CN111485010A - Preparation method of protein for lowering cholesterol - Google Patents
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- CN111485010A CN111485010A CN202010408766.9A CN202010408766A CN111485010A CN 111485010 A CN111485010 A CN 111485010A CN 202010408766 A CN202010408766 A CN 202010408766A CN 111485010 A CN111485010 A CN 111485010A
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- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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Abstract
The invention discloses a preparation method of a protein for reducing cholesterol, belonging to the field of preparation of functional proteins. Preheating a protein solution or suspension, and modifying the protein by using high-intensity ultrasound; then carrying out low-intensity ultrasonic-enzymolysis coupling modification on the protein, adding neutral protease into the ultrasonically modified protein for enzymolysis modification, taking the highest cholesterol-reducing activity of the product after gastrointestinal digestion as a hydrolysis end point, stopping enzymolysis, inactivating enzyme, cooling, concentrating and spray drying to prepare the functional protein for reducing cholesterol. Under the condition, the cholesterol reducing activity of the zymolyte is improved by more than 4 times compared with that of the raw material, is improved by more than 2 times compared with that of high-intensity ultrasonic modified protein, and is increased by more than 44.90 percent compared with that of low-intensity ultrasonic-enzymolysis coupling modified protein.
Description
Technical Field
The invention belongs to the field of functional protein preparation, and particularly relates to a method for preparing functional protein for reducing cholesterol through protein modification.
Background
Food-borne proteins in the nature except a few proteins such as immunoglobulin can resist the digestion of the stomach and intestine, and further play a physiological role in improving the immunity and the like, and most of the proteins are degraded into small molecular peptides and amino acids through the stomach and intestine digestion to play a nutritional function. In short, after most of proteins are digested by gastrointestinal tract, the high-level structure of the proteins is damaged, and the physiological function value cannot be embodied. If the protein is modified by adopting a proper treatment method, some beneficial high-level structures can be reserved before complete digestion after the protein enters the gastrointestinal tract, and partial physiological functions can be realized. The enzyme method modified protein can carry out enzymolysis on long-chain protein into short-chain protein, and the short-chain protein can theoretically generate peptide fragments with smaller molecular weight and physiological functions under the action of gastrointestinal digestion, so as to further exert the physiological functions.
The physiological function of the protein depends on the high-order structure of the protein, and in addition to the enzymatic modification of the protein, other physical methods can modify the protein, for example, ultrasonication can change the conformation of the protein, ultrasonication can increase the surface hydrophobicity, the enzymatic hydrolysis, the solubility, the thiol content of the protein, and change the composition of the secondary structure of the protein (Yang et al, Effects of low power sensitivity multi-frequency ultrasound analysis on the enzymatic and structural catalysis of protein gel protein, 2017; Yang et al, Effects and mechanical interaction 2020), and the function of the protein can be further improved by modifying the conformation of the protein and modifying the function of the protein by ultrasonication.
Hypercholesterolemia refers to the increase of blood fat due to the increase of cholesterol in blood beyond the normal range, which leads to the formation of arterial plaque in coronary artery wall, thus causing atherosclerosis and increasing the risk of cardiovascular and cerebrovascular diseases. At present, researches on the function of reducing cholesterol by using ultrasonic modification and combining with an enzymatic modification technology to prepare functional protein after the functional protein is digested by gastrointestinal protease in a digestive tract are not reported.
Disclosure of Invention
The invention aims to provide a preparation method of protein for reducing cholesterol, which is to adopt ultrasonic-enzymolysis coupling modification on the basis of ultrasonic modification to further obtain functional protein for reducing cholesterol activity.
In order to achieve the above purpose, the specific technical solution is as follows: a preparation method of protein for reducing cholesterol comprises the following steps:
(1) high-intensity ultrasonic modification of protein, namely preheating a protein solution or suspension (with the concentration of 10-100 g/L), and then modifying by using ultrasonic, wherein the ultrasonic processing parameters are that the ultrasonic power density is 50-500W/L, and the ultrasonic processing time is 5-60 min.
(2) Adding protease into the ultrasonically modified protein for enzymolysis modification, then adjusting the temperature to 50 ℃, adjusting the pH to 7.0, adding 5% neutral protease (E/S) for enzymolysis, keeping the enzymolysis condition constant, performing the enzymolysis modification process in a low-intensity ultrasonic field, controlling the ultrasonic power density to be 5-20W/L, taking the highest cholesterol-reducing activity of the product obtained after in vitro simulation of gastrointestinal tract digestion as a hydrolysis end point, stopping enzymolysis, killing enzyme, cooling, concentrating, and performing spray drying to prepare the functional protein for reducing cholesterol.
Wherein the protein in the step (1) is water-soluble protein, such as milk protein and egg white protein. It may also be a protein that is not readily soluble in water, such as zein, rice protein.
The invention has the advantages that: the invention adopts high-intensity ultrasound to modify the protein structure, then adopts low-intensity ultrasound-enzymolysis coupling to modify the protein, and takes the highest cholesterol-reducing activity of the zymolyte after the digestion of the gastrointestinal tract is simulated in vitro as the judgment basis of the enzymolysis end point. Under the condition, the cholesterol-reducing activity of the zymolyte is greatly increased, compared with the raw material, the cholesterol-reducing activity is improved by more than 4 times, compared with the high-intensity ultrasonic modified protein, the cholesterol-reducing activity is improved by more than 2 times, and compared with the low-intensity ultrasonic-enzymolysis coupling modified protein, the cholesterol-reducing activity is increased by more than 44.90%.
The technical solution of the present invention is further described in detail by the following examples.
Detailed Description
Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified. The present invention is described in further detail below with reference to specific examples and with reference to the data. It is to be understood that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention, which is intended to be covered by the appended claims.
In the following examples, various procedures and methods not described in detail are conventional methods well known in the art. The source, trade name and composition of the reagents used are indicated at the first appearance and the same reagents used thereafter are the same as indicated for the first time unless otherwise specified.
Proteases of the present example and the comparative example were purchased from Sigma.
The water-soluble proteins of the present example and the comparative example, taking milk protein as an example, are all bright best pasteurized fresh milk (protein content 3.4%, fat content 0%).
The water-insoluble proteins of the present example and the comparative example, taking rice protein as an example, were provided by biological technologies of Beijing Yangshan, lake (crude protein content 57.90%, starch content 11.73%).
The degree of hydrolysis in the examples and the comparative examples was measured by the pH-stat method.
The ultrasonic equipment of the present example and the comparative example was a KQ-300DE type numerical control ultrasonic cleaning apparatus, which was purchased from kunshan ultrasonic instruments ltd.
The following methods were used for simulating gastrointestinal digestion in vitro in both the present example and the control example: adding pepsin into the enzymolysis product according to the enzyme adding amount of 2% (E/S), carrying out enzymolysis for 2 hours, inactivating enzyme after the enzymolysis is finished, adding pancreatin according to the enzyme adding amount of 4% (E/S), carrying out enzymolysis for 4 hours, inactivating enzyme after the enzymolysis is finished, centrifuging, and measuring the cholesterol-reducing activity.
Measurement of Cholesterol lowering Activity in the present example and comparative example refer to the measurement method of the inhibition rate of cholesterol micelle solubility (Nagaoka et al, Biochemical and Biophysical Research Communications,2001,281: 11-17).
Comparative example 1
Weighing 3L milk, adjusting its temperature to 50 deg.C, preheating, and reducing cholesterol activity to 7.24% after in vitro gastrointestinal tract digestion simulation, concentrating and spray drying, adding water for redissolving (keeping its protein content unchanged), wherein the reducing cholesterol activity is 0.43% after in vitro gastrointestinal tract digestion simulation, and the activity is 5.94% after spray drying.
Comparative example 2
Weighing 3L milk, adjusting the temperature of the milk to 50 ℃ for preheating, modifying the protein by adopting high-intensity ultrasound, wherein the modification time is 5min, and the power density is 50W/L. after the modified protein is subjected to in vitro gastrointestinal tract digestion simulation, the cholesterol-reducing activity reaches 11.35 percent, and after concentration and spray drying, a modified protein product is obtained, after water is added for redissolution (the protein content is kept unchanged), the cholesterol-reducing activity of the modified protein after in vitro gastrointestinal tract digestion simulation is measured to be 0.90 percent, and the activity of the modified protein after spray drying is kept to be 7.93 percent.
Comparative example 3
Weighing 3L milk, adjusting the temperature to 50 ℃ for preheating, adjusting the pH value to 7.0, adding 5% E/S neutral protease to carry out low-intensity ultrasonic-enzymolysis coupling modification on the protein, keeping the temperature and the pH constant, and keeping the ultrasonic power density at 5W/L. when the cholesterol reduction activity of the modified protein reaches the highest value of 28.73% after the modified protein is subjected to in vitro simulated gastrointestinal tract digestion, stopping the enzymolysis reaction, carrying out enzyme inactivation, concentration and spray drying to obtain a modified protein product, adding water for redissolution (keeping the protein content unchanged), measuring the cholesterol reduction activity of the modified protein after the in vitro simulated gastrointestinal tract digestion to be 6.24%, and keeping the activity of the modified protein after the spray drying to be 21.72%.
Example 1
Weighing 3L milk, adjusting the temperature to 50 ℃ for preheating, modifying the protein by adopting high-intensity ultrasound, the modification time is 5min, the power density is 50W/L, adjusting the temperature to 50 ℃ after the completion, the pH value is 7.0, adding 5% E/S neutral protease to perform low-intensity ultrasound-enzymolysis coupling modification on the protein, maintaining the temperature and the pH value constant, and the ultrasound power density is 5W/L. when the cholesterol reduction activity of the modified protein reaches the maximum value of 41.63% after the digestion of the in vitro simulated gastrointestinal tract, terminating the enzymolysis reaction, performing enzyme inactivation, concentration and spray drying to obtain a modified protein product, adding water for redissolving (keeping the protein content unchanged), determining the cholesterol reduction activity of the modified protein after the digestion of the in vitro simulated gastrointestinal tract to be 21.56%, and keeping the activity of the modified protein after the spray drying to be 51.79%.
Example 2
Weighing 3L milk, adjusting the temperature to 50 ℃, preheating, modifying the protein by adopting high-intensity ultrasound, wherein the modification time is 20min, the power density is 200W/L, adjusting the temperature to 50 ℃, the pH value is 7.0, adding 5% E/S neutral protease to perform low-intensity ultrasound-enzymolysis coupling modification on the protein, keeping the temperature and the pH value constant, and keeping the ultrasound power density at 10W/L. when the cholesterol-reducing activity of the modified protein reaches the maximum value of 53.28% after the modified protein is digested by simulating gastrointestinal tracts in vitro, terminating the enzymolysis reaction, inactivating enzyme, concentrating and spray drying to obtain a modified protein product, adding water for redissolving (keeping the protein content unchanged), measuring the cholesterol-reducing activity of the modified protein after the digested by simulating gastrointestinal tracts in vitro to be 32.43%, and keeping the activity of the modified protein after the spray drying to be 60.87%.
Example 3
Weighing 3L milk, adjusting the temperature to 50 ℃, preheating, modifying the protein by adopting high-intensity ultrasound, wherein the modification time is 60min, the power density is 500W/L, adjusting the temperature to 50 ℃, the pH value is 7.0 after the modification is finished, adding 5% E/S neutral protease to perform low-intensity ultrasound-enzymolysis coupling modification on the protein, maintaining the temperature and the pH value constant, and maintaining the ultrasound power density to be 20W/L. when the cholesterol reduction activity of the modified protein reaches the maximum value of 48.29% after the modified protein is digested by simulating gastrointestinal tract in vitro, terminating the enzymolysis reaction, performing enzyme deactivation, concentration and spray drying to obtain a modified protein product, adding water for redissolving (keeping the protein content unchanged), measuring the cholesterol reduction activity of the modified protein after the digestion of the simulated gastrointestinal tract in vitro to be 26.72%, and keeping the activity of the modified protein after the spray drying to be 55.33%.
Comparative example 4
Weighing 10g of rice protein, adding 1L distilled water to prepare a material with the concentration of 10 g/L, uniformly stirring, adjusting the temperature to 50 ℃ for preheating, wherein the cholesterol-reducing activity of the rice protein is 3.37% after in vitro gastrointestinal tract digestion simulation, adding water for redissolution (keeping the protein content unchanged) after concentration and spray drying, the cholesterol-reducing activity of the rice protein is 0.21% after in vitro gastrointestinal tract digestion simulation, and the activity of the rice protein is kept 6.24% after spray drying.
Comparative example 5
Weighing 10g of rice protein, adding 1L distilled water to prepare a material with the concentration of 10 g/L, uniformly stirring, adjusting the temperature to 50 ℃ for preheating, performing protein modification by adopting high-intensity ultrasound, wherein the modification time is 5min, and the power density is 50W/L. the cholesterol-reducing activity of the modified protein reaches 6.17 percent after the modified protein is subjected to in-vitro gastrointestinal tract digestion simulation, and a modified protein product is obtained by concentration and spray drying, and after water is added for redissolution (the protein content is kept unchanged), the cholesterol-reducing activity of the modified protein after the in-vitro gastrointestinal tract digestion simulation is measured to be 0.54 percent, and the activity of the modified protein after the spray drying is kept to be 8.75 percent.
Comparative example 6
Weighing 10g of rice protein, adding 1L distilled water to prepare a material with the concentration of 10 g/L, uniformly stirring, adjusting the temperature to 50 ℃ for preheating, adjusting the pH to 7.0, adding 5% E/S neutral protease to perform low-intensity ultrasonic-enzymolysis coupling modification on the protein, maintaining the temperature and the pH constant, and performing ultrasonic power density of 5W/L. when the cholesterol-reducing activity of the modified protein reaches the maximum value of 14.56% after the modified protein is digested by simulating gastrointestinal tract in vitro, stopping the enzymolysis reaction, performing enzyme inactivation, concentration and spray drying to obtain a modified protein product, adding water for redissolving (keeping the protein content unchanged), determining the cholesterol-reducing activity of the modified protein after the digested by simulating gastrointestinal tract in vitro to be 3.38%, and keeping the activity of the modified protein to be 23.21% after the spray drying.
Example 4
Weighing 10g of rice protein, adding 1L distilled water to prepare a material with the concentration of 10 g/L, uniformly stirring, adjusting the temperature to 50 ℃ for preheating, performing protein modification by adopting high-intensity ultrasound for 5min, wherein the power density is 50W/L, adjusting the temperature to 50 ℃ after the completion of the stirring, adjusting the pH to 7.0, adding 5% E/S neutral protease to perform low-intensity ultrasound-enzymolysis coupling modification on the protein, keeping the temperature and the pH constant, and performing ultrasound with the power density of 5W/L. when the cholesterol-reducing activity of the modified protein reaches the maximum value of 26.35% after the digestion of an in-vitro simulated gastrointestinal tract, stopping the enzymolysis reaction, performing enzyme deactivation, concentration and spray drying to obtain a modified protein product, adding water for redissolution (keeping the protein content unchanged), measuring the cholesterol-reducing activity of the modified protein after the digestion of the in-vitro simulated gastrointestinal tract to be 11.63%, and keeping the activity of the modified protein after the spray drying to be 44.13%.
Example 5
Weighing 50g of rice protein, adding 1L distilled water to prepare a material with the concentration of 50 g/L, uniformly stirring, adjusting the temperature to 50 ℃ for preheating, performing protein modification by adopting high-intensity ultrasound for 20min, wherein the power density is 200W/L, adjusting the temperature to 50 ℃ after the completion of the temperature, adjusting the pH to 7.0, adding 5% E/S neutral protease to perform low-intensity ultrasound-enzymolysis coupling modification on the protein, maintaining the temperature and the pH constant, and performing ultrasound power density of 10W/L.
Example 6
Weighing 100g of rice protein, adding 1L of distilled water to prepare a material with the concentration of 100 g/L, uniformly stirring, adjusting the temperature to 50 ℃ for preheating, performing protein modification by adopting high-intensity ultrasound for 60min, wherein the power density is 500W/L, adjusting the temperature to 50 ℃ after the completion of the modification, adjusting the pH to 7.0, adding 5% E/S neutral protease to perform low-intensity ultrasound-enzymolysis coupling modification on the protein, maintaining the temperature and the pH constant, and performing ultrasonic power density of 20W/L.
Claims (2)
1. A method for preparing protein for reducing cholesterol is characterized by comprising the following steps:
(1) high-intensity ultrasonic modification of protein, namely preheating a protein solution or suspension (with the concentration of 10-100 g/L), and then modifying by using ultrasonic, wherein the ultrasonic processing parameters are that the ultrasonic power density is 50-500W/L, and the ultrasonic processing time is 5-60 min;
(2) adding protease into the ultrasonically modified protein for enzymolysis modification, then adjusting the temperature to 50 ℃, adjusting the pH to 7.0, adding 5% neutral protease (E/S) for enzymolysis, keeping the enzymolysis condition constant, performing the enzymolysis modification process in a low-intensity ultrasonic field, controlling the ultrasonic power density to be 5-20W/L, taking the highest cholesterol-reducing activity of the product obtained after in vitro simulation of gastrointestinal tract digestion as a hydrolysis end point, stopping enzymolysis, killing enzyme, cooling, concentrating, and performing spray drying to prepare the functional protein for reducing cholesterol.
2. The method for preparing protein capable of lowering cholesterol according to claim 1, wherein the protein in step (1) is water soluble protein such as milk protein, egg white protein, or protein not easily soluble in water such as corn protein, rice protein.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116391787A (en) * | 2023-05-05 | 2023-07-07 | 安徽农业大学 | Method for preparing egg white protein cholesterol-reducing peptide through enzymolysis modification |
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Cited By (1)
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| CN116391787A (en) * | 2023-05-05 | 2023-07-07 | 安徽农业大学 | Method for preparing egg white protein cholesterol-reducing peptide through enzymolysis modification |
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