the centrifugal 30min of 9500rpm is except precipitation; Again be adjusted to pH 8.0-10.0, the centrifugal 30min of 9500rpm, collect supernatant liquor, obtain test soybean whey liquid; PH is regulated by pH adjusting agent;
(2) primary separation of soybean whey protein: the above-mentioned soybean whey liquid measuring certain volume, regulate pH to 4.5-5.0, at 4-30 DEG C, adding ammonium sulfate to whole saturation ratio is the centrifugal 30min of 30%-40%, 9500rpm, collecting precipitation, add deionized water dissolving, make protein mass final concentration be 1%-10%, molecular weight cut-off 3500 dialysis desalting 36-48h, trapped fluid vacuum lyophilization, obtains the soybean whey protein component containing Kunitz and Bowman Birk type two kinds of trypsin inhibitors;
(3) soybean whey protein and polyanionic polysaccharide complex coacervation: soybean whey protein component prepared by step (2), with the soybean whey protein solution that deionized water preparation w/v final concentration is 0.1%-0.4%, regulate pH to 7.0, and prepare the polyanionic polysaccharide solution of w/v 0.1%-0.4% simultaneously, regulate pH to 7.0; In above-mentioned soybean whey protein solution, slowly add the polyanionic polysaccharide solution of v/v 2.5%-10%, regulate the centrifugal 20min of pH to 5.0-3.0,4000rpm, collecting precipitation; To supernatant liquor again addition polymerization anionic polysaccharide soln repeat precipitation 1-2 time, merging throw out, obtain the complex coacervation thing of KSTI and polyanionic polysaccharide;
Merge above-mentioned go precipitate after supernatant liquor, continue the polyanionic polysaccharide solution slowly adding v/v 5%-10%, regulate the centrifugal 20min of pH to 4.5-3.8,4000rpm, collect supernatant liquor, obtain the complex solution containing Bowman Birk type trypsin inhibitor and polyanionic polysaccharide;
(4) Kunitz and Bowman Birk type trypsin inhibitor is reclaimed: be suspended in the deionized water of 2-3 times of volume by the complex coacervation thing of the KSTI/polyanionic polysaccharide obtained, regulate pH to 7.0-9.0, cross ultra-filtration membrane 100kD-300kD except sugar, collect filtered solution; Molecular weight cut-off 3000-4000 ultrafiltration desalination, trapped fluid vacuum lyophilization, obtains the KSTI of purifying;
By the complex solution of Bowman Birk type trypsin inhibitor/polyanionic polysaccharide obtained, regulate pH to 7.0-9.0, cross ultra-filtration membrane 00kD-300kD except sugar, collect filtered solution; Molecular weight cut-off 3000-4000 ultrafiltration desalination, trapped fluid vacuum lyophilization, obtains the Bowman Birk type trypsin inhibitor of purifying.
2. the method reclaiming Kunitz and Bowman Birk type trypsin inhibitor from soybean whey according to claim 1, is characterized in that: described pH adjusting agent is: acid: organic acid: include but not limited to formic acid, acetic acid; Mineral acid: include but not limited to hydrochloric acid, sulfuric acid; Prioritizing selection mineral acid; Alkalescence: include but not limited to sodium hydroxide, potassium hydroxide, sodium bicarbonate, prioritizing selection sodium hydroxide;
Described polyanionic polysaccharide includes but not limited to: carrageenin, sulfated galactan and other controlling sulfate polyose, gum arabic, sodium alginate, pectin; Preferential carrageenin; Its average molecular weight range is greater than 300kD, but is no more than 2000kD.
3. the method reclaiming Kunitz and Bowman Birk type trypsin inhibitor from soybean whey according to claim 1, it is characterized in that the source of step (1) soybean whey comprises: conventional soybean protein isolate produces the by product formed, and laboratory utilizes defatted soybean meal, after alcohol wash, the whey liquid after extraction and isolation albumen; The pre-treatment of soybean whey, its condition is: the soybean whey producing soybean protein isolate need through the static 1-2d of pH4.5, and centrifugal de-precipitation, to deviate from residual soybean protein isolate.
4. the method reclaiming Kunitz and Bowman Birk type trypsin inhibitor from soybean whey according to claim 1, it is characterized in that the primary separation of step (2) soybean whey protein, its condition is: before process of carrying out saltouing, and is preferentially readjusted by soybean whey liquid within the scope of pH to acid 4.5-4.8; Temperature range is the most suitable with low temperature, includes but not limited to 4-30 DEG C; Its ammonium sulfate saturation ratio should be not less than 30%, but not higher than 40%, prioritizing selection high saturation; Its corresponding ammonium sulfate saturation ratio need according to ammonium sulfate saturation table, according to actual temperature, and red-tape operati; Resolution of precipitate need add the deionized water of 2-4 times of volume, dissolves completely; Desalting treatment includes but not limited to dialysis, and ultrafiltration, nanofiltration, need ensure that desalination is complete.
5. the method reclaiming Kunitz and Bowman Birk type trypsin inhibitor from soybean whey according to claim 1, it is characterized in that step (3) soybean whey protein and polyanionic polysaccharide complex coacervation, its condition is: the isocyatic protein solution of prioritizing selection mixes according to aforementioned proportion with polysaccharide soln; If select not isoconcentration solution to mix, need ensure that ultimate density is than consistent with above ratio of mixture.
6. the method reclaiming Kunitz and Bowman Birk type trypsin inhibitor from soybean whey according to claim 1, it is characterized in that step (4) reclaims Kunitz and Bowman Birk type trypsin inhibitor, its condition is: except sugared ultra-filtration membrane molecular weight cut-off scope is not less than 100kD, and be not more than 300kD; Desalting treatment includes but not limited to ultrafiltration, dialysis, and nanofiltration, need ensure that desalination is complete; The KSTI KTI reclaimed and Bowman Birk type trypsin inhibitor BBI detects sample purity through polyacrylamide gel electrophoresis SDS-PAGE and high performance liquid chromatography SEC-HPLC, protein content is measured through biuret method, sugar degree is measured through Phenol sulfuric acid procedure GB/T 15672-2009, moisture is measured, through calcination gravimetric determination ash content through desiccating method GB/T 5497-1985.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410742435.3A CN104513307B (en) | 2014-12-09 | 2014-12-09 | A method of recycling Kunitz and bowman-Birk trypsin inhibitor from soy-bean whey |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410742435.3A CN104513307B (en) | 2014-12-09 | 2014-12-09 | A method of recycling Kunitz and bowman-Birk trypsin inhibitor from soy-bean whey |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104513307A true CN104513307A (en) | 2015-04-15 |
| CN104513307B CN104513307B (en) | 2018-09-21 |
Family
ID=52789201
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410742435.3A Active CN104513307B (en) | 2014-12-09 | 2014-12-09 | A method of recycling Kunitz and bowman-Birk trypsin inhibitor from soy-bean whey |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104513307B (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105941115A (en) * | 2016-05-13 | 2016-09-21 | 晶叶（青岛）生物科技有限公司 | Rice coleoptiles and content extraction method and application thereof |
| CN107373012A (en) * | 2017-08-15 | 2017-11-24 | 临邑禹王植物蛋白有限公司 | A kind of production technology of soybean whey protein |
| CN110483634A (en) * | 2019-08-27 | 2019-11-22 | 合肥天汇孵化科技有限公司 | A kind of purification process of soybean trypsin inhibitor crude extract |
| CN110551209A (en) * | 2019-08-27 | 2019-12-10 | 合肥天汇孵化科技有限公司 | soybean clear water-based SBTI protein crude extraction process based on soybean deep processing waste liquid |
| CN110551208A (en) * | 2019-08-27 | 2019-12-10 | 合肥天汇孵化科技有限公司 | method for extracting soybean trypsin inhibitor |
| CN110590940A (en) * | 2019-08-27 | 2019-12-20 | 合肥天汇孵化科技有限公司 | Process for refining SBTI from soybean deep processing waste liquid by ASP dry powder |
| CN112553274A (en) * | 2020-12-02 | 2021-03-26 | 江南大学 | Method for cutting DNA by soybean extract Bowman-Birk inhibitor |
| CN112913962A (en) * | 2021-01-27 | 2021-06-08 | 江西恒顶食品有限公司 | Preparation method of protein powder |
| CN113243448A (en) * | 2021-05-12 | 2021-08-13 | 东北农业大学 | Method for recovering soybean whey protein by using dextran sulfate |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103965352A (en) * | 2014-05-09 | 2014-08-06 | 江南大学 | Method for purifying Kunitz-type trypsin inhibitor from soybean whey waste water |
-
2014
- 2014-12-09 CN CN201410742435.3A patent/CN104513307B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103965352A (en) * | 2014-05-09 | 2014-08-06 | 江南大学 | Method for purifying Kunitz-type trypsin inhibitor from soybean whey waste water |
Non-Patent Citations (2)
| Title |
|---|
| XINGFEI LI,ET AL.: "2", <JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY> * |
| 张维娟，王玉兰: "《生物化学与分子生物学实验教程》", 28 February 2014, 河南大学出版社 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105941115A (en) * | 2016-05-13 | 2016-09-21 | 晶叶（青岛）生物科技有限公司 | Rice coleoptiles and content extraction method and application thereof |
| CN107373012A (en) * | 2017-08-15 | 2017-11-24 | 临邑禹王植物蛋白有限公司 | A kind of production technology of soybean whey protein |
| CN110483634A (en) * | 2019-08-27 | 2019-11-22 | 合肥天汇孵化科技有限公司 | A kind of purification process of soybean trypsin inhibitor crude extract |
| CN110551209A (en) * | 2019-08-27 | 2019-12-10 | 合肥天汇孵化科技有限公司 | soybean clear water-based SBTI protein crude extraction process based on soybean deep processing waste liquid |
| CN110551208A (en) * | 2019-08-27 | 2019-12-10 | 合肥天汇孵化科技有限公司 | method for extracting soybean trypsin inhibitor |
| CN110590940A (en) * | 2019-08-27 | 2019-12-20 | 合肥天汇孵化科技有限公司 | Process for refining SBTI from soybean deep processing waste liquid by ASP dry powder |
| CN114560928A (en) * | 2019-08-27 | 2022-05-31 | 合肥天汇孵化科技有限公司 | Soybean clear water-based SBTI protein crude extraction process based on soybean deep processing waste liquid |
| CN112553274A (en) * | 2020-12-02 | 2021-03-26 | 江南大学 | Method for cutting DNA by soybean extract Bowman-Birk inhibitor |
| WO2022116378A1 (en) * | 2020-12-02 | 2022-06-09 | 江南大学 | Method for cleaving dna using soybean extract bowman-birk inhibitor |
| CN112913962A (en) * | 2021-01-27 | 2021-06-08 | 江西恒顶食品有限公司 | Preparation method of protein powder |
| CN113243448A (en) * | 2021-05-12 | 2021-08-13 | 东北农业大学 | Method for recovering soybean whey protein by using dextran sulfate |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104513307B (en) | 2018-09-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104513307A (en) | Method for recovery of Kunitz and Bowman-Birk trypsin inhibitors from soybean whey | |
| CN104004806B (en) | One kind has anticoagulation and thrombus dissolving earthworm polypeptide and its enzymolysis preparation and application | |
| CN104480092B (en) | A kind of method that urease is extracted from sword bean | |
| CN106309280B (en) | A kind of Ginger P.E and its preparation method and application | |
| CN104530207A (en) | Method for separating and purifying soybean agglutinin from soybean whey | |
| CN102775466A (en) | Preparation method of selenium-containing protein in selenium-enriched yeast | |
| CN105734099A (en) | Preparation method of cordyceps militaris polypeptide | |
| CN109527270A (en) | Leech specific nutrient induces the extracting method of liquid and natural hirudin | |
| CN103965352B (en) | A kind of method of KSTI of purifying from soya whey wastewater | |
| CN108929891A (en) | A kind of preparation method of horseshoe crab blood blood cell protein active peptide | |
| CN104292352A (en) | Method for extracting and separating eucommia ulmoides flavor powder, eucommia ulmoides oliv polysaccharides and eucommea rubber through coproduction | |
| CN103275948B (en) | Production process for extracting four enzymes from cattle or pig pancreas | |
| CN103740797B (en) | Method for preparing high-hydrolysis degree functional oligopeptide by use of high-temperature peanut meal | |
| CN104531813B (en) | A kind of preparation method of cornu caprae hircus active peptides | |
| CN103695513B (en) | A kind of method improving yield of soybean peptide with low molecular weight | |
| CN105085605B (en) | A kind of extracting method of rice albumin and globulin mixed protein | |
| CN104945495A (en) | Production process for separating and purifying fiber linking proteins from pig blood | |
| CN104055702B (en) | A kind of skin antiallergic wetting agent | |
| WO2020181784A1 (en) | Moringa oleifera leaf extract having uric acid lowering activity, preparation method therefor and application thereof | |
| CN106834399A (en) | A kind of Antihypertensive Peptides from Trachyostracous mussel closed shell flesh | |
| CN113813291B (en) | Preparation method of animal medicinal material freeze-dried powder | |
| CN102220296A (en) | Method for extracting garlic superoxide dismutase from garlic sheet processing waste water | |
| CN102512463B (en) | Extraction method of nettle extract | |
| CN108841200A (en) | A kind of preparation method of water solubility agaric melanin | |
| CN102746413A (en) | Method for preparing bee pollen polysaccharide through combining enzymolytic wall-breaking with hot-water ultrasonic extracting |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |