CN103627767B - A kind of method preparing Rice Bran Polypeptides from rice bran high temperature meal - Google Patents
A kind of method preparing Rice Bran Polypeptides from rice bran high temperature meal Download PDFInfo
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Abstract
The present invention relates to a kind of method preparing Rice Bran Polypeptides from rice bran high temperature meal, step is as follows: (1) alkali is carried: rice bran high temperature meal being added water according to feed liquid mass ratio 1:10 ~ 12 stirs, regulate pH to 11 ~ 11.5,2h is carried in 50 DEG C of alkali, centrifugal supernatant liquor, supernatant liquor pH value is adjusted to 4.6, leaves standstill 1h, and recentrifuge obtains rice bran protein; (2) primary enzymolysis: rice bran protein adds suitable quantity of water and stirs, regulate pH to 6.8 ~ 7.0, be heated to 50 DEG C all dissolve to rice bran protein, add appropriate flavor protease in 50 ~ 55 DEG C of enzymolysis 2.5 ~ 3h, enzymolysis solution pH value 6.8 ~ 7.0 is maintained, the enzyme that goes out centrifugal acquisition primary enzymolysis supernatant liquor in enzymolysis process; (3) secondary enzymolysis: primary enzymolysis supernatant liquor adjust pH, to alkalescence, adds Sumizyme MP in 50 ~ 60 DEG C of enzymolysis 2.5 ~ 3h, maintains enzymolysis solution pH value in alkalescence, the enzyme that goes out centrifugal acquisition secondary enzymolysis supernatant liquor in enzymolysis process; (4) concentrated and dry.Rice Bran Polypeptides rich amino acids prepared by the method, extraction yield is high, and foreign matter content is low, and solubleness is high.
Description
Technical field
The present invention relates to a kind of method preparing polypeptide, relate to a kind of method preparing Rice Bran Polypeptides from rice bran high temperature meal specifically.
Background technology
Rice bran high temperature meal is that rice bran carries the product after oil through high temperature, because being subject to temperatures involved, the content of its soluble proteins reduces, result in the reduction of its extraction yield, and impurity is more in the albumen extracted, namely protein content is not high, in addition, the albumen solubility extracted is not high, and can the solubleness of protein affect one of its important factor be used widely in food.For making full use of rice bran high temperature meal resource, further improving rice bran protein solvability, strengthening its functional performance, widen its application in food, rice bran high temperature meal should be utilized to prepare Rice Bran Polypeptides.
Summary of the invention
The object of the invention is to solve prior art, from rice bran high temperature meal, prepare the method extraction yield of Rice Bran Polypeptides low, and the deficiency that polypeptide impurities is many and solubleness is not high extracted, a kind of method preparing Rice Bran Polypeptides from rice bran high temperature meal is newly provided.The method utilizes 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:
From rice bran high temperature meal, prepare a method for Rice Bran Polypeptides, described preparation method comprises the following steps:
(1) alkali is carried: rice bran high temperature meal being added water according to feed liquid mass ratio 1:10 ~ 12 stirs, and regulates pH to 11 ~ 11.5, carries 2h in 50 DEG C of alkali, then centrifugal supernatant liquor, described supernatant liquor pH value is adjusted to 4.6, leaves standstill 1h, recentrifuge, removes supernatant liquor and obtains rice bran protein;
(2) primary enzymolysis: the rice bran protein that step (1) obtains is added suitable quantity of water and stirs, regulate pH to 6.8 ~ 7.0, be heated to 50 DEG C all dissolve to rice bran protein, add appropriate flavor protease in 50 ~ 55 DEG C of enzymolysis 2.5 ~ 3h, enzymolysis solution pH value 6.8 ~ 7.0 is maintained in enzymolysis process, go out after enzymolysis terminates enzyme, and then centrifugal segregation precipitation, obtains primary enzymolysis supernatant liquor;
(3) secondary enzymolysis: by the primary enzymolysis supernatant liquor adjust pH of step (2) to alkalescence, add Sumizyme MP in 50 ~ 60 DEG C of enzymolysis 2.5 ~ 3h, maintain enzymolysis solution pH value in enzymolysis process in alkalescence, go out after enzymolysis completes enzyme, then centrifugal segregation precipitation, obtains secondary enzymolysis supernatant liquor;
(4) concentrated and dry: after the secondary enzymolysis supernatant liquor that step (3) obtains is carried out desalting treatment, then through vacuum concentration postlyophilization, to obtain rice bran protein polypeptide.
As preferably, in step (2), rice bran protein (in butt) with the mass ratio of 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.
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 DEG C, and pH is 9.4, and enzymolysis time is 3.0h.PH is on the impact of enzyme reaction, main manifestations is on the impact at enzyme activity, peracid or excessively alkali all can affect its activity, contriver through experimental study find when secondary enzymolysis pH value from 8 to 9.5 time, enzymolysis efficiency is gradually high, increase pH value again, its enzymolysis efficiency reduces on the contrary, the significant enzymic activity of this surface alkalinty proteolytic enzyme only occurs in very narrow pH value range, the protein of Sumizyme MP and substrate is all containing the group that dissociates, when only having these groups that dissociate to be in specific dissociated state, Sumizyme MP and substrate protein just combine 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 the best dissociated state combining and be converted into product, the best secondary enzymolysis pH value that the present invention determines is 9.4, now the enzymolysis efficiency of Sumizyme MP to rice bran protein substrate is the highest.
As preferably, in step (2) primary enzymolysis, enzyme-removal temperature is 90 DEG C, 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 DEG C 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, and then prepare Rice Bran Polypeptides further by double-enzyme method enzymolysis rice bran protein, technological design is reasonable, reaction conditions is gentle, there is enzymolysis efficiency high, Rice Bran Polypeptides productive rate be high, advantage that production cost is low, be easy to realize industrialization.
(2) the present invention's Rice Bran Polypeptides rich amino acids of utilizing double-enzyme method to prepare, containing 17 seed amino acids, removing 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) molecular weight of the present invention's Rice Bran Polypeptides of utilizing double-enzyme method to prepare mainly concentrates between 180Da ~ 500Da, accounts for about 50.38% of whole Rice Bran Polypeptides; Polypeptide between molecular weight 500Da ~ 1000Da accounts for about 21.25% of whole Rice Bran Polypeptides, and the molecular weight of Rice Bran Polypeptides is little, and absorption rate is high, further enhances the nutritive value of Rice Bran Polypeptides.
Embodiment
Below by specific embodiment, be described in further detail technical scheme of the present invention, the plant and instrument that each embodiment uses is all commercial conventional equipment, and the reagent that each embodiment uses 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), the molecular weight distribution of the Rice Bran Polypeptides of preparation is measured: chromatographic column selects TSKgel2000SWXL300mm × 7.8mm, moving phase: acetonitrile: water: trifluoroacetic acid, 45:55:0.1(V/V), determined wavelength: 220nm, flow velocity: 0.5mL/min; Column temperature: 30 DEG C, sampling volume: 10 μ l.With cytochrome C (Mw12500); Bacillus enzyme (Mw1450); Glycocoll-glycocoll-Tyr-Arg (Mw451); Glycocoll-glycocoll-glycocoll (Mw189) is molecular weight references.
(1) drafting of molecular mass standard curve
With the logarithm of above-mentioned 4 standard protein molecular masses for ordinate zou, retention time is the lgMw mobility figure of X-coordinate, i.e. molecular mass standard curve, and sets up equation of linear regression.
(2) Rice Bran Polypeptides molecular weight distribution determination
Adopt HPLC measure its molecular weight distribution, condition determination and sample preparation the same.The mass profile binding molecule amount typical curve GPC software determined is analyzed, with the peak area percent in different molecular weight ranges for the per-cent in this range of molecular weight distributions shared by polypeptide.
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 uses: the protein content 79.56% of rice bran high temperature meal, moisture content 7.55%, ash oontent 1.79%.
Embodiment 1:
From rice bran high temperature meal, prepare a method for Rice Bran Polypeptides, described preparation method comprises the following steps:
(1) alkali is carried: rice bran high temperature meal being added water according to feed liquid mass ratio 1:10 stirs, regulate pH to 11,2h is carried in 50 DEG C of alkali, then centrifugal supernatant liquor, centrifugal rotating speed is 4000rpm, and centrifugation time is 25min, described supernatant liquor pH value is adjusted to 4.6, leave standstill 1h, again with the centrifugal 25min of the rotating speed of 4000rpm, remove supernatant liquor and obtain rice bran protein;
(2) primary enzymolysis: the rice bran protein that step (1) obtains is added suitable quantity of water and stirs, the massfraction of rice bran protein is made to be 33.3%, regulate pH to 6.8, be heated to 50 DEG C all dissolve to rice bran protein, add the flavor protease of rice bran protein quality 0.5% in 50 DEG C of enzymolysis 3h, enzymolysis solution pH value 6.8 is maintained in enzymolysis process, in 90 DEG C of enzyme 10min that go out after enzymolysis terminates, then centrifugal segregation precipitation, 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 DEG C of enzymolysis 3h, enzymolysis solution pH value 9.0 is maintained in enzymolysis process, in 85 DEG C of enzyme 10min that go out after enzymolysis completes, then under rotating speed is 4000rpm condition, centrifugal 10min removes precipitation, obtains secondary enzymolysis supernatant liquor;
(4) concentrated and dry: the secondary enzymolysis supernatant liquor that step (3) obtains to be crossed after anion-cation exchange resin carries out desalting treatment, then through vacuum concentration postlyophilization, obtain rice bran protein polypeptide.
Embodiment 2:
From rice bran high temperature meal, prepare a method for Rice Bran Polypeptides, described preparation method comprises the following steps:
(1) alkali is carried: rice bran high temperature meal being added water according to feed liquid mass ratio 1:12 stirs, regulate pH to 11.5,2h is carried in 50 DEG C of alkali, then centrifugal supernatant liquor, centrifugal rotating speed is 4500rpm, and centrifugation time is 20min, described supernatant liquor pH value is adjusted to 4.65, leave standstill 1h, again with the centrifugal 20min of the rotating speed of 4500rpm, remove supernatant liquor and obtain rice bran protein;
(2) primary enzymolysis: the rice bran protein that step (1) obtains is added suitable quantity of water and stirs, the massfraction of rice bran protein is made to be 33.3%, regulate pH to 7.0, be heated to 50 DEG C all dissolve to rice bran protein, add the flavor protease of rice bran protein quality 0.5% in 55 DEG C of enzymolysis 2.5h, enzymolysis solution pH value 7.0 is maintained in enzymolysis process, in 90 DEG C of enzyme 10min that go out after enzymolysis terminates, then centrifugal segregation precipitation, 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 DEG C of enzymolysis 2.5h, enzymolysis solution pH value 10.0 is maintained in enzymolysis process, in 85 DEG C of enzyme 10min that go out after enzymolysis completes, then under rotating speed is 4500rpm condition, centrifugal 15min removes precipitation, obtains secondary enzymolysis supernatant liquor;
(4) concentrated and dry: the secondary enzymolysis supernatant liquor that step (3) obtains to be crossed after anion-cation exchange resin carries out desalting treatment, then through vacuum concentration postlyophilization, obtain rice bran protein polypeptide.
Embodiment 3:
From rice bran high temperature meal, prepare a method for Rice Bran Polypeptides, described preparation method comprises the following steps:
(1) alkali is carried: rice bran high temperature meal being added water according to feed liquid mass ratio 1:11 stirs, regulate pH to 11.2,2h is carried in 50 DEG C of alkali, then centrifugal supernatant liquor, centrifugal rotating speed is 4200rpm, and centrifugation time is 22min, described supernatant liquor pH value is adjusted to 4.62, leave standstill 1h, again with the centrifugal 21min of the rotating speed of 4200rpm, remove supernatant liquor and obtain rice bran protein;
(2) primary enzymolysis: the rice bran protein that step (1) obtains is added suitable quantity of water and stirs, the massfraction of rice bran protein is made to be 33.3%, regulate pH to 6.9, be heated to 50 DEG C all dissolve to rice bran protein, add the flavor protease of rice bran protein quality 0.5% in 52 DEG C of enzymolysis 3h, enzymolysis solution pH value 6.9 is maintained in enzymolysis process, in 90 DEG C of enzyme 10min that go out after enzymolysis terminates, then centrifugal segregation precipitation, 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 DEG C of enzymolysis 3h, enzymolysis solution pH value 9.4 is maintained in enzymolysis process, in 85 DEG C of enzyme 10min that go out after enzymolysis completes, then under rotating speed is 4300rpm condition, centrifugal 12min removes precipitation, obtains secondary enzymolysis supernatant liquor;
(4) concentrated and dry: the secondary enzymolysis supernatant liquor that step (3) obtains to be crossed after anion-cation exchange resin carries out desalting treatment, then through vacuum concentration postlyophilization, obtain rice bran protein polypeptide.
The molecular weight distribution of the Rice Bran Polypeptides that embodiment 1 ~ 3 is obtained is in table 1.
The molecular weight distribution of table 1 Rice Bran Polypeptides
Show that the molecular weight of the Rice Bran Polypeptides that the present invention utilizes double-enzyme method to prepare mainly is concentrated between 180Da ~ 500Da by table 1, account 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, further enhances the nutritive value of Rice Bran Polypeptides.
Below with the obtained Rice Bran Polypeptides of embodiment 3 for analytic target, analyze its amino acid composition and content, the results are shown in Table 2, the quality of the Rice Bran Polypeptides simultaneously aminoacids content of Rice Bran Polypeptides obtained for embodiment 3 and WHO recommended contrasts, and the results are shown in Table 3.
The amino acid composition of the Rice Bran Polypeptides that table 2 embodiment 3 is obtained and content
The Rice Bran Polypeptides aminoacids content that table 3 embodiment 2 is obtained and WHO recommend pattern contrast
The Rice Bran Polypeptides that table 2 and table 3 item obtain after showing to utilize flavor protease and Sumizyme MP double-enzyme hydrolysis, its amino acid classes is very abundant, containing 17 seed amino acids; Removing 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 classes of the Rice Bran Polypeptides prepared by double-enzyme method and content recommend pattern close to WHO, and nutritive value is higher.
Above-described embodiment is one of the present invention preferably scheme, not does any pro forma restriction to the present invention, also has other variant and remodeling under the prerequisite not exceeding the technical scheme described in claim.
Claims (5)
1. from rice bran high temperature meal, prepare a method for Rice Bran Polypeptides, it is characterized in that, described preparation method comprises the following steps:
(1) alkali is carried: rice bran high temperature meal being added water according to feed liquid mass ratio 1:10 ~ 12 stirs, and regulates pH to 11 ~ 11.5, carries 2h in 50 DEG C of alkali, then centrifugal supernatant liquor, described supernatant liquor pH value is adjusted to 4.6, leaves standstill 1h, recentrifuge, removes supernatant liquor and obtains rice bran protein;
(2) primary enzymolysis: the rice bran protein that step (1) obtains is added suitable quantity of water and stirs, regulate pH to 6.8 ~ 7.0, be heated to 50 DEG C all dissolve to rice bran protein, add appropriate flavor protease in 50 ~ 55 DEG C of enzymolysis 2.5 ~ 3h, enzymolysis solution pH value 6.8 ~ 7.0 is maintained in enzymolysis process, go out after enzymolysis terminates enzyme, and then centrifugal segregation precipitation, obtains primary enzymolysis supernatant liquor;
(3) secondary enzymolysis: by the primary enzymolysis supernatant liquor adjust pH of step (2) to alkalescence, add Sumizyme MP in 50 ~ 60 DEG C of enzymolysis 2.5 ~ 3h, enzymolysis solution pH value is maintained in alkalescence in enzymolysis process, go out after enzymolysis completes enzyme, then centrifugal segregation precipitation, obtains secondary enzymolysis supernatant liquor;
(4) concentrated and dry: after the secondary enzymolysis supernatant liquor that step (3) obtains is carried out desalting treatment, then through vacuum concentration postlyophilization, to obtain rice bran protein polypeptide;
In step (2), the mass ratio of rice bran protein and water is: 1:2, wherein the quality of rice bran protein is in butt, and the addition of flavor protease is 0.5% of rice bran protein butt quality, and the activity of flavor protease is 20000U/g.
2. a kind of method preparing Rice Bran Polypeptides from rice bran high temperature meal 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.
3. a kind of method preparing Rice Bran Polypeptides from rice bran high temperature meal according to claim 2, is characterized in that: in step (3), the temperature of secondary enzymolysis is 54 DEG C, and pH is 9.4, enzyme concentration is 0.28%, and enzymolysis time is 3.0h.
4. a kind of method preparing Rice Bran Polypeptides from rice bran high temperature meal according to claim 1, it is characterized in that: in step (2) primary enzymolysis, enzyme-removal temperature is 90 DEG C, time is 10min, and centrifugal rotating speed is 4000 ~ 4500rpm, and centrifugation time is 20 ~ 25min.
5. a kind of method preparing Rice Bran Polypeptides from rice bran high temperature meal according to claim 1, it is characterized in that: in step (3) secondary enzymolysis, enzyme-removal temperature is 85 DEG C, time is 10min, and centrifugal rotating speed is 4000 ~ 4500rpm, and centrifugation time is 10 ~ 15min.
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| CN104073540B (en) * | 2014-07-05 | 2016-08-24 | 广东润科生物工程有限公司 | A kind of production process of polypeptide improving protein utilization |
| CN105063149B (en) * | 2015-09-01 | 2018-10-23 | 武汉轻工大学 | A kind of method that enzyme process auxiliary prepares cottonseed protein |
| CN108823274A (en) * | 2018-07-13 | 2018-11-16 | 贵州大学 | A method of preparing adlay Rice Bran Polypeptides from adlay rice bran |
| 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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| CN102030773A (en) * | 2010-10-27 | 2011-04-27 | 江南大学 | Technique for coproducing phytic acid and oligopeptide from defatted rice bran |
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Inventor after: He Dongping Inventor after: Liu Jingwei Inventor after: Hu Chuanrong Inventor after: Zhang Sihong Inventor after: Liu Lingyi Inventor before: He Dongping Inventor before: Yao Xingquan Inventor before: Hu Chuanrong Inventor before: Yao Xingjiang Inventor before: Zhang Shihong Inventor before: Zou Li Inventor before: You Mengyuan Inventor before: Wu Jianbao Inventor before: Liu Jingwei |
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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. |