CN102174627A - Method for preparing rapeseed bioactive peptide - Google Patents
Method for preparing rapeseed bioactive peptide Download PDFInfo
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Abstract
The invention provides a method for preparing a rapeseed bioactive peptide, comprising the following steps: taking degreased rapeseed dregs as a raw material, preparing a rapeseed protein by an alkali dissolving and acid precipitation method; adding protease hydrolyzed proteins; carrying out activated carbon decoloration, ultrafiltration grading, and cation-anion exchange resin desalination in sequence; and finally concentrating, spray drying or freeze drying to obtain the rapeseed bioactive peptide.
Description
Technical field
The invention belongs to biological technical field, be specifically related to utilize rapeseed meal to be raw material, adopt protease hydrolysis technology, prepare the method for vegetable seed biologically active peptides.
Background technology
Contain 20%～25% protein in the vegetable seed, contain 32%～40% protein in the rapeseed meal after the degreasing.Rape seed protein mainly is made up of the 12S albumen of high molecular weight and the 2S albumen of low relative molecular mass, rape seed protein is a kind of high-quality protein, its amino acid is formed and is met FAO/WHO recommendation mode value, the PER value is higher than other vegetable-protein, even being higher than casein, maximum characteristics are that the content of sulfur-containing amino acid and Methionin is higher.Rape seed protein is except having preferably nutritive property, and its functional other albumen, particularly oil absorbency, water-retentivity and emulsifying property of also being better than is better than other albumen.The enzymic hydrolysis of rape seed protein is a kind of incomplete, halfway hydrolysis, and its product mainly is a biologically active peptides.The vegetable seed biologically active peptides is except the advantage with rape seed protein, and it is easier of little intestinal absorption than rape seed protein and amino acid also to have, and has more physiologically active than vegetable seed protein and amino acid.
The preparation technology of existing vegetable seed biologically active peptides mainly contains three kinds: (1) gene engineering method; (2) chemical synthesis process; (3) enzymolysis process.Wherein, enzymolysis process has less investment, and is simple to operate, the product content advantages of higher.Existing utilize enzymolysis process to prepare in the technology of rapeseed peptide (rsp), all add the potential of hydrogen of a large amount of alkali lye or acid solution solution when regulating enzyme digestion reaction in the enzymolysis process, and do not have desalting treatment in the technology, make finished product salinity too high levels; And, contain a large amount of pigment compositions in the enzymolysis solution, influence the color and luster of finished product.In addition, because the difference of enzymatic hydrolysis condition, the molecular weight size distribution of enzymolysis after product is inhomogeneous, has influenced the physiologically active of product.
Summary of the invention
The objective of the invention is for provide a kind of with rapeseed meal as raw material, prepare the method for vegetable seed biologically active peptides.
For achieving the above object, the preparation method of vegetable seed biologically active peptides provided by the invention: with the degreasing rapeseed meal is raw material, earlier be equipped with rape seed protein with the alkali extraction and acid precipitation legal system, then add proteolytic enzyme protolysate matter, pass through activated carbon decolorizing, ultrafiltration classification, anion-cation exchange resin desalination successively, after concentrate, spraying drying or lyophilize obtain the vegetable seed biologically active peptides.
The method for preparing the vegetable seed biologically active peptides of the present invention comprises the steps:
A kind of preparation method of vegetable seed biologically active peptides, this method comprises the steps:
(1) degreasing rapeseed meal and water are dissolved in the water by 1: 5～1: 15 (mass ratio), pulverize, regulate pH to 9.0～13.0, do not stop to stir 15min, 3000～4000rpm, 15min, centrifuging and taking supernatant liquor with NaOH reagent with colloidal mill;
(2) supernatant liquor with step 1 is heated to 70～90 ℃, with lemon acid for adjusting pH value to 3.0～5.0,40～60 ℃ of temperature, stirring velocity 30～50r/min makes protein precipitation 15～45min, then 3000r/min centrifugation 5～15min, remove supernatant liquid, collect solid phase, add the water mixing by above step again, make protein at isoelectric precipitation, triplicate, wash twice at last, centrifugal solid phase promptly gets and is rape seed protein;
(3) rape seed protein and the water with step 2 is dissolved in the water by 1: 5～1: 15 (mass ratio), be heated to 80～95 ℃, keep 15～30min, be cooled to 35～50 ℃ then, regulating and keep pH 6.0～8.0 with NaOH reagent, is 3.0%～5.0% to add neutral protease As1.398, enzymolysis 2～4h by the E/S ratio, use the adjusting of NaOH reagent in the enzymolysis process and keep pH, constantly stir 6.0～8.0;
(4) treat that enzymolysis finishes after, with lemon acid for adjusting pH value to 3.0～5.0,3000～4000rpm, 15min, the centrifuging and taking supernatant liquor is the rough liquid of rapeseed peptide (rsp);
(5) account for the rape seed protein quality than the gac that is 3%～5% in the supernatant liquor adding, stir 2～3h, gac is removed with vacuum filtration with agitator;
(6) the rapeseed peptide (rsp) destainer with step 5 gained injects storage tank, solution is the 1000Da ultra-filtration membrane by the molecular weight interception behind micro-filtration, small-molecule substance in the solution infiltrates through the inwall of hollow-fibre membrane becomes ultrafiltrated, collects ultrafiltrated, is the rapeseed peptide (rsp) ultrafiltrated; Macromolecular substance such as the albumen in the solution are kept and are concentrated, and concentrated solution is recycled in the step 3 enzymolysis once more;
(7) exchange resin immersion treatment in deionized water was adorned post after 24 hours, with 7.5% hydrochloric acid and 10%NaOH it was soaked respectively then, was converted into H
+Type Zeo-karb and OH
-Type anionite-exchange resin is 7.0 stand-by with washed with de-ionized water to pH again;
With the rapeseed peptide (rsp) ultrafiltrated of gained in the step 6, pass through H with the flow velocity of 5～10 times of column volume/h
+The type Zeo-karb removes positively charged ion, stops application of sample when treating the effluent liquid pH=4.0 left and right sides, afterwards this effluent liquid is passed through OH with same flow velocity
-Type anionite-exchange resin removes negatively charged ion, is pH to effluent liquid and stops application of sample at 7.0 o'clock, is collected as the rapeseed peptide (rsp) refined liquid;
(8) by the rapeseed peptide (rsp) refined liquid that obtains in the step 7, be evaporated to solid content 20%～30%;
(9) after purified rapeseed peptide (rsp) liquid concentrated, dry powder was made in spray-dried or lyophilize.
The present invention compared with prior art has following advantage and effect:
The purpose of above-mentioned steps (1) is: utilize protein dissolved characteristic under alkaline condition, rape seed protein and alkali insoluble substance are separated, to improve the content of rape seed protein.Simultaneously, contain anti-nutrition components such as sulphur glucoside, phytic acid in the rapeseed meal, these compositions dissolution rate under alkaline condition is very low, adopts alkali solution technique can effectively remove these compositions.
The heavy purpose of above-mentioned steps (2) acid is: utilize protein sedimentary characteristic under acid iso-electric point condition, rape seed protein and acid soluble material are separated, to improve the content of rape seed protein.
The purpose of above-mentioned steps (1), (2) is in order to remove the impurity component in the rape seed protein, to improve the content of rape seed protein, for enzymolysis is supplied raw materials.Proteic content is about 30% in the general rapeseed meal, and after handling through aforesaid method, protein content can reach more than 65%.
The purpose of above-mentioned steps (3) enzymolysis is: with the macro-molecular protein enzymolysis in the vegetable seed is small protein and peptide;
The purpose of above-mentioned steps (4) acid adjustment is in order to make enzyme deactivation, to stop enzyme digestion reaction; The centrifugal purpose is for solid-liquid separation, collects enzymolysis solution.
The purpose of activated carbon decolorizing is in order to utilize the characteristic of charcoal absorption pigment, to remove the pigment composition in the rapeseed peptide (rsp) in the above-mentioned steps (5).
The purpose of ultrafiltration is the product that needs molecular weight in order to intercept in the above-mentioned steps (6); The purpose of secondary enzymolysis is for the high molecular weight protein more than the molecular weight 1000Da is hydrolyzed into small molecules, and the molecular weight that makes the finished product is all below 1000Da.
The purpose of desalination is in order to remove in the enzymolysis solution because of constantly regulating the salt ion that pH brings in the enzymolysis process in the above-mentioned steps (7).The product salt content that does not pass through desalting treatment is greater than 8%, and the research and development of products content of process desalting treatment is less than 2%.
The present invention compared with prior art has following advantage and effect:
(1) adopts the alkali extraction and acid precipitation method to improve to be used for the content of the rape seed protein of enzymolysis, remove the anti-nutrition component of sulphur glucoside, phytic acid in the rapeseed meal simultaneously;
(2) utilize gac to slough pigment in the rapeseed peptide (rsp);
(3) adopt ultrafiltration process that rapeseed peptide (rsp) is carried out stage treatment, guarantee 100% rapeseed peptide (rsp) molecular weight below 1000Da, and carry out secondary enzymolysis for the rice bran peptide more than the molecular weight 1000Da, the molecular weight distribution that makes the finished product is all below 1000Da;
(4) utilize the negative and positive exchange resin that the rapeseed peptide (rsp) crude product is carried out desalination, the product salt content that does not pass through desalting treatment is greater than 8%, and the product salt content of process present method desalting treatment is less than 2%;
The rapeseed peptide (rsp) of present method preparation, its product is to comprise following characteristics:
(1) rapeseed peptide (rsp) of present method preparation is white or light green powder, the visible foreign matter of no naked eyes;
(2) rapeseed peptide (rsp) of present method preparation produces total nitrogen content (in butt) 〉=80.0%, peptide content (in butt) 〉=70.0%, the relative molecular mass of gained is all below 1000Da, moisture content≤5.0%, ash oontent (in butt)≤2.0%, phase lipid content (in butt)≤2.0%.
Description of drawings
The present invention will be further described in detail below in conjunction with the drawings and specific embodiments.
Fig. 1 is the preparation method's of vegetable seed biologically active peptides of the present invention process flow sheet.
Embodiment
E/S described in the present invention is than the mass ratio of consumption that is meant enzyme and rape seed protein; The enzymic activity of neutral protease As1.398 described in the present invention is 130000U/g; Ratio among the present invention all refers to mass ratio if no special instructions.
Embodiment 1
(1) extracting degreasing rapeseed meal 100g is dissolved in the 800mL water, pulverizes with colloidal mill, regulates pH to 10.0 with NaOH reagent, does not stop to stir 15min, 3000rpm, 15min, centrifuging and taking supernatant liquor;
(2) supernatant liquor with step 1 is heated to 90 ℃, with lemon acid for adjusting pH value to 3.5,45 ℃ of temperature, stirring velocity 45r/min makes protein precipitation 30min, then 3000r/min centrifugation 10min, remove supernatant liquid, collect solid phase, add the water mixing by above step again, with lemon acid for adjusting pH value to 3.5, make protein precipitation, triplicate, wash at last twice, centrifugal solid phase promptly gets and is rape seed protein, is weighed as 58g;
(3) with the precipitation of step 2 by pouring in the 600mL water, be heated to 90 ℃, keep 30min, be cooled to 50 ℃ then, add neutral protease As1.398 (enzymic activity is 130000U/g) 2.32g, use the adjusting of NaOH reagent and keep pH 7.0, enzymolysis 2h constantly stirs in the enzymolysis process;
(4) treat that enzymolysis finishes after, with lemon acid for adjusting pH value to 3.0, the centrifugal 15min of 3000rpm gets supernatant liquor after centrifugal, is the rough liquid of rapeseed peptide (rsp);
(5) add the 1.74g gac, stir 2h, gac is removed with vacuum filtration with agitator;
(6) the rough liquid of the rapeseed peptide (rsp) in the step 5 is injected storage tank, solution is the 1000Da ultra-filtration membrane by the molecular weight interception behind micro-filtration, small-molecule substance in the solution infiltrates through the inwall of hollow-fibre membrane becomes ultrafiltrated, collects ultrafiltrated, is the rapeseed peptide (rsp) ultrafiltrated; Macromolecular substance such as the albumen in the solution are kept and are concentrated, and concentrated solution is recycled to the hydrolysis of step 3 secondary;
(7) exchange resin immersion treatment in deionized water was adorned post after 24 hours, with 7.5% hydrochloric acid and 10%NaOH it was soaked respectively then, was converted into H
+Type Zeo-karb and OH
-Type anionite-exchange resin is 7.0 stand-by with washed with de-ionized water to pH again;
Rapeseed peptide (rsp) ultrafiltrated with gained in the step 6, flow velocity with 5 times of column volume/h removes positively charged ion by H+ type Zeo-karb, stop application of sample when treating the effluent liquid pH=4.0 left and right sides, afterwards this effluent liquid is removed negatively charged ion with same flow velocity by OH-type anionite-exchange resin, when effluent liquid is slightly acidic, stop application of sample, be collected as the rapeseed peptide (rsp) refined liquid;
(8) by the rapeseed peptide (rsp) refined liquid that obtains in the step 7, be evaporated to solid content 30%;
(9) after purified rapeseed peptide (rsp) liquid concentrated, dry powder was made in spray-dried or lyophilize, is weighed as 50.6g.
The rapeseed peptide (rsp) of present method preparation produces total nitrogen content (in butt) 87.6%, peptide content (in butt) 78.4%, the relative molecular mass of gained all below 1000Da, moisture content 8%, ash oontent (in butt) 1.8%, crude fat content (in butt) 1.5%;
Embodiment 2
(1) gets rapeseed meal 500g and be dissolved in the 5L water, pulverize, regulate pH to 12.0, do not stop to stir 15min, 3000rpm, 15min, centrifuging and taking supernatant liquor with NaOH reagent with colloidal mill;
(2) supernatant liquor with step 1 is heated to 90 ℃, with lemon acid for adjusting pH value to 4.2,50 ℃ of temperature, stirring velocity 30r/min makes protein precipitation 45min, then 3000r/min centrifugation 5min, remove supernatant liquid, collect solid phase, add the water mixing by above step again, with lemon acid for adjusting pH value to 4.2, make protein precipitation, triplicate, wash at last twice, centrifugal solid phase promptly gets and is rape seed protein, is weighed as 326g;
(3) precipitation of step 2 is poured in the 4L water, be heated to 95 ℃, keep 30min, be cooled to 45 ℃ then, add neutral protease As1.398 (enzymic activity is 130000U/g) 14.67g, use the adjusting of NaOH reagent and keep pH 7.5, enzymolysis 3h constantly stirs in the enzymolysis process;
(4) treat that enzymolysis finishes after, with lemon acid for adjusting pH value to 4.0, the centrifugal 15min of 3000rpm gets supernatant liquor after centrifugal, is the rough liquid of rapeseed peptide (rsp);
(5) supernatant liquor adds the 13.04g gac, stirs 2h with agitator, with vacuum filtration gac is removed;
(6) the rough liquid of the rapeseed peptide (rsp) in the step 5 is injected storage tank, solution is the 1000Da ultra-filtration membrane by the molecular weight interception behind micro-filtration, small-molecule substance in the solution infiltrates through the inwall of hollow-fibre membrane becomes ultrafiltrated, collects ultrafiltrated, is the rapeseed peptide (rsp) ultrafiltrated; Macromolecular substance such as the albumen in the solution are kept and are concentrated, and concentrated solution is recycled to the hydrolysis of step 3 secondary;
(7) exchange resin immersion treatment in deionized water was adorned post after 24 hours, with 7.5% hydrochloric acid and 10%NaOH it was soaked respectively then, was converted into H
+Type Zeo-karb and OH
-Type anionite-exchange resin is 7.0 stand-by with washed with de-ionized water to pH again;
Rapeseed peptide (rsp) ultrafiltrated with gained in the step 6, flow velocity with 10 times of column volume/h removes positively charged ion by H+ type Zeo-karb, stop application of sample when treating the effluent liquid pH=4.0 left and right sides, afterwards this effluent liquid is removed negatively charged ion with same flow velocity by OH-type anionite-exchange resin, when effluent liquid is slightly acidic, stop application of sample, be collected as the rapeseed peptide (rsp) refined liquid;
(8) by the rapeseed peptide (rsp) refined liquid that obtains in the step 8, be evaporated to solid content 30%;
(9) after purified rapeseed peptide (rsp) liquid concentrated, dry powder was made in spray-dried or lyophilize, is weighed as 231.7g.
The rapeseed peptide (rsp) of present method preparation produces total nitrogen content (in butt) 84.5%, peptide content (in butt) 75.1%, the relative molecular mass of gained all below 1000Da, moisture content 8.2%, ash oontent (in butt) 1.8%, crude fat content (in butt) 1.9%.
Embodiment 3
(1) gets rapeseed meal 1000g and be dissolved in the 12L water, pulverize, regulate pH to 9.0, do not stop to stir 15min, 3000rpm, 15min, centrifuging and taking supernatant liquor with NaOH reagent with colloidal mill;
(2) supernatant liquor with step 1 is heated to 90 ℃, with lemon acid for adjusting pH value to 4.5,40 ℃ of temperature, stirring velocity 40r/min makes protein precipitation 20min, then 3000r/min centrifugation 10min, remove supernatant liquid, collect solid phase, add the water mixing by above step again, make protein at isoelectric precipitation, triplicate washes twice at last, the centrifugal solid phase that gets, promptly get and be rape seed protein, be weighed as 589g;
(3) precipitation of step 2 is poured in the 6L water, be heated to 90 ℃, keep 30min, be cooled to 50 ℃ then, add neutral protease As1.398 (enzymic activity is 130000U/g) 29.45g, use the adjusting of NaOH reagent and keep pH 8.0, enzymolysis 3h constantly stirs in the enzymolysis process;
(4) treat that enzymolysis finishes after, with lemon acid for adjusting pH value to 4.2, the centrifugal 15min of 3000rpm gets supernatant liquor after centrifugal, is the rough liquid of rapeseed peptide (rsp);
(5) add the 29.45g gac at supernatant liquor, stir 2h, gac is removed with vacuum filtration with agitator;
(6) the rough liquid of the rapeseed peptide (rsp) in the step 5 is injected storage tank, be adjusted to a fixed flow velocity, solution is the 1000Da ultra-filtration membrane by the molecular weight interception behind micro-filtration, small-molecule substance in the solution infiltrates through the inwall of hollow-fibre membrane becomes ultrafiltrated, collect ultrafiltrated, be the rapeseed peptide (rsp) ultrafiltrated; Macromolecular substance such as the albumen in the solution are kept and are concentrated, and concentrated solution is recycled to the hydrolysis of step 3 secondary;
(7) exchange resin immersion treatment in deionized water was adorned post after 24 hours, with 7.5% hydrochloric acid and 10%NaOH it was soaked respectively then, was converted into H
+Type Zeo-karb and OH
-Type anionite-exchange resin is 7.0 stand-by with washed with de-ionized water to pH again;
Rapeseed peptide (rsp) ultrafiltrated with gained in the step 6, flow velocity with 10 times of column volume/h removes positively charged ion by H+ type Zeo-karb, stop application of sample when treating the effluent liquid pH=4.0 left and right sides, afterwards this effluent liquid is removed negatively charged ion with same flow velocity by OH-type anionite-exchange resin, when effluent liquid is slightly acidic, stop application of sample, be collected as the rapeseed peptide (rsp) refined liquid;
(8) by the rapeseed peptide (rsp) refined liquid that obtains in the step 8, be evaporated to solid content 30%;
(9) after purified rapeseed peptide (rsp) liquid concentrated, dry powder was made in spray-dried or lyophilize, is weighed as 503.9g.
The rapeseed peptide (rsp) of present method preparation produces total nitrogen content (in butt) 81.6%, peptide content (in butt) 72.9%, the relative molecular mass of gained all below 1000Da, moisture content 9.1%, ash oontent (in butt) 1.9%, crude fat content (in butt) 1.9%.
Claims (1)
1. the preparation method of a vegetable seed biologically active peptides, this method comprises the steps:
(1) degreasing rapeseed meal and water are dissolved in the water by 1: 5～1: 15 (mass ratio), pulverize, regulate pH to 9.0～13.0, do not stop to stir 15min, 3000～4000rpm, 15min, centrifuging and taking supernatant liquor with NaOH reagent with colloidal mill;
(2) supernatant liquor with step 1 is heated to 70～90 ℃, with lemon acid for adjusting pH value to 3.0～5.0,40～60 ℃ of temperature, stirring velocity 30～50r/min makes protein precipitation 15～45min, then 3000r/min centrifugation 5～15min, remove supernatant liquid, collect solid phase, add the water mixing by above step again, make protein at isoelectric precipitation, triplicate, wash twice at last, centrifugal solid phase promptly gets and is rape seed protein;
(3) rape seed protein and the water with step 2 is dissolved in the water by 1: 5～1: 15 (mass ratio), be heated to 80～95 ℃, keep 15～30min, be cooled to 35～50 ℃ then, regulating and keep pH 6.0～8.0 with NaOH reagent, is 3.0%～5.0% to add neutral protease As1.398, enzymolysis 2～4h by the E/S ratio, use the adjusting of NaOH reagent in the enzymolysis process and keep pH, constantly stir 6.0～8.0;
(4) treat that enzymolysis finishes after, with lemon acid for adjusting pH value to 3.0～5.0,3000～4000rpm, 15min, the centrifuging and taking supernatant liquor is the rough liquid of rapeseed peptide (rsp);
(5) account for the rape seed protein quality than the gac that is 3%～5% in the supernatant liquor adding, stir 2～3h, gac is removed with vacuum filtration with agitator;
(6) the rapeseed peptide (rsp) destainer with step 5 gained injects storage tank, solution is the 1000Da ultra-filtration membrane by the molecular weight interception behind micro-filtration, small-molecule substance in the solution infiltrates through the inwall of hollow-fibre membrane becomes ultrafiltrated, collects ultrafiltrated, is the rapeseed peptide (rsp) ultrafiltrated; Macromolecular substance such as the albumen in the solution are kept and are concentrated, and concentrated solution is recycled in the step 3 enzymolysis once more;
(7) exchange resin immersion treatment in deionized water was adorned post after 24 hours, with 7.5% hydrochloric acid and 10%NaOH it was soaked respectively then, was converted into H
+Type Zeo-karb and OH
-Type anionite-exchange resin is 7.0 stand-by with washed with de-ionized water to pH again;
With the rapeseed peptide (rsp) ultrafiltrated of gained in the step 6, pass through H with the flow velocity of 5～10 times of column volume/h
+The type Zeo-karb removes positively charged ion, stops application of sample when treating the effluent liquid pH=4.0 left and right sides, afterwards this effluent liquid is passed through OH with same flow velocity
-Type anionite-exchange resin removes negatively charged ion, is pH to effluent liquid and stops application of sample at 7.0 o'clock, is collected as the rapeseed peptide (rsp) refined liquid;
(7) by the rapeseed peptide (rsp) refined liquid that obtains in the step 7, be evaporated to solid content 20%～30%;
(9) after purified rapeseed peptide (rsp) liquid concentrated, dry powder was made in spray-dried or lyophilize.
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