CN111440837A - High F value oligopeptide and preparation method thereof - Google Patents
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Abstract
The invention provides a high F value oligopeptide and a preparation method thereof, relating to the technical field of active peptide preparation; the method comprises the steps of taking degreased rice bran as a raw material, adding cellulase for wall breaking, extracting rice bran protein by an alkaline method, adding neutral protease for hydrolysis of protein, removing aromatic amino acid by using active carbon static adsorption chromatography, and separating and drying to obtain the rice bran high-F-value oligopeptide. The high F value oligopeptide has an F value of 28-32. The preparation method of the invention opens up a new direction for the application of the rice bran which is the grain leftovers.
Description
Technical Field
The invention belongs to the technical field of preparation of active peptides, and particularly relates to a high F value oligopeptide and a preparation method thereof.
Background
The rice bran is a byproduct in fine milling of rice after rice husking, and consists of an exocarp, a mesocarp, a cross-linked layer, a seed coat and an aleurone layer, the rice bran concentrates 64% of nutritional functional components of the rice, and is rich in high-quality protein, fat, mineral nutrients, B vitamins, vitamin E and the like, wherein the content of the rice bran protein is almost twice as high as that of common fine rice (tomon. a new way of comprehensive utilization of the rice bran [ J ]. practical technology: 2003, (5): 21.). the rice bran protein is vegetable protein with abundant nutritional values, mainly albumin, globulin, glutelin and alcohol soluble protein, the ratio of the 4 proteins is approximately 37: 36: 22: 5. the rice bran protein is the protein with the lowest anaphylaxis in known grains and is the only cereal protein capable of avoiding anaphylaxis tests, so the rice bran protein is always considered as an ideal infant food raw material, is widely used in infant food (Wang, 1999; Helm, 1996; Yaohao, 2002) and is an ideal infant food for supplementing the amino acid mixture obtained after the rice bran protein is hydrolyzed, especially rice bran protein, the rice bran protein mixture is a special dietary protein mixture which is capable of supplementing the high in the dietary supplement of the dietary deficiency of the high-rich amino acid digestion of infant food (WHO), and the dietary supplement of the high-rich nutrition of the infant food, such as the food, and the food for the health care food, and the food for the infant, the food for which is not capable of supplementing the high-care food for the high.
At present, the preparation technology of the rice bran active peptide mainly aims at antioxidation, for example, Chinese patent application with application publication number of CN101434980B discloses a method for preparing rice bran short peptide, and the invention hydrolyzes protein by adding compound protease to obtain the rice bran short peptide; chinese patent application with application publication number CN10208011A discloses a preparation technology of rice bran antioxidant peptide, which adopts an alkaline method to prepare rice bran protein, and then uses compound protease for enzymolysis to obtain the rice bran protein peptide with antioxidant activity; the Chinese patent application with the application publication number of CN10174818A discloses a preparation method of rice bran antioxidant peptide, wherein an alkaline protease method is adopted. The preparation of rice bran high F value oligopeptides has not been reported yet.
Disclosure of Invention
In view of the above, the invention aims to provide a high-F-value oligopeptide and a preparation method thereof, which can significantly improve the F value, yield and purity of the rice bran oligopeptide and provide a new idea for the application of rice bran.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of high F value oligopeptide, which comprises the following steps: mixing defatted rice bran and cellulase, performing enzymolysis and wall breaking to obtain protein liquid, and extracting rice bran protein in the protein liquid; sequentially carrying out neutral protease enzymolysis and alkaline protease enzymolysis on the rice bran protein to obtain enzymolysis polypeptide, and carrying out active carbon static adsorption chromatography on the enzymolysis polypeptide, centrifuging and drying to obtain the high F value oligopeptide.
Preferably, the enzymolysis wall breaking comprises mixing the defatted rice bran with distilled water, adjusting the pH value to 4.5-5.5, and adding cellulase; the mass ratio of the defatted rice bran to the distilled water is 1: 8-12; the mass ratio of the defatted rice bran to the cellulase is 100: 0.3-0.5.
Preferably, the temperature of the enzymolysis wall breaking is 45-55 ℃, and the time of the enzymolysis wall breaking is 3.5-4.5 h.
Preferably, the rice bran protein in the protein liquid is extracted by an alkaline method, wherein the alkaline method comprises the steps of adjusting the pH value of the protein liquid to 9.0, stirring for reaction, performing first centrifugation, and taking supernatant; and performing PI precipitation on the supernatant, performing second centrifugation, collecting the precipitate, washing and drying to obtain the rice bran protein.
Preferably, the neutral protease comprises AS1.398 neutral protease; when the neutral protease is subjected to enzymolysis, the rice bran protein and water are mixed according to the mass ratio of 1: 10-12, the pH value is adjusted to 6-7, and the neutral protease is added.
Preferably, the mass of the neutral protease is 5-6% of the mass of the rice bran protein; the hydrolysis temperature of the neutral protease is 40-50 ℃, and the hydrolysis time is 3-4 h.
Preferably, the alkaline protease comprises alcalase2.4fg alkaline protease; when alkaline protease enzymolysis is carried out, adjusting the pH of hydrolysate hydrolyzed by the neutral protease to 11-12, and adding the alkaline protease; the mass of the alkaline protease is 6.5-7.5% of that of the rice bran protein; the hydrolysis temperature of the alkaline protease is 50-60 ℃, and the hydrolysis time is 5-6 h.
Preferably, the temperature of the active carbon static adsorption chromatography is 35-45 ℃, and the time is 4.5-5.5 h.
Preferably, the method further comprises detecting separation after the centrifugationOD of post-cardiac supernatant220And OD260By OD220/OD260The F value is obtained by calculating the ratio of (A) to (B).
The invention also provides the high F value oligopeptide prepared by the preparation method, wherein the F value of the high F value oligopeptide is 28-32.
The invention provides a preparation method of high F value oligopeptide, which takes defatted rice bran as a raw material, adds cellulase for wall breaking, extracts rice bran protein, sequentially adds neutral protease and alkaline protease for protein hydrolysis, removes aromatic amino acid by using active carbon static adsorption chromatography, and obtains the rice bran high F value oligopeptide after separation and drying.
Before the rice bran protein is extracted, the rice bran protein is hydrolyzed by using the cellulase to release the rice bran protein, so that the yield of the rice bran protein is improved, the hydrolysis efficiency of neutral protease and alkaline protease is improved, and the improvement of the F value, yield and purity of the rice bran peptide is facilitated.
The invention adopts the steps of firstly adding neutral protease and then adding alkaline protease to hydrolyze the rice bran protein, although both the neutral protease and the alkaline protease are endoproteases, the alkaline protease belongs to serine protease and mainly acts on peptide bonds containing tyrosine, phenylalanine and tryptophan. In the first step of enzymolysis, the degree of hydrolysis of the neutral protease added firstly is higher, the formed peptide chain is shorter, so that the alkaline protease used in the second stage serves as an exonuclease, and aromatic amino acids at the tail end of the peptide chain are specifically hydrolyzed into free amino acids, so that the F value is improved.
The invention provides a preparation method of rice bran high F value oligopeptide, and opens up a new direction for the application of rice bran which is a grain leftover.
Detailed Description
The invention provides a preparation method of high F value oligopeptide, which comprises the following steps: mixing defatted rice bran and cellulase, performing enzymolysis and wall breaking to obtain protein liquid, and extracting rice bran protein in the protein liquid; sequentially carrying out neutral protease enzymolysis and alkaline protease enzymolysis on the rice bran protein to obtain enzymolysis polypeptide, and carrying out active carbon static adsorption chromatography on the enzymolysis polypeptide, centrifuging and drying to obtain the high F value oligopeptide.
The method comprises the steps of mixing the defatted rice bran with distilled water, adjusting the pH value to 4.5-5.5, and adding cellulase, wherein the mass ratio of the defatted rice bran to the distilled water is preferably 1: 8-12, the mass ratio of the defatted rice bran to the cellulase is preferably 100: 0.3-0.5, the type and source of the cellulase are not particularly limited, the cellulase is available in the conventional market in the field, the pH value is preferably adjusted to 4.5-5.5 by using 1.0-2.0 mol/L hydrochloric acid solution, the temperature of the wall breaking enzymolysis is preferably 45-55 ℃, the time is preferably 3.5-4.5 h, the enzymolysis is preferably carried out in a water bath environment, and the cellulase is released, so that the rice bran protein is released, the yield of neutral protease and the hydrolysis efficiency of alkaline protease are improved, and the yield of the rice bran protease and the hydrolysis efficiency of the alkaline protease are improved, and the protein yield, the F value of the rice bran and the purity of the rice bran and the peptide are improved.
The method preferably comprises the steps of adjusting the pH value of the protein liquid to 9.0, performing stirring reaction, performing first centrifugation, collecting supernatant, performing PI precipitation on the supernatant, performing second centrifugation, collecting precipitate, washing and drying to obtain the rice bran protein, wherein the stirring reaction time is preferably 2-3 h, the stirring speed of the stirring reaction is not particularly limited, and the stirring reaction can be performed by using a conventional magnetic stirrer in the field.
The rice bran protein is sequentially subjected to neutral protease enzymolysis and alkaline protease enzymolysis to obtain enzymolysis polypeptide. The neutral protease of the present invention preferably includes AS1.398 neutral protease. When the neutral protease is subjected to enzymolysis, the rice bran protein and water are preferably mixed according to the mass ratio of 1: 10-12, the pH value is adjusted to 6-7, and the neutral protease is added. The neutral protease of the present invention is 5 to 6% by mass (abbreviated as [ E ]/[ S ] is 5 to 6% by mass) of the rice bran protein. The temperature of the neutral protease hydrolysis is preferably 40-50 ℃, and the time is preferably 3-4 h. The neutral protease enzymolysis of the invention is preferably carried out in a water bath environment.
The pH of the hydrolysate subjected to the neutral protease enzymolysis is preferably adjusted to 11-12, and alkaline protease is added. The alkaline protease of the present invention preferably comprises Alcalase2.4FG alkaline protease. The alkaline protease of the present invention is present in an amount of 6.5 to 7.5% by mass of the rice bran protein ([ E ]/[ S ] is 6.5 to 7.5%). The temperature of the alkaline protease enzymolysis is preferably 50-60 ℃, and the time is preferably 5-6 h. The alkaline protease enzymatic hydrolysis of the present invention is preferably carried out in a water bath environment.
The invention carries out active carbon static adsorption chromatography on the obtained hydrolyzed protein. In the invention, when the activated carbon static adsorption chromatography is carried out, the hydrolyzed protein is preferably diluted by a factor of 5-6. Before the active carbon static adsorption chromatography, the method preferably further comprises the steps of adjusting the pH value to 3 and then adding active carbon to perform the static adsorption chromatography. The temperature of the active carbon static adsorption chromatography is preferably 35-45 ℃, and the time is preferably 4.5-5.5 h. The activated carbon static adsorption chromatography of the present invention is preferably carried out in a water bath environment.
The invention centrifugally dries the adsorption solution after the active carbon static adsorption chromatography to obtain the high F value oligopeptide. The centrifugation is preferably carried out for 10-15 min at the rotating speed of 170-200 rpm. The invention also includes detecting the separation after said centrifugationOD of post-cardiac supernatant220And OD260By OD220/OD260The F value is obtained by calculating the ratio of (A) to (B). The present invention is not limited to any particular manner of concentration and drying.
In the present invention, the F value (Fischer Ratio) is the molar Ratio of branched chain amino acids (BCAA: L eu, Ile, Val) to aromatic amino acids (AAA: Phe, Tyr, Try), the high F value should be greater than 20, the maximum absorption at 220nm is determined by the oligopeptide with high branched chain amino acid content, the maximum absorption at 260nm is determined by the oligopeptide with aromatic amino acid content, and OD can be used220/OD260The F value is preliminarily judged according to the ratio of (A) to (B).
The invention also provides the high F value oligopeptide prepared by the preparation method, wherein the F value of the high F value oligopeptide is 28-32.
The high F value oligopeptide and the preparation method thereof provided by the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Adjusting pH of defatted rice bran to 4.5 with 1.0 mol/L hydrochloric acid, adding 0.3 g cellulase into per 100 g rice bran, hydrolyzing in 45 deg.C water bath for 3.5h, adjusting pH to 9.0 with 1.0 mol/L sodium hydroxide at room temperature, stirring for reaction for 3h, centrifuging at 4500rpm for 20min, collecting supernatant, adjusting pH to 4.0 (isoelectric point) with 1.0 mol/L hydrochloric acid to perform PI precipitation, centrifuging at 8000rpm in a centrifuge for 20min after precipitation is completed, washing and drying to obtain rice bran protein, adding 10 times weight of distilled water into the obtained protein, adjusting pH to 6 with 1.0 mol/L sodium hydroxide, adding AS1.398 neutral protease, and hydrolyzing in 40 deg.C water bath for 4h, wherein [ E]/[S]Regulating pH to 11.5% with 1.0 mol/L sodium hydroxide, adding Alcalase2.4FG alkaline protease, and hydrolyzing in 50 deg.C water bath for 6.5 hr, wherein [ E%]/[S]6.5 percent, diluting the hydrolysate by 5 times, adding hydrochloric acid to adjust the pH value to 3, adding active carbon, performing static adsorption chromatography in a water bath at 35 ℃ to remove aromatic amino acid in the hydrolysate, retaining branched chain amino acid, wherein the volume ratio of the mass of the active carbon to the volume of the hydrolysate is 2g/100m L, placing the adsorption solution in a centrifuge for separationThe centrifugation conditions were 180rpm for 15 minutes. Collecting supernatant, concentrating, drying to obtain rice bran high F value oligopeptide, and detecting OD with ultraviolet spectrophotometer220/OD260＝28.4。
Example 2
Adjusting pH of defatted rice bran to 5 with 10 times weight of distilled water, adding 0.4 g cellulase into per 100 g rice bran, hydrolyzing in 50 deg.C water bath for 4 hr, adjusting pH to 9.0 with 1.0 mol/L sodium hydroxide at room temperature, stirring for reaction for 2.5 hr, centrifuging at 5000rpm for 25min, collecting supernatant, adjusting pH to 4.5 (isoelectric point) with 1.0 mol/L hydrochloric acid to perform PI precipitation, centrifuging at 8500rpm in a centrifuge for 15min after precipitation is complete, washing and drying to obtain rice bran protein, adding 11 times weight of distilled water into the obtained protein, adjusting pH to 6.5 with 1.0 mol/L sodium hydroxide, adding AS1.398 neutral protease, and hydrolyzing in 50 deg.C water bath for 3.5 hr, wherein [ E]/[S]5.5%, adjusting pH to 12 with 1.0 mol/L sodium hydroxide, adding Alcalase2.4FG alkaline protease, and hydrolyzing in 55 deg.C water bath for 5.5h [ E ]]/[S]7 percent, diluting the hydrolysate by 5 times, adding hydrochloric acid to adjust the pH value to 3, adding active carbon to perform static adsorption chromatography in water bath at 40 ℃ for 4.5 to remove aromatic amino acid in the hydrolysate, reserving branched chain amino acid, wherein the volume ratio of the mass of the active carbon to the hydrolysate is 1.5g/100m L, placing the adsorption solution in a centrifuge for separation, centrifuging at the centrifugal speed of 170rpm for 15 minutes, collecting supernatant, concentrating and drying to obtain rice bran high-F-value oligopeptide, and simultaneously detecting OD (optical density) by using an ultraviolet spectrophotometer220/OD260＝29.5。
Example 3
Taking 12 times of distilled water of defatted rice bran, adjusting pH to 5.5 with 1.0 mol/L hydrochloric acid, adding 0.3 g cellulase into per 100 g rice bran, hydrolyzing in 55 deg.C water bath for 3.5h, adjusting pH to 9.0 with 1.0 mol/L sodium hydroxide at room temperature, stirring for reaction for 2h, centrifuging at 5500rpm for 15min, taking supernatant, adjusting pH to 4.5 (isoelectric point) with 1.0 mol/L hydrochloric acid to perform PI precipitation, centrifuging at 8500rpm for 15min after precipitation is completed, washing and drying to obtain rice bran protein, adding 12 times of distilled water into the obtained protein, and using 1.0mol of distilled water/L adjusting pH to 6.5 with sodium hydroxide, adding AS1.398 neutral protease, and hydrolyzing in water bath at 55 deg.C for 3 hr, wherein [ E]/[S]Regulating pH to 12 with 1.0 mol/L sodium hydroxide, adding Alcalase2.4FG alkaline protease, and hydrolyzing in 55 deg.C water bath for 5.5 hr [ E ]]/[S]7.5 percent, diluting the hydrolysate by 5 times, adding hydrochloric acid to adjust the pH value to 3, adding active carbon to remove aromatic amino acid in the hydrolysate by static adsorption chromatography and 4 in a water bath at 45 ℃, reserving branched chain amino acid, wherein the volume ratio of the mass of the active carbon to the hydrolysate is 1g/100m L, placing the adsorption solution in a centrifuge for separation, centrifuging at the medium-centrifugal condition of 200rpm for 10min, collecting supernatant, concentrating and drying to obtain rice bran high-F-value oligopeptide, and simultaneously detecting OD (optical density) by using an ultraviolet spectrophotometer220/OD260＝29.8。
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A preparation method of high F value oligopeptide is characterized by comprising the following steps: mixing defatted rice bran and cellulase, performing enzymolysis and wall breaking to obtain protein liquid, and extracting rice bran protein in the protein liquid; sequentially carrying out neutral protease enzymolysis and alkaline protease enzymolysis on the rice bran protein to obtain enzymolysis polypeptide, and carrying out active carbon static adsorption chromatography on the enzymolysis polypeptide, centrifuging and drying to obtain the high F value oligopeptide.
2. The preparation method according to claim 1, wherein the enzymatic wall breaking comprises mixing defatted rice bran with distilled water, adjusting pH to 4.5-5.5, and adding cellulase; the mass ratio of the defatted rice bran to the distilled water is 1: 8-12; the mass ratio of the defatted rice bran to the cellulase is 100: 0.3-0.5.
3. The preparation method according to claim 1 or 2, wherein the temperature of the enzymatic wall breaking is 45-55 ℃, and the time of the enzymatic wall breaking is 3.5-4.5 h.
4. The method according to claim 1, wherein the rice bran protein in the protein liquid is extracted by an alkaline process, the alkaline process comprises adjusting the pH of the protein liquid to 9.0, performing a first centrifugation after the stirring reaction, and collecting a supernatant; and performing PI precipitation on the supernatant, performing second centrifugation, collecting the precipitate, washing and drying to obtain the rice bran protein.
5. The method of claim 1, wherein the neutral protease comprises AS1.398 neutral protease; when the neutral protease is subjected to enzymolysis, the rice bran protein and water are mixed according to the mass ratio of 1: 10-12, the pH value is adjusted to 6-7, and the neutral protease is added.
6. The method according to claim 1 or 5, wherein the neutral protease is 5 to 6% by mass of the rice bran protein; the hydrolysis temperature of the neutral protease is 40-50 ℃, and the hydrolysis time is 3-4 h.
7. The production method according to claim 1, wherein the alkaline protease comprises alcalase2.4fg alkaline protease; when alkaline protease enzymolysis is carried out, adjusting the pH of hydrolysate hydrolyzed by the neutral protease to 11-12, and adding the alkaline protease; the mass of the alkaline protease is 6.5-7.5% of that of the rice bran protein; the hydrolysis temperature of the alkaline protease is 50-60 ℃, and the hydrolysis time is 5-6 h.
8. The preparation method according to claim 1, wherein the temperature of the activated carbon static adsorption chromatography is 35-45 ℃ and the time is 4.5-5.5 h.
9. The method of claim 1, further comprising, after the centrifugation, detecting the OD of the supernatant after the centrifugation220And OD260By OD220/OD260The F value is obtained by calculating the ratio of (A) to (B).
10. The high F oligopeptide according to any one of claims 1 to 9, wherein the high F oligopeptide has an F value of 28 to 32.
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| CN113881561A (en) * | 2021-05-07 | 2022-01-04 | 东北农业大学 | Method for producing rice bran protein polypeptide by using adjustable enzyme membrane reactor |
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Application publication date: 20200724 |