CN102295700B - 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 of mung bean trypsin and the application in gefillte fish is produced.
Background technology
Mung bean trypsin (Mung Bean Trypsin Inhibitor, MBTI) be from mung bean (
phaseolus radiatus L.) separate in mature seed belong to Bowman-Birk type serpin.The 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 is altogether containing 72 amino-acid residues, and molecular weight is 8,883 Da, containing 7 pairs of disulfide linkage, very high to the stability of heat, acid, alkali, enzymic digestion.It has two structural domains that inhibition vigor is identical, and two are suppressed avtive spot (Lys20-Ser21, Arg47-Ser48) and all trypsinase worked.The former is formed by connecting by two pairs of disulfide linkage containing the long-chain of 26 residues and the short chain containing 9 residues by one.And the latter is made up of the strand containing 27 residues.The standard that the mechanism of MBTI and enzyme effect is followed other oroteins type serpin suppresses mechanism.Nearest research discovery, MBTI also has the antitumor medicine effect of Denging.
At present, the separation and purification of mung bean trypsin mainly adopts the methods such as sulfuric acid extracting, ammonium sulfate precipitation, membrane ultrafiltration, ion-exchange chromatography, affinity chromatography or high performance liquid chromatography, and separation and purification process is loaded down with trivial details, complicated operation, length consuming time, yield is low, and cost is high.
Summary of the invention
The object of the present invention is to provide a kind of technique simple, the separation purification method of the mung bean trypsin of good separating effect with and to gefillte fish elasticity strengthen improvement effect.
Process design principle of the present invention is: under the effect of low-concentration sulfuric acid, in mung bean, part foreign protein is destroyed by acid and is degraded into small-molecule substance, and heat treated has further been removed foreign protein.Substantially unaffected and in supernatant liquor after centrifugal to acid, heat stable mung bean trypsin (MBTI).Salt and the little peptide component of macromole (>30 kDa) albumen and small molecules (<5 kDa) can be effectively removed in film processing.In 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 pH value and the salt concn of solution.In solution, the variation of salt concn directly affects the adsorptive power of anionite to protein.Therefore the present invention adopts salt concn linear gradient elution mode to carry out purifying to MBTI.
In addition,, in gefillte fish production process, the sarcostyle mating type serine protease (myofibril-bound serine proteinase, MBSP) in muscle is the principal element that causes gefillte fish flexibility decrease.High purity MBTI prepared by above method or partially purified MBTI add to taking sea water fish or fresh-water fishes 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:
The separation purification method of a kind of mung bean trypsin (Mung bean trypsin inhibitor, MBTI), comprises the steps:
1) extract: dry mung bean is ground into powder with pulverizer; In 1:4-6 m/v ratio vitriolization, stir after immersion, its supernatant liquor of centrifuging and taking is MBTI crude extract;
2) neutralization: be neutral to the pH of solution to adding alkali in the MBTI crude extract of step 1) gained;
3) thermal treatment: by step 2) the MBTI crude extract of gained heats in water-bath, cooling immediately after heating, centrifugal collection supernatant liquor;
4) film processing: 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; The ultra-filtration membrane ultrafiltration desalination of further holding back with 5 kDa molecular weight makes MBTI concentrated solution;
5) fractionation by adsorption desorb: the MBTI concentrated solution after step 4) desalination (is purchased to An Ma West Asia company or DEAE-Sepharose anionite-exchange resin (being purchased from An Ma West Asia company) fractionation by adsorption through Q-Sepharose, MBTI is adsorbed on resin, resin after absorption, through gradient elution desorb, obtains high purity mung bean trypsin (MBTI).
In described step 1), sulfuric acid concentration is 0.05-0.1 mol/L, and stirring soak time is 18-36 h, and after tissue mashing, with the centrifugal 15-30 min of 8000-12000 g, supernatant liquor is MBTI crude extract.
Described step 2) in alkali adopt the NaOH of 1-2 mol/L.
In described step 3), heating condition adopts 60 ° of C, 90 min or 80 ° of C, 30 min or 90 ° of C, the one in 15 min; The centrifugal 10-15 min of 10000-12000 g, collecting supernatant liquor is partially purified mung bean trypsin.
The ultra-filtration membrane ultrafiltration that supernatant liquor in described step 4) is held back with 30 kDa molecular weight, holds back large molecular weight protein, the MBTI(of molecular weight approximately 9 kDa) flow out to outer liquid; The ultra-filtration membrane ultrafiltration desalination of further holding back with 5 kDa molecular weight, adds 20 mmol/L Tris-HCl buffered soln or the phosphoric acid buffer of pH 7.5-8.0 to make MBTI concentrated solution simultaneously in ultrafiltrated.
In described step 5), anionite-exchange resin adopts diethyl aminoethyl (DEAE-) or season amino (Q-) anionite-exchange resin; Resin after absorption, through the salt drip washing desorb of 0-0.5 mol/L NaCl linear gradient, obtains high purity mung bean trypsin (MBTI).
The application of mung bean trypsin in gefillte fish is produced, adds mung bean trypsin taking sea water fish or fresh-water fishes in the gefillte fish of main raw material, mixes, for strengthening elasticity of minced fish.
The final concentration that described mung bean trypsin is added in gefillte fish is more than 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 low-concentration sulfuric acid extracting, heat treated, and membrane sepn, the means such as ion-exchange, obtain highly purified mung bean trypsin.Especially utilize the feature of mung bean trypsin molecular weight little (9 kDa), adopt the ultra-filtration membrane that 30 kDa molecular weight are held back to remove macromolecule foreign protein.The ultra-filtration membrane that further adopts 5 kDa molecular weight to hold back is removed the little peptide of small molecules amount and salt.Compared with longer dialysis desalting consuming time, membrane concentration has reduced sample volume in realizing quick desalination, has saved the loading time of next step ion exchange column; Film processing has also been removed molecular weight and has been greater than 30 kDa and the foreign protein that is less than 5 kDa, has significantly improved purification efficiency.Separate fast effectively mung bean trypsin because the present invention utilizes anionite-exchange resin, thereby it is simple to have technique, good separating effect, the advantage that can prepare on a large scale, has simplified original needs and has carried out repeatedly the purification step of column chromatography greatly.
In sum, the present invention mainly adopts low-concentration sulfuric acid extracting, and heating and membrane technique are processed desalination except foreign protein, further utilize 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.
Brief description of the 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 respectively under differing temps to 30 min (●), after 1h () and 2h (△) to tryptic inhibition activity residual.
Fig. 4 is that the mung bean trypsin of purifying of the present invention is on the result that affects of gefillte fish gel-strength.
Embodiment
Embodiment 1
The separation purification method of a kind of mung bean trypsin (Mung bean trypsin inhibitor, MBTI) of the present embodiment, comprises the steps:
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 supernatant liquor approximately 700 mL, are MBTI crude extract.
2, neutralization: add 1 mol/L NaOH in 700 mL crude extracts, be neutral to the pH of solution.
3, thermal treatment: the MBTI crude extract after neutralization is heated to 90 min in 60 ° of C water-baths, and cooling immediately after heating, centrifugal 10 min of 12000 g, collect supernatant liquor.
4, film processing: by 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.The ultra-filtration membrane of further holding back with 5 kDa molecular weight, to collecting outer liquid desalination and concentration by ultrafiltration, adds 0.02 mol/L Tris-HCl simultaneously in ultrafiltrated, pH 8.0, and the final pH that makes solution is 8.0.
5, fractionation by adsorption: by 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, fully rinse absorbancy under pillar to 280 nm to baseline with 0.02 mol/L Tris-HCl damping fluid (pH 8.0).
6, desorb: the resin after 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, containing 0.5 mol/L NaCl) mix the linear gradient elution liquid drip washing desorb forming, collect elutriant (2 mL/ pipe) and obtain high purity mung bean trypsin (MBTI).
7, resin regeneration: after desorb, resin is first used 2 mol/L NaCl solution drip washing 4 hours, then resin is pulled down is soaked 0.5 hour in 0.1 mol/L NaOH solution, then cleans resin near neutral with a large amount of distilled water, and resin regeneration is complete, can be recycled.
The MBTI of purifying is splined on MALDI-TOF mass spectrograph (Shimadzu/Kratos, Kyoto, Japan) and carries out molecular weight determination, 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, and as shown in Figure 2, running gel is silver dyeing to result, and electrophoresis result is single band, and molecular weight is consistent with mass spectrometry results, shows that present method can obtain high purity MBTI.
Embodiment 2
The separation purification method of a kind of mung bean trypsin (Mung bean trypsin inhibitor, MBTI) of the present embodiment, comprises the steps:
1, extract: get 100g Powder Phaseoli radiati, add 500 mL 0.1 mol/L H
2sO
4stir and place 20 hours, fully tissue mashing, centrifugal 20 min of 10000 g, collect supernatant liquor approximately 170 mL, are MBTI crude extract.
2, neutralization: with 2 mol/L NaOH adjustment crude extract pH, make pH value of solution be stabilized in 7 left and right.
3, thermal treatment: the MBTI crude extract after neutralization is heated to 80 ° of C in water-bath, 30 min, cooling immediately after heating, centrifugal 15 min of 10000g, collect supernatant liquor.
4, film processing: by above-mentioned ultra-filtration membrane ultrafiltration of holding back with 30 kDa molecular weight through the MBTI of heat treated gained supernatant liquor, hold back large molecular weight protein, target protein MBTI(approximately 9 kDa of molecular weight) flow out to outer liquid.The external liquid desalination and concentration by ultrafiltration of ultra-filtration membrane of further holding back with 5 kDa molecular weight adds in right amount 0.02 mol/L Tris-HCl(pH 8.0 simultaneously 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 desalination is pumped in post, coutroi velocity is 1 mL/min.
6, desorb: the resin after absorption arrives baseline by the absorbancy that 0.02 mol/L Tris-HCl damping fluid (pH 8.0) fully rinses under pillar to 280 nm.Resin after absorption mixes the gradient eluent drip washing desorb of composition with 100 mL 0.02 mol/L Tris-HCl damping fluids (pH 8.0) and 100 mL 0.02 mol/L Tris-HCl damping fluids (pH 8.0 is containing 0.2 mol/L NaCl), collect elutriant (2 mL/ pipe), obtain high purity mung bean trypsin (MBTI).
7, resin regeneration: after desorb, resin is first used 2 mol/L NaCl solution drip washing 4 hours, then resin is pulled down is soaked 0.5 hour in 0.1 mol/L NaOH solution, then cleans resin near neutral with a large amount of distilled water, and resin regeneration is complete, can be recycled.
High purity MBTI prepared by above method carries out thermal stability analysis under differing temps, and acquired results as shown in Figure 3.The MBTI of purifying is 100
oc heating still had 85 % activity after 1 hour, still had the activity of 55 % after 2 hours, showed that it is to thermally-stabilised.
High purity MBTI prepared by above method or partially purified MBTI add in the gefillte fish taking fresh water silver carp as 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 muscle, MBSP) activity, makes the gel-strength of goods increase by 10%.
Embodiment 3
The separation purification method of a kind of mung bean trypsin (Mung bean trypsin inhibitor, MBTI) of the present embodiment, comprises the steps:
1, extracting: mung bean dry 500 g is milled to powder, and adding 3000 ml concentration is the sulfuric acid of 0.075 mol/L, stirs 36 h, after tissue mashing 30 min under 12000 g centrifugal 20 min, collect supernatant liquor approximately 1250 mL, be MBTI crude extract.
2, neutralization: add 1.2 mol/L NaOH in 1250 mL crude extracts, be neutral to the pH of solution.
3, thermal treatment: the MBTI crude extract after neutralization is heated to 30 min in 80 DEG C of water-baths, cooling immediately after heating, at centrifugal 15 min of 12000 g, collect supernatant liquor.
4, film processing: by 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.The external liquid desalination and concentration by ultrafiltration of ultra-filtration membrane of further holding back with 5 kDa molecular weight adds 20 mmol/L Tris-HCl simultaneously in ultrafiltrated, adjusts 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 arrives baseline by the absorbancy that 0.02 mol/L Tris-HCl damping fluid (pH 8.0) fully rinses under pillar to 280 nm.
6, desorb: the resin after absorption is respectively through 0.02 mol/L Tris-HCl (pH 8.0) with containing the 0.02 mol/L Tris-HCl(pH 8.0 of different concns NaCl) elutriant drip washing desorb.NaCl concentration is followed successively by respectively 0.1,0.2,0.3,0.4 and 0.5 mol/L, 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 desorb, resin is first used 2 mol/L NaCl solution drip washing 4 hours, then resin is pulled down is soaked 0.5 hour in 0.1 mol/L NaOH solution, then cleans resin near neutral with a large amount of distilled water, and resin regeneration is complete, can be recycled.
High purity MBTI prepared by above method or partially purified MBTI add to taking the blue circle of sea water fish Scad 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 muscle, MBSP) activity, make the gel-strength of goods increase by 21%, result as shown in Figure 4.
Embodiment 4
The separation purification method of a kind of mung bean trypsin (Mung bean trypsin inhibitor, MBTI) of the present embodiment, comprises the steps:
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, fully tissue mashing, and centrifugal 15 min under 10000 g, collect supernatant liquor approximately 870 mL, are MBTI crude extract.
2, neutralization: add 2 mol/L NaOH in 870 mL crude extracts, be neutral to the pH of solution.
3, thermal treatment: the 90 ° of C in water-bath of the MBTI crude extract after neutralization are processed 15 minutes, be placed in frozen water coolingly immediately, centrifugal 15 min under 10000 g, collect supernatant liquor.
4, film processing: by above-mentioned ultra-filtration membrane ultrafiltration of holding back with 30 kDa molecular weight through the MBTI of heat treated gained supernatant liquor, hold back large molecular weight protein, target protein MBTI(approximately 9 kDa of molecular weight) flow out to outer liquid.The external liquid desalination and concentration by ultrafiltration of ultra-filtration membrane of further holding back with 5 kDa molecular weight, adds in right amount 20 mmol/L phosphoric acid buffers (pH 8.0) to guarantee that desalination is complete simultaneously in ultrafiltrated.
5, fractionation by adsorption: the MBTI albumen mother liquor after desalination is pumped in the Q-Sepharose anion-exchange column (1.5 × 10 cm) by 0.02 mol/L phosphoric acid buffer (pH 8.0) balance, and coutroi velocity is 1 mL/min.
6, desorb: the absorbancy under abundant drip washing pillar to 280 nm of 0.02 mol/L phosphoric acid buffer for the resin after absorption (pH 8.0) is to baseline.Resin after absorption mixes the gradient eluent desorb of composition with 200 mL 0.02 mol/L phosphoric acid buffers (pH=8.0) and 200 mL 0.02 mol/L phosphoric acid buffers (pH 8.0 is containing 0.4 mol/L NaCl), collect elutriant (3 mL/ pipe), obtain high purity mung bean trypsin (MBTI).
7, resin regeneration: after desorb, resin is first used 2 mol/L NaCl solution drip washing 4 hours, then resin is pulled down is soaked 0.5 hour in 0.1 mol/L NaOH solution, then cleans resin near neutral with a large amount of distilled water, and resin regeneration is complete, can be recycled.
High purity MBTI prepared by above method or partially purified MBTI add to taking the blue circle of sea water fish Scad 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 muscle, MBSP) activity, makes the gel-strength of goods increase by 21%.
Embodiment 5
The separation purification method of a kind of mung bean trypsin (Mung bean trypsin inhibitor, MBTI) of the present embodiment, comprises the steps:
1, extracting: mung bean dry 500 g is milled to powder, and adding 2000 ml concentration is the sulfuric acid of 0.1 mol/L, stirs 24 h, tissue mashing 30 min; Continue to stir 10 hours, then tissue mashing after 10 minutes under 12000 g centrifugal 15 min, collect supernatant liquor approximately 900 mL, be MBTI crude extract.
2, neutralization: add 1 mol/L NaOH in 900 mL crude extracts, be neutral to the pH of solution.
3, thermal treatment: the MBTI crude extract after neutralization is heated to 90 min in 60 DEG C of water-baths, cooling immediately after heating, at centrifugal 12 min of 12000 g, collect supernatant liquor.
4, film processing: by 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.The external liquid desalination and concentration by ultrafiltration of ultra-filtration membrane of further holding back with 5 kDa molecular weight, 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 resin by the MBTI concentrated solution after step 4 desalination.Resin after absorption arrives baseline by the absorbancy under abundant drip washing to 280 nm of 0.02 mol/L Tris-HCl damping fluid (pH 8.0).
6, desorb: 200 mL 0.02 mol/L Tris-HCl (pH 8.0) and 200 mL 0.02 mol/L Tris-HCl(pH 8.0 for the resin after absorption, containing 0.5 mol/L NaCl) mix the linear gradient elution liquid wash-out of composition, collect elutriant (2 mL/ pipe), can obtain high purity mung bean trypsin (MBTI).
7, resin regeneration: after desorb, resin is first used 2 mol/L NaCl solution drip washing 4 hours, then resin is pulled down is soaked 0.5 hour in 0.1 mol/L NaOH solution, then cleans resin near neutral with a large amount of distilled water, and resin regeneration is complete, can be recycled.
High purity MBTI prepared by above method or partially purified MBTI add in the gefillte fish taking fresh water silver carp as 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 muscle, MBSP) activity, makes the gel-strength of goods increase by 14.5%.
Embodiment 6
The separation purification method of a kind of mung bean trypsin (Mung bean trypsin inhibitor, MBTI) of the present embodiment, comprises the steps:
1, extract: by Powder Phaseoli radiati dry 1000 g, adding 4000 ml concentration is the sulfuric acid of 0.05 mol/L, stir 24 h, be divided into 5 parts, every part of tissue mashing 30 min.Centrifugal 15 min under 12000 g, collect supernatant liquor approximately 1750 mL, are MBTI crude extract.
2, neutralization: add 1.5 mol/L NaOH in 1750 mL crude extracts, be neutral to the pH of solution.
3, thermal treatment: the MBTI crude extract after neutralization is heated to 30 min in 80 ° of C of water-bath, and cooling immediately after heating, centrifugal 15 min of 10000 g, collect supernatant liquor.
4, film processing: process the ultra-filtration membrane ultrafiltration that the MBTI supernatant liquors of gained are held back with 30 kDa molecular weight by above-mentioned 3, hold back macromole, collect and contain target protein MBTI(approximately 9 kDa) outer liquid.The external liquid desalination and concentration by ultrafiltration of ultra-filtration membrane of further holding back with 5 kDa molecular weight, 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 to the DEAE-Sepharose Fast Flow anion-exchange column (2.5 × 18 cm) by 0.02 mol/L phosphoric acid buffer (pH 7.5) balance, flow velocity 2 mL/min.After absorption, with 0.02 mol/L phosphoric acid buffer (pH=7.5) fully the absorbancy under drip washing pillar to 280 nm to baseline.
6, desorb: 400 mL 0.02 mol/L phosphoric acid buffers (pH 7.5) and 400 mL 0.02 mol/L phosphoric acid buffer (pH 7.5 for the resin after absorption, containing 0.5 mol/L NaCl) the linear gradient elution lyolysis of composition inhales, collects elutriant (4 mL/ pipe) and obtain high purity mung bean trypsin (MBTI).
7, resin regeneration: after desorb, resin is first used 2 mol/L NaCl solution drip washing 4 hours, then resin is pulled down is soaked 0.5 hour in 0.1 mol/L NaOH solution, then cleans resin near neutral with a large amount of distilled water, and resin regeneration is complete, can be recycled.
High purity MBTI prepared by above method or partially purified MBTI add to taking the blue circle of sea water fish Scad 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 muscle, MBSP) activity, makes the gel-strength of end article increase by 17.6%.
Above are only specific embodiments of the invention, but design concept of the present invention is not limited to this, allly utilizes this design to carry out the change of unsubstantiality to the present invention, all should belong to the behavior of invading protection domain of the present invention.
Claims (8)
1. the separation purification method of mung bean trypsin, is characterized in that step is as follows:
The first step, extracting: 500g mung bean is milled to powder, and adding 2000ml concentration is the sulfuric acid of 0.1mol/L, stirs extracting 22h, fully tissue mashing, centrifugal 15min under 10000g, collects the about 870mL of supernatant liquor, is MBTI crude extract;
Second step, neutralization: add 2mol/LNaOH in 870mL crude extract, be neutral to the pH of solution;
The 3rd step, thermal treatment: the 90 ° of C in water-bath of the MBTI crude extract after neutralization are processed 15 minutes, be placed in frozen water coolingly immediately, centrifugal 15min under 10000g, collects supernatant liquor;
The 4th step, film processing: by above-mentioned ultra-filtration membrane ultrafiltration of holding back with 30kDa molecular weight through the MBTI of heat treated gained supernatant liquor, hold back large molecular weight protein, the target protein MBTI of 9kDa molecular weight flows out to outer liquid; The external liquid desalination and concentration by ultrafiltration of ultra-filtration membrane of further holding back with 5kDa molecular weight, adds in right amount pH8.0,20mmol/L phosphoric acid buffer to guarantee that desalination is complete simultaneously in ultrafiltrated;
The 5th step, fractionation by adsorption: the MBTI albumen mother liquor after desalination is pumped in the Q-Sepharose anion-exchange column by pH8.0,0.02mol/L phosphoric acid buffer balance, and coutroi velocity is 1mL/min; The size of described anion-exchange column is 1.5 × 10cm;
The 6th step, desorb: pH8.0, the abundant drip washing pillar of 0.02mol/L phosphoric acid buffer to the absorbancy under 280nm for resin after absorption arrive baseline; 200mL, pH8.0,0.02mol/L phosphoric acid buffer and 200mL, pH8.0,0.02mol/L phosphoric acid buffer containing 0.4mol/L NaCl for resin after absorption mix the gradient eluent desorb forming, collect elutriant according to 3mL/ pipe, obtain high purity mung bean trypsin;
The 7th step, resin regeneration: after desorb, resin is first used 2mol/L NaCl solution drip washing 4 hours, again resin is pulled down, in 0.1mol/L NaOH solution, soaked 0.5 hour, then clean resin near neutral with a large amount of distilled water, resin regeneration is complete, can be recycled.
2. a mung bean trypsin as claimed in claim 1 application in gefillte fish is produced, it is characterized in that: mung bean trypsin claimed in claim 1 is added to taking the blue circle of sea water fish Scad in the gefillte fish of main raw material, make its final concentration reach 15 μ g/kg, stir.
3. the separation purification method of mung bean trypsin, is characterized in that step is as follows:
The first step, extracting: mung bean dry 500g is milled to powder, and adding 3000ml concentration is the sulfuric acid of 0.075mol/L, stirs 36h, after tissue mashing 30min under 12000g centrifugal 20min, collect supernatant liquor about 1250mL, be MBTI crude extract;
Second step, neutralization: add 1.2mol/L NaOH in 1250mL crude extract, be neutral to the pH of solution;
The 3rd step, thermal treatment: the MBTI crude extract after neutralization is heated to 30min in 80 DEG C of water-baths, cooling immediately after heating, at the centrifugal 15min of 12000g, collect supernatant liquor;
The 4th step, film processing: by above-mentioned ultra-filtration membrane ultrafiltration of holding back with 30kDa molecular weight through the MBTI of heat treated gained supernatant liquor, collect outer liquid; The external liquid desalination and concentration by ultrafiltration of ultra-filtration membrane of further holding back with 5kDa molecular weight adds 20mmol/L Tris-HCl simultaneously in ultrafiltrated, adjusts the pH to 7.5 of solution;
The 5th step, fractionation by adsorption: the MBTI concentrated solution after step 4 desalination is pumped in the adsorption column of DEAE-Sepharose Fast Flow anionite-exchange resin, flow velocity 1.5mL/min, the Tris-HCl damping fluid of pH0.8,0.02mol/L for resin after absorption fully rinses pillar and arrives baseline to the absorbancy under 280nm, and the size of the adsorption column of described anionite-exchange resin is 2.5 × 12cm;
The 6th step, desorb: the resin after absorption is respectively through pH8.0,0.02mol/L Tris-HCl with containing pH8.0, the 0.02mol/L Tris-HCl elutriant drip washing desorb of different concns NaCl; NaCl concentration is followed successively by respectively 0.1,0.2,0.3,0.4 and 0.5mol/L, and each concentration is drip washing 80mL respectively; Collect elutriant according to 2mL/ pipe and obtain high purity mung bean trypsin;
The 7th step, resin regeneration: after desorb, resin is first used 2mol/L NaCl solution drip washing 4 hours, again resin is pulled down, in 0.1mol/L NaOH solution, soaked 0.5 hour, then clean resin near neutral with a large amount of distilled water, resin regeneration is complete, can be recycled.
4. a mung bean trypsin as claimed in claim 3 application in gefillte fish is produced, it is characterized in that: mung bean trypsin claimed in claim 3 is added to taking the blue circle of sea water fish Scad in the gefillte fish of main raw material, make its final concentration reach 50 μ g/kg, stir.
5. the separation purification method of mung bean trypsin, is characterized in that step is as follows:
The first step, extracting: mung bean dry 500g is milled to powder, and adding 2000ml concentration is the sulfuric acid of 0.1mol/L, stirs 24h, tissue mashing 30min; Continue to stir 10 hours, then tissue mashing after 10 minutes under 12000g centrifugal 15min, collect supernatant liquor about 900mL, be MBTI crude extract;
Second step, neutralization: add 1mol/L NaOH in 900mL crude extract, be neutral to the pH of solution;
The 3rd step, thermal treatment: the MBTI crude extract after neutralization is heated to 90min in 60 DEG C of water-baths, cooling immediately after heating, at the centrifugal 12min of 12000g, collect supernatant liquor;
The 4th step, film processing: by above-mentioned ultra-filtration membrane ultrafiltration of holding back with 30kDa molecular weight through the MBTI of heat treated gained supernatant liquor, collect outer liquid; The external liquid desalination and concentration by ultrafiltration of ultra-filtration membrane of further holding back with 5kDa molecular weight, adds 0.02mol/L Tris-HCl simultaneously in ultrafiltrated, and pH8.0, makes the final pH of ultrafiltrated reach 8.0;
The 5th step, fractionation by adsorption: through Q-Sepharose Fast Flow anionite-exchange resin fractionation by adsorption, MBTI is adsorbed on resin by the MBTI concentrated solution after step 4 desalination; PH8.0, the abundant drip washing of 0.02mol/L Tris-HCl damping fluid to the absorbancy under 280nm for resin after absorption arrive baseline; The size of described anionite-exchange resin is 2.5 × 8cm;
The 6th step, desorb: pH8.0, the 200mL 0.02mol/L Tris-HCl for resin after absorption and the linear gradient elution liquid wash-out forming containing pH8.0, the 200mL 0.02mol/L Tris-HCl mixing of 0.5mol/L NaCl, collect elutriant according to 2mL/ pipe, can obtain high purity mung bean trypsin;
The 7th step, resin regeneration: after desorb, resin is first used 2mol/L NaCl solution drip washing 4 hours, again resin is pulled down, in 0.1mol/L NaOH solution, soaked 0.5 hour, then clean resin near neutral with a large amount of distilled water, resin regeneration is complete, can be recycled.
6. a mung bean trypsin as claimed in claim 5 application in gefillte fish is produced, it is characterized in that: mung bean trypsin claimed in claim 5 is added to taking the blue circle of sea water fish Scad in the gefillte fish of main raw material, make its final concentration reach 25 μ g/kg, stir.
7. the separation purification method of mung bean trypsin, is characterized in that step is as follows:
The first step, extracts: by Powder Phaseoli radiati dry 1000g, adding 4000ml concentration is the sulfuric acid of 0.05mol/L, stirs 24h, is divided into 5 parts, every part of tissue mashing 30min; Centrifugal 15min under 12000g, collects the about 1750mL of supernatant liquor, is MBTI crude extract;
Second step, neutralization: add 1.5mol/L NaOH in 1750mL crude extract, be neutral to the pH of solution;
The 3rd step, thermal treatment: the MBTI crude extract after neutralization is heated to 30min in 80 ° of C of water-bath, and cooling immediately after heating, the centrifugal 15min of 10000g, collects supernatant liquor;
The 4th step, film processing: the ultra-filtration membrane ultrafiltration that the MBTI supernatant liquor of step 3 gained is held back with 30kDa molecular weight, hold back macromole, collect the outer liquid containing the target protein MBTI of 9kDa molecular weight; The external liquid desalination and concentration by ultrafiltration of ultra-filtration membrane of further holding back with 5kDa molecular weight, adds pH7.5,0.02mol/L phosphoric acid buffer simultaneously in ultrafiltrated, makes desalination complete, and volume is about 300mL;
The 5th step, fractionation by adsorption: the MBTI concentrated solution after step 4 desalination is splined on to the DEAE-Sepharose Fast Flow anion-exchange column by pH7.5,0.02mol/L phosphoric acid buffer balance, flow velocity 2mL/min; After absorption, use pH7.5, the abundant drip washing pillar of 0.02mol/L phosphoric acid buffer to the absorbancy under 280nm to arrive baseline; The size of described anion-exchange column is 2.5 × 18cm;
The 6th step, desorb: 400mL, pH7.5 for resin, the 0.02mol/L phosphoric acid buffer after absorption and inhaling containing 400mL, the pH7.5 of 0.5mol/L NaCl, the linear gradient elution lyolysis of 0.02mol/L phosphoric acid buffer composition, collect elutriant according to 4mL/ pipe and obtain high purity mung bean trypsin;
The 7th step, resin regeneration: after desorb, resin is first used 2mol/L NaCl solution drip washing 4 hours, again resin is pulled down, in 0.1mol/L NaOH solution, soaked 0.5 hour, then clean resin near neutral with a large amount of distilled water, resin regeneration is complete, can be recycled.
8. a mung bean trypsin as claimed in claim 7 application in gefillte fish is produced, it is characterized in that: mung bean trypsin claimed in claim 7 is added to taking the blue circle of sea water fish Scad in the gefillte fish of main raw material, make its final concentration reach 30 μ g/kg, stir.
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Non-Patent Citations (10)
| Title |
|---|
| Extraction, purification and properties of trypsin inhibitor from Thai mung bean (Vigna radiata (L.) R. Wilczek);Sappasith Klomklao et al;《Food Chemistry》;20110511;1348–1354 * |
| 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》.1993, |
| 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.》.2010, |
| Mung Bean Trypsin Inhibitor Is Effective in Suppressing the Degradation of Myofibrillar Proteins in the Skeletal Muscle of Blue Scad (Decapterus maruadsi);LE-CHANG SUN et al;《J. Agric. Food Chem.》;20101119;12986–12992 * |
| Purification, characterization and evaluation of insecticidal potential of trypsin inhibitor from mungbean (Vigna radiata L. Wilczek) seeds;Rekha Kansal et al;《Acta Physiol Plant》;20080522;761–768 * |
| Rekha Kansal et al.Purification, characterization and evaluation of insecticidal potential of trypsin inhibitor from mungbean (Vigna radiata L. Wilczek) seeds.《Acta Physiol Plant》.2008, |
| Sappasith Klomklao et al.Extraction, purification and properties of trypsin inhibitor from Thai mung bean (Vigna radiata (L.) R. Wilczek).《Food Chemistry》.2011, |
| The 0.25-nm X-ray structure of the Bowman-Birk-type inhibitor from mung bean in ternary complex with porcine trypsin;Guangda Lin et al;《Eur. J. Biochem》;19931231;549-555 * |
| 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》.2007,第28卷(第3期), |
| 绿豆胰蛋白酶抑制剂的分离和纯化;刘同祥等;《中国生化药物杂志》;20071231;第28卷(第3期);145-148 * |
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