CN111485010B - Preparation method of protein for lowering cholesterol - Google Patents
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- CN111485010B CN111485010B CN202010408766.9A CN202010408766A CN111485010B CN 111485010 B CN111485010 B CN 111485010B CN 202010408766 A CN202010408766 A CN 202010408766A CN 111485010 B CN111485010 B CN 111485010B
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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 a protein depends on the higher order structure of the protein and, in addition to 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 condensation multi-stage chromatography on the enzymatic and structural characterization of degraded protein 2017; Yang et al, Effects and mechanism of adsorbed prediction of protein on the metallic reaction of protein-hydrolysis protein, 2020), ultrasonication can modify the protein by changing the conformation of the protein, and further improve the functional activity of the functional protein.
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: preheating a protein solution or suspension (the concentration is 10-100 g/L), and modifying by using ultrasound, wherein the ultrasound processing parameters are as follows: the ultrasonic power density is 50-500W/L, and the ultrasonic treatment time is 5-60 min.
(2) Low-intensity ultrasonic-enzymolysis coupling modification of protein: adding protease into the ultrasonically modified protein for enzymolysis modification, then adjusting the temperature to 50 ℃, adjusting the pH value 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 reduction activity of a product obtained after in vitro simulation of gastrointestinal tract digestion as a hydrolysis end point, stopping enzymolysis, inactivating 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 of milk, adjusting the temperature to 50 deg.C, preheating, and simulating gastrointestinal digestion in vitro to obtain cholesterol-lowering activity of 7.24%. After concentration and spray drying, the mixture is added with water for redissolution (keeping the protein content of the mixture unchanged), the cholesterol-reducing activity of the mixture after in vitro simulation of gastrointestinal tract digestion is 0.43 percent, and the activity of the mixture after spray drying is kept 5.94 percent.
Comparative example 2
Weighing 3L of milk, adjusting the temperature of the milk to 50 ℃, preheating, and carrying out protein modification by adopting high-intensity ultrasound, wherein the modification time is 5min, and the power density is 50W/L. The cholesterol-lowering activity of the modified protein reaches 11.35 percent after the modified protein is subjected to in vitro gastrointestinal tract digestion simulation, and the modified protein product is obtained after concentration and spray drying. After being reconstituted by adding water (keeping the protein content unchanged), the cholesterol-lowering activity of the composition after in vitro simulated gastrointestinal digestion is measured to be 0.90%, and the activity after spray drying is kept to be 7.93%.
Comparative example 3
Weighing 3L of milk, adjusting the temperature of the milk to 50 ℃, preheating, adjusting the pH to 7.0, adding 5% E/S neutral protease to perform low-intensity ultrasonic-enzymolysis coupling modification on the protein, and maintaining the temperature and the pH constant, wherein the ultrasonic power density is 5W/L. When the cholesterol-reducing activity of the modified protein reaches the highest value of 28.73 percent after the modified protein is subjected to in vitro gastrointestinal tract digestion simulation, the enzymolysis reaction is stopped, and the modified protein product is obtained after enzyme deactivation, concentration and spray drying. After being reconstituted by adding water (keeping the protein content unchanged), the cholesterol-lowering activity of the composition after in vitro simulated gastrointestinal digestion is measured to be 6.24%, and the activity after spray drying is kept to be 21.72%.
Example 1
Weighing 3L of milk, adjusting the temperature of the milk to 50 ℃, preheating, and carrying out protein modification by adopting high-intensity ultrasound, wherein the modification time is 5min, and the power density is 50W/L; after the temperature is adjusted to 50 ℃, the pH value is 7.0, 5 percent of E/S neutral protease is added to carry out low-intensity ultrasonic-enzymolysis coupling modification on the protein, the temperature and the pH value are kept constant, and the ultrasonic power density is 5W/L. When the cholesterol-lowering activity of the modified protein reaches 41.63% of the highest value after in vitro gastrointestinal tract digestion simulation, the enzymolysis reaction is stopped, and the modified protein product is obtained after enzyme deactivation, concentration and spray drying. After being reconstituted with water (keeping the protein content unchanged), the cholesterol-lowering activity of the composition after in vitro simulated gastrointestinal digestion is determined to be 21.56%, and the activity after spray drying is kept to be 51.79%.
Example 2
Weighing 3L of milk, adjusting the temperature of the milk to 50 ℃, preheating, and performing protein modification by adopting high-intensity ultrasound, wherein the modification time is 20min and the power density is 200W/L; after the temperature is adjusted to 50 ℃, the pH value is 7.0, 5 percent of E/S neutral protease is added to carry out low-intensity ultrasonic-enzymolysis coupling modification on the protein, the temperature and the pH value are kept constant, and the ultrasonic power density is 10W/L. When the cholesterol-lowering activity of the modified protein reaches 53.28% of the highest value after in vitro gastrointestinal tract digestion simulation, the enzymolysis reaction is stopped, and the modified protein product is obtained after enzyme deactivation, concentration and spray drying. After being reconstituted with water (keeping the protein content unchanged), the cholesterol-lowering activity of the composition after in vitro simulated gastrointestinal digestion is determined to be 32.43%, and the activity after spray drying is maintained at 60.87%.
Example 3
Weighing 3L of milk, adjusting the temperature of the milk to 50 ℃, preheating, and performing protein modification by adopting high-intensity ultrasound, wherein the modification time is 60min, and the power density is 500W/L; after the temperature is adjusted to 50 ℃, the pH value is 7.0, 5 percent of E/S neutral protease is added to carry out low-intensity ultrasonic-enzymolysis coupling modification on the protein, the temperature and the pH value are kept constant, and the ultrasonic power density is 20W/L. When the cholesterol-reducing activity of the modified protein reaches the highest value of 48.29 percent after the modified protein is subjected to in vitro gastrointestinal tract digestion simulation, the enzymolysis reaction is stopped, and the modified protein product is obtained after enzyme deactivation, concentration and spray drying. After being reconstituted with water (keeping the protein content constant), the cholesterol-lowering activity of the composition after in vitro simulated gastrointestinal digestion was determined to be 26.72%, and the activity after spray drying was determined to be 55.33%.
Comparative example 4
Weighing 10g of rice protein, adding 1L of distilled water to prepare a material with the concentration of 10g/L, uniformly stirring, adjusting the temperature to 50 ℃ for preheating, and simulating gastrointestinal tract digestion in vitro to obtain the cholesterol-reducing activity of 3.37%. After concentration and spray drying, the mixture is added with water for redissolution (keeping the protein content of the mixture unchanged), the cholesterol-reducing activity of the mixture after in vitro simulation of gastrointestinal tract digestion is 0.21 percent, and the activity of the mixture after spray drying is kept 6.24 percent.
Comparative example 5
Weighing 10g of rice protein, adding 1L of distilled water to prepare a material with the concentration of 10g/L, uniformly stirring, adjusting the temperature to 50 ℃ for preheating, and performing protein modification by adopting high-intensity ultrasound for 5min with the power density of 50W/L. The cholesterol-lowering 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 after concentration and spray drying. After being reconstituted with water (keeping the protein content unchanged), the cholesterol-lowering activity of the composition after in vitro simulated gastrointestinal digestion is determined to be 0.54%, and the activity after spray drying is kept to be 8.75%.
Comparative example 6
Weighing 10g of rice protein, adding 1L of distilled water to prepare a material with the concentration of 10g/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, and maintaining the temperature and the pH constant, wherein the ultrasonic power density is 5W/L. When the cholesterol-reducing activity of the modified protein reaches the highest value of 14.56 percent after the modified protein is subjected to in vitro gastrointestinal tract digestion simulation, the enzymolysis reaction is stopped, and the modified protein product is obtained after enzyme deactivation, concentration and spray drying. After being reconstituted by adding water (keeping the protein content unchanged), the cholesterol-lowering activity of the composition after in vitro simulated gastrointestinal digestion is measured to be 3.38%, and the activity after spray drying is kept to be 23.21%.
Example 4
Weighing 10g of rice protein, adding 1L of distilled water to prepare a material with the concentration of 10g/L, uniformly stirring, adjusting the temperature of the material to 50 ℃ for preheating, and performing protein modification by adopting high-intensity ultrasound, wherein the modification time is 5min and the power density is 50W/L; after the temperature is adjusted to 50 ℃, the pH value is 7.0, 5 percent of E/S neutral protease is added to carry out low-intensity ultrasonic-enzymolysis coupling modification on the protein, the temperature and the pH value are kept constant, and the ultrasonic power density is 5W/L. When the cholesterol-reducing activity of the modified protein reaches the highest value of 26.35 percent after the modified protein is subjected to in vitro gastrointestinal tract digestion simulation, the enzymolysis reaction is stopped, and the modified protein product is obtained after enzyme deactivation, concentration and spray drying. After being reconstituted with water (keeping the protein content unchanged), the cholesterol-lowering activity of the composition after in vitro simulated gastrointestinal digestion is measured to be 11.63%, and the activity after spray drying is kept to be 44.13%.
Example 5
Weighing 50g of rice protein, adding 1L of distilled water to prepare a material with the concentration of 50g/L, uniformly stirring, adjusting the temperature of the material to 50 ℃ for preheating, and performing protein modification by adopting high-intensity ultrasound, wherein the modification time is 20min and the power density is 200W/L; after the temperature is adjusted to 50 ℃, the pH value is 7.0, 5 percent of E/S neutral protease is added to carry out low-intensity ultrasonic-enzymolysis coupling modification on the protein, the temperature and the pH value are kept constant, and the ultrasonic power density is 10W/L. When the cholesterol-lowering activity of the modified protein reaches 43.20% of the highest value after in vitro gastrointestinal tract digestion simulation, the enzymolysis reaction is stopped, and the modified protein product is obtained after enzyme deactivation, concentration and spray drying. After being reconstituted with water (keeping the protein content unchanged), the cholesterol-lowering activity of the composition after in vitro simulated gastrointestinal digestion is measured to be 25.42%, and the activity after spray drying is kept to be 58.84%.
Example 6
Weighing 100g of rice protein, adding 1L of distilled water to prepare a material with the concentration of 100g/L, uniformly stirring, adjusting the temperature of the material to 50 ℃ for preheating, and performing protein modification by adopting high-intensity ultrasound, wherein the modification time is 60min and the power density is 500W/L; after the temperature is adjusted to 50 ℃, the pH value is 7.0, 5 percent of E/S neutral protease is added to carry out low-intensity ultrasonic-enzymolysis coupling modification on the protein, the temperature and the pH value are kept constant, and the ultrasonic power density is 20W/L. When the cholesterol-lowering activity of the modified protein reaches 54.13% of the highest value after in vitro gastrointestinal tract digestion simulation, the enzymolysis reaction is stopped, and the modified protein product is obtained after enzyme deactivation, concentration and spray drying. After being reconstituted with water (keeping the protein content unchanged), the cholesterol-lowering activity of the composition after in vitro simulated gastrointestinal digestion is determined to be 27.94%, and the activity after spray drying is kept to be 51.62%.
Claims (1)
1. A method for preparing protein for reducing cholesterol is characterized by comprising the following steps:
(1) high-intensity ultrasonic modification of protein: preheating a protein solution or suspension with the concentration of 10-100 g/L, and modifying by using ultrasound, wherein the ultrasound processing parameters are as follows: the ultrasonic power density is 50-500W/L, and the ultrasonic treatment time is 5-60 min;
(2) low-intensity ultrasonic-enzymolysis coupling modification of protein: adding protease into the ultrasonically modified protein for enzymolysis modification, then adjusting the temperature to 50 ℃, adjusting the pH to 7.0, adding neutral protease with the E/S of 5% to start enzymolysis, maintaining 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, stopping enzymolysis by taking the highest cholesterol-reducing activity of a product obtained after in vitro simulation of gastrointestinal tract digestion as a hydrolysis end point, inactivating enzyme, cooling, concentrating, and performing spray drying to prepare the functional protein for reducing cholesterol;
wherein the protein in the step (1) is milk protein or rice protein.
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| CN103695518B (en) * | 2013-12-30 | 2016-04-06 | 东北农业大学 | Whey protein peptide is utilized to reduce product and the preparation method of serum cholesterol level |
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