CN111330514A - Preparation method of oleosin - Google Patents
Preparation method of oleosin Download PDFInfo
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- CN111330514A CN111330514A CN201811552854.5A CN201811552854A CN111330514A CN 111330514 A CN111330514 A CN 111330514A CN 201811552854 A CN201811552854 A CN 201811552854A CN 111330514 A CN111330514 A CN 111330514A
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Abstract
The invention relates to a method for preparing oil-holding protein, which takes water-soluble protein as raw material, uses peroxide to controllably open disulfide bonds in protein molecules to obtain water-soluble protein with partially opened disulfide bonds to prepare protein solution, adds the protein solution into an oil-water system, and enables the water-soluble protein to fully absorb oily substances by stirring, homogenizing or ultrasonic methods under the premise of ensuring no emulsion breaking and oil separation and keeping the protein molecules to be uniformly dispersed in water to prepare oil-holding protein molecular emulsion. According to the invention, the proportion and the performance of oily substance particles penetrating into the hydrophobic interior of protein molecules are improved by opening the disulfide bonds in the protein molecules, the prepared oleosin has large oil holding capacity and stable performance, can replace the traditional emulsifier, and can be used for preparing a cream product which has stable oil-water performance and can release fragrance for a long time, and the cream product is widely applied to the industries of food, medicine, chemical industry and daily chemicals.
Description
Technical Field
The invention relates to a preparation method of oil-holding protein molecule emulsion, in particular to a method for improving the oil holding capacity of protein molecules by opening protein molecule disulfide bonds in a controlled manner.
Background
Proteins are important components constituting living organisms, are basic organic substances constituting cells, and are main players of life activities. Proteins can be divided into simple proteins and binding proteins, wherein the binding proteins are further divided into glycoproteins, lipoproteins, phosphorylated proteins, metal binding proteins, and the like. Although a complex such as glycoprotein, lipoprotein, phosphorylated protein and the like can be formed between a protein and other cell components such as nucleic acid, carbohydrate, lipid and the like through covalent bonds, or a complex such as glycoprotein, lipoprotein, phosphorylated protein and the like can be formed through secondary bonds such as hydrogen bonds, hydrophobic interaction forces, van der waals forces and the like, the existence form of the protein molecules basically exists independently in the traditional knowledge system, the protein molecules are only partially combined with other cell components, or a plurality of protein molecules and other cell components form a complex together, namely the protein molecules are not completely fused with other cell components, or the protein molecules do not completely contain other cell components.
However, the results of the previous experiments show that the protein molecules are dispersed in the form of single molecules in the aqueous solution. Even water-soluble protein without surface activity or with poor surface activity can still show certain oil-holding performance in an oil-water system. In an oil-water system, a small amount of particles such as oil and fat, fat-soluble vitamins, fat-soluble pigments, essences and flavors can be infiltrated into the hydrophobic interior of the water-soluble protein molecules by methods such as stirring, homogenizing, ultrasonic treatment and the like until the oil-holding capacity of the water-soluble protein molecules is limited. The oil-holding capacity of water-soluble proteins is influenced by the internal structure and properties of the protein molecule, so that the oil-holding properties of common water-soluble proteins are extremely limited.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the invention provides a method for preparing oleosin, which takes water-soluble protein as a raw material, controllably opens disulfide bonds in protein molecules by oxidizing the disulfide bonds in the protein molecules, and controllably changes the plastic property of the internal space of the protein molecules, thereby improving and improving the proportion and the performance of particles such as grease, fat-soluble vitamins, fat-soluble pigments, essences and fragrances and the like in an oil-water system penetrating into the hydrophobic internal part of the protein molecules, and preparing the oleosin with larger capacity.
The technical scheme for solving the technical problems is as follows: a process for preparing the oil-holding protein includes such steps as using water-soluble protein as raw material, using peroxide to open the internal disulfide bonds of protein molecule to obtain the water-soluble protein with partially opened disulfide bonds, dissolving in water to obtain protein solution, adding the protein solution to oil-water system, stirring, homogenizing or ultrasonic treating to make the water-soluble protein with partially opened disulfide bonds fully absorb oily substance to obtain the oil-holding protein emulsion.
Further, the method comprises the following specific steps:
A. preparing a water-soluble protein solution;
B. opening of protein disulfide bonds: adding the peroxide solution into the water-soluble protein solution according to the volume ratio
The calculation is as follows: the ratio of the peroxide solution to the water-soluble protein solution is 0.005-0.03: 1, and the concentration of the peroxide solution is 0.5-1.5 mol/L; the protein content of the water-soluble protein solution is controlled to be 0.01 g/mL-0.02 g/mL;
placing the obtained mixture in an ice bath at the temperature of minus 15-minus 5 ℃, reacting for 1-5 hours in the dark, and adding a terminator into the mixture to terminate the reaction;
C. purifying to obtain water-soluble protein with partially opened protein disulfide bonds;
D. and D, adding the water-soluble protein obtained in the step C into an oil-water system, wherein the oil-water system is composed of an oily substance and water, and the volume ratio of the oily substance to the water is = 20-80: 80-20 wt% of a protein solution with the mass concentration of 1-10% formed by the water-soluble protein and water in an oil-water system, wherein the oily substances are grease, fat-soluble vitamins, fat-soluble pigments or essences and fragrances; under the premise of ensuring no demulsification and oil separation and keeping protein molecules uniformly dispersed in water, the water-soluble protein with partially opened protein disulfide bonds fully absorbs oily substances by stirring, homogenizing or ultrasonic methods, and the oil-retaining protein molecular emulsion is prepared.
The terminating agent is ethanol, and the using amount of the terminating agent is calculated according to the volume ratio: terminator: mixture = 0.005-0.013: 1.
the peroxide is peroxyformic acid, peroxyacetic acid, peroxypropionic acid, hydrogen peroxide or sodium peroxide.
The poor oil retention of common water-soluble proteins is due to the restriction of the spatial structure of the protein, in particular the intramolecular disulfide bonds. Therefore, on the premise of keeping the structure of the protein molecule relatively stable, the disulfide bonds in the protein molecule are controllably opened by oxidizing the disulfide bonds in the protein molecule, the plasticity of the internal space of the protein molecule is controllably changed, and the proportion and the performance of the particles such as grease, fat-soluble vitamins, fat-soluble pigments, essences and fragrances and the like in an oil-water system penetrating into the hydrophobic internal part of the protein molecule can be improved.
Proteins are generally biological macromolecules composed of one or more polypeptide chains of 20 amino acids joined end to end by peptide bonds, wherein the polypeptide chains are further coiled and folded to form secondary, tertiary and quaternary structures of the protein molecules. Naturally occurring protein molecules all have tertiary or quaternary molecular structures. Although the 20 kinds of amino acids constituting a protein can be basically classified into two major groups, namely polar amino acids and nonpolar amino acids, due to further coiling and folding of the protein polypeptide chain, most of the polar amino acid side chain groups of almost all proteins (even including insoluble keratin, silk fibroin, collagen, elastin, and the like) are distributed on the surface of the protein molecule, and most of the nonpolar amino acid side chain groups are wrapped inside the dense and stable protein molecule. Almost all protein molecules are hydrophilic on the surface, while most of the interior of the protein molecule is hydrophobic. Therefore, by stirring, homogenizing, ultrasonic treatment, etc., fine particles of fats and oils, fat-soluble vitamins, fat-soluble pigments, flavors, etc. in an oil-water system comprising fats and oils, fat-soluble vitamins, fat-soluble pigments, flavors, etc. can be made to penetrate into the hydrophobic interior of protein molecules, thereby preparing an oil-retaining protein molecule (see fig. 1).
Due to the internal disulfide bonds of most protein molecules, the disulfide bonds are the key forces for maintaining the spatial structure of the protein molecules and limit the plasticity of the internal space of the protein molecules. Therefore, the internal disulfide bonds of the protein molecules are further oxidized, the internal disulfide bonds of the protein molecules are controllably opened, and the internal space plasticity of the protein molecules is controllably changed, so that the proportion and the performance of oil, fat-soluble vitamins, fat-soluble pigments, flavors, fragrances and other particles in an oil-water system penetrating into the hydrophobic internal parts of the protein molecules are improved, and the oil-holding protein molecules with larger capacity are prepared (see figure 2).
The present applicant has succeeded in preparing a novel protein-based surfactant by oxidizing disulfide bonds in a protein molecule with an oxidizing agent to convert the disulfide bonds into sulfonic acid groups, controllably opening the folded coiled structure of the protein, and thereby exhibiting the surface active properties of the protein molecule (see "method for preparing a protein-based surfactant by opening protein disulfide bonds", patent No. 200810166640.4).
The invention firstly adopts the method of opening disulfide bonds in protein molecules by oxidation, and then adopts the methods of stirring, homogenizing, ultrasound and the like to disperse soluble protein in an oil-water system in a single molecular form in an aqueous solution, and the single protein molecular form holds particles of grease, fat-soluble vitamins, fat-soluble pigments, essences, spices and the like, thereby preparing the protein molecule with large capacity of holding oil. The oil-holding protein molecule prepared by the invention has large oil holding amount and stable performance, can replace the traditional emulsifier, can prepare a cream product with stable oil-water performance and lasting fragrance release, and can be widely applied to the industries of food, medicine, chemical industry and daily chemicals.
Drawings
FIG. 1: schematic representation of the protein molecule oil holding process.
FIG. 2: schematic representation of the oil holding process of protein molecules with partially opened disulfide bonds.
Detailed Description
Example 1:
200mL of water-soluble soybean protein solution with the concentration of 0.01246g/mL is added into a flask to prepare three portions, 1 mL, 2 mL and 3 mL of peroxyacetic acid with the concentration of 1mol/L are respectively added, the mixture is placed in an ice bath at the temperature of minus 10 ℃ after being mixed, and 2 mL of ethanol is added to stop the reaction after the reaction is carried out for 3 hours in the dark. After the content of disulfide bonds of the reaction product is detected to be changed, drying treatment (namely purification) is carried out by reduced pressure distillation and the like to obtain the water-soluble soybean protein with oxidized partial opened disulfide bonds, and the water-soluble soybean protein solution with partial opened disulfide bonds is prepared according to the concentration requirement of 0.01246g/mL, namely the soybean protein with partial opened disulfide bonds is added into an oil-water system consisting of soybean oil and water, and the soybean protein and the water in the oil-water system form the soybean protein solution with the concentration of 0.01246 g/mL. The concentration of the water-soluble protein solution was analyzed by the Kjeldahl method, and the disulfide bond content thereof was determined.
Stirring for 1min under high speed emulsifying machine, placing into 100mL graduated cylinder, standing for 1hr, and measuring the emulsifying height to express the change of oil-holding protein molecular property. The properties of the oil-holding protein molecular emulsion prepared are shown in Table 1.
The specific operation of the purification step in this example is: and (3) distilling the mixture added with the terminating agent in a water bath at 40-50 ℃ under reduced pressure, adding a proper amount of water after the solvent is evaporated to dryness, and evaporating the water to dryness again for 2-3 times. As an alternative, the purification step may also be carried out by removing excess oxygen from the mixture and neutralizing excess peroxide by alkali neutralization.
The raw material water-soluble protein of the invention is various edible proteins such as soybean protein, milk protein, peanut protein, collagen and the like, and can be prepared by self or purchased in the market.
In the process of preparing the water-soluble protein solution, some special proteins need some special reagents for assisting dissolution, for example, casein needs NaOH solution for assisting dissolution before being dissolved and dispersed well.
Control experiment:
the method comprises the steps of taking degreased low-temperature soybean meal as a raw material, dissolving 50g of degreased low-temperature soybean meal in deionized water according to a feed-liquid ratio of 1:10 (w/v), adjusting the pH value to 8.5 by 1mol/L NaOH, placing the degreased low-temperature soybean meal in a constant-temperature magnetic stirrer, leaching for 2 hours at 45 ℃ and at a stirring speed of 40 r/min, and then adjusting the pH value to 4.5 by 1mol/L HCl. Centrifuging the precipitate, and removing the supernatant to obtain the soybean protein isolate. Re-dissolving the soybean protein isolate precipitate, and re-precipitating for 2 times to obtain the pure soybean protein isolate product. The concentration of the water-soluble protein solution was analyzed by the Kjeldahl method, and the disulfide bond content thereof was determined.
Adding water soluble soybean protein into oil-water system composed of soybean oil and fat and water, wherein the soybean protein and water in the oil-water system constitute 0.01246g/mL soybean protein solution. Stirring at high speed for 1min, placing into 100mL graduated cylinder, standing for 1hr, measuring emulsion height, and expressing oleophilic protein molecular property and size by emulsion layer height, with the results as shown in Table 1.
Table 1 summary of the relevant experimental results
As can be seen from Table 1, the emulsion layer volume of the oil-retaining protein molecular emulsion prepared in example 1 is significantly higher than that of the control experiment. The protein with opened disulfide bonds through peroxide has oil retention property obviously higher than that of protein without opened disulfide bonds, and the large-capacity oil retention protein can be prepared.
The protein disulfide bond content in the invention is measured according to the method introduced by Lilina and the like [ Lilina, Lifan, Hayasi; measuring the content of disulfide bonds in the food protein by a spectrophotometry method; food science, 2008, 29(8), 562- "564 ].
Claims (4)
1. A method for preparing oleosin, which is characterized in that: the method comprises the steps of taking water-soluble protein as a raw material, controllably opening disulfide bonds in protein molecules by using peroxide to obtain water-soluble protein with partially opened disulfide bonds, dissolving the water-soluble protein into water to prepare a protein solution, adding the protein solution into an oil-water system, and enabling the water-soluble protein with the partially opened protein disulfide bonds to fully absorb oily substances by stirring, homogenizing or ultrasonic methods on the premise of ensuring that emulsion breaking and oil separation are not carried out and the protein molecules are uniformly dispersed in the water to prepare the oil-holding protein molecular emulsion.
2. The method of claim 1, wherein the step of preparing the oleosin comprises: the method comprises the following specific steps:
A. preparing a water-soluble protein solution;
B. opening of protein disulfide bonds: adding the peroxide solution into the water-soluble protein solution according to the volume ratio
The calculation is as follows: the ratio of the peroxide solution to the water-soluble protein solution is 0.005-0.03: 1, and the concentration of the peroxide solution is 0.5-1.5 mol/L; the protein content of the water-soluble protein solution is controlled to be 0.01 g/mL-0.02 g/mL;
placing the obtained mixture in an ice bath at the temperature of minus 15-minus 5 ℃, reacting for 1-5 hours in the dark, and adding a terminator into the mixture to terminate the reaction;
C. purifying to obtain water-soluble protein with partially opened protein disulfide bonds;
D. and D, adding the water-soluble protein obtained in the step C into an oil-water system, wherein the oil-water system is composed of an oily substance and water, and the volume ratio of the oily substance to the water is = 20-80: 80-20 wt% of a protein solution with the mass concentration of 1-10% formed by the water-soluble protein and water in an oil-water system, wherein the oily substances are grease, fat-soluble vitamins, fat-soluble pigments or essences and fragrances; under the premise of ensuring no demulsification and oil separation and keeping protein molecules uniformly dispersed in water, the water-soluble protein with partially opened protein disulfide bonds fully absorbs oily substances by stirring, homogenizing or ultrasonic methods, and the oil-retaining protein molecular emulsion is prepared.
3. The method of claim 2, wherein the step of preparing the oleosin comprises: the terminating agent is ethanol, and the using amount of the terminating agent is calculated according to the volume ratio: terminator: mixture = 0.005-0.013: 1.
4. the method of claim 1, 2 or 3, wherein the step of preparing the oleosin comprises: the peroxide is peroxyformic acid, peroxyacetic acid, peroxypropionic acid, hydrogen peroxide or sodium peroxide.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101228249A (en) * | 2005-04-01 | 2008-07-23 | 巴斯福股份公司 | Drilling fluid containing hydrophobin |
| CN101590382A (en) * | 2008-05-29 | 2009-12-02 | 广西工学院 | By opening the method that protein disulfide prepares protein-based surfactant |
| CN102731796A (en) * | 2012-07-06 | 2012-10-17 | 广西工学院 | Preparation method of protein-based surfactant fibroin acid calcium |
| CN103636915A (en) * | 2013-12-13 | 2014-03-19 | 广西科技大学 | Method for improving utilization rate of fish meal protein |
| CN104323235A (en) * | 2014-09-28 | 2015-02-04 | 中山大学 | Double-protein co-emulsification system, preparation method and application thereof |
| CN107118358A (en) * | 2017-05-25 | 2017-09-01 | 中原工学院 | The method that disulfide bond reconstruction method prepares native protein composite micelle |
-
2018
- 2018-12-19 CN CN201811552854.5A patent/CN111330514A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101228249A (en) * | 2005-04-01 | 2008-07-23 | 巴斯福股份公司 | Drilling fluid containing hydrophobin |
| CN101590382A (en) * | 2008-05-29 | 2009-12-02 | 广西工学院 | By opening the method that protein disulfide prepares protein-based surfactant |
| CN102731796A (en) * | 2012-07-06 | 2012-10-17 | 广西工学院 | Preparation method of protein-based surfactant fibroin acid calcium |
| CN103636915A (en) * | 2013-12-13 | 2014-03-19 | 广西科技大学 | Method for improving utilization rate of fish meal protein |
| CN104323235A (en) * | 2014-09-28 | 2015-02-04 | 中山大学 | Double-protein co-emulsification system, preparation method and application thereof |
| CN107118358A (en) * | 2017-05-25 | 2017-09-01 | 中原工学院 | The method that disulfide bond reconstruction method prepares native protein composite micelle |
Non-Patent Citations (9)
| Title |
|---|
| 朱卫星等: "蛋白质氧化机制及其评价技术研究进展", 《食品工业科技》 * |
| 李丽娜等: "分光光度法测定食品蛋白质中二硫键的含量", 《食品科学》 * |
| 江波等: "《普通高等教育"十五"国家级规划教材 食品化学 第2版》", 30 September 2018, 北京：中国轻工业出版社 * |
| 董文秀等: "乙醇对丝素蛋白结构及功能特性的影响", 《现代食品科技》 * |
| 蔡勇建等: "过氧自由基氧化对大豆蛋白功能性质影响", 《粮食与油脂》 * |
| 蔡武城: "二硫键在人血清白蛋白空间构型中的作用", 《复旦学报(自然科学版)》 * |
| 计红芳等: "豌豆蛋白对牛肉盐溶蛋白理化性质及二级结构的影响", 《食品与发酵工业》 * |
| 钟新等: "二硫键在蛋白质中的作用及其氧化改性研究进展", 《中国饲料》 * |
| 钟新等: "氧化变构制备大豆分离蛋白质基表面活性剂", 《中国饲料》 * |
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