CN114921517A - Preparation method of millet antioxidant active peptide - Google Patents
Preparation method of millet antioxidant active peptide Download PDFInfo
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- 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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- 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
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Abstract
The invention relates to a preparation method of millet antioxidant active peptide, which comprises the following steps: extracting and purifying millet protein, and performing enzymolysis and polypeptide separation on the millet protein. The antioxidant activity test result shows that the millet polypeptide prepared by the invention has high clearance rate on ABTS free radicals, and the clearance rate is up to 84% when the concentration of the millet polypeptide is 1 mg/ml. The invention aims to lay a solid foundation for the subsequent research and development of the antioxidant polypeptide into functional health-care food, further improve the additional value of millet, improve the comprehensive utilization level of millet and promote the development of the millet industry.
Description
Technical Field
The invention relates to the technical field of polypeptide preparation, and particularly relates to a preparation method of millet antioxidant active peptide.
Background
The bioactive peptide is a polypeptide substance which naturally exists in organisms such as plants, animals, microorganisms and the like, or is obtained by carrying out protease enzymolysis on animal and plant proteins, artificial chemical synthesis or a biological engineering method, has various special physiological activities, and is a general name of low molecular peptides or polypeptide substances with 20 natural amino acids in proteins, wherein the low molecular peptides or polypeptide substances are composed of dipeptide and complex linear and annular structures by amido bonds. Bioactive peptides have multiple physiological functions, and the bioactivity of the bioactive peptides changes along with the difference of the composition and the arrangement sequence of amino acids. Studies have shown that the physiological functions of antiproliferative, antimicrotubulin and cytotoxic bioactive peptides make them potentially anticancer, which would have immeasurable effects in cancer therapy. Researches show that the polypeptide obtained by hydrolyzing the protein has better nutrition than the original protein; the protein is not necessarily absorbed in the form of free amino acids but mainly in the form of low peptides (especially di-, tri-peptides) after enzymatic action in the digestive tract. Biological metabolism experiments also prove that the absorptivity of the low peptide is really better than that of free amino acid, and the low peptide is easier to be absorbed and utilized by organisms through small intestinal mucosa than the amino acid. The osmotic pressure of the low peptide is lower than that of free amino acid, and adverse reactions such as diarrhea and the like can not be caused after eating; meanwhile, the oligopeptide has lower antigenicity and cannot cause anaphylactic reaction. Thus, low peptides can be used to supplement proteins for special populations that do not adequately meet the protein needs of their diet. Therefore, the low peptide has extremely high edible safety and is the hottest research topic and the functional factor with great development prospect in the current global food world.
Millet is a traditional crop in China, has a long history and a wide planting area which is far higher than that of other grains. Millet is rich in various nutrients, such as protein, minerals, dietary fibers, antioxidants and other nutrients, and has a content which is equivalent to or even higher than that of other traditional grains. The millet essence has the effects of nourishing yin, tonifying kidney, clearing heat, promoting urination and nourishing stomach, and is popular with people in China. However, the processing mode of millet is single at home and abroad at present, and the research on the functionality of millet is deficient, and the research aims to explore the physiological function of millet, improve the nutritional value of millet and develop the market of millet food.
The millet protein resource in China is rich, but the development and utilization of the high added value of the millet protein are far from enough due to the limitation of the technology and the like, so that the deep research and development of a new technology for preparing the millet protein peptide and the overcoming of main influence factors in the millet protein peptide preparation process have important significance for the development of the whole millet processing industry. The development and application of the new process and the new technology can greatly improve the deep processing and comprehensive utilization level of the millet at home and abroad. The industrialization technical level of millet protein and millet peptide is improved, so that the economic benefit and competitiveness of processing enterprises are greatly improved, the acquisition of millet by the enterprises is driven, and the price of millet is stabilized and improved.
Disclosure of Invention
In order to make up for the defects of the prior art, the invention aims to provide a preparation method of millet antioxidant polypeptide.
In order to solve the problems, the invention provides the following technical scheme:
1. a preparation method of millet antioxidant active peptide comprises the following steps: extracting and purifying millet protein, and performing enzymolysis and polypeptide separation on the millet protein; it is characterized in that the preparation method is characterized in that,
the method for extracting and purifying the millet protein comprises the following steps:
weighing a certain amount of millet, crushing by using a tissue triturator, sieving by using a 80-mesh sieve, and mixing according to a material-liquid ratio of 1: 3-5 adding n-hexane, mixing, stirring at room temperature for 2-4h, standing for 1-3h, repeating for 2-3 times, pouring back supernatant, and air drying the precipitate in a fume hood to obtain defatted millet flour; adding the rice flour into millet flour according to the proportion of 1: 10-15, adding distilled water, adjusting the pH to 8-10 by using 1mol/L NaOH solution, then carrying out ultrasonic treatment for 30-50min, magnetically stirring for 1-2h, centrifuging for 10-15min at 8000r/min with 5000-;
the enzymolysis and polypeptide separation of the millet protein comprise the following steps:
dissolving millet protein powder into 4-6% (w/v) solution, adjusting the pH value to 9-10, heating in boiling water bath for 15-30min, cooling to 55-65 ℃, adding Alcalase3.0T alkaline protease with 8000u/g substrate of 5000 plus materials, reacting for 1-2h, rapidly heating to 100 ℃ to inactivate enzyme for 10-15min, cooling to 40-50 ℃, adjusting the pH value to 7.5-8.5, adding 3000u/g substrate of neutral protease, reacting for 2-4h, rapidly heating to 100 ℃ to inactivate enzyme for 10-15min, cooling to 7000r/min of 5000 plus materials, centrifuging for 15-25min, centrifuging, taking supernatant, and freeze-drying to obtain the millet antioxidant active peptide.
Preferably, each time before freeze-drying, the freeze-dried powder needs to be pre-frozen at-80-60 ℃ for 18-24h, and then freeze-dried for 24-30 h.
The millet active peptide prepared by the preparation method of the millet antioxidant active peptide has a peptide segment of 0.5-1 KD.
The combination reaction of two enzymes according to the invention can produce millet protein degradation polypeptide with good oxidation resistance in the range of 0.5-1KD, and other combination conditions are not feasible.
The millet is used as a raw material, the protein in the millet is extracted by an alkali dissolution and acid precipitation method and ultrasonic assistance, the millet protein is hydrolyzed by a compound enzyme method to obtain millet polypeptide, a four-factor three-level orthogonal test is designed by taking the polypeptide yield as an index on the basis of protease screening and single-factor influence determination experiments to optimize the process for preparing the millet polypeptide by carrying out enzymolysis on the millet protein by neutral protease and Alcalase3.0T alkaline protease, the optimal process parameters are selected, and finally the obtained millet active peptide segment is 0.5-1 KD.
The invention has the advantages that:
(1) the millet protein is hydrolyzed in multiple stages by adopting the compound enzyme method, so that the hydrolysis degree of the millet protein and the utilization rate of enzyme are improved.
(2) The millet polypeptide prepared by the invention has the advantages of simple preparation method and operation and mild treatment conditions.
(3) According to the enzymology characteristics of the hydrolytic protease, the pH value, the hydrolysis temperature and the hydrolysis time of the protein solution are adjusted, and the hydrolysis environment of the enzyme is controlled in stages. The method not only improves the hydrolysis activity of the enzyme, but also reduces the enzyme dosage, shortens the enzymolysis time and saves the energy consumption.
(4) The millet polypeptide with high antioxidant activity can be obtained by double-enzyme compound hydrolysis of millet protein, and is a novel natural antioxidant, and the method combines the measurement of reducibility, so that the yield of the millet polypeptide reaches 28.5 percent.
The millet polypeptide prepared by the invention has high clearance rate on ABTS free radicals, and when the concentration of the millet polypeptide is 1mg/ml, the clearance rate is as high as 84%.
Drawings
FIG. 1 is a process for preparing millet polypeptide
FIG. 2 shows millet polypeptide powder prepared in example 1 of the present invention
FIG. 3 shows the reducing power of millet polypeptide and Vc
FIG. 4 shows the effect of millet polypeptide and Vc on the elimination of ABTS free radicals.
Detailed Description
The following examples are presented to enable one of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way.
Example 1
(1) Extraction of millet protein
Step 1: weighing 500g of millet, crushing by using a tissue triturator, and sieving by using a 80-mesh sieve;
step 2: adding the millet flour treated in the step 1 into the mixture according to the material-liquid ratio of 1: 3 adding n-hexane, mixing, stirring at room temperature for at least 2h, standing for 1h after stirring, removing supernatant (yellow, containing n-hexane, water and fat), adding n-hexane, stirring, repeating the above steps, removing supernatant, and air drying the precipitate in a fume hood to obtain defatted millet flour.
And step 3: adding the millet flour treated in the step 1 into a mixture according to the material-liquid ratio of 1: adding distilled water into 10, adjusting the pH value of the system to 10 by using 1mol/L NaOH solution, then carrying out ultrasonic treatment for 30min, magnetically stirring for 2h at the constant temperature by using a digital display, centrifuging for 15min at 5000r/min, taking supernatant, adjusting the pH value to 3 (the isoelectric point of millet protein) by using 1mol/L hydrochloric acid, collecting precipitate, dialyzing to remove salt, pre-freezing for 24h at-60 ℃, and freeze-drying for 30 h.
(2) Preparation of millet antioxidant active peptide
And (2) adding distilled water into the millet protein powder obtained in the first step to prepare a millet protein solution with the concentration of 4% (w/v), fully stirring, adjusting the pH value to 10 by using a 1mol/LNaOH solution, heating to 100 ℃, and maintaining for 15min to ensure that the millet protein structure becomes loose and is convenient for enzymolysis.
Cooling to the appropriate reaction temperature of alkaline protease 65 ℃, adding 5000u/g Alcalase3.0T alkaline protease, reacting for 2h, rapidly raising the temperature to 100 ℃ to inactivate the enzyme for 10min, then cooling to 50 ℃, adjusting the pH to 8.5, adding 2000u/g neutral protease, reacting for 2h, rapidly raising the temperature to 100 ℃ to inactivate the enzyme for 10min, centrifuging for 25min at 5000r/min, taking the supernatant, pre-freezing for 24h at-60 ℃, and then freeze-drying for 30 h. Measuring the millet polypeptide peptide segment by 0.5-1 KD.
Example 2
(1) Extraction of millet protein
Step 1: weighing 500g of millet, crushing by using a tissue triturator, and sieving by using a 80-mesh sieve;
step 2: adding the millet flour treated in the step 1 into a mixture according to the material-liquid ratio of 1: 4 adding n-hexane, mixing, stirring at room temperature for 5h, standing for 3h after stirring, decanting the supernatant, repeating for 2 times, decanting the supernatant, and air drying the precipitate in a fume hood to obtain defatted millet flour.
And step 3: adding the millet flour treated in the step 1 into a mixture according to the material-liquid ratio of 1: 15 adding distilled water, then adjusting the pH value of the system to 9 by using 1mol/L NaOH solution, then carrying out ultrasonic treatment for 50min, stirring for 1h by using constant-temperature magnetic force at normal temperature, then centrifuging for 10min at 8000r/min, taking supernate, adjusting the pH value to 4 (isoelectric point of millet protein) by using 1mol/L hydrochloric acid, then collecting precipitate, dialyzing to remove salt, prefreezing for 18h at-80 ℃, and then freeze-drying for 24 h.
(2) Preparation of millet antioxidant active peptide
And (2) adding distilled water into the millet protein powder obtained in the first step to prepare a millet protein solution with the concentration of 6% (w/v), fully stirring, adjusting the pH value to 9 by using a 1mol/LNaOH solution, heating to 100 ℃, and maintaining for 30min to ensure that the millet protein structure becomes loose and is convenient for enzymolysis.
Cooling to 55 ℃ which is suitable for the alkaline protease, adding 8000u/g Alcalase3.0T alkaline protease, reacting for 1h, quickly raising the temperature to 100 ℃ to inactivate the enzyme for 15min, then cooling to 40 ℃, adjusting the pH to 7.5, adding 3000u/g neutral protease, reacting for 4h, quickly raising the temperature to 100 ℃ to inactivate the enzyme for 15min, centrifuging at 7000r/min for 15min, taking the supernatant, pre-freezing at-80 ℃ for 18h, and freeze-drying for 24 h. Measuring millet polypeptide peptide segment 0.5-1 KD.
The invention prepares the polypeptide of 0.5-1 peptide fragment with high yield for the first time, and other methods have not high peptide fragment yield in the range, and the polypeptide with antioxidant activity in the range has the best effect and high free radical clearance rate.
Functional evaluation
1. Determination of reduction power of millet antioxidant active peptide
The millet polypeptide prepared in example 1 and Vc were adjusted to concentrations of 0.05, 0.1, 0.5, 1.0, 5.0, 10.0, 25.0, and 50.0mg/mL, 1mL of millet peptide solutions of different concentrations (blank was replaced with 1mL of distilled water) were taken, 2.5mL of 1% potassium ferricyanide solution and 2.5mL of phosphate buffer (0.2mol/L) with pH6.6 were added, and the mixture was reacted in a water bath at 50 ℃ for 20 min. Then, 2.5mL of 10% trichloroacetic acid (TCA) solution was added to the mixture, and after mixing, the mixture was centrifuged at 3000r/min for 10 min. 2.5mL of the supernatant was taken, and 2.5mL of distilled water and 0.5mL of 0.1% ferric chloride were added. The solution was mixed well and left to stand for 10min, and then the absorbance was measured at 700 nm. Higher absorbance indicates higher reducing power. All assays were repeated three times.
It can be seen from fig. 3 that the control group Vc has a strong antioxidant effect. In the concentration range selected by the experiment, the millet polypeptide has weaker reducing power at low concentration, and the reducing power is gradually increased along with the increase of the concentration, which shows that the reducing power of the millet polypeptide has better dose-effect relationship with the concentration.
2. Determination of ABTS free radical clearance rate of millet antioxidant active peptide
5mL of 7mmol/L ABTS solution and 88 μ L of 140mmol potassium permanganate are taken. Standing overnight at room temperature in the dark to form ABTS free radical stock solution, and diluting with phosphate buffer solution of pH7.4 to A 734 0.700 +/-0.002 to prepare ABTS + Working fluid. Then taking ABTS + 4mL of working solution, 1mL of millet peptide solution was added, and after thorough mixing, the mixture was allowed to stand at 25 ℃ for 6min, and the absorbance A was measured at 734nm Sample (A) Simultaneously fetch 4mLABTS + Working solution, 1mL of ethanol solution was added thereto, and the absorbance A was measured at 734nm Control . Then ABTS + The clearance rate was:
the ability of the millet antioxidant active peptide to ABTS free radicals is shown in figure 4, and the millet polypeptide and Vc both have good effect of removing ABTS free radicals, the removal rate of the millet polypeptide and Vc to ABTS free radicals is higher and higher along with the increase of the concentration in the concentration range selected by experiments, and the concentration of the millet polypeptide reaches 84% when the concentration is 1mg/ml, so that the millet polypeptide has strong effect of removing ABTS free radicals.
The above examples are preferred embodiments of the present invention, but the present invention is not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (3)
1. A preparation method of millet antioxidant active peptide comprises the following steps: the extraction and purification of the millet protein, the enzymolysis and the polypeptide separation of the millet protein are characterized in that,
the method for extracting and purifying the millet protein comprises the following steps:
weighing a certain amount of millet, crushing by using a tissue triturator, sieving by using a 80-mesh sieve, and mixing according to a material-liquid ratio of 1: 3-5, adding n-hexane, mixing, fully stirring at room temperature for 2-4h, standing for 1-3h, repeating for 2-3 times, pouring out supernatant, and air drying the precipitate in a fume hood to obtain defatted millet flour; adding the rice flour into millet flour according to the proportion of 1: 10-15, adding distilled water, adjusting the pH to 8-10 by using 1mol/L NaOH solution, performing ultrasonic treatment for 30-50min, magnetically stirring for 1-2h, centrifuging for 10-15min at 8000r/min, taking supernatant, adjusting the pH to 3-4 by using 1mol/L hydrochloric acid, collecting precipitate, dialyzing to remove salt, and finally performing freeze drying to obtain millet protein powder;
the enzymolysis and polypeptide separation of the millet protein comprise the following steps:
dissolving millet protein powder into 4-6% (w/v) solution, adjusting the pH value to 9-10, heating in boiling water bath for 15-30min, cooling to 55-65 ℃, adding Alcalase3.0T alkaline protease with 8000u/g substrate of 5000 plus materials, reacting for 1-2h, rapidly heating to 100 ℃ to inactivate enzyme for 10-15min, cooling to 40-50 ℃, adjusting the pH value to 7.5-8.5, adding 3000u/g substrate of neutral protease, reacting for 2-4h, rapidly heating to 100 ℃ to inactivate enzyme for 10-15min, cooling to 7000r/min of 5000 plus materials, centrifuging for 15-25min, centrifuging, taking supernatant, and freeze-drying to obtain the millet antioxidant active peptide.
2. The preparation method of the millet antioxidant active peptide according to claim 1, characterized in that: before each freeze drying, pre-freezing at-80-60 deg.C for 18-24 hr, and freeze-drying for 24-30 hr.
3. The millet active peptide prepared by the method for preparing millet antioxidant active peptide according to any one of claims 1 or 2 has a peptide fragment of 0.5-1 KD.
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| CN101168764A (en) * | 2007-11-05 | 2008-04-30 | 中华全国供销合作总社南京野生植物综合利用研究院 | Method for producing anti-oxidation active peptide by glutelin powder biological enzyme method |
| CN106148468A (en) * | 2016-09-20 | 2016-11-23 | 湖南农业大学 | The method that the double enzyme enzymolysis grain germ of a kind of irradiation auxiliary prepares anti-oxidation peptide |
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| US20200140913A1 (en) * | 2018-11-01 | 2020-05-07 | Kansas State University Research Foundation | Antioxidant protein hydrolysates and peptides from cereal grain crops |
| CN114106083A (en) * | 2021-12-09 | 2022-03-01 | 南开大学 | Preparation method of millet protein and millet protein hydrolysate and application of millet protein hydrolysate |
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| US20200140913A1 (en) * | 2018-11-01 | 2020-05-07 | Kansas State University Research Foundation | Antioxidant protein hydrolysates and peptides from cereal grain crops |
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