CN103627767A - Method for preparing rice bran polypeptide from high temperature rice bran meal - Google Patents
Method for preparing rice bran polypeptide from high temperature rice bran meal Download PDFInfo
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Abstract
The invention relates to a method for preparing rice bran polypeptide from high temperature rice bran meal. The method comprises the following steps: (1) alkaline extraction: the high temperature rice bran meal is added with water for uniform stirring according to a feed liquid mass ratio of 1 to (10-12), the pH value is adjusted to be 11 to 11.5, alkaline extraction is performed for 2 hours at the temperature of 50 DEG C, a supernatant fluid is obtained through centrifugation, the pH value of the supernatant fluid is adjusted to be 4.6, standing for 1 hour is performed, and then rice bran protein is obtained through centrifugation again; (2), first enzymolysis: the rice bran protein is added with appropriate water for stirring uniformly, the pH value is adjusted to be 6.8 to 7.0, heating is performed so as to reach a temperature of 50 DEG C until the rice bran protein is completely dissolved, appropriate flavourzyme is added at the temperature of 50 to 55 DEG C for enzymolysis for 2.5 to 3 hours, the pH value is maintained to be 6.8 to 7.0 during the enzymolysis process, and one-time enzymolysis supernatant fluid is obtained through enzyme deactivation centrifugation; (3), secondary enzymolysis: the pH value of the first enzymolysis supernatant fluid is adjusted to be alkaline, alkaline protease is added at the temperature of 50 to 60 DEG C for enzymolysis for 2.5 to 3 hours, the pH value of the enzymatic hydrolysate is maintained to be alkaline during the enzymolysis process, and secondary enzymolysis supernatant fluid is obtained through enzyme deactivation centrifugation; (4), concentration and drying are performed. The rice bran polypeptide prepared by the method provided by the invention has rich amino acid species, is high in extraction ratio, low in impurity content and high in solvability.
Description
Technical field
The present invention relates to a kind of method of preparing polypeptide, relate to specifically a kind of method of preparing Rice Bran Polypeptides from the rice bran high temperature dregs of rice.
Background technology
The rice bran high temperature dregs of rice are rice bran products after high temperature is carried oil, because being subject to temperatures involved, the content of its soluble proteins reduces, caused reducing of its extraction yield, and in the albumen extracting, impurity is more, and protein content is not high, in addition, the albumen solubility extracting is not high, and can the solubleness of protein affect one of its important factor that be used widely in food.For making full use of rice bran high temperature dregs of rice resource, further improve rice bran protein solvability, strengthen its functional performance, widen its application in food, should utilize the rice bran high temperature dregs of rice to prepare Rice Bran Polypeptides.
Summary of the invention
The object of the invention is to solve prior art, from the rice bran high temperature dregs of rice, to prepare the method extraction yield of Rice Bran Polypeptides low, and the polypeptide impurity extracting is many and the not high deficiency of solubleness, and a kind of new method of Rice Bran Polypeptides of preparing from the rice bran high temperature dregs of rice is provided.The method is utilized flavor protease and Sumizyme MP fractional hydrolysis, the Rice Bran Polypeptides of Preparation of amino acid abundant species and comprehensive nutrition, and extraction yield is high, and in the Rice Bran Polypeptides of acquisition, foreign matter content is low, and solubleness is high, can be widely used in field of food.
The technical solution adopted for the present invention to solve the technical problems is:
A method of preparing Rice Bran Polypeptides from the rice bran high temperature dregs of rice, described preparation method comprises the following steps:
(1) alkali is carried: the rice bran high temperature dregs of rice are added to water according to feed liquid mass ratio 1:10～12 and stir, regulate pH to 11～11.5, in 50 ℃ of alkali, carry 2h, then centrifugal supernatant liquor, described supernatant liquor pH value is adjusted to 4.6, standing 1h, recentrifuge, removes supernatant liquor and obtains rice bran protein;
(2) primary enzymolysis: the rice bran protein that step (1) is obtained adds suitable quantity of water to stir, regulate pH to 6.8～7.0, being heated to 50 ℃ all dissolves to rice bran protein, add appropriate flavor protease in 50～55 ℃ of enzymolysis 2.5～3h, in enzymolysis process, maintain enzymolysis solution pH value 6.8～7.0, the enzyme that goes out after enzymolysis finishes, then centrifugal removal precipitation, obtains primary enzymolysis supernatant liquor;
(3) secondary enzymolysis: the primary enzymolysis supernatant liquor adjust pH of step (2) is extremely alkaline, add Sumizyme MP in 50～60 ℃ of enzymolysis 2.5～3h, maintain enzymolysis solution pH value and be alkalescence in enzymolysis process, enzyme goes out after enzymolysis completes, then centrifugal removal precipitation, obtains secondary enzymolysis supernatant liquor;
(4) concentrated and dry: the secondary enzymolysis supernatant liquor that step (3) is obtained carries out, after desalting treatment, then passing through vacuum concentration postlyophilization, obtains rice bran protein polypeptide.
As preferably, the mass ratio of rice bran protein in step (2) (take butt) and water is 1:2, and the addition of flavor protease is 0.5% of rice bran protein butt quality, and the activity of flavor protease is 20000U/g.
As preferably, the addition of step (3) secondary enzymolysis neutral and alkali proteolytic enzyme is 0.28% of rice bran protein butt quality, and the activity of Sumizyme MP is 200,000 U/g.
More preferably, in step (3), the temperature of secondary enzymolysis is 54 ℃, and pH is 9.4, and enzymolysis time is 3.0h.The impact of pH on enzyme reaction, main manifestations is on the impact at enzyme activity, peracid or excessively alkali all can affect its activity, contriver finds when the pH of the secondary enzymolysis value from 8 to 9.5 through experimental study, enzymolysis efficiency is gradually high, increase again pH value, its enzymolysis efficiency reduces on the contrary, the significant enzymic activity of this surface alkalinty proteolytic enzyme only occurs within the scope of very narrow pH value, the protein of Sumizyme MP and substrate all contains the group that dissociates, only have these to dissociate group when specific dissociated state, Sumizyme MP and substrate protein are just in conjunction with the fastest, generate the fastest of product, the pH value of system directly affects the dissociated state of some group that dissociates of enzyme and substrate protein white matter molecule, only under specific pH value condition, the group that dissociates of enzyme and substrate protein white matter is just in combination be converted into the best dissociated state of product, the best secondary enzymolysis pH value that the present invention determines is 9.4, now Sumizyme MP is the highest to the enzymolysis efficiency of rice bran protein substrate.
As preferably, in step (2) primary enzymolysis, enzyme-removal temperature is 90 ℃, and the time is 10min, and centrifugal rotating speed is 4000～4500rpm, and centrifugation time is 20～25min.
As preferably, in step (3) secondary enzymolysis, enzyme-removal temperature is 85, and ℃ time is 10min, and centrifugal rotating speed is 4000～4500rpm, and centrifugation time is 10～15min.
The invention has the beneficial effects as follows:
(1) the present invention first obtains rice bran protein by alkali extraction and acid precipitation, then further by double-enzyme method enzymolysis rice bran protein, prepares Rice Bran Polypeptides, and technological design is reasonable, reaction conditions is gentle, have enzymolysis efficiency high, the advantage that Rice Bran Polypeptides productive rate is high, production cost is low, is easy to realize industrialization.
(2) the present invention utilizes Rice Bran Polypeptides rich amino acids prepared by double-enzyme method, contain 17 seed amino acids, remove tryptophane (destroyed during detection), also contain indispensable amino acid in other 7, and its first limiting amino acids lysine content is fine, account for 7.2% of total amino acid content, methionine(Met) and cysteine content also account for 6.7% of total amino acid content, and nutritive value is very abundant.
(3) the present invention utilizes the molecular weight of Rice Bran Polypeptides prepared by double-enzyme method mainly to concentrate between 180Da～500Da, accounts for 50.38% left and right of whole Rice Bran Polypeptides; Polypeptide between molecular weight 500Da～1000Da accounts for 21.25% left and right of whole Rice Bran Polypeptides, and the molecular weight of Rice Bran Polypeptides is little, and absorption rate is high, has further strengthened the nutritive value of Rice Bran Polypeptides.
Embodiment
Below by specific embodiment, technical scheme of the present invention is described in further detail, the plant and instrument that each embodiment is used is all commercial conventional equipment, the reagent that each embodiment is used is all commercial conventional reagent.
Sumizyme MP enzyme is lived: 200,000 U/g; Flavor protease enzyme is lived: 20000U/g.
Rice Bran Polypeptides molecular weight determination: adopt Waters600 high performance liquid chromatograph (joining 2487 UV-detector, Empower workstation and GPC software), molecular weight distribution to the Rice Bran Polypeptides of preparation is measured: chromatographic column is selected TSKgel2000SWXL300mm * 7.8mm, moving phase: acetonitrile: water: trifluoroacetic acid, 45:55:0.1(V/V), detect wavelength: 220nm, flow velocity: 0.5mL/min; Column temperature: 30 ℃, sampling volume: 10 μ l.With cytochrome C (Mw12500); Bacillus enzyme (Mw1450); Glycocoll-glycocoll-tyrosine-arginine (Mw451); Glycocoll-glycocoll-glycocoll (Mw189) is molecular weight object of reference.
(1) drafting of molecular weight standard curve
The logarithm of above-mentioned 4 standard protein molecular masses of take is ordinate zou, the lgMw mobility figure that retention time is X-coordinate, i.e. and molecular weight standard curve, and set up equation of linear regression.
(2) Rice Bran Polypeptides molecular weight distribution determination
Adopt HPLC to measure its molecular weight distribution, condition determination and sample preparation are the same.The molecular mass distribution plan binding molecule amount typical curve determining is analyzed with GPC software, peak area per-cent shared per-cent of polypeptide in this range of molecular weight distributions of take in different molecular weight ranges.
Rice Bran Polypeptides Contents of Amino Acids: test center of oil crops institute of Chinese agriculture Research Center.
The raw material that embodiment 1～3 is used: the protein content 79.56% of the rice bran high temperature dregs of rice, moisture content 7.55%, ash oontent 1.79%.
Embodiment 1:
A method of preparing Rice Bran Polypeptides from the rice bran high temperature dregs of rice, described preparation method comprises the following steps:
(1) alkali is carried: the rice bran high temperature dregs of rice are added to water according to feed liquid mass ratio 1:10 and stir, regulate pH to 11, in 50 ℃ of alkali, carry 2h, then centrifugal supernatant liquor, centrifugal rotating speed is 4000rpm, centrifugation time is 25min, described supernatant liquor pH value is adjusted to 4.6, standing 1h, again with the centrifugal 25min of rotating speed of 4000rpm, removes supernatant liquor and obtains rice bran protein;
(2) primary enzymolysis: the rice bran protein that step (1) is obtained adds suitable quantity of water to stir, the massfraction that makes rice bran protein is 33.3%, regulate pH to 6.8, being heated to 50 ℃ all dissolves to rice bran protein, add the flavor protease of rice bran protein quality 0.5% in 50 ℃ of enzymolysis 3h, in enzymolysis process, maintain enzymolysis solution pH value 6.8, after enzymolysis finishes in 90 ℃ of enzyme 10min that go out, then centrifugal removal precipitates, obtain primary enzymolysis supernatant liquor, centrifugal rotating speed is 4000rpm, and centrifugation time is 25min;
(3) secondary enzymolysis: by the primary enzymolysis supernatant liquor adjust pH to 9.0 of step (2), add the Sumizyme MP of rice bran protein quality 0.2% in 50 ℃ of enzymolysis 3h, in enzymolysis process, maintain enzymolysis solution pH value 9.0, after enzymolysis completes in 85 ℃ of enzyme 10min that go out, then at rotating speed, be that under 4000rpm condition, centrifugal 10min removes precipitation, obtain secondary enzymolysis supernatant liquor;
(4) concentrated and dry: the secondary enzymolysis supernatant liquor that step (3) is obtained is crossed anion-cation exchange resin and carried out, after desalting treatment, then passing through vacuum concentration postlyophilization, obtains rice bran protein polypeptide.
Embodiment 2:
A method of preparing Rice Bran Polypeptides from the rice bran high temperature dregs of rice, described preparation method comprises the following steps:
(1) alkali is carried: the rice bran high temperature dregs of rice are added to water according to feed liquid mass ratio 1:12 and stir, regulate pH to 11.5, in 50 ℃ of alkali, carry 2h, then centrifugal supernatant liquor, centrifugal rotating speed is 4500rpm, centrifugation time is 20min, described supernatant liquor pH value is adjusted to 4.65, standing 1h, again with the centrifugal 20min of rotating speed of 4500rpm, removes supernatant liquor and obtains rice bran protein;
(2) primary enzymolysis: the rice bran protein that step (1) is obtained adds suitable quantity of water to stir, the massfraction that makes rice bran protein is 33.3%, regulate pH to 7.0, being heated to 50 ℃ all dissolves to rice bran protein, add the flavor protease of rice bran protein quality 0.5% in 55 ℃ of enzymolysis 2.5h, in enzymolysis process, maintain enzymolysis solution pH value 7.0, after enzymolysis finishes in 90 ℃ of enzyme 10min that go out, then centrifugal removal precipitates, obtain primary enzymolysis supernatant liquor, centrifugal rotating speed is 4500rpm, and centrifugation time is 20min;
(3) secondary enzymolysis: by the primary enzymolysis supernatant liquor adjust pH to 10.0 of step (2), add the Sumizyme MP of rice bran protein quality 0.3% in 60 ℃ of enzymolysis 2.5h, in enzymolysis process, maintain enzymolysis solution pH value 10.0, after enzymolysis completes in 85 ℃ of enzyme 10min that go out, then at rotating speed, be that under 4500rpm condition, centrifugal 15min removes precipitation, obtain secondary enzymolysis supernatant liquor;
(4) concentrated and dry: the secondary enzymolysis supernatant liquor that step (3) is obtained is crossed anion-cation exchange resin and carried out, after desalting treatment, then passing through vacuum concentration postlyophilization, obtains rice bran protein polypeptide.
Embodiment 3:
A method of preparing Rice Bran Polypeptides from the rice bran high temperature dregs of rice, described preparation method comprises the following steps:
(1) alkali is carried: the rice bran high temperature dregs of rice are added to water according to feed liquid mass ratio 1:11 and stir, regulate pH to 11.2, in 50 ℃ of alkali, carry 2h, then centrifugal supernatant liquor, centrifugal rotating speed is 4200rpm, centrifugation time is 22min, described supernatant liquor pH value is adjusted to 4.62, standing 1h, again with the centrifugal 21min of rotating speed of 4200rpm, removes supernatant liquor and obtains rice bran protein;
(2) primary enzymolysis: the rice bran protein that step (1) is obtained adds suitable quantity of water to stir, the massfraction that makes rice bran protein is 33.3%, regulate pH to 6.9, being heated to 50 ℃ all dissolves to rice bran protein, add the flavor protease of rice bran protein quality 0.5% in 52 ℃ of enzymolysis 3h, in enzymolysis process, maintain enzymolysis solution pH value 6.9, after enzymolysis finishes in 90 ℃ of enzyme 10min that go out, then centrifugal removal precipitates, obtain primary enzymolysis supernatant liquor, centrifugal rotating speed is 4200rpm, and centrifugation time is 25min;
(3) secondary enzymolysis: by the primary enzymolysis supernatant liquor adjust pH to 9.4 of step (2), add the Sumizyme MP of rice bran protein quality 0.28% in 54 ℃ of enzymolysis 3h, in enzymolysis process, maintain enzymolysis solution pH value 9.4, after enzymolysis completes in 85 ℃ of enzyme 10min that go out, then at rotating speed, be that under 4300rpm condition, centrifugal 12min removes precipitation, obtain secondary enzymolysis supernatant liquor;
(4) concentrated and dry: the secondary enzymolysis supernatant liquor that step (3) is obtained is crossed anion-cation exchange resin and carried out, after desalting treatment, then passing through vacuum concentration postlyophilization, obtains rice bran protein polypeptide.
The molecular weight distribution of the Rice Bran Polypeptides that embodiment 1～3 makes is in Table 1.
The molecular weight distribution of table 1 Rice Bran Polypeptides
By table 1, show that the present invention utilizes the molecular weight of Rice Bran Polypeptides prepared by double-enzyme method mainly to concentrate between 180Da～500Da, accounts for 50.17～51.2% of whole Rice Bran Polypeptides; Polypeptide between molecular weight 500Da～1000Da accounts for 20.38～21.25% of whole Rice Bran Polypeptides, what molecular weight was less than 180Da accounts for 20.01～20.93% of whole Rice Bran Polypeptides, the molecular weight of Rice Bran Polypeptides prepared by the present invention is little, absorption rate is high, has further strengthened the nutritive value of Rice Bran Polypeptides.
The Rice Bran Polypeptides that the embodiment 3 of take below makes is analytic target, analyzes its amino acid and forms and content, the results are shown in Table 2, and the quality of the Rice Bran Polypeptides that the aminoacids content of the Rice Bran Polypeptides simultaneously embodiment 3 being made and WHO recommend contrasts, and the results are shown in Table 3.
The amino acid of the Rice Bran Polypeptides that table 2 embodiment 3 makes forms and content
The Rice Bran Polypeptides aminoacids content that table 3 embodiment 2 makes and the contrast of WHO recommendation pattern
Table 2 and table 3 item show to utilize the Rice Bran Polypeptides obtaining after flavor protease and Sumizyme MP double-enzyme hydrolysis, and its amino acid kind is very abundant, contains 17 seed amino acids; Remove tryptophane (destroyed during detection), also contain indispensable amino acid in other 7, and its first limiting amino acids lysine content is fine, account for 7.2% of total amino acid content, methionine(Met) and halfcystine also account for 6.7% of total amino acid content, its amino acid kind of the Rice Bran Polypeptides of preparing by double-enzyme method and content approach WHO and recommend pattern, and nutritive value is higher.
Above-described embodiment is a kind of preferably scheme of the present invention, not the present invention is done to any pro forma restriction, also has other variant and remodeling under the prerequisite that does not exceed the technical scheme that claim records.
Claims (6)
1. from the rice bran high temperature dregs of rice, prepare a method for Rice Bran Polypeptides, it is characterized in that, described preparation method comprises the following steps:
(1) alkali is carried: the rice bran high temperature dregs of rice are added to water according to feed liquid mass ratio 1:10～12 and stir, regulate pH to 11～11.5, in 50 ℃ of alkali, carry 2h, then centrifugal supernatant liquor, described supernatant liquor pH value is adjusted to 4.6, standing 1h, recentrifuge, removes supernatant liquor and obtains rice bran protein;
(2) primary enzymolysis: the rice bran protein that step (1) is obtained adds suitable quantity of water to stir, regulate pH to 6.8～7.0, being heated to 50 ℃ all dissolves to rice bran protein, add appropriate flavor protease in 50～55 ℃ of enzymolysis 2.5～3h, in enzymolysis process, maintain enzymolysis solution pH value 6.8～7.0, the enzyme that goes out after enzymolysis finishes, then centrifugal removal precipitation, obtains primary enzymolysis supernatant liquor;
(3) secondary enzymolysis: the primary enzymolysis supernatant liquor adjust pH of step (2) is extremely alkaline, add Sumizyme MP in 50～60 ℃ of enzymolysis 2.5～3h, maintain enzymolysis solution pH value and be alkalescence in enzymolysis process, enzyme goes out after enzymolysis completes, then centrifugal removal precipitation, obtains secondary enzymolysis supernatant liquor;
(4) concentrated and dry: the secondary enzymolysis supernatant liquor that step (3) is obtained carries out, after desalting treatment, then passing through vacuum concentration postlyophilization, obtains rice bran protein polypeptide.
2. a kind of method of preparing Rice Bran Polypeptides from the rice bran high temperature dregs of rice according to claim 1, it is characterized in that: the mass ratio of rice bran protein in step (2) (take butt) and water is 1:2, the addition of flavor protease is 0.5% of rice bran protein butt quality, and the activity of flavor protease is 20000U/g.
3. a kind of method of preparing Rice Bran Polypeptides from the rice bran high temperature dregs of rice according to claim 1, it is characterized in that: the addition of step (3) secondary enzymolysis neutral and alkali proteolytic enzyme is 0.2～0.3% of rice bran protein butt quality, and the activity of Sumizyme MP is 200,000 U/g.
4. a kind of method of preparing Rice Bran Polypeptides from the rice bran high temperature dregs of rice according to claim 3, is characterized in that: in step (3), the temperature of secondary enzymolysis is 54 ℃, and pH is 9.4, enzyme concentration is 0.28%, and enzymolysis time is 3.0h.
5. a kind of method of preparing Rice Bran Polypeptides from the rice bran high temperature dregs of rice according to claim 1, it is characterized in that: in step (2) primary enzymolysis, enzyme-removal temperature is 90 ℃, time is 10min, and centrifugal rotating speed is 4000～4500rpm, and centrifugation time is 20～25min.
6. a kind of method of preparing Rice Bran Polypeptides from the rice bran high temperature dregs of rice according to claim 1, it is characterized in that: in step (3) secondary enzymolysis, enzyme-removal temperature is 85 ℃, time is 10min, and centrifugal rotating speed is 4000～4500rpm, and centrifugation time is 10～15min.
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| CN104073540A (en) * | 2014-07-05 | 2014-10-01 | 广东润科生物工程有限公司 | Polypeptide production method for enhancing utilization ratio of protein |
| CN105063149A (en) * | 2015-09-01 | 2015-11-18 | 武汉轻工大学 | Method for auxiliary preparation of cottonseed protein by means of enzymatic method |
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| CN111440837A (en) * | 2020-04-01 | 2020-07-24 | 沈阳农业大学 | High F value oligopeptide and preparation method thereof |
| CN112262954A (en) * | 2020-10-27 | 2021-01-26 | 山西大学 | Functional wheat self-raising flour rich in samara polypeptide and preparation method thereof |
| CN113981029A (en) * | 2021-11-26 | 2022-01-28 | 南昌大学 | Method for efficiently preparing micromolecular rice residue peptide |
| CN115669794A (en) * | 2022-11-01 | 2023-02-03 | 河南工业大学 | Method for improving solubility of rice bran protein |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104073540A (en) * | 2014-07-05 | 2014-10-01 | 广东润科生物工程有限公司 | Polypeptide production method for enhancing utilization ratio of protein |
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| CN111440837A (en) * | 2020-04-01 | 2020-07-24 | 沈阳农业大学 | High F value oligopeptide and preparation method thereof |
| CN112262954A (en) * | 2020-10-27 | 2021-01-26 | 山西大学 | Functional wheat self-raising flour rich in samara polypeptide and preparation method thereof |
| CN113981029A (en) * | 2021-11-26 | 2022-01-28 | 南昌大学 | Method for efficiently preparing micromolecular rice residue peptide |
| CN115669794A (en) * | 2022-11-01 | 2023-02-03 | 河南工业大学 | Method for improving solubility of rice bran protein |
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Effective date of registration: 20170527 Address after: 324000 Wujiang East Road, Shen Shen Economic Development Zone, Qujiang District, Zhejiang, Quzhou Co-patentee after: Wuhan Polytechnic University Patentee after: Quzhou Liu Jia Fragrant Food Co., Ltd. Address before: 324000 Wujiang East Road, Shen Shen Economic Development Zone, Qujiang District, Zhejiang, Quzhou Patentee before: Quzhou Liu Jia Fragrant Food Co., Ltd. |