CN102295700A - Separation and purification method of mung bean trypsin inhibitor (MBTI) and application of MBTI in production of surimi products - Google Patents
Separation and purification method of mung bean trypsin inhibitor (MBTI) and application of MBTI in production of surimi products Download PDFInfo
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Abstract
The invention discloses a separation and purification method of an MBTI. The method comprises the following steps: 1, extraction; 2, neutralization; 3, heat treatment; 4, membrane treatment; and 5, adsorptive separation, and desorption. So the high purity MBTI is obtained. The invention also discloses an application of the MBTI, and the application is that the MBTI, which is added to surimi products treating seawater fish or freshwater fish as a main raw material and is uniformly mixed, is used for enhancing the elasticity of the surimi products. The separation and purification method of the invention has the advantages of simple technology and good separation effect, and the MBTI has a great improvement effect on the elasticity enhancement of the surimi products.
Description
Technical field
The present invention relates to a kind of separation purification method and the application in gefillte fish is produced of mung bean trypsin.
Background technology
Mung bean trypsin (Mung Bean Trypsin Inhibitor, MBTI) be from mung bean (
Phaseolus radiatus L.) the isolating Bowman-Birk type serpin that belongs in the mature seed.People such as the positive force of Chinese scholar relative have deeply systematically studied physical chemistry and the biochemical property of MBTI, have measured its primary structure.MBTI contains 72 amino-acid residues altogether, and molecular weight is 8,883 Da, contains 7 pairs of disulfide linkage, and is very high to the stability of heat, acid, alkali, enzymic digestion.It has two structural domains that the inhibition vigor is identical, and (Lys20-Ser21's two inhibition avtive spots Arg47-Ser48) works to trypsinase.The former is formed by connecting by two pairs of disulfide linkage with the short chain that contains 9 residues by a long-chain that contains 26 residues.And the latter is made up of the strand that contains 27 residues.The standard that the mechanism of MBTI and enzyme effect is followed other protein type serpin suppresses mechanism.Nearest discovers that MBTI also has the antitumor medicine effect of Denging.
At present, methods such as sulfuric acid extracting, ammonium sulfate precipitation, membrane ultrafiltration, ion-exchange chromatography, affinity chromatography or high performance liquid chromatography are mainly adopted in the separation and purification of mung bean trypsin, and the separation and purification process is loaded down with trivial details, complicated operation, length consuming time, yield is low, the cost height.
Summary of the invention
The object of the present invention is to provide a kind of technology simple, the separation purification method of the mung bean trypsin of good separating effect with and to gefillte fish elasticity enhanced improvement effect.
Process design principle of the present invention is: under the effect of low-concentration sulfuric acid, the part foreign protein is destroyed by acid and is degraded into small-molecule substance in the mung bean, and heat treated has further been removed foreign protein.To acid, heat stable mung bean trypsin (MBTI) is then unaffected substantially and in the supernatant liquor after centrifugal.Film is handled and can effectively be removed macromole (〉 30 kDa) salt and the little peptide component of albumen and small molecules (＜5 kDa).In the ion exchange chromatography, MBTI depends on the electrostatic attraction of opposite charges group to each other to the bonding force of anionite, and this magnetism is relevant with the pH value and the salt concn of solution.The variation of salt concn directly influences anionite to the absorption of proteins ability in the solution.So the present invention adopts salt concn linear gradient elution mode that MBTI is carried out purifying.
In addition, in the gefillte fish production process, (myofibril-bound serine proteinase MBSP) is the principal element that causes the gefillte fish flexibility decrease to the sarcostyle mating type serine protease in the muscle.Adding to the high purity MBTI of above method preparation or partially purified MBTI with sea water fish or fresh-water fishes is in the gefillte fish of main raw material, make its final concentration reach 10-50 mg/kg or more than, stir, can make the gel-strength of end article increase 10-20%, reach the effect of improving quality of fish meat.
In order to reach above-mentioned purpose, solution of the present invention is:
(Mung bean trypsin inhibitor, separation purification method MBTI) comprises the steps: a kind of mung bean trypsin
1) extracts: the exsiccant mung bean is ground into powder with pulverizer; In 1:4-6 m/v ratio vitriolization, after stirring was soaked, its supernatant liquor of centrifuging and taking was the MBTI crude extract;
2) neutralization: in the MBTI of step 1) gained crude extract, add alkali and be neutral to the pH of solution;
3) thermal treatment: with step 2) the MBTI crude extract of gained heats in water-bath, cooling immediately after the heating, centrifugal collection supernatant liquor;
4) film is handled: get outer liquid after the ultra-filtration membrane ultrafiltration that the supernatant liquor of step 3) gained is held back with 30 kDa molecular weight; Further the ultra-filtration membrane ultrafiltration desalination of holding back with 5 kDa molecular weight makes the MBTI concentrated solution;
5) fractionation by adsorption desorb: the MBTI concentrated solution after the step 4) desalination (is purchased in peace agate West Asia company or DEAE-Sepharose anionite-exchange resin (purchasing in peace agate West Asia company) fractionation by adsorption through Q-Sepharose, MBTI is adsorbed on the resin, resin after the absorption obtains high purity mung bean trypsin (MBTI) through the gradient elution desorb.
Sulfuric acid concentration is 0.05-0.1 mol/L in the described step 1), and stirring soak time is 18-36 h, and after tissue was smashed to pieces, with the centrifugal 15-30 min of 8000-12000 g, supernatant liquor was the MBTI crude extract.
Described step 2) alkali adopts the NaOH of 1-2 mol/L in.
Heating condition adopts 60 ° of C in the described step 3), 90 min or 80 ° of C, 30 min or 90 ° of C, a kind of among 15 min; The centrifugal 10-15 min of 10000-12000 g, collecting supernatant liquor is partially purified mung bean trypsin.
Large molecular weight protein is held back in the ultra-filtration membrane ultrafiltration that supernatant liquor in the described step 4) is held back with 30 kDa molecular weight, about 9 kDa of MBTI(that molecular weight is less) then flow out to outer liquid; The further ultra-filtration membrane ultrafiltration desalination of holding back with 5 kDa molecular weight, the 20 mmol/L Tris-HCl buffered soln or the phosphoric acid buffer that add pH 7.5-8.0 simultaneously in ultrafiltrated make the MBTI concentrated solution.
Anionite-exchange resin adopts diethyl aminoethyl (DEAE-) or season amino (Q-) anionite-exchange resin in the described step 5); Resin after the absorption obtains high purity mung bean trypsin (MBTI) through the salt drip washing desorb of 0-0.5 mol/L NaCl linear gradient.
The application of a kind of mung bean trypsin in gefillte fish is produced added to mung bean trypsin with sea water fish or fresh-water fishes and is in the gefillte fish of main raw material, mixes, and is used to strengthen elasticity of minced fish.
The final concentration that described mung bean trypsin is added in gefillte fish is more than the 10-50 mg/kg, makes the gel-strength of gefillte fish improve 10-20%.
Beneficial effect of the present invention is: the present invention is by carrying out the low-concentration sulfuric acid extracting, heat treated, and membrane sepn, means such as ion-exchange obtain highly purified mung bean trypsin.Especially utilize the characteristics of mung bean trypsin molecular weight little (9 kDa), the ultra-filtration membrane that adopts 30 kDa molecular weight to hold back is removed the macromolecule foreign protein.The ultra-filtration membrane that further adopts 5 kDa molecular weight to hold back is removed little peptide of small molecular weight and salt.Compare with long dialysis desalting consuming time, membrane concentration has reduced sample volume when realizing quick desalination, saved the last sample time of next step ion exchange column; Film is handled and also have been removed molecular weight greater than 30 kDa with less than the foreign protein of 5 kDa, has significantly improved purification efficiency.Because the present invention utilizes anionite-exchange resin effectively to separate mung bean trypsin fast, thereby it is simple to have technology, good separating effect, but the advantage of mass preparation have been simplified the purification step that original needs carry out repeatedly column chromatography greatly.
In sum, the present invention mainly adopts the low-concentration sulfuric acid extracting, and heating and membrane technique are handled desalination and removed foreign protein, further utilize the anion exchange chromatography method, fast and effeciently separate mung bean trypsin.The present invention adopts membrane technique to accelerate desalination speed and has effectively removed high molecular and lower molecular weight foreign protein.
Description of drawings
Fig. 1 is the mass spectrum of purifying mung bean trypsin of the present invention, M: molecular weight of albumen standard, 1: the MBTI of purifying;
Fig. 2 is the Tricine-SDS-PAGE electrophorogram of purifying mung bean trypsin of the present invention, and running gel is silver dyeing;
Fig. 3 is the thermostability figure of purifying mung bean trypsin of the present invention; MBTI is heated 30 min (●) respectively under differing temps, 1h () and 2h (△) back are to tryptic inhibition activity residual.
Fig. 4 is the influence result of the mung bean trypsin of purifying of the present invention to the gefillte fish gel-strength.
Embodiment
(Mung bean trypsin inhibitor, separation purification method MBTI) comprises the steps: a kind of mung bean trypsin of present embodiment
1, extracting: 400 g mung beans are milled to powder, and adding 1600 ml concentration is the sulfuric acid of 0.1 mol/L, stirs 24 h.Using Kinematica(Switzerland) tissue mashing machine smashs 30 min to pieces, and centrifugal 20 min under 12000 g collect about 700 mL of supernatant liquor, are the MBTI crude extract.
2, neutralization: in 700 mL crude extracts, add 1 mol/L NaOH, be neutral to the pH of solution.
3, thermal treatment: the MBTI crude extract after will neutralizing heats 90 min in 60 ° of C water-baths, cooling immediately after the heating, and centrifugal 10 min of 12000 g collect supernatant liquor.
4, film is handled: with above-mentioned ultra-filtration membrane ultrafiltration of holding back with 30 kDa molecular weight through the MBTI of heat treated gained supernatant liquor, collect outer liquid.Further the ultra-filtration membrane of holding back with 5 kDa molecular weight adds 0.02 mol/L Tris-HCl simultaneously to collecting outer liquid desalination and concentration by ultrafiltration in ultrafiltrated, and pH 8.0, and the final pH that makes solution is 8.0.
5, fractionation by adsorption: with the MBTI concentrated solution after step 4 desalination through being splined on Q-Sepharose Fast Flow anionite-exchange resin (2.5 * 9 cm), flow velocity 1mL/min.After absorption finished, the absorbancy of fully washing under pillar to 280 nm with 0.02 mol/L Tris-HCl damping fluid (pH 8.0) arrived baseline.
6, desorb: the resin after the absorption is through 200 mL, 0.02 mol/L Tris-HCl (pH 8.0) and 200 mL, 0.02 mol/L Tris-HCl(pH 8.0, contain 0.5 mol/L NaCl) mix the linear gradient elution liquid drip washing desorb of forming, collect elutriant (2 mL/ pipe) and obtain high purity mung bean trypsin (MBTI).
7, resin regeneration: after the desorb, resin is pulled down resin more earlier with 2 mol/L NaCl solution drip washing 4 hours, soaks 0.5 hour in 0.1 mol/L NaOH solution, cleans resin near neutral with a large amount of distilled water then, and resin regeneration finishes, and can be recycled.
The MBTI of purifying is splined on MALDI-TOF mass spectrograph (Shimadzu/Kratos, Kyoto, Japan) and carries out molecular weight determination, the result as shown in Figure 1, peak value the molecular weight of corresponding MBTI be 8887.25Da.The MBTI of purifying is analyzed by Tricine-SDS-PAGE, the result as shown in Figure 2, running gel is silver dyeing, electrophoresis result is single band, molecular weight is consistent with mass spectrometry results, shows that present method can obtain high purity MBTI.
Embodiment 2
(Mung bean trypsin inhibitor, separation purification method MBTI) comprises the steps: a kind of mung bean trypsin of present embodiment
1, extracts: get the 100g Powder Phaseoli radiati, add 500 mL, 0.1 mol/L H
2SO
4Stir and placed 20 hours, fully tissue is smashed to pieces, and centrifugal 20 min of 10000 g collect about 170 mL of supernatant liquor, are the MBTI crude extract.
2, neutralization: adjust crude extract pH with 2 mol/L NaOH, make pH value of solution be stabilized in about 7.
3, thermal treatment: the MBTI crude extract after will neutralizing heats 80 ° of C in water-bath, 30 min, and cooling immediately after the heating, centrifugal 15 min of 10000g collect supernatant liquor.
4, film processing: with above-mentioned ultra-filtration membrane ultrafiltration of holding back with 30 kDa molecular weight, hold back large molecular weight protein, about 9 kDa of target protein MBTI(that molecular weight is less) then flow out to outer liquid through the MBTI of heat treated gained supernatant liquor.Further the external liquid desalination and concentration by ultrafiltration of holding back with 5 kDa molecular weight of ultra-filtration membrane adds 0.02 mol/L Tris-HCl(pH 8.0 simultaneously in right amount in ultrafiltrated) guarantee that desalination is complete.
5, fractionation by adsorption: with 0.02 mol/L Tris-HCl damping fluid (pH=8.0) balance Q-Sepharose anionite-exchange resin (1.5 * 8 cm), the MBTI albumen mother liquor after the desalination is pumped in the post, the control flow velocity is 1 mL/min.
6, desorb: the resin after absorption finishes arrives baseline with the absorbancy that 0.02 mol/L Tris-HCl damping fluid (pH 8.0) fully washes under pillar to 280 nm.Resin after the absorption mixes the gradient eluent drip washing desorb of forming with 100 mL, 0.02 mol/L Tris-HCl damping fluid (pH 8.0) and 100 mL, 0.02 mol/L Tris-HCl damping fluids (pH 8.0 contains 0.2 mol/L NaCl), collect elutriant (2 mL/ pipe), obtain high purity mung bean trypsin (MBTI).
7, resin regeneration: after the desorb, resin is pulled down resin more earlier with 2 mol/L NaCl solution drip washing 4 hours, soaks 0.5 hour in 0.1 mol/L NaOH solution, cleans resin near neutral with a large amount of distilled water then, and resin regeneration finishes, and can be recycled.
The high purity MBTI of above method preparation is carried out thermal stability analysis under differing temps, the gained result as shown in Figure 3.The MBTI of purifying is 100
oThe C heating still had 85 % activity after 1 hour, still had the activity of 55 % after 2 hours, showed that it is to thermally-stabilised.
Adding to the high purity MBTI of above method preparation or partially purified MBTI with the fresh water silver carp is in the gefillte fish of main raw material, make its final concentration reach 40 mg/kg or more than, stir, can effectively suppress sarcostyle mating type serine protease (the myofibril-bound serine proteinase in the muscle, MBSP) activity makes the gel-strength of goods increase by 10%.
Embodiment 3
(Mung bean trypsin inhibitor, separation purification method MBTI) comprises the steps: a kind of mung bean trypsin of present embodiment
1, extracting: 500 g exsiccant mung beans are milled to powder, and adding 3000 ml concentration is the sulfuric acid of 0.075 mol/L, stirs 36 h, and tissue is smashed behind 30 min centrifugal 20 min under 12000 g to pieces, collects about 1250 mL of supernatant liquor, is the MBTI crude extract.
2, neutralization: in 1250 mL crude extracts, add 1.2 mol/L NaOH, be neutral to the pH of solution.
3, thermal treatment: the MBTI crude extract after will neutralizing heats 30 min in 80 ℃ of water-baths, and cooling immediately after the heating at centrifugal 15 min of 12000 g, is collected supernatant liquor.
4, film is handled: with above-mentioned ultra-filtration membrane ultrafiltration of holding back with 30 kDa molecular weight through the MBTI of heat treated gained supernatant liquor, collect outer liquid.Further the external liquid desalination and concentration by ultrafiltration of holding back with 5 kDa molecular weight of ultra-filtration membrane adds 20 mmol/L Tris-HCl simultaneously in ultrafiltrated, transfers the pH to 7.5 of solution.
5, fractionation by adsorption: the MBTI concentrated solution after step 4 desalination is pumped in the adsorption column (2.5 * 12 cm) of DEAE-Sepharose Fast Flow anionite-exchange resin, flow velocity 1.5 mL/min, the resin after absorption finishes arrives baseline with the absorbancy that 0.02 mol/L Tris-HCl damping fluid (pH 8.0) fully washes under pillar to 280 nm.
6, desorb: the resin after the absorption respectively through 0.02 mol/L Tris-HCl (pH 8.0) and contain the 0.02 mol/L Tris-HCl(pH 8.0 of different concns NaCl) elutriant drip washing desorb.NaCl concentration is followed successively by 0.1,0.2,0.3,0.4 and 0.5 mol/L respectively, and each concentration is drip washing 80 mL respectively.Collect elutriant (2 mL/ pipe) and obtain high purity mung bean trypsin (MBTI).
7, resin regeneration: after the desorb, resin is pulled down resin more earlier with 2 mol/L NaCl solution drip washing 4 hours, soaks 0.5 hour in 0.1 mol/L NaOH solution, cleans resin near neutral with a large amount of distilled water then, and resin regeneration finishes, and can be recycled.
Adding to the high purity MBTI of above method preparation or partially purified MBTI with the blue circle of sea water fish Scad is in the gefillte fish of main raw material, make its final concentration reach 50 μ g/kg, stir, can effectively suppress sarcostyle mating type serine protease (the myofibril-bound serine proteinase in the muscle, MBSP) activity, make the gel-strength of goods increase by 21%, the result as shown in Figure 4.
Embodiment 4
(Mung bean trypsin inhibitor, separation purification method MBTI) comprises the steps: a kind of mung bean trypsin of present embodiment
1, extracting: 500 g mung beans are milled to powder, and adding 2000 ml concentration is the sulfuric acid of 0.1 mol/L, stirs extracting 22 h, and fully tissue is smashed to pieces, and centrifugal 15 min under 10000 g collect about 870 mL of supernatant liquor, are the MBTI crude extract.
2, neutralization: in 870 mL crude extracts, add 2 mol/L NaOH, be neutral to the pH of solution.
3, thermal treatment: the 90 ° of C in water-bath of the MBTI crude extract after will neutralizing handled 15 minutes, were placed on cooling immediately in the frozen water, and centrifugal 15 min under 10000 g collect supernatant liquor.
4, film processing: with above-mentioned ultra-filtration membrane ultrafiltration of holding back with 30 kDa molecular weight, hold back large molecular weight protein, about 9 kDa of target protein MBTI(that molecular weight is less) then flow out to outer liquid through the MBTI of heat treated gained supernatant liquor.Further the external liquid desalination and concentration by ultrafiltration of holding back with 5 kDa molecular weight of ultra-filtration membrane adds 20 mmol/L phosphoric acid buffers (pH 8.0) simultaneously in right amount and guarantees that desalination is complete in ultrafiltrated.
5, fractionation by adsorption: the MBTI albumen mother liquor after the desalination is pumped into in 0.02 mol/L phosphoric acid buffer (pH 8.0) the equilibrated Q-Sepharose anion-exchange column (1.5 * 10 cm), and the control flow velocity is 1 mL/min.
6, desorb: the resin after absorption finishes arrives baseline with the absorbancy under abundant drip washing pillar to 280 nm of 0.02 mol/L phosphoric acid buffer (pH 8.0).Resin after the absorption mixes the gradient eluent desorb of forming with 200 mL, 0.02 mol/L phosphoric acid buffer (pH=8.0) and 200 mL, 0.02 mol/L phosphoric acid buffer (pH 8.0 contains 0.4 mol/L NaCl), collect elutriant (3 mL/ pipe), obtain high purity mung bean trypsin (MBTI).
7, resin regeneration: after the desorb, resin is pulled down resin more earlier with 2 mol/L NaCl solution drip washing 4 hours, soaks 0.5 hour in 0.1 mol/L NaOH solution, cleans resin near neutral with a large amount of distilled water then, and resin regeneration finishes, and can be recycled.
Adding to the high purity MBTI of above method preparation or partially purified MBTI with the blue circle of sea water fish Scad is in the gefillte fish of main raw material, make its final concentration reach 15 μ g/kg, stir, can effectively suppress sarcostyle mating type serine protease (the myofibril-bound serine proteinase in the muscle, MBSP) activity makes the gel-strength of goods increase by 21%.
Embodiment 5
(Mung bean trypsin inhibitor, separation purification method MBTI) comprises the steps: a kind of mung bean trypsin of present embodiment
1, extracting: 500 g exsiccant mung beans are milled to powder, and adding 2000 ml concentration is the sulfuric acid of 0.1 mol/L, stirs 24 h, and tissue is smashed 30 min to pieces; Continue to stir 10 hours, tissue is smashed after 10 minutes centrifugal 15 min under 12000 g to pieces again, collects about 900 mL of supernatant liquor, is the MBTI crude extract.
2, neutralization: in 900 mL crude extracts, add 1 mol/L NaOH, be neutral to the pH of solution.
3, thermal treatment: the MBTI crude extract after will neutralizing heats 90 min in 60 ℃ of water-baths, and cooling immediately after the heating at centrifugal 12 min of 12000 g, is collected supernatant liquor.
4, film is handled: with above-mentioned ultra-filtration membrane ultrafiltration of holding back with 30 kDa molecular weight through the MBTI of heat treated gained supernatant liquor, collect outer liquid.Further the external liquid desalination and concentration by ultrafiltration of holding back with 5 kDa molecular weight of ultra-filtration membrane adds 0.02 mol/L Tris-HCl simultaneously in ultrafiltrated, and pH8.0 makes the final pH of ultrafiltrated reach 8.0.
5, fractionation by adsorption: through Q-Sepharose Fast Flow anionite-exchange resin (2.5 * 8 cm) fractionation by adsorption, MBTI is adsorbed on the resin with the MBTI concentrated solution after step 4 desalination.Resin after absorption finishes arrives baseline with the absorbancy under abundant drip washing to 280 nm of 0.02 mol/L Tris-HCl damping fluid (pH 8.0).
6, desorb: the resin after the absorption is with 200 mL, 0.02 mol/L Tris-HCl (pH 8.0) and 200 mL, 0.02 mol/L Tris-HCl(pH 8.0, contain 0.5 mol/L NaCl) linear gradient elution liquid wash-out that mix to form, collect elutriant (2 mL/ pipe), can obtain high purity mung bean trypsin (MBTI).
7, resin regeneration: after the desorb, resin is pulled down resin more earlier with 2 mol/L NaCl solution drip washing 4 hours, soaks 0.5 hour in 0.1 mol/L NaOH solution, cleans resin near neutral with a large amount of distilled water then, and resin regeneration finishes, and can be recycled.
Adding to the high purity MBTI of above method preparation or partially purified MBTI with the fresh water silver carp is in the gefillte fish of main raw material, make its final concentration reach 25 μ g/kg, stir, can effectively suppress sarcostyle mating type serine protease (the myofibril-bound serine proteinase in the muscle, MBSP) activity makes the gel-strength of goods increase by 14.5%.
Embodiment 6
(Mung bean trypsin inhibitor, separation purification method MBTI) comprises the steps: a kind of mung bean trypsin of present embodiment
1, extract: with 1000 g exsiccant Powder Phaseoli radiatis, adding 4000 ml concentration is the sulfuric acid of 0.05 mol/L, stirs 24 h, is divided into 5 parts, and every part of tissue is smashed 30 min to pieces.Centrifugal 15 min under 12000 g collect about 1750 mL of supernatant liquor, are the MBTI crude extract.
2, neutralization: in 1750 mL crude extracts, add 1.5 mol/L NaOH, be neutral to the pH of solution.
3, thermal treatment: the MBTI crude extract after will neutralizing heats 30 min in 80 ° of C of water-bath, cooling immediately after the heating, and centrifugal 15 min of 10000 g collect supernatant liquor.
4, film is handled: the ultra-filtration membrane ultrafiltration of will the above-mentioned 3 MBTI supernatant liquors of handling gained holding back with 30 kDa molecular weight, hold back macromole, collection contains about 9 kDa of target protein MBTI() outer liquid.Further the external liquid desalination and concentration by ultrafiltration of holding back with 5 kDa molecular weight of ultra-filtration membrane adds 0.02 mol/L phosphoric acid buffer (pH 7.5) simultaneously in ultrafiltrated, makes desalination complete, and volume is about 300 mL.
5, fractionation by adsorption: the MBTI concentrated solution after step 4 desalination is splined on 0.02 mol/L phosphoric acid buffer (pH 7.5) equilibrated DEAE-Sepharose Fast Flow anion-exchange column (2.5 * 18 cm), flow velocity 2 mL/min.After absorption finishes, arrive baseline with the absorbancy under abundant drip washing pillar to 280 nm of 0.02 mol/L phosphoric acid buffer (pH=7.5).
6, desorb: (pH 7.5 with 400 mL, 0.02 mol/L phosphoric acid buffers (pH 7.5) and 400 mL, 0.02 mol/L phosphoric acid buffer for the resin after the absorption, containing 0.5 mol/L NaCl) the linear gradient elution lyolysis formed inhales, collects elutriant (4 mL/ pipe) and obtain high purity mung bean trypsin (MBTI).
7, resin regeneration: after the desorb, resin is pulled down resin more earlier with 2 mol/L NaCl solution drip washing 4 hours, soaks 0.5 hour in 0.1 mol/L NaOH solution, cleans resin near neutral with a large amount of distilled water then, and resin regeneration finishes, and can be recycled.
Adding to the high purity MBTI of above method preparation or partially purified MBTI with the blue circle of sea water fish Scad is in the gefillte fish of main raw material, make its final concentration reach 30 mg/kg, beating bursts stirs, can effectively suppress sarcostyle mating type serine protease (the myofibril-bound serine proteinase in the muscle, MBSP) activity makes the gel-strength of end article increase by 17.6%.
Above-mentioned only is specific embodiments of the invention, but design concept of the present invention is not limited thereto, and allly utilizes this design that the present invention is carried out the change of unsubstantiality, all should belong to the behavior of invading protection domain of the present invention.
Claims (8)
1. the separation purification method of a mung bean trypsin is characterized in that comprising the steps:
1) extracts: the exsiccant mung bean is ground into powder with pulverizer; In 1:4-6 m/v ratio vitriolization, after stirring was soaked, its supernatant liquor of centrifuging and taking was a crude extract;
2) neutralization: in the crude extract of step 1) gained, add alkali and be neutral to the pH of solution;
3) thermal treatment: with step 2) crude extract of gained heats in water-bath, cooling immediately after the heating, centrifugal collection supernatant liquor;
4) film is handled: get outer liquid after the ultra-filtration membrane ultrafiltration that the supernatant liquor of step 3) gained is held back with 30 kDa molecular weight; Further the ultra-filtration membrane ultrafiltration desalination of holding back with 5 kDa molecular weight makes concentrated solution;
5) fractionation by adsorption desorb: with the concentrated solution after the step 4) desalination through Q-Sepharose or DEAE-Sepharose anionite-exchange resin fractionation by adsorption, mung bean trypsin is adsorbed on the resin, resin after the absorption obtains the high purity mung bean trypsin through the gradient elution desorb.
2. the separation purification method of a kind of mung bean trypsin according to claim 1, it is characterized in that: sulfuric acid concentration is 0.05-0.1 mol/L in the described step 1), stirring soak time is 18-36 h, after tissue is smashed to pieces, with the centrifugal 15-30 min of 8000-12000 g, supernatant liquor is a crude extract.
3. the separation purification method of a kind of mung bean trypsin according to claim 1 is characterized in that: the NaOH of alkali employing 1-2 mol/L described step 2).
4. the separation purification method of a kind of mung bean trypsin according to claim 1 is characterized in that: heating condition adopts 60 ° of C in the described step 3), 90 min or 80 ° of C, 30 min or 90 ° of C, a kind of among 15 min; The centrifugal 10-15 min of 10000-12000 g, collecting supernatant liquor is partially purified mung bean trypsin.
5. the separation purification method of a kind of mung bean trypsin according to claim 1, it is characterized in that: the ultra-filtration membrane ultrafiltration that the supernatant liquor in the described step 4) is held back with 30 kDa molecular weight, hold back large molecular weight protein, the less albumen of molecular weight then flows out to outer liquid; The further ultra-filtration membrane ultrafiltration desalination of holding back with 5 kDa molecular weight, the 20 mmol/L Tris-HCl buffered soln or the phosphoric acid buffer that add pH 7.5-8.0 simultaneously in ultrafiltrated make concentrated solution.
6. the separation purification method of a kind of mung bean trypsin according to claim 1 is characterized in that: in the described step 5) anionite-exchange resin adopt diethyl aminoethyl or season amino anionite-exchange resin; Resin after the absorption obtains the high purity mung bean trypsin through the salt drip washing desorb of 0-0.5 mol/L NaCl gradient.
7. the application of mung bean trypsin as claimed in claim 1 in gefillte fish is produced, it is characterized in that: adding to mung bean trypsin with sea water fish or fresh-water fishes is in the gefillte fish of main raw material, mix, be used to strengthen elasticity of minced fish.
8. the application of a kind of mung bean trypsin as claimed in claim 7 in gefillte fish is produced, it is characterized in that: the final concentration that described mung bean trypsin is added in gefillte fish is more than the 10-50 mg/kg, makes the gel-strength of gefillte fish improve 10-20%.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104473231A (en) * | 2014-12-01 | 2015-04-01 | 福建安井食品股份有限公司 | Multi-target inhibiting base material for aquatic muscle endogenous enzyme and preparation method and application of multi-target inhibiting base material |
| CN104522740A (en) * | 2014-11-24 | 2015-04-22 | 宁波大学 | Method used for increasing gel strength of mackerel minced fillet |
| CN105360275A (en) * | 2015-11-13 | 2016-03-02 | 集美大学 | Sea cucumber preservative and use thereof |
| CN105566491A (en) * | 2016-03-08 | 2016-05-11 | 山西大学 | Method for extracting trypsin inhibitor from flax protein powder |
| CN106565839A (en) * | 2016-10-13 | 2017-04-19 | 浙江万里学院 | Method for extracting trypsin inhibitor from pitaya seeds |
| CN113785873A (en) * | 2021-09-09 | 2021-12-14 | 集美大学 | Natural anti-blackening agent for shrimps as well as preparation method and application thereof |
-
2011
- 2011-07-29 CN CN201110215634.5A patent/CN102295700B/en active Active
Non-Patent Citations (5)
| Title |
|---|
| GUANGDA LIN ET AL: "The 0.25-nm X-ray structure of the Bowman-Birk-type inhibitor from mung bean in ternary complex with porcine trypsin", 《EUR. J. BIOCHEM》 * |
| LE-CHANG SUN ET AL: "Mung Bean Trypsin Inhibitor Is Effective in Suppressing the Degradation of Myofibrillar Proteins in the Skeletal Muscle of Blue Scad (Decapterus maruadsi)", 《J. AGRIC. FOOD CHEM.》 * |
| REKHA KANSAL ET AL: "Purification, characterization and evaluation of insecticidal potential of trypsin inhibitor from mungbean (Vigna radiata L. Wilczek) seeds", 《ACTA PHYSIOL PLANT》 * |
| SAPPASITH KLOMKLAO ET AL: "Extraction, purification and properties of trypsin inhibitor from Thai mung bean (Vigna radiata (L.) R. Wilczek)", 《FOOD CHEMISTRY》 * |
| 刘同祥等: "绿豆胰蛋白酶抑制剂的分离和纯化", 《中国生化药物杂志》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104522740A (en) * | 2014-11-24 | 2015-04-22 | 宁波大学 | Method used for increasing gel strength of mackerel minced fillet |
| CN104522740B (en) * | 2014-11-24 | 2018-01-30 | 宁波大学 | A kind of method for improving mackerel minced fish gel intensity |
| CN104473231A (en) * | 2014-12-01 | 2015-04-01 | 福建安井食品股份有限公司 | Multi-target inhibiting base material for aquatic muscle endogenous enzyme and preparation method and application of multi-target inhibiting base material |
| CN104473231B (en) * | 2014-12-01 | 2017-06-13 | 福建安井食品股份有限公司 | Many targeting endogenous enzyme level base-materials of aquatic products muscle and its preparation method and application |
| CN105360275A (en) * | 2015-11-13 | 2016-03-02 | 集美大学 | Sea cucumber preservative and use thereof |
| CN105360275B (en) * | 2015-11-13 | 2019-04-19 | 集美大学 | A kind of sea cucumber antistaling agent and purposes |
| CN105566491A (en) * | 2016-03-08 | 2016-05-11 | 山西大学 | Method for extracting trypsin inhibitor from flax protein powder |
| CN106565839A (en) * | 2016-10-13 | 2017-04-19 | 浙江万里学院 | Method for extracting trypsin inhibitor from pitaya seeds |
| CN106565839B (en) * | 2016-10-13 | 2020-01-03 | 浙江万里学院 | Method for extracting trypsin inhibitor from pitaya seeds |
| CN113785873A (en) * | 2021-09-09 | 2021-12-14 | 集美大学 | Natural anti-blackening agent for shrimps as well as preparation method and application thereof |
| CN113785873B (en) * | 2021-09-09 | 2023-10-27 | 集美大学 | Natural shrimp blackening preventing agent and preparation method and application thereof |
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