CN113234786B - Method for extracting selenium-enriched polypeptide from selenium-enriched rice processing byproducts - Google Patents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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Abstract
The invention discloses a method for extracting selenium-enriched polypeptide from selenium-enriched rice processing byproducts, which belongs to the technical field of natural product extraction and specifically comprises the following steps: (1) Drying selenium-enriched rice bran, pulverizing, sieving, mixing with rice polishing powder, and homogenizing with water to obtain mixed solution; (2) Sieving the mixed solution, performing ultrasonic treatment, centrifuging, and collecting a solid precipitated substrate; (3) Adding water into the solid precipitate until the mass fraction of the solid in the mixture is 6-16%, and then carrying out primary homogenization and secondary homogenization to obtain a dispersion liquid; (4) Injecting the dispersion liquid into an immobilized enzymolysis tank for enzymolysis reaction to obtain a reaction liquid; (5) And (3) regulating the pH value of the reaction solution to be neutral, centrifuging, retaining supernatant, desalting the supernatant, and drying to obtain the selenium-enriched polypeptide. Compared with the traditional alkali extraction and acid precipitation method, the extraction recovery rate is high, the quality of the extracted selenoprotein is good, the oxidation resistance and the hypoglycemic activity are high, and the economic value effect is obvious.
Description
Technical Field
The invention relates to the technical field of natural product extraction, in particular to a method for extracting selenium-enriched polypeptide from selenium-enriched rice processing byproducts.
Background
Rice proteins are superior to other sources of proteins such as soy protein, potato protein, peanut protein, wheat protein, kidney bean protein, and peanut protein in terms of chemical properties, functional characteristics, and biological activity. The proteins in rice, especially rice bran, are considered as one of important vegetable proteins, have low allergy, high nutritive value, good functional properties, etc., and have become a protein resource with great potential in the food industry, and can be used as or as an important constituent of many products, such as infant foods, gluten-free products, and cosmetics. The rice is used as the crop with the highest proportion of the Guangxi selenium-rich crops (accounting for 17 percent of the yield of the selenium-rich crops), is not only one of key staple food sources of daily diet, but also a safe, economic and effective method for improving the selenium intake of residents in China by taking the extraction and processing of selenoprotein as raw materials to develop related selenium-enriched functional products.
Selenium is a trace element necessary for human body, and participates in synthesizing various selenase and selenoprotein in human body. In recent years, selenium-enriched foods are becoming more popular, and selenium-enriched rice is used as a crop with the highest proportion of Guangxi selenium-enriched crops (accounting for 17 percent of the yield of the selenium-enriched crops), and the processed rice is a key staple food source of daily diet. Earlier researches show that the selenium content in rice bran and polished rice powder which are by-products of selenium-enriched rice processing is higher than that in rice, for example, when the selenium content in rice is 150 mug/kg, the selenium content in rice bran is more than or equal to 220 mug/kg, and the selenium content in polished rice is more than or equal to 200 mug/kg. However, rice bran and the like are often discarded as byproducts in the rice processing process, and are not utilized well, so that great waste is caused, and therefore, a method capable of fully utilizing the organic selenium in the rice processing byproducts is needed to be found. The rice processing byproducts contain a large amount of selenoprotein, have good antioxidant and hypoglycemic activities, and can be extracted and processed and used as raw materials for developing related selenium-enriched functional products.
In the prior art, the protein extraction method has the advantages of multipurpose alkali extraction and acid precipitation, low efficiency and large organic selenium loss. Therefore, how to provide a method for reducing the loss rate of organic selenium with high protein extraction rate is a problem that needs to be solved by those skilled in the art.
Disclosure of Invention
In view of the above, the invention provides a method for extracting selenium-binding protein from rice processing byproducts, which has high extraction efficiency, high extraction recovery rate of selenium-rich polypeptide, good quality of obtained selenium protein and remarkable economic value effect.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
A method for extracting selenium-enriched polypeptide from selenium-enriched rice processing byproducts, comprising the following steps:
(1) Drying selenium-enriched rice bran, crushing and sieving to obtain selenium-enriched rice bran powder, mixing the selenium-enriched rice bran powder with rice polishing powder, adding clear water, and homogenizing to obtain mixed solution;
(2) Sieving the mixed solution, performing ultrasonic treatment, centrifuging, and collecting a solid precipitated substrate;
(3) Adding water into the solid precipitate until the solid mass fraction is 6-16%, homogenizing for the first time, sieving, heating to 60 ℃ and homogenizing for the second time to obtain a dispersion liquid;
(4) Injecting the dispersion liquid into an immobilized enzymolysis tank for enzymolysis reaction for 30-180 min to obtain a reaction liquid;
(5) And adding HCl into the reaction solution to adjust the pH value to be neutral, centrifuging, reserving supernatant, desalting the supernatant, and drying to obtain the selenium-enriched polypeptide.
The beneficial effects are that: the invention is suitable for industrial production, omits partial unnecessary steps such as extraction and concentration of protein, directly exposes the protein from plant cells by means of homogenization and the like from seeds, directly carries out enzymolysis, and other components which cannot be hydrolyzed by protease, such as cellulose hemicellulose, starch and the like, are removed by a centrifugal precipitation method after the reaction is finished, so that the steps of repeatedly extracting the protein are saved, meanwhile, the loss caused in the protein extraction process is avoided, on the other hand, the loss caused by the temperature is one of main factors of selenium loss in the processing process, and the total reaction temperature in the invention is not higher than 60 ℃, thereby avoiding the loss of selenium element caused by overhigh temperature in the reaction process.
Preferably, in the step (1), the drying temperature is 50 ℃ and the drying time is 8-12 h; the number of the sieving meshes is 60 meshes.
Preferably, in the step (1), the mass ratio of the selenium-enriched rice bran powder to the rice polishing powder to the clear water is 1:1:10.
Preferably, the homogenization in the step (1) is carried out by using a colloid mill, the gap of the colloid mill is 0.1-1 mm, the homogenization temperature is 25 ℃, and the homogenization time is 5-10 min.
The beneficial effects are that: proteins are exposed from plant cells by homogenization treatment, while sieving to remove broken cell walls, cellulose and other plant tissues.
Preferably, the number of the sieving meshes in the step (2) is 200 meshes; the ultrasonic treatment frequency is 20-40 kHz, the ultrasonic treatment power is 500-1200W, and the ultrasonic treatment time is 10-20 min; the centrifugal speed is 8000-10000 r/min, and the centrifugal time is 10-20 min.
The beneficial effects are that: the invention exposes the protein from the plant cells by homogenization and ultrasound-assisted treatment.
Preferably, in the step (3), the primary homogenization is carried out for 2-3 times by using a conventional homogenizer, the primary homogenization temperature is 55-60 ℃, and the primary homogenization pressure is 5-30 MPa; the secondary homogenization is carried out for 2-3 times by utilizing an ultrahigh pressure micro-jet homogenizer, and the secondary homogenization pressure is 100-150 MPa.
The beneficial effects are that: the ultrahigh pressure micro-jet homogenizer can further crush the cells which cannot be crushed in the previous step, and simultaneously partially shear and crush the plant proteins in the solution, so that the protein suspension property and the water retention property in the solution are further enhanced, and the subsequent enzymolysis step is facilitated.
Preferably, in the step (4), the pH value in the immobilized enzymolysis tank is 8-9.0, and the extraction temperature is 45-60 ℃;
3-4 immobilized enzyme reaction bars are arranged in the immobilized enzymolysis tank; the mass ratio of the dispersion liquid to the immobilized enzyme is as follows: 80-200:1; the immobilized enzyme comprises papain, alkaline protease and flavourzyme, and the mass ratio is (1-2) 1 (0-1); in the immobilized enzymolysis tank, the mass ratio of immobilized enzyme to carrier is (0.01-0.030): 1, the rotation speed of the immobilized enzyme reaction rod is 5-20 r/min, and the enzymolysis reaction time is 30-180 min.
The beneficial effects are that: the three proteases are subjected to compound enzymolysis, so that plant proteins can be degraded into short peptides with proper sizes most effectively, selenium-amino acid binding sites are fully exposed, and the in-vivo and in-vitro effective rate of selenium-amino acid is improved.
The enzymolysis reaction is carried out in an immobilized enzymolysis tank, and compared with a mixed enzyme-substrate system, the immobilized enzyme has higher reaction efficiency. The immobilized enzyme reaction rod is loaded in the stainless steel sleeve core and rotates under the drive of the rotating device, so that compared with a common mixed enzyme-substrate system, the immobilized enzyme reaction rod has the advantages of larger contact area of enzyme and substrate, longer contact time, more complete reaction, no residual enzyme in the product and easy purification.
Preferably, the centrifugation speed in the step (5) is 6000-8000 r/min, the centrifugation time is 5-10 min, and the desalination in the step (5) is as follows: and (3) desalting by using 001X 7 cation exchange resin and 201X 7 anion resin, wherein the flow rate of the ion exchange resin is 10BV/h, and the mass ratio of the anion resin to the cation resin is 2:1.
The beneficial effects are that: the desalination rate of the desalination treatment was 87.5%, and the polypeptide recovery rate was 73.5%.
Preferably, the drying in step (5) comprises low temperature spray drying or vacuum freeze drying;
Wherein the air inlet temperature in the low-temperature spray drying process is 50-105 ℃, and the vacuum degree is-0.05-0.06 MPa;
The vacuum freeze-drying temperature is-40 ℃, the vacuum degree is 5Pa, and the freeze-drying time is 8 hours.
The beneficial effects are that: the low-temperature spray drying has the advantages of low energy consumption, short operation time, large treatment capacity, 60-90 min of polypeptide recovery time of 1kg and 84-87% of polypeptide recovery rate. The energy consumption of vacuum freeze drying is larger, the operation time is longer, the polypeptide recovery rate is more than or equal to 95%, and the organic selenium loss rate is less than or equal to 5%. Can be selected according to actual needs.
Compared with the traditional alkali extraction and acid precipitation method, the method provided by the invention has the advantages that the recovery rate of the selenium-rich polypeptide is high, the quality of the extracted selenium-rich protein is good, the oxidation resistance and the hypoglycemic activity are high, and the economic value effect is obvious.
Detailed Description
The following description will clearly and fully describe the technical solutions of the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
A method for extracting selenium-enriched polypeptide from selenium-enriched rice processing byproducts, comprising the following steps:
(1) Drying selenium-enriched rice bran in a 50 ℃ drying box for 8 hours, crushing by a Chinese herbal medicine crusher, sieving by a 60-mesh sieve, and mixing the sieved powder according to a weight ratio of 1:1 mixing rice polishing powder in a ratio of 1:10 (g/mL), adding clear water, homogenizing by a colloid mill at 25 ℃ with a gap of 0.1-1 mm for 5-10 min to obtain a mixed solution;
(2) Sieving the mixed solution with a 200-mesh sieve, performing ultrasonic treatment for 10-20 min under the conditions of ultrasonic frequency of 20-40kHz and ultrasonic power of 1200W, centrifuging at a speed of 8000-10000 r/min for 10-20 min by using a large-capacity centrifugal machine, and then removing the supernatant and collecting a solid sediment substrate;
(3) Adding water into the solid precipitate until the mass fraction of the solid in the mixture is 6-16%, homogenizing by a homogenizer at 57 ℃ under 20MPa for 3 times, sieving the homogenized material with a 600 mesh sieve, heating to 60 ℃ and homogenizing for the second time, wherein the homogenizing is carried out by an ultrahigh pressure micro-jet homogenizer, the treatment pressure is 100-150 MPa, and the treatment time is 2-3 times to obtain a dispersion liquid;
(4) Injecting the dispersion liquid into an immobilized enzymolysis tank for reaction, wherein the reaction system conditions are as follows: the mass concentration of the substrate is 6-16%, the pH value is 8, the enzymolysis temperature is 45 ℃,3 immobilized enzyme reaction bars are arranged in an immobilized enzymolysis tank, 1 enzyme is immobilized each, and the immobilized enzyme comprises 1 papain, 1 alkaline protease and 1 flavourzyme, and the mass ratio of the immobilized enzyme to the carrier is as follows: 1:0.010, the rotation speed of the immobilized enzyme reaction rod is 5r/min in the enzymolysis process, and the enzymolysis reaction time is 30min.
(5) And (3) regulating the pH value of the reaction solution to be neutral, centrifuging for 5-10 min at the speed of 6000-8000 r/min, reserving supernatant, desalting the supernatant, and then performing low-temperature spray drying (the air inlet temperature is 50-105 ℃ and the vacuum degree is-0.05-0.06 MPa) to obtain the selenium-enriched polypeptide.
Example 2
A method for extracting selenium-enriched polypeptide from selenium-enriched rice processing byproducts, comprising the following steps:
(1) Drying selenium-enriched rice bran in a 50 ℃ drying box for 10 hours, crushing by a Chinese herbal medicine crusher, sieving by a 60-mesh sieve, and mixing the sieved powder according to a weight ratio of 1: mixing rice polishing powder in a ratio of 1:10 (g/mL), adding clear water, and homogenizing with colloid mill. The homogenization temperature is 25 ℃, the clearance of a colloid mill is 0.1-1 mm, and the homogenization time is 5-10 min, so as to obtain mixed liquid;
(2) Sieving the mixed solution with a 200-mesh sieve, performing ultrasonic treatment for 10-20 min under the conditions of ultrasonic frequency of 20-40kHz and ultrasonic power of 800W, centrifuging at a speed of 8000-10000 r/min for 10-20 min by using a large-capacity centrifugal machine, and then removing the supernatant and collecting a solid sediment;
(3) Adding water into the solid precipitate until the mass fraction of the solid in the mixture is 6-16%, homogenizing by a homogenizer at 55 ℃ under 30MPa for 2 times, sieving the homogenized material with a 600 mesh sieve, heating to 60 ℃ and homogenizing for the second time, wherein the homogenizing is carried out by an ultrahigh pressure micro-jet homogenizer, the treating pressure is 100-150 MPa, and the treating time is 2-3 times, so as to obtain a dispersion liquid;
(4) Injecting the dispersion liquid into an immobilized enzymolysis tank for reaction, wherein the reaction system conditions are as follows: the mass concentration of the substrate is 6-16%, the pH value is 8, the enzymolysis temperature is 50 ℃,4 immobilized enzyme reaction bars are arranged in an immobilized enzymolysis tank, each immobilized enzyme is 2 papain and 1 alkaline protease, the mass ratio of the immobilized enzyme reaction bars to the carrier is 2:1, and the mass ratio of the immobilized enzyme to the carrier is: 1:0.020, the rotation speed of the immobilized enzyme reaction rod in the enzymolysis process is 15r/min, and the enzymolysis reaction time is 120min.
(5) And (3) regulating the pH value of the reaction solution to be neutral, centrifuging for 5-10 min at the speed of 6000-8000 r/min, reserving supernatant, desalting the supernatant, and then performing low-temperature spray drying (the air inlet temperature is 50-105 ℃ and the vacuum degree is-0.05-0.06 MPa) to obtain the selenium-enriched polypeptide.
Example 3
A method for extracting selenium-enriched polypeptide from selenium-enriched rice processing byproducts, comprising the following steps:
(1) Drying selenium-enriched rice bran in a 50 ℃ drying box for 12 hours, crushing by a Chinese herbal medicine crusher, sieving by a 60-mesh sieve, and mixing the sieved powder according to a weight ratio of 1: mixing rice polishing powder in a ratio of 1:10 (g/mL), adding clear water, and homogenizing with colloid mill. The homogenization temperature is 25 ℃, the clearance of a colloid mill is 0.1-1 mm, and the homogenization time is 5-10min, so as to obtain mixed liquid;
(2) Sieving the mixed solution with a 200-mesh sieve, performing ultrasonic treatment for 10-20 min under the conditions of ultrasonic frequency of 20-40kHz and ultrasonic power of 500W, centrifuging at a speed of 8000-10000 r/min for 10-20 min by using a large-capacity centrifugal machine, and then removing the supernatant and collecting a solid sediment substrate;
(3) Adding water into the solid precipitate until the mass fraction of the solid in the mixture is 6-16%, homogenizing by a homogenizer at 60 ℃ under 5MPa for 3 times, sieving the homogenized material with a 600 mesh sieve, heating to 60 ℃ and homogenizing for the second time, wherein the homogenizing is carried out by an ultrahigh pressure micro-jet homogenizer, the treating pressure is 100-150 MPa, and the treating time is 2-3 times, so as to obtain a dispersion liquid;
(4) Injecting the dispersion liquid into an immobilized enzymolysis tank for reaction, wherein the reaction system conditions are as follows: the mass concentration of the substrate is 6-16%, the pH value is 9.0, the enzymolysis temperature is 60 ℃,4 immobilized enzyme reaction bars are arranged in an immobilized enzymolysis tank, 1 enzyme is immobilized in each immobilized enzyme reaction bar, and the immobilized enzyme reaction bars are respectively 2 papain, 1 alkaline protease and 1 flavourzyme, and the mass ratio of the immobilized enzyme to the carrier is as follows: 1:0.030, the quantity ratio is 2:1:1, the rotating speed of the immobilized enzyme reaction rod in the enzymolysis process is 20r/min, and the enzymolysis reaction time is 90min.
(5) And (3) regulating the pH value of the reaction solution to be neutral, centrifuging at the speed of 6000-8000 r/min for 5-10 min, retaining supernatant, desalting the supernatant, and performing vacuum freeze drying to obtain the selenium-enriched polypeptide.
Comparative example 1
The method for extracting selenium polypeptide from the selenium-enriched malt comprises the following steps:
(1) Drying selenium-enriched malt at 50deg.C for 9h, pulverizing, sieving with a 60 mesh sieve, homogenizing the sieved powder with water in a colloid mill at 25deg.C for 10min, and mixing the sieved powder with water at a feed liquid ratio of 1:10g/mL to obtain a mixture;
(2) Sieving the mixture, performing ultrasonic treatment at ultrasonic frequency of 20-40kHz and ultrasonic power of 1200W for 20min, centrifuging at a speed of 10000r/min for 20min, and removing supernatant to obtain precipitate;
(3) Diluting the precipitate with water to a mass concentration of 16%, homogenizing at 60deg.C under 30MPa for 3 times, and sieving with 600 mesh sieve to obtain solution;
(4) Heating the solution to 60 ℃, and homogenizing by an ultrahigh pressure micro-jet homogenizer, wherein the homogenizing pressure is 150Mpa, and the homogenizing times are 3 times;
(5) Adding protease into the homogenized solution for enzymolysis in a glass container, wherein the enzymolysis conditions are as follows: the mass ratio of the protease addition amount is 2.5%, and the protease system is as follows: papain, alkaline protease and flavourzyme in a mass ratio of 2:1:1, wherein the enzymolysis reaction temperature is 50 ℃, and the reaction time is4 hours;
(6) After the reaction is completed, regulating the pH of the mixed solution to be neutral, centrifuging to obtain supernatant, wherein the centrifuging speed is 8000r/min, and the centrifuging time is 10min;
(7) Desalting the supernatant, spray drying at low temperature, air inlet temperature of 105deg.C, air outlet temperature of 80deg.C, and vacuum degree of 0.06Mpa to obtain purified malt selenium polypeptide mixture.
The enzymatic hydrolysis results in the enzymatic hydrolysis tanks of comparative example 1 and example 1 are shown in Table 1:
TABLE 1
| Enzymolysis method | Single enzyme consumption | Enzymolysis time | Recovery rate |
| Comparative example 1 | 4500～8000U/g | 2～4h | 70～85％ |
| Example 1 | 500～1600U/g | 0.5～3h | 75～90％ |
As can be seen from the data in Table 1, the immobilized enzyme can be repeatedly used, the enzyme consumption for a single reaction is far lower than that for a common enzymolysis reaction, the enzymolysis efficiency is higher, the enzyme consumption in the enzymolysis of the immobilized enzymolysis method is higher than that for the common enzymolysis, the enzymolysis time is short, the efficiency is high, and the recovery rate of the polypeptide is higher.
Comparative example 2
A method for extracting selenium-enriched polypeptide from selenium-enriched rice processing byproducts, which is different from example 1:
In the step (3), secondary homogenization is not performed.
(1) Drying selenium-rich rice byproducts at 50 ℃ for a drying time of crushing, sieving with a 60-mesh sieve, adding water into the sieved powder for homogenization by a colloid mill, wherein the homogenization temperature is 25 ℃, the homogenization time is 10min, and the feed liquid ratio of the sieved powder to water is 1g to 10mL to obtain a mixture;
(2) Sieving the mixture, performing ultrasonic treatment with power of 800W for 20min, centrifuging at 10000r/min for 20min, and removing supernatant to obtain precipitate;
(3) Diluting the precipitate with water to a mass concentration of 16%, homogenizing at 60deg.C under 30MPa for 3 times, and sieving with 600 mesh sieve to obtain solution;
(4) Setting 3 immobilized enzyme reaction bars for an immobilized enzyme special enzymolysis tank, wherein each immobilized enzyme is composed of 2 papain and 1 alkaline protease, the mass ratio of the papain to the alkaline protease is 2:1, injecting the solution into the immobilized enzyme special enzymolysis tank, adjusting pH to be 8, adding the immobilized enzyme and a carrier for reaction, wherein the enzyme feeding amount of each 1g carrier is 20mg, the immobilized enzyme consists of the immobilized enzyme, the rotating speed of the reaction bar is 15r/min, the reaction temperature is 50 ℃, and the reaction time is 120min;
(5) After the reaction is finished, pumping out the mixed solution from an enzymolysis tank special for immobilized enzyme, regulating the pH to be neutral, centrifuging to obtain supernatant, wherein the centrifuging speed is 8000r/min, and the centrifuging time is 10min;
(6) Desalting the supernatant, spray drying at low temperature, air inlet temperature being 105deg.C, air outlet temperature being 85deg.C, and vacuum degree being 0.06Mpa to obtain purified rice byproduct selenium polypeptide mixture.
Comparative example 3
A method for extracting selenium-enriched polypeptide from selenium-enriched rice processing byproducts, which is different from example 1:
in the step (4), a common enzymolysis method is adopted, and an immobilized enzymolysis method is not used for enzymolysis reaction, and the specific steps are as follows:
(1) Drying selenium-rich rice byproducts at 50 ℃ for 12 hours, crushing, sieving with a 60-mesh sieve, adding water into the sieved powder for homogenization by a colloid mill, wherein the homogenization temperature is 25 ℃, the homogenization time is 10 minutes, and the feed liquid ratio of the sieved powder to water is 1 g/10 mL to obtain a mixture;
(2) Sieving the mixture, performing ultrasonic treatment with power of 1000W for 20min, centrifuging at 10000r/min for 20min, and removing supernatant to obtain precipitate;
(3) Diluting the precipitate with water to a mass concentration of 16%, homogenizing at 60deg.C under 30MPa for 3 times, and sieving with 600 mesh sieve to obtain solution;
(4) Heating the solution to 60 ℃, and homogenizing by an ultrahigh pressure micro-jet homogenizer at a homogenizing pressure of 150Mpa for 3 times;
(5) Adding alkaline protease into the homogenized solution for enzymolysis, wherein the enzymolysis conditions are as follows: the mass ratio of the addition amount of the alkaline protease is 1.5%, the reaction temperature is 50 ℃, and the reaction time is 3.5h. ;
(6) After the reaction is completed, the pH is regulated to be neutral, the supernatant is centrifugally taken, the centrifugal speed is 8000r/min, and the centrifugal time is 10min;
(7) Desalting the supernatant, spray drying at low temperature, air inlet temperature 105 deg.C, air outlet temperature 80 deg.C, vacuum degree 0.06Mpa, or vacuum freeze drying, cold trap temperature-40 deg.C, vacuum degree 5Pa, freeze drying for 8 hr to obtain purified rice selenium polypeptide mixture.
Comparative example 4
The method for extracting rice byproduct selenoprotein and polypeptide by adopting the traditional alkali extraction and acid precipitation method comprises the following steps:
(1) Drying selenium-rich rice byproduct at 50deg.C for 8 hr, pulverizing, sieving with 60 mesh sieve,
(2) Adding water into the sieved powder to prepare solution to extract protein, wherein the reaction system is as follows: the feed liquid ratio is 1:10, the pH is 10.5, the extraction temperature is 50 ℃, and the extraction time is4 hours.
(3) Centrifuging the solution to obtain supernatant after the reaction is completed, wherein the centrifuging speed is 8000r/min, and the centrifuging time is 10min;
(4) Regulating pH of the supernatant to 4.5, precipitating overnight, centrifuging to obtain precipitate, centrifuging at 8000r/min for 10min;
(5) And (3) performing vacuum freeze drying on the precipitate, wherein the cold trap temperature is-40 ℃, the vacuum degree is 5Pa, and the freeze drying time is 8 hours, so as to obtain the rice byproduct selenium polypeptide mixture.
Comparative example 5
Ultrasonic assisted extraction of rice byproduct selenoprotein and polypeptide, which comprises the following steps:
(1) Drying selenium-rich rice byproduct at 50deg.C for 11 hr, pulverizing, sieving with 60 mesh sieve,
(2) Adding water into the sieved powder to prepare a solution, wherein the reaction system is as follows: the ratio of the feed liquid to the water is 1:10, the pH value is 10.5, and the ultrasonic treatment conditions are as follows: ultrasonic power is 60W, ultrasonic time is 20min, and after ultrasonic extraction is finished, the extraction temperature is adjusted to 60 ℃ and the extraction time is 4 hours.
(3) Centrifuging the solution to obtain supernatant after the reaction is completed, wherein the centrifuging speed is 8000r/min, and the centrifuging time is 10min;
(4) Regulating pH of the supernatant to 4.5, precipitating overnight, centrifuging to obtain precipitate, centrifuging at 8000r/min for 10min;
(5) And (3) performing vacuum freeze drying on the precipitate, wherein the cold trap temperature is-40 ℃, the vacuum degree is 5Pa, and the freeze drying time is 8 hours, so as to obtain the rice byproduct selenium polypeptide mixture.
Technical effects
TABLE 2 comparison of selenium extraction levels in examples 1-3 and comparative examples 1-2
Wherein examples 1-3 were conducted according to the procedures of the present application, comparative example 1 was conducted without the ultra-high pressure micro-jet treatment, and comparative example 2 was conducted without the use of immobilized enzyme for enzymolysis, and it can be seen from Table 2 that the recovery rates of selenium polypeptide and organic selenium extracted by the method of the present application in examples 1-3 were far higher than those in comparative examples 1-2, and that the selenium polypeptide in the by-product of selenium-enriched rice processing could be efficiently and highly extracted and prepared only by the procedure of the present application.
Actual application effect 1: in vitro oxidation resistance of extracted selenium polypeptides
The products obtained in examples 1-3 and comparative examples 4-5 were examined for DPPH radical scavenging ability, hydroxyl radical scavenging ability and superoxide anion radical scavenging ability.
Technical effects
In vitro antioxidant comparison of selenium Polypeptides
The specific test method is as follows:
① Determination of ability of rice byproduct rice bran selenoprotein to remove hydroxyl radical (OH)
1ML of 8.8mmol/L FeSO 4 solution and 1mL of 9mmol/L salicylic acid-ethanol solution were added, 1mg of each of the rice bran selenoprotein obtained in examples 1-3 and comparative examples 4-5 was measured, and finally H 2O2 solution was added to start the reaction, and the reaction was carried out at 37℃for 30min, and absorbance at 510nm was measured at each concentration.
Wherein: w 0 is the absorbance of the sample replaced with salicylic acid-ethanol solution; w i is the absorbance of the added sample solution; w j is the absorbance of the sample solution without the addition of the color reagent H 2O2.
② Determination of DPPH free radical removal ability of rice bran selenoprotein
3.0ML of a methanol solution of 0.1mmol/LDPPH was taken, and the rice bran selenoprotein and vitamin C obtained in examples 1-3 and comparative examples 4-5 were added to the mixture, and after the mixture was sufficiently mixed, the mixture was allowed to stand for 30 minutes, and then the OD 515 nm was measured by a spectrophotometer.
Wherein: a1 is the absorbance value in the reaction system; a2 is the absorbance of the negative control; a0 is the absorbance of the sample without the addition of the reagent.
③ Determination of activity of rice bran selenoprotein for eliminating superoxide anion free radical
A48-well plate was taken, and 450. Mu.L of LTris-HCl solution (50 mmol/L, pH 8.2) was added to each well to preheat for 25min, then 100. Mu.L of sample solution and 40. Mu.L of 6mmol/L of pyrogallol solution were added to react accurately at 25℃for 4min, then 50. Mu.L of 8mol/L of hydrochloric acid solution was added to terminate the reaction, and the OD value of the solution system was measured at 320nm by using an enzyme-labeled instrument. Distilled water is used as a blank instead of a sample solution, distilled water is used as a sample control instead of a pyrogallol solution, and ascorbic acid is used as a positive control. Superoxide anion radical scavenging was calculated as follows. Each sample was run 3 times in parallel and the results averaged.
Wherein: OD 0 is OD 320nm;OD1 of distilled water instead of the sample solution system, OD 320nm;OD2 of distilled water instead of the pyrogallol solution system is OD 320nm of the reaction system of the sample solution and the pyrogallol solution.
TABLE 3 comparison of in vitro antioxidant properties of selenium polypeptides in examples 1-3 and comparative examples 1-2
As can be seen from table 3:
Examples 1-3 were conducted according to the procedure of the present invention, and comparative example 1 was conducted without the ultrahigh pressure microfluidic treatment, and comparative example 2 was conducted without the use of immobilized enzymes, so that DPPH radical scavenging ability, hydroxyl radical scavenging ability, superoxide anion radical scavenging ability of the selenium polypeptides prepared in examples 1-3 were superior to those of comparative examples. In particular, in example 3, each of the oxidation resistance indexes was higher than that of the comparative example and other examples.
Actual application effect 2: in vivo oxidation resistance of extracted selenium polypeptides
Grouping the test mice, wherein each group comprises 10 blank groups, a control group 1, a control group 2, low, medium and high dosage groups, wherein the blank groups are filled with distilled water for stomach 10mg/kg every day, the control groups and the test groups are filled with selenium polypeptide serving as a by-product of processing the hundred-rice, and the daily dosage of selenium polypeptide serving as a by-product of processing the hundred-rice is 10mg/kg of the control group 1 (the selenium polypeptide serving as the by-product of processing the rice is extracted by adopting a traditional alkali extraction and acid precipitation method, the feed liquid ratio is 1:10-15, the extraction temperature is 50 ℃, the pH is 10.5, the extraction time is 4 hours, the supernatant is subjected to centrifugal precipitation removal, the pH is adjusted to 4.5, the precipitation is overnight, and the precipitate is obtained by centrifugation and dried); control group 2, ultrasonic-assisted extraction of rice processing by-product selenium polypeptide (feed liquid ratio 1:10-15, pH10.5, ultrasonic power 60W, ultrasonic time 20min, after ultrasonic extraction is finished, extraction temperature 60 ℃, extraction time 4 hours, centrifugation to remove sediment, supernatant adjustment of pH4.5, sediment overnight, centrifugation to take sediment, drying) 10mg/kg; the experimental group (adopting the method) respectively has low, medium and high dosages of 10, 20 and 30mg/kg for 30 days continuously, other conditions refer to normal mouse feeding conditions, and after 30 days, a mouse liver tissue test is taken to detect 3 indexes of SOD activity, MDA content and GSH-Px activity.
TABLE 4 Table 4
| SOD(U/mg) | MAD(nmol/mg) | GSH-Px(U/mg) | |
| Blank space | 54.2±2.1 | 15.2±1.0 | 60.2±0.9 |
| Comparative example 4 | 51.6±1.8 | 11.1±2.3 | 53.9±2.4 |
| Comparative example 5 | 60.3±0.9 | 10.4±1.0 | 64.5±1.3 |
| Low dose | 65.0±0.5 | 9.2±0.6 | 67.2±1.1 |
| Medium dosage | 83.2±1.1 | 7.6±0.8 | 83.2±2.3 |
| High dose | 94.6±2.7 | 5.6±0.3 | 97.4±1.7 |
As can be seen from table 4: mice in daily gastric lavage selenium-rich polypeptide (low, medium and high) experimental groups have higher in-vivo antioxidant capacity than mice in blank groups and control groups, wherein the high-dose group mice have the best in-vivo antioxidant effect, which indicates that a certain dose of selenium polypeptide has a positive effect on improving the in-vivo antioxidant capacity of the mice, and the dose is positively correlated with the in-vivo antioxidant effect.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (3)
1. A method for extracting selenium-enriched polypeptide from selenium-enriched rice processing byproducts, which is characterized by comprising the following steps:
(1) Drying selenium-enriched rice bran, crushing and sieving to obtain selenium-enriched rice bran powder, mixing the selenium-enriched rice bran powder with rice polishing powder, adding clear water, and homogenizing to obtain mixed solution;
The drying temperature is 50 ℃, and the drying time is 8-12 hours; the number of the sieving meshes is 60 meshes;
the mass ratio of the selenium-enriched rice bran powder to the rice polishing powder to the clear water is 1:1:10;
the homogenization is carried out by using a colloid mill, the gap of the colloid mill is 0.1-1 mm, the homogenization temperature is 25 ℃, and the homogenization time is 5-10 min;
(2) Sieving the mixed solution, performing ultrasonic treatment, centrifuging, and collecting solid precipitate;
the number of the sieving meshes is 200 meshes; the ultrasonic treatment frequency is 20-40 kHz, the ultrasonic treatment power is 500-1200W, and the ultrasonic treatment time is 10-20 min; the centrifugal speed is 8000-10000 r/min, and the centrifugal time is 10-20 min;
(3) Adding water into the solid precipitate until the solid mass fraction is 6-16%, homogenizing for the first time, sieving, heating to 60 ℃ and homogenizing for the second time to obtain a dispersion liquid;
The primary homogenization is carried out for 2-3 times by using a conventional homogenizer, the primary homogenization temperature is 55-60 ℃, and the primary homogenization pressure is 5-30 MPa; the secondary homogenization is carried out for 2-3 times by utilizing an ultrahigh pressure micro-jet homogenizer, and the secondary homogenization pressure is 100-150 MPa;
(4) Injecting the dispersion liquid into an immobilized enzymolysis tank for enzymolysis reaction for 30-180 min to obtain a reaction liquid;
the pH value in the immobilized enzymolysis tank is 8-9.0, and the extraction temperature is 45-60 ℃;
3-4 immobilized enzyme reaction bars are arranged in the immobilized enzymolysis tank; the mass ratio of the dispersion liquid to the immobilized enzyme is as follows: 80-200:1; the immobilized enzyme comprises papain, alkaline protease and flavourzyme, and the mass ratio is (1-2) 1 (0-1); in the immobilized enzymolysis tank, the mass ratio of immobilized enzyme to carrier is (0.01-0.030): 1, the rotation speed of an immobilized enzyme reaction rod is 5-20 r/min, and the enzymolysis reaction time is 30-180 min;
(5) Adding HCl into the reaction solution to adjust the pH to be neutral, centrifuging, reserving supernatant, desalting the supernatant, and drying to obtain selenium-enriched polypeptide;
The desalination is as follows: and (3) desalting by using 001X 7 cation exchange resin and 201X 7 anion resin, wherein the flow rate of the ion exchange resin is 10BV/h, and the mass ratio of the anion resin to the cation resin is 2:1.
2. The method for extracting selenium-enriched polypeptide from selenium-enriched rice processing byproducts as recited in claim 1, wherein the centrifugation speed in the step (5) is 6000-8000 r/min and the centrifugation time is 5-10 min.
3. A method for extracting selenium-enriched polypeptide from a selenium-enriched rice processing byproduct as recited in claim 1, wherein the drying in step (5) comprises low temperature spray drying or vacuum freeze drying;
Wherein the air inlet temperature in the low-temperature spray drying process is 50-105 ℃, and the vacuum degree is-0.05-0.06 MPa;
The vacuum freeze-drying temperature is-40 ℃, the vacuum degree is 5Pa, and the freeze-drying time is 8 hours.
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