CN118318910A - Preparation method and application of colored rice bran protein hydrolysate - Google Patents
Preparation method and application of colored rice bran protein hydrolysate Download PDFInfo
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Abstract
The invention discloses a preparation method of colored rice bran protein hydrolysate, which comprises the steps of placing defatted colored rice bran in alkaline solution, stirring and extracting at normal temperature after ultrahigh pressure treatment, carrying out solid-liquid separation, adjusting the pH of an extracting solution to 4-5, standing, carrying out solid-liquid separation, and washing and drying solids to obtain colored rice bran protein; sequentially hydrolyzing the colored rice bran protein by using pepsin, neutral protease and trypsin, separating the hydrolysate by using an ultrafiltration membrane, and drying the separated liquid to obtain colored rice bran protein hydrolysate; the colored rice bran protein hydrolysate prepared by the method has the functional characteristics of resisting oxidation and reducing blood sugar, and the extraction rate and the hydrolysis degree of the purple rice bran protein and the red rice bran protein are higher, so that the colored rice bran protein hydrolysate can be used for developing milk powder, protein powder and other protein functional foods for middle-aged and elderly people, and can also be used for preparing an antioxidant additive and preparing an alpha-glycosidase inhibitor.
Description
Technical Field
The invention relates to a preparation method and application of a colored rice bran protein hydrolysate, belonging to the field of functional foods.
Background
Rice bran is a byproduct of the rice milling process and accounts for about 10% of the total weight of the brown rice. A large amount of rice bran is used as animal feed cheaply per year without being effectively utilized. Rice bran is mainly composed of five parts: the nutritional ingredients of the epicarp, the mesocarp, the crosslinked layer, the seed coat and the aleurone layer are mainly protein, fat, dietary fiber, vitamins and minerals, and the research reports that the nutritional ingredients in the rice bran account for about 64% of the total nutritional ingredients in the rice. The rice bran protein is a high-quality plant protein, mainly comprises albumin, globulin, prolamin and glutelin, and has the nutrition characteristics of reasonable amino acid composition structure, high biological potency and low anaphylaxis. Furthermore, colored rice varieties exhibit different colors due to the high amount of anthocyanin contained in their bran, purple and red being relatively most common. Compared with common rice bran, the colored rice bran contains a large amount of anthocyanin and other substances, and the anthocyanin has good biological activity in the aspects of cardiovascular disease resistance, inflammation resistance, oxidation resistance, tumor resistance, obesity prevention, blood sugar reduction and the like, so the functional activity of the colored rice bran protein is relatively better, and the colored rice bran protein is more beneficial to the health of organisms.
The rice bran protein has strong hydrophobicity and is not easy to be absorbed and utilized by organisms, but under the action of enzyme, the rice bran protein can be decomposed into small molecular polypeptide chains with different lengths, and the hydrolysate can be absorbed and utilized by the organisms better and can also show better biological activity.
Disclosure of Invention
The invention provides a preparation method of a colored rice bran protein hydrolysate; the research of the invention discovers that after the purple rice bran and red rice bran proteins are continuously hydrolyzed by three proteases, the hydrolysate contains various functional peptides, such as antioxidant peptides and hypoglycemic peptides. Specifically, the defatted purple rice bran and red rice bran are extracted by an super-alkaline method to obtain purple rice bran protein and red rice bran protein, then pepsin, neutral protease and trypsin are used for continuous hydrolysis, after the hydrolysis is completed, an ultrafiltration membrane is used for filtration, and the filtrate is freeze-dried to obtain the hydrolysate of the purple rice bran and red rice bran protein. The detection shows that the components of the hydrolysate are mainly polypeptides, the content of free amino acids is very small, the hydrolysate does not need to be separated to remove the free amino acids, and the hydrolysate is also called polypeptides. The purple rice bran polypeptide and the red rice bran protein polypeptide have various functional characteristics such as antioxidation, blood sugar reduction and the like, and the extraction rate and the hydrolysis degree of the purple rice bran and the red rice bran protein are high, so that the purple rice bran polypeptide and the red rice bran protein polypeptide can be used for developing infant milk powder, protein powder and other protein functional foods.
The invention is realized by the following steps:
(1) Ultrahigh pressure assisted extraction of protein: degreasing rice bran (adopting a conventional cyclohexane degreasing method), weighing defatted rice bran (defatted purple rice bran and defatted red rice bran), respectively placing into a composite bag, adding NaOH solution with pH of 11-12, sealing, placing into an ultrahigh pressure treatment bin, performing pressure maintaining treatment under the pressure of 450-550 MPa for 10-20 min for ultrahigh pressure treatment, taking out after treatment, extracting for 2-3 h under stirring at normal temperature, filtering to obtain filtrate, centrifuging the filtrate to remove sediment, and collecting supernatant; regulating pH of the supernatant to 4-5 with HCl solution, standing for 25-35 min, separating solid from liquid, removing liquid, washing the solid with distilled water, centrifuging, collecting precipitate, pre-freezing at-40deg.C, and lyophilizing with a lyophilizing machine to obtain purple rice bran protein or red rice bran protein;
the ratio of the mass (g) of the defatted rice bran to the volume (mL) of the NaOH solution is 1:13-17;
。
(2) Preparing colored rice bran protein into a protein solution with the mass concentration of 2% -6% by taking water as a solvent, then adjusting the pH of the protein solution to 2-3 by using an HCl solution, adding pepsin (250U/mg) with the volume of 0.004% -0.008% of the protein solution, and vibrating in a constant-temperature water bath oscillator at 37 ℃ for 80-120 min under the condition of avoiding light. After the shaking is finished, the pH of the hydrolysis solution is quickly adjusted to 7.0 by using 1mol/L NaOH solution, then neutral protease (50U/mg) accounting for 0.04-0.06% of the volume of the protein solution is added, and the hydrolysis solution is shaken for 80-120 min in a constant-temperature water bath under the condition of 50 ℃. After neutral proteinase digestion is completed, the pH value of the solution is adjusted to 8.5, when the temperature of the solution is reduced to 37 ℃, trypsin (the enzyme activity is 6500U/mL) accounting for 0.001-0.002 percent of the volume of the protein solution is added, and the solution is oscillated in a constant temperature water bath oscillator at 37 ℃ to 80-120 min. After digestion, inactivating enzyme by boiling in water bath, cooling to normal temperature, centrifuging, collecting supernatant, passing through 3000Da membrane, collecting permeate, pre-freezing at-20deg.C, and lyophilizing with a lyophilizing machine to obtain purple rice bran protein hydrolysate or red rice bran protein hydrolysate;
。
functional determination of the colored rice bran protein peptide:
(1) Measurement of antioxidant Activity: and (3) determining DPPH and ABTS free radical clearance rate of the rice bran protein polypeptide of purple rice and the rice bran protein polypeptide of red rice by referring to the existing experimental method, and calculating the IC 50 value.
(2) Determination of alpha-glucosidase: and determining the inhibition rate of the purple rice bran and red rice bran protein polypeptide to alpha-glycosidase by referring to the existing experimental method, and calculating the IC 50 value.
The invention also aims to apply the colored rice bran protein hydrolysate prepared by the method to the preparation of an antioxidant additive or the preparation of an alpha-glycosidase inhibitor.
The invention has the beneficial effects that:
(1) The extraction rate of the purple rice bran protein or the red rice bran protein is improved to a certain extent by the ultra-high pressure auxiliary alkaline extraction;
(2) The digestion rate of the purple rice bran protein or the red rice bran protein is improved through continuous sequential hydrolysis of three proteases;
(3) The protein polypeptide has good antioxidant and hypoglycemic activities, and meanwhile, the extraction rate of the purple rice bran and red rice bran protein is high, so that the purple rice bran and red rice bran protein polypeptide has wide market application prospect; therefore, the invention has innovation, economy and practicability.
Detailed Description
The invention is further illustrated by the following examples, but the scope of the invention is not limited to the details, and the methods in the examples are conventional unless otherwise specified;
Example 1
(1) Weighing 100g of defatted purple rice bran and 100g of defatted red rice bran respectively, placing into a composite bag, adding 1300mL of NaOH solution with pH of 11, sealing, placing into an ultrahigh pressure treatment bin, treating for 20min under 450MPa, taking out, stirring at normal temperature, extracting for 2h, filtering to obtain filtrate, centrifuging the filtrate to remove precipitate, and collecting supernatant; regulating pH of the supernatant to 4.7 with HCl solution, standing for 30min, centrifuging, and discarding supernatant; shaking the precipitate with distilled water, centrifuging, collecting precipitate, pre-freezing at-40deg.C, and lyophilizing with a freeze dryer to obtain purple rice bran protein and red rice bran protein with 24.1% and 16.5% of the obtained red rice bran protein;
(2) Respectively weighing purple rice bran protein and red rice bran protein, preparing 500mL of protein solution with the mass concentration of 2%, adjusting the pH of the protein solution to 2 by using HCl solution, adding pepsin (namely 0.02 mL) with the volume of 0.004% of the protein solution, and vibrating in a constant-temperature water bath oscillator at 37 ℃ for 80min under the condition of avoiding light. After the shaking is finished, the pH of the hydrolysis solution is quickly adjusted to 7.0 by using 1mol/L NaOH solution, then neutral protease (namely 0.2 mL) accounting for 0.04 percent of the volume of the protein solution is added, and the hydrolysis solution is subjected to shaking for 80 minutes in a constant-temperature water bath at 50 ℃; after neutral proteinase digestion is completed, regulating the pH value of the solution to 8.5, when the temperature of the solution is reduced to 37 ℃, adding trypsin (0.005 mL) accounting for 0.001% of the volume of the protein solution, vibrating in a constant-temperature water bath vibrator for 80min, after the hydrolysis is completed, boiling in a water bath to inactivate enzymes, cooling to normal temperature after the inactivation is completed, centrifugally separating, collecting supernatant fluid, passing through a 3000Da membrane, collecting permeate liquid, pre-freezing in a refrigerator at-20 ℃, and freeze-drying by a freeze dryer to obtain purple rice bran protein and red rice bran protein polypeptide, wherein the yield of the purple rice bran protein polypeptide is 15.6%, and the yield of the red rice bran protein polypeptide is 7.4%.
Example 2
(1) Weighing 100g of defatted purple rice bran and defatted red rice bran respectively, putting into a composite bag, adding 1500mL of NaOH solution with pH of 11.5, sealing, putting into an ultrahigh pressure treatment bin, treating for 15min under 500MPa, taking out after treatment, extracting for 2.5h under stirring at normal temperature, filtering to obtain filtrate, centrifuging the filtrate to remove precipitate, and collecting supernatant; regulating pH of the supernatant to 4.7 with HCl solution, standing for 30min, centrifuging, and discarding supernatant; shaking the precipitate with distilled water, centrifuging, collecting precipitate, pre-freezing at-40deg.C, and lyophilizing with a freeze dryer to obtain purple rice bran protein and red rice bran protein, wherein the yield of purple rice bran protein is 26.3% and the yield of red rice bran protein is 17.7%;
(2) Respectively weighing purple rice bran protein and red rice bran protein, preparing 500mL of protein solution with the mass concentration of 4%, adjusting the pH of the protein solution to 2.5 by using HCl solution, adding pepsin (namely 0.03 mL) with the volume of 0.006% of the protein solution, vibrating 90 min in a constant temperature water bath vibrator at 37 ℃ under the condition of avoiding light, quickly adjusting the pH of a hydrolysis solution to 7.0 by using 1mol/L NaOH solution after the vibration is finished, then adding neutral protease (namely 0.25 mL) with the volume of 0.05% of the protein solution, vibrating 90 min in a constant temperature water bath at 50 ℃, adjusting the pH of the solution to 8.5 after digestion of the neutral protease is finished, adding trypsin (namely 0.0075 mL) with the volume of 0.0015% of the protein solution when the temperature of the solution is reduced to 37 ℃, and vibrating 90 min in the constant temperature water bath vibrator at 37 ℃. After digestion, inactivating enzyme by boiling in water bath, cooling to normal temperature, centrifuging, collecting supernatant, passing through 3000Da membrane, collecting permeate, pre-freezing at-20deg.C, and lyophilizing with a lyophilizing machine to obtain purple rice bran protein polypeptide and red rice bran protein polypeptide; the yield of the purple rice bran protein polypeptide is 18.6%, and the yield of the red rice bran protein polypeptide is 8.9%.
Example 3
(1) Weighing defatted rice bran and defatted red rice bran respectively, placing into a composite bag, adding 1700mL of NaOH solution with pH of 12, sealing, placing into an ultrahigh pressure treatment bin, performing ultrahigh pressure treatment under 550MPa for 10min, taking out after treatment, extracting under stirring at normal temperature for 3h, filtering to obtain filtrate, centrifuging the filtrate to remove precipitate, and collecting supernatant; regulating pH of the supernatant to 4.7 with HCl solution, standing for 30min, centrifuging, and discarding supernatant; shaking the precipitate with distilled water, centrifuging, collecting precipitate, pre-freezing at-40deg.C, and lyophilizing with a freeze dryer to obtain purple rice bran protein and red rice bran protein with yield of 28.7% and 18.2%;
(2) Respectively weighing purple rice bran protein and red rice bran protein, preparing 500mL of protein solution with the mass concentration of 6%, adjusting the pH of the protein solution to 3 by using HCl solution, adding pepsin (namely 0.04 mL) with the volume of 0.008% of the protein solution, and vibrating for 120min in a constant-temperature water bath oscillator at 37 ℃ under the condition of avoiding light. After the shaking, the pH of the hydrolysis solution was rapidly adjusted to 7.0 with 1 mol/L NaOH solution, followed by the addition of neutral protease (i.e., 0.3 mL) in an amount of 0.06% by volume of the protein solution, and shaking in a constant temperature water bath at 50℃for 120min. After the neutral protease digestion is completed, the pH of the solution is adjusted to 8.5, when the temperature of the solution is reduced to 37 ℃, trypsin (namely 0.01 mL) solution with the volume of 0.002% of the protein solution is added, and the solution is vibrated for 120min in a constant-temperature water bath vibrator at 37 ℃. After digestion is completed, the mixture is boiled in water bath for 10min for inactivation, after the inactivation is completed, the mixture is cooled to normal temperature, centrifugal separation is carried out, supernatant fluid is collected, the supernatant fluid passes through a 3000Da membrane, permeate fluid is collected, the permeate fluid is pre-frozen in a refrigerator at the temperature of minus 20 ℃, and freeze-drying is carried out by a freeze dryer, so that the purple rice bran protein polypeptide and red rice bran protein polypeptide are obtained, the yield of the purple rice bran protein polypeptide is 19.5%, and the yield of the red rice bran protein polypeptide is 9.3%.
Examples
The method of the embodiment is the same as that of the embodiment 1, except that the mass volume ratio of defatted rice bran to NaOH solution is 1:15, the pH=12 of the extracting solution, the ultrahigh pressure is 550MPa, the ultrahigh pressure treatment time is 20min, the stirring extraction time is 3h at normal temperature, the yield of purple rice and rice bran protein is 29.5%, and the yield of red rice and rice bran protein is 19.2%;
The hydrolysis method is the same as in example 1, except that the mass concentration of the protein solution is 3.6%, the pH is 2, the added amount of pepsin is 0.005% of the volume of the protein solution, and the hydrolysis method is carried out for 90min in a constant-temperature water bath oscillator at 37 ℃ under the condition of avoiding light; the addition amount of neutral protease is 0.06% of the volume of the protein solution, the optimal temperature is 50 ℃, the pH is 7, and the water bath is oscillated for 120min; the optimal pH of trypsin is 8.5, the adding amount is 0.002% of the volume of protein solution at the proper temperature of 37 ℃, and the trypsin is vibrated for 120min in a constant-temperature water bath vibrator. Under the secondary condition, the yield of the purple rice bran protein polypeptide is 20.7 percent, and the yield of the red rice bran protein polypeptide is 9.8 percent.
Example 5: functional assay of colored rice bran proteolytic peptides
The rice bran protein hydrolysates obtained in the above examples were mixed uniformly and their functional activities were measured. Determining DPPH and ABTS free radical clearance rate of the colored rice bran protein polypeptide by referring to the existing method, and calculating IC 50 value by the inhibition rate of alpha-glycosidase;
The results of DPPH and ABTS radical clearance IC 50 of the purple rice bran polypeptide and the red rice bran protein polypeptide are shown in Table 1, and the results show that the purple rice bran polypeptide and the red rice bran protein polypeptide have excellent antioxidant capacity;
TABLE 1 IC 50 values for purple rice bran polypeptide and red rice bran protein polypeptide DPPH and ABTS
The different superscript letters in the same column indicate that the difference is statistically significant (p < 0.05).
The IC50 values of the inhibition rates of alpha-glycosidases of the purple rice bran polypeptide and the red rice bran protein polypeptide are shown in Table 2, and the results show that the purple rice bran polypeptide and the red rice bran protein polypeptide have a certain inhibition rate of alpha-glycosidases;
2. IC 50 values of inhibition ratios of purple Rice bran polypeptide and Red Rice bran protein polypeptide alpha-glycosidases of different examples
The different superscript letters in the same column indicate that the difference is statistically significant (p < 0.05).
Claims (6)
1. A preparation method of colored rice bran protein hydrolysate is characterized by comprising the following steps: placing defatted colored rice bran in an alkaline solution, performing ultrahigh pressure treatment, stirring and extracting at normal temperature, performing solid-liquid separation, adjusting the pH of an extracting solution to 4-5, standing, performing solid-liquid separation, and washing and drying solids to obtain colored rice bran protein; sequentially hydrolyzing the colored rice bran protein by using pepsin, neutral protease and trypsin, separating the hydrolysate by using an ultrafiltration membrane, and drying the separating liquid to obtain the colored rice bran protein hydrolysate.
2. The method for preparing colored rice bran protein hydrolysate according to claim 1 wherein: the alkaline solution is NaOH solution with pH value of 11-12.
3. The method for preparing colored rice bran protein hydrolysate according to claim 1 wherein: the ultrahigh pressure treatment is carried out for 10-20 min under the pressure of 450-550 MPa.
4. The method for preparing colored rice bran protein hydrolysate according to claim 1 wherein: preparing a solution with the mass concentration of 2% -6% from colored rice bran protein, regulating the pH value of the protein solution to 2-3, adding pepsin with the volume of 0.004% -0.008% into the protein solution, and oscillating for 80-120 min in a constant-temperature water bath at 37 ℃ in the absence of light; then adjusting the pH of the hydrolysate to 7.0, adding neutral protease accounting for 0.04-0.06% of the volume of the protein solution, and oscillating in a constant-temperature water bath at 50 ℃ for 80-120 min; regulating the pH of the hydrolysate to 8.5, adding trypsin with the volume of 0.001-0.002% of the protein solution when the temperature of the solution is reduced to 37 ℃, oscillating 80-120 min in a constant-temperature water bath at 37 ℃, boiling the solution in the water bath to inactivate enzymes, cooling to normal temperature, centrifugally separating, collecting supernatant, passing the supernatant through a 3000Da membrane, and collecting the permeate for freeze drying to obtain the colored rice bran protein hydrolysate.
5. Use of the colored rice bran protein hydrolysate prepared by the method for preparing colored rice bran protein hydrolysate of any one of claims 1 to 4 for preparing an antioxidant additive.
6. Use of the colored rice bran protein hydrolysate prepared by the method for preparing colored rice bran protein hydrolysate of any one of claims 1 to 4 for preparing an alpha-glucosidase inhibitor.
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118318910A true CN118318910A (en) | 2024-07-12 |
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