Method for separating hordein B-component by using iota-carrageenan

Abstract

The invention discloses a method for separating a hordein B-component by utilizing iota-carrageenan. The method comprises the following steps: step 1, extracting hordein; step 2, pretreating protein and polysaccharide; 3, preparing a protein / polysaccharide complex coacervation precipitate based on electrostatic interaction; step 4, removing iota-carrageenan, removing iota-carrageenan precipitate at the bottom layer, and collecting supernate, so as to obtain a B-hordein solution; and step five, freeze-drying B-hordein: collecting a B-hordein solution, removing ethanol by using a rotary evaporator, freezing a sample, and freeze-drying to obtain B-hordein protein powder. According to the method, the B-hordein can be separated in a low-loss and high-selectivity manner, the operation process is simple, the equipment requirement is low, large-scale production is easy, the dosage of iota-carrageenan is small, the iota-carrageenan can be recycled through membrane separation, and the C-hordein in the hordein can be recycled at the same time.

Description

Technical Field

[0001] The invention relates to the technical field of protein separation, in particular to a method for separating hordein B-component by utilizing iota-carrageenan. Background Art

[0002] Based on amino acid composition and molecular weight, barley alcohol-soluble protein can be divided into four components, namely A-Hordein, B-Hordein, C-Hordein and D-Hordein, among which B and C components account for the vast majority of the total amount of hordein (more than 95%), and A and D components are very small (less than 5%). B-Hordein is the main component of hordein, accounting for 70%-80% of the total alcohol-soluble protein, with a molecular weight of about 36~45kDa. B-Hordein is composed of at least 10 polypeptides, of which glutamine and glutamic acid content is 35%, proline content is 21%, glycine content is 1.5%, and cysteine ​​content is 2.5%.

[0003] In recent years, more and more researchers have developed nutrient delivery systems based on plant alcohol-soluble proteins by taking advantage of their low cost, strong hydrophobicity, and self-assembly ability. Since B-Hordein contains a relatively high proportion of sulfur amino acids, it has great application prospects in the construction of delivery systems, such as nanoparticles and edible films. In addition, B-Hordein also has excellent surface properties, such as emulsification, foaming, film-forming properties, as well as good biocompatibility, biodegradability, and antioxidant properties. Therefore, it can be developed into an encapsulation delivery carrier for food and medicine, an edible packaging film, and a natural antioxidant. Therefore, obtaining B-Hordein from barley and using it in industries such as food and medicine can not only improve the utilization of barley resources, but also make up for the high cost of obtaining other alcohol-soluble proteins (such as zein and wheat alcohol-soluble proteins), so it has great development and application potential.

[0004] However, the molecular weights of components B and C are very close, and protein separation cannot be achieved directly by ultrafiltration. The traditional method uses the Gilson PLC2020 purification chromatography system to separate B-hordein. Although it has high selectivity, the separation efficiency is low and the cost is high. The comprehensive cost is close to 200 yuan / gram of B-hordein, and organic solvents such as trifluoroacetic acid are used in the separation process, and their safety is questionable. Therefore, it is necessary to develop a fast, cheap and efficient separation method. Summary of the invention

[0005] The present invention aims to provide a method for separating the B-component of hordein by using iota-carrageenan. Hordein is used as a raw material, and a negatively charged polysaccharide iota-carrageenan of a certain concentration is selected as a recovery material. The iota-carrageenan is added to the hordein according to a certain mass ratio to carry out a selective complex coacervation reaction. The iota-carrageenan is used to preferentially bind to B-hordein to form a complex coacervate precipitate, while C-hordein is retained in the supernatant. The amount of iota-carrageenan and the pH value of the reaction are controlled to obtain a complex coacervate precipitate of B-hordein and iota-carrageenan, and then the pH value is adjusted to separate B-hordein from iota-carrageenan, and finally the B-hordein component is recovered by centrifugation.

[0006] In order to solve the above technical problems, the technical solution of the present invention is: a method for separating hordein B-component using iota-carrageenan, the specific steps are as follows: Step 1, extraction of hordein: weigh 40g barley flour, add n-hexane at a mass-to-volume ratio of 1:5, stir at 25°C, centrifuge, discard the supernatant, and take out the precipitate; add ultrapure water at a ratio of 1:5, stir, centrifuge, discard the supernatant, and take out the precipitate; add 0.1M sodium chloride solution at a ratio of 1:5, stir, centrifuge, discard the supernatant, and take out the precipitate; add 75% ethanol at a ratio of 1:10, stir, centrifuge, and discard the precipitate. The supernatant is hordein, and the ethanol is removed by a rotary evaporator. The heating temperature is 45°C, and the turbid liquid containing the protein is placed in a 4°C refrigerator. After standing and freeze-drying, a protein sample is obtained, and the sample is crushed into powder using a mortar and placed in a desiccator for storage; Step 2: Pretreatment of proteins and polysaccharides: prepare a 1.0 mg / mL barley alcohol-soluble protein solution using 75% ethanol, and adjust the pH of the barley alcohol-soluble protein solution to 11.0 using 0.1 M sodium hydroxide solution, stir evenly, dissolve iota carrageenan in deionized water in advance, and after the iota carrageenan is completely dissolved, add a certain volume of anhydrous ethanol to obtain an iota carrageenan solution with a final concentration of 1.0 mg / mL, forming an ethanol aqueous solution with a solvent of 30-50%, and adjust the pH of the iota carrageenan solution to 11.0 using 0.5 M sodium hydroxide solution; Step 3, preparation of protein / polysaccharide complex coacervate precipitate based on electrostatic interaction: 10 mL of polysaccharide solution was measured, and the two were mixed according to the protein: polysaccharide mass ratio of 20:1, 15:1, 10:1, 5:1, 2:1, and 1:1, respectively, and the pH was adjusted to 11.0 with 0.02 M sodium hydroxide solution, and stirred evenly. Then, the pH value was adjusted to 6.4, 6.2, 6.0, 4.8, 4.2, and 4.0 with 0.05 M hydrochloric acid, stirred, centrifuged, and the supernatant was discarded. The centrifuge tube was inverted and dried at room temperature for 5 min, and the bottom precipitate was taken out to obtain the complex coacervate of B-hordein and ι-carrageenan; Step 4, removing iota-carrageenan: 30 mL of deionized water was added to the complex coacervate, and the pH of the deionized water was 7.0. The mixture was freeze-dried for 48 h. Further, 30 mL of 75% ethanol was added to the obtained powder sample. The pH of the dispersion was adjusted to 10.0-12.0 using a 1 M sodium hydroxide solution. The mixture was stirred at 37° C. to completely disperse the protein. At this time, the iota-carrageenan was not dissolved. The mixture was centrifuged to remove the bottom iota-carrageenan precipitate, and the supernatant was collected to obtain a B-hordein solution. Step 5. Freeze-drying of B-hordein: Collect the B-hordein solution, remove the ethanol using a rotary evaporator, freeze the sample, and freeze-dry to obtain B-hordein protein powder.

[0007] Further, in step one, after adding n-hexane, stirring was continued for 2 hours at 25°C, centrifuged at 2000×g for 5 minutes, the supernatant was discarded, and the precipitate was taken out; ultrapure water was added at a ratio of 1:5, stirring was continued for 0.5 hours, centrifuged at 2000×g for 5 minutes, the supernatant was discarded, and the precipitate was taken out; 0.1M sodium chloride solution was added at a ratio of 1:5, stirring was continued for 0.5 hours, centrifuged at 2000×g for 5 minutes, the supernatant was discarded, and the precipitate was taken out; 75% ethanol was added at a ratio of 1:10, stirring was continued for 2 hours, centrifuged at 2000×g for 5 minutes, and the precipitate was discarded.

[0008] Furthermore, in step 3, the pH values ​​were adjusted to 6.4, 6.2, 6.0, 4.8, 4.2, and 4.0, respectively, using 0.05 M hydrochloric acid, and then magnetically stirred at 500 rpm for 5-20 min, and then centrifuged at 800-2000×g for 5-20 min, and the supernatant was discarded.

[0009] Further, in step 4, the pH of the dispersion is adjusted to 10.0-12.0 using 1M sodium hydroxide solution, and magnetic stirring is carried out at 500 rpm for 10-30 min at 37°C to thoroughly disperse the protein. At this time, ι-carrageenan is not dissolved, and centrifugation is carried out at 800-2000×g for 5-20 min to remove the ι-carrageenan precipitate at the bottom.

[0010] Furthermore, in step five, the sample is frozen at -80°C and further freeze-dried for 48 hours to obtain B-hordein protein powder.

[0011] Furthermore, the protein solution in step 2 is placed at room temperature of 25° C. for more than 1 hour and stirred again before use.

[0012] Furthermore, the solvent of the polysaccharide solution in step three should be 50% ethanol aqueous solution.

[0013] Furthermore, in step three, the mass ratio of protein:polysaccharide is 15:1, the pH value of the mixture is adjusted to 6.2, the magnetic stirring time is 10 min, and the centrifugation conditions are: 800×g, 5 min.

[0014] Furthermore, in step 4, the pH value of the dispersion is 12.0, the stirring time is 20 min, and the centrifugal conditions are: 2000×g, 10 min.

[0015] Furthermore, the protein purity of B-hordein obtained in step 5 was analyzed by polyacrylamide gel electrophoresis.

[0016] In the present invention, since the isoelectric point of hordein is concentrated at about 6.4-6.6, when the pH value of the mixed solution of hordein and iota-carrageenan is adjusted to be below the isoelectric point of the protein, the positively charged B-hordein will preferentially combine with the negatively charged iota-carrageenan through electrostatic interaction to form a complex coacervate insoluble in ethanol aqueous solution, and the complex coacervate can be quickly precipitated by low-speed centrifugation.

[0017] The invention uses natural edible negatively charged polysaccharide iota-carrageenan as a material for recovering and separating proteins, utilizes the principle that target protein B-hordein and iota-carrageenan generate complex coacervation precipitation under the condition of carrying opposite charges, and non-target protein C-hordein does not participate in the formation of complex coacervates, thereby obtaining relatively single B-hordein. In addition, the prepared complex coacervates can be directly used as food materials. From the perspective of resource recovery, the invention can not only separate B-hordein with low loss and high selectivity, but also has a simple operation process, low equipment requirements, and is easy to scale up. The amount of iota-carrageenan used is small, and it can be recycled and utilized through membrane separation, and C-hordein in hordein can also be recovered at the same time. According to preliminary calculations, only 1 gram of iota-carrageenan is needed to separate B-hordein and C-hordein in 15 grams of total protein, and the operation can be completed by using only ethanol and ordinary containers, with a comprehensive cost of about 10 yuan per gram of B-hordein. Therefore, the method provides technical support for subsequent development and research. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a separation process flow chart of the present invention; Figure 2 It is a polyacrylamide gel electrophoresis detection spectrum of the protein purification of the present invention; Explanation of the accompanying drawings: Lane M represents protein marker; Lane 1 represents total hordein; Lane 2 represents B-hordein after separation; Lane 3 represents the protein components in the supernatant after separation (mainly C-hordein). DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with specific embodiments.

[0020] like Figure 1-2 As shown, a method for separating hordein B-component using iota-carrageenan, the specific steps are as follows: Step 1, extraction of hordein: weigh 40g barley flour, add n-hexane at a mass to volume ratio of 1:5, stir continuously for 2h at 25°C, centrifuge at 2000×g for 5min, discard the supernatant, and take out the precipitate; add ultrapure water at a ratio of 1:5, stir continuously for 0.5h, centrifuge at 2000×g for 5min, discard the supernatant, and take out the precipitate; add 0.1M sodium chloride solution at a ratio of 1:5, stir continuously for 0.5h, centrifuge at 2000×g for 5min, discard the supernatant, and take out the precipitate; add 75% ethanol at a ratio of 1:10, stir continuously for 2h, centrifuge at 2000×g for 5min, discard the precipitate, the supernatant is hordein, use a rotary evaporator to remove ethanol, heat at 45°C, place the turbid liquid containing protein in a 4°C refrigerator, stand and freeze-dry to obtain a protein sample, use a mortar to grind the sample into powder, and place it in a desiccator for storage; Step 2: Pretreatment of proteins and polysaccharides: Use 75% ethanol to prepare a 1.0 mg / mL barley alcohol-soluble protein solution, and use 0.1 M sodium hydroxide solution to adjust the pH of the barley alcohol-soluble protein solution to 11.0, stir evenly, use deionized water to dissolve ι-carrageenan in advance, and after the ι-carrageenan is completely dissolved, add a certain volume of anhydrous ethanol to prepare a final concentration of 1.0 mg / mL ι-carrageenan solution, forming a solvent of 30-50% ethanol aqueous solution, and use 0.5 M sodium hydroxide solution to adjust the pH of the ι-carrageenan solution to 11.0, and the protein solution is placed at room temperature 25°C for more than 1 hour, and stirred evenly again before use; Step 3, preparation of protein / polysaccharide complex coacervate precipitate based on electrostatic interaction: 10 mL of polysaccharide solution, the polysaccharide solution solvent is 50% ethanol aqueous solution, the two are mixed at a protein: polysaccharide mass ratio of 15:1, and the pH is adjusted to 11.0 with 0.02 M sodium hydroxide solution, stirred evenly, and then, the pH value is adjusted to 6.2 with 0.05 M hydrochloric acid, after magnetic stirring at 500 rpm for 10 min, centrifuged at 800 × g for 5 min, the supernatant is discarded, the centrifuge tube is inverted, and dried at room temperature for 5 min, and the bottom precipitate is taken out to obtain the complex coacervate of B-hordein and ι-carrageenan; Step 4, removing ι-carrageenan: 30 mL of deionized water was added to the complex coacervate, and the pH of the deionized water was 7.0. The mixture was freeze-dried for 48 h. Further, 30 mL of 75% ethanol was added to the obtained powder sample. The pH of the dispersion was adjusted to 12.0 using a 1 M sodium hydroxide solution. The mixture was magnetically stirred at 500 rpm for 20 min at 37 ° C to thoroughly disperse the protein. At this time, the ι-carrageenan was not dissolved. The mixture was centrifuged at 2000 × g for 10 min to remove the bottom ι-carrageenan precipitate. The supernatant was collected to obtain a B-hordein solution. Step 5. Freeze-drying of B-hordein: Collect the B-hordein solution, remove the ethanol using a rotary evaporator, freeze the sample at -80°C, and further freeze-dry for 48 hours to obtain B-hordein protein powder.

[0021] The protein content of the obtained B-hordein was determined by the BCA method, the residual polysaccharide content was determined by the phenol-sulfuric acid method GB / T 15672-2009, the ash content was determined by the ignition weight method, and the protein purity was analyzed by high performance liquid chromatography-quadrupole / electrostatic field orbital trap high resolution mass spectrometry (HPLC-Q-Exactive-MS / MS) and polyacrylamide gel electrophoresis (SDS-PAGE).

[0022] Table 1 shows the HPLC-Q-Exactive-MS / MS results of the abundance of purified B-hordein protein: No Accession Description Mascot score Mr Seq Cov (%) emPAI Num. of significant matches Num. of significant sequences 1 ALA63868.1 B-hordein precursor 8026 34929 0.63 235692.9 342 86 2 prf||1103203A hordein B 12497 30840 0.79 26661.1 500 70 3 AFM77743.1 B1 hordein 7804 29564 0.76 3260.3 299 51 4 CAA25509.1 unnamed protein product, partial 3492 21086 0.62 2225.2 155 33 5 CAA25912.1 unnamed protein product, partial 10617 30632 0.72 2128.6 424 52 6 AFM77746.1 B3 hordein 11928 32254 0.63 1640.5 450 52 7 ALA63867.1 B-hordein precursor 7052 34857 0.55 1553.9 243 57 8 KAE8807270.1 B3 hordein 9324 37127 0.8 1412.5 405 58 9 AFM77742.1 B1 hordein 7648 33233 0.75 1047.1 291 50 10 AFM77738.1 gamma 1 hordein 2806 35211 0.94 1013.7 138 50 11 ALA63866.1 B-hordein precursor 3449 34947 0.56 938.8 180 52 12 ACU09490.1 B hordein 8369 30621 0.78 935.9 358 45 13 AFM37566.1 B-hordein 6825 31340 0.6 708.7 253 43 14 AFM77733.1 B-hordein 7361 33880 0.69 556.6 278 46 15 ACU09493.1 B hordein 4042 30651 0.82 411.2 175 39 16 ACU09491.1 B hordein 3969 30728 0.84 402.4 169 39 17 CAA25913.1 unnamed protein product, partial 5455 28177 0.6 379.5 189 37 18 AFM77747.1 B3 hordein 4111 35859 0.7 310.3 176 42 19 prf||1103203B hordein B 5612 30850 0.55 261.7 191 38 20 prf||1604464A gamma hordein 2071 35356 0.77 183.7 112 39 21 KAE8807289.1 gamma 1 hordein 1865 35420 0.73 163.1 103 38 22 CAE45747.1 putative gamma 2 hordein, partial 1695 29471 0.59 142.7 89 31 23 ALA63864.1 B-hordein precursor 5587 34464 0.41 130.7 193 37 24 ABA06537.1 B hordein 3935 34013 0.52 20.9 151 24 25 KAE8775013.1 B hordein precursor 1054 24857 0.28 16.4 70 15 26 ALA63865.1 B-hordein precursor 1773 33747 0.29 9.6 85 17 27 sp|P80198.1|HOG3_HORVU RecName: Full=Gamma-hordein-3 960 33624 0.43 8.5 35 18 28 AAA32944.1 C-hordein storage protein, partial 141 8040 0.79 6.6 7 4 29 AAA92333.1 C hordein 104 40522 0.15 1.3 8 8 30 AAU06228.1 B hordein 96 34918 0.21 1.1 9 6 31 AFM77750.1 D hordein 466 79874 0.14 1.0 21 12 32 AAU06229.1 B hordein 89 34197 0.16 0.9 8 5 34 AAB28161.1 C-hordein 47 36485 0.19 0.3 2 2 The content of B-hordein was 91.17% on dry basis, the residual polysaccharide was 1.94%, and the ash content was 4.79%. The mass spectrometry results are shown in Table 1. The protein abundance of B-hordein after purification was 97.78%, indicating that its purity was high. The SDS-PAGE detection spectrum is shown in Figure 2 As shown, the results showed that the protein purity of B-hordein was about 96.72%.

[0023] The above description is only an illustrative embodiment of the present invention and is not intended to limit the scope of the present invention. Any equivalent changes and modifications made by any person skilled in the art without departing from the concept and principle of the present invention shall fall within the scope of protection of the present invention.

Claims

1. A method for separating hordein B-component using iota-carrageenan, characterized in that: The specific steps are as follows: Step 1, extraction of hordein: weigh 40g barley flour, add n-hexane at a mass-to-volume ratio of 1:5, stir at 25°C, centrifuge, discard the supernatant, and take out the precipitate; add ultrapure water at a ratio of 1:5, stir, centrifuge, discard the supernatant, and take out the precipitate; add 0.1M sodium chloride solution at a ratio of 1:5, stir, centrifuge, discard the supernatant, and take out the precipitate; add 75% ethanol at a ratio of 1:10, stir, centrifuge, and discard the precipitate. The supernatant is hordein, and the ethanol is removed by a rotary evaporator. The heating temperature is 45°C, and the turbid liquid containing the protein is placed in a 4°C refrigerator. After standing and freeze-drying, a protein sample is obtained, and the sample is crushed into powder using a mortar and placed in a desiccator for storage; Step 2: Pretreatment of proteins and polysaccharides: prepare a 1.0 mg / mL barley alcohol-soluble protein solution using 75% ethanol, and adjust the pH of the barley alcohol-soluble protein solution to 11.0 using 0.1 M sodium hydroxide solution, stir evenly, dissolve iota carrageenan in deionized water in advance, and after the iota carrageenan is completely dissolved, add anhydrous ethanol to obtain an iota carrageenan solution with a final concentration of 1.0 mg / mL, forming an ethanol aqueous solution with a solvent of 30-50%, and adjust the pH of the iota carrageenan solution to 11.0 using 0.5 M sodium hydroxide solution; Step 3, preparation of protein / polysaccharide complex coacervate precipitate based on electrostatic interaction: 10 mL of polysaccharide solution was measured, and the two were mixed according to the protein: polysaccharide mass ratio of 20:1, 15:1, 10:1, 5:1, 2:1, and 1:1, respectively, and the pH was adjusted to 11.0 with 0.02 M sodium hydroxide solution, and stirred evenly. Then, the pH value was adjusted to 6.4, 6.2, 6.0, 4.8, 4.2, and 4.0 with 0.05 M hydrochloric acid, stirred, centrifuged, and the supernatant was discarded. The centrifuge tube was inverted and dried at room temperature for 5 min, and the bottom precipitate was taken out to obtain the complex coacervate of B-hordein and ι-carrageenan; Step 4, removing iota-carrageenan: 30 mL of deionized water was added to the complex coacervate, and the pH of the deionized water was 7.

0. The mixture was freeze-dried for 48 h. Further, 30 mL of 75% ethanol was added to the obtained powder sample. The pH of the dispersion was adjusted to 10.0-12.0 using a 1 M sodium hydroxide solution. The mixture was stirred at 37° C. to completely disperse the protein. At this time, the iota-carrageenan was not dissolved. The mixture was centrifuged to remove the bottom iota-carrageenan precipitate, and the supernatant was collected to obtain a B-hordein solution. Step 5. Freeze-drying of B-hordein: Collect the B-hordein solution, remove the ethanol using a rotary evaporator, freeze the sample, and freeze-dry to obtain B-hordein protein powder.

2. A method for separating hordein B-component using iota-carrageenan as claimed in claim 1, characterized in that: In step 1, after adding n-hexane, stirring was continued for 2 h at 25°C, centrifuged at 2000×g for 5 min, the supernatant was discarded, and the precipitate was taken out; ultrapure water was added at a ratio of 1:5, stirring was continued for 0.5 h, centrifuged at 2000×g for 5 min, the supernatant was discarded, and the precipitate was taken out; 0.1M sodium chloride solution was added at a ratio of 1:5, stirring was continued for 0.5 h, centrifuged at 2000×g for 5 min, the supernatant was discarded, the precipitate was taken out, 75% ethanol was added at a ratio of 1:10, stirring was continued for 2 h, centrifuged at 2000×g for 5 min, and the precipitate was discarded.

3. A method for separating hordein B-component using iota-carrageenan as claimed in claim 1, characterized in that: In step 3, the pH values ​​were adjusted to 6.4, 6.2, 6.0, 4.8, 4.2, and 4.0 respectively using 0.05 M hydrochloric acid, and then magnetically stirred at 500 rpm for 5-20 min, centrifuged at 800-2000×g for 5-20 min, and the supernatant was discarded.

4. A method for separating hordein B-component using iota-carrageenan as claimed in claim 1, characterized in that: In step 4, the pH of the dispersion is adjusted to 10.0-12.0 using 1M sodium hydroxide solution, and magnetic stirring is performed at 500 rpm for 10-30 min at 37°C to thoroughly disperse the protein. At this time, ι-carrageenan is not dissolved, and centrifugation is performed at 800-2000×g for 5-20 min to remove the ι-carrageenan precipitate at the bottom.

5. A method for separating hordein B-component using iota-carrageenan as claimed in claim 1, characterized in that: In step 5, the sample is frozen at -80°C and further freeze-dried for 48 hours to obtain B-hordein protein powder.

6. A method for separating hordein B-component using iota-carrageenan as claimed in claim 1, characterized in that: The protein solution in step 2 was placed at room temperature (25°C) for more than 1 hour and stirred again before use.

7. A method for separating hordein B-component using iota-carrageenan as claimed in claim 1, characterized in that: The polysaccharide solution described in step 3 has a solvent of 50% ethanol aqueous solution.

8. A method for separating hordein B-component using iota-carrageenan as claimed in claim 3, characterized in that: In step 3, the mass ratio of protein:polysaccharide is 15:1, the pH value of the mixture is adjusted to 6.2, the magnetic stirring time is 10 min, and the centrifugation conditions are: 800×g, 5 min.

9. A method for separating hordein B-component using iota-carrageenan as claimed in claim 4, characterized in that: The pH value of the dispersion in step 4 is 12.0, the stirring time is 20 min, and the centrifugation conditions are: 2000×g, 10 min.

10. A method for separating hordein B-component using iota-carrageenan as claimed in claim 1, characterized in that: The protein purity of B-hordein prepared in step 5 was analyzed by polyacrylamide gel electrophoresis.

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