阅读说明：本技术 一种提高乳铁蛋白热稳定性的方法 (Method for improving heat stability of lactoferrin ) 是由 于景华 王淑晨 韩翼宇 刘晓辉 于 2020-12-14 设计创作，主要内容包括：本发明涉及一种提高乳铁蛋白热稳定性的方法,所述方法包括如下步骤：(1)以乳铁蛋白为原料,经过透析利用所述原料制备出无铁乳铁蛋白；(2)向所述无铁乳铁蛋白中加入铁氮基三乙酸来制备出高铁饱和度的乳铁蛋白。本发明方法利用乳铁蛋白在酸性缓冲溶液中透析制备出无铁乳铁蛋白,然后向无铁乳铁蛋白中铁氮基三乙酸溶液制备出了高铁饱和度的乳铁蛋白,所述高铁饱和度的乳铁蛋白加热后的稳定性得到提高。(The invention relates to a method for improving the heat stability of lactoferrin, which comprises the following steps: (1) taking lactoferrin as a raw material, and preparing the iron-free lactoferrin by using the raw material through dialysis; (2) adding iron nitrilotriacetic acid into the iron-free lactoferrin to prepare the lactoferrin with high iron saturation. The method prepares the iron-free lactoferrin by dialyzing the lactoferrin in an acidic buffer solution, then prepares the lactoferrin with high iron saturation from a solution of iron nitrilotriacetic acid in the iron-free lactoferrin, and improves the stability of the lactoferrin with high iron saturation after being heated.)

1. A method for increasing the thermal stability of lactoferrin, comprising the steps of:

(1) taking lactoferrin as a raw material, and preparing the iron-free lactoferrin by using the raw material through dialysis;

(2) adding iron nitrilotriacetic acid into the iron-free lactoferrin to prepare the lactoferrin with high iron saturation.

2. The method according to claim 1, wherein in step (1), the lactoferrin is dissolved in water to prepare an aqueous solution, the aqueous solution is dialyzed, and the dialyzed lactoferrin solution is dried to obtain the iron-free lactoferrin.

3. The method according to claim 1 or 2, characterized in that the drying is vacuum drying.

4. The method according to any one of claims 1 to 3, wherein the dialysis is performed using dialysis bags with a cut-off in the range of 8-14 kDa.

5. The method according to any one of claims 1 to 4, wherein the dialysis time for dialysis is 24 hours, wherein the buffer solution for the first 12 hours is an acidic buffer solution with pH 3-5; the buffer solution after the last 12 hours was water.

6. The method according to any one of claims 1 to 5, wherein the dialysis is carried out at a temperature below 37 ℃ and a constant rotational speed is maintained during the dialysis.

7. The method according to any one of claims 1 to 6, wherein the acidic buffer is one selected from the group consisting of a citric acid-sodium citrate buffer solution, a disodium hydrogen phosphate-citric acid buffer, a citric acid-sodium hydroxide-hydrochloric acid buffer, and an acetic acid-sodium acetate buffer.

8. The method according to any one of claims 1 to 7, wherein step (2) comprises the sub-steps of:

(A) dissolving the iron-free lactoferrin in a Tris-HCl buffer solution containing NaCl to prepare a 1% solution, and adjusting the pH value to 7.0-7.8 by using HCl to prepare an iron-free lactoferrin solution;

(B) and adding a FeNTA solution into the iron-free lactoferrin solution for reaction, and then drying to obtain the lactoferrin with high iron saturation.

9. The method of claim 8, wherein in step (B), the FeNTA solution is formulated using (i) iron nitrate and (ii) nitrilotriacetic acid or a disodium salt of nitrilotriacetic acid; preferably, the FeNTA solution is prepared using (i) a 9.9mM iron nitrate solution and (ii)8.5mM nitrilotriacetic acid or disodium nitrilotriacetic acid salt solution.

10. The process according to any one of claims 1 to 9, characterized in that the lactoferrin is native lactoferrin.

Technical Field

The invention belongs to the technical field of food, and particularly relates to a method for improving heat stability of lactoferrin.

Background

Lactoferrin is mainly present in mammalian exudates, such as milk, tears, saliva, sweat, pancreatic juice, bile, and the like. In the case of dairy cattle, bovine lactoferrin is one of the major glycoproteins in mammary gland secretions during the colostrum formation phase, lactation phase, dry milk phase and during mastitis. Lactoferrin has a variety of bioactive functions: broad-spectrum antibacterial property, and can enhance antiviral, antioxidant and immunity of organism, prevent and treat infectious diseases, stimulate Bacillus bifidus growth, maintain intestinal flora balance, and enhance iron transfer and absorption. Have become a concern in the food industry, particularly the dairy industry.

Lactoferrin is an ideal functional food ingredient. However, for a long time, researchers have considered lactoferrin to be easily inactivated by heat, which is detrimental to dairy processing. For example, bovine lactoferrin starts to inactivate at a pH of 6.6 and a temperature of 65-69 ℃; the human lactoferrin is completely inactivated after being heated at 70 ℃ for 15-30 minutes. The thermostability of lactoferrin molecules is generally increased by covalent binding of sugar molecules to proteins. Peinado et al, which make submicron biopolymer particles by electrostatic complexation of lactoferrin particles with anionic polysaccharides (alginate, carrageenan or pectin), found that anionic polysaccharides such as alginate, carrageenan and pectin can prevent heat-induced denaturation of lactoferrin, thereby retaining its iron-binding capacity. Kang Xu et al bound okra polysaccharide and lactoferrin to form a complex to prevent loss of secondary and tertiary structures after lactoferrin is heated. The addition of these polysaccharides changes the particle size of lactoferrin, and the addition of these polysaccharides, which are mostly emulsifiers and thickeners, affects the use of lactoferrin in the process. The invention improves the heat stability of lactoferrin by changing the lactoferrin. The amount of iron binding in lactoferrin affects the molecular conformation of lactoferrin, and lactoferrin with bound iron has a more compact structure than lactoferrin without iron. Lactoferrin is generally inactivated by aggregation through a combination of non-covalent interactions and intermolecular thiol/disulfide reactions of free thiol residues. Lactoferrin has two lobes which close upon iron saturation, thereby hiding the noncovalent sites of the two protein lobe cores and reducing intermolecular interactions and the formation of insoluble aggregates. Lactoferrin with high iron saturation will be stable at higher temperatures and one of the manifestations of this stability is to maintain the integrity of the disulfide bonds. Therefore, the thermal stability of lactoferrin can be increased by increasing the iron saturation of lactoferrin.

Disclosure of Invention

The invention aims to solve the problems of heat instability and the like of lactoferrin.

The invention provides a method for improving the thermal stability of lactoferrin.

Specifically, the invention provides a method for improving the heat stability of lactoferrin, which comprises the following steps: (1) taking lactoferrin as a raw material, and preparing the iron-free lactoferrin by using the raw material through dialysis; (2) adding iron nitrilotriacetic acid into the iron-free lactoferrin to prepare the lactoferrin with high iron saturation.

Preferably, in the step (1), the lactoferrin is dissolved in water to prepare an aqueous solution, then the aqueous solution is dialyzed, and the lactoferrin solution obtained by dialysis is dried to obtain the iron-free lactoferrin.

Further preferably, the drying is vacuum drying.

It is further preferred that the dialysis is performed using dialysis bags with a cut-off in the range of 8-14 kDa.

It is also preferred that the dialysis time for dialysis is 24 hours, wherein the buffer solution for the first 12 hours is an acidic buffer solution having a PH of 3 to 5; the buffer solution after the last 12 hours is water;

it is also preferred that the dialysis is carried out at a temperature below 37 ℃ and that a constant rotational speed is maintained during the dialysis.

It is also preferable that the acidic buffer is one selected from the group consisting of a citric acid-sodium citrate buffer solution, a disodium hydrogen phosphate-citric acid buffer solution, a citric acid-sodium hydroxide-hydrochloric acid buffer solution, and an acetic acid-sodium acetate buffer solution.

It is also preferable that the step (2) includes the sub-steps of: (A) dissolving the iron-free lactoferrin in a Tris-HCl buffer solution containing NaCl to prepare a 1% solution, and adjusting the pH value to 7.0-7.8 by using HCl to prepare an iron-free lactoferrin solution; (B) and adding a FeNTA solution into the iron-free lactoferrin solution for reaction, and then drying to obtain the lactoferrin with high iron saturation.

Further preferably, in step (B), the FeNTA solution is formulated using (i) iron nitrate and (ii) nitrilotriacetic acid or a disodium salt of nitrilotriacetic acid; preferably, the FeNTA solution is prepared using (i) a 9.9mM iron nitrate solution and (ii)8.5mM nitrilotriacetic acid or disodium nitrilotriacetic acid salt solution.

It is further preferred that the lactoferrin is native lactoferrin.

In some embodiments, the methods of the invention comprise the steps of:

(1) dissolving natural lactoferrin in ultrapure water to prepare a solution (50-100mg/mL), fully dissolving on a magnetic stirrer, and placing the solution in a dialysis bag with the interception range of 8-14 kDa;

(2) the first 12 hours of the buffer solution is acidic buffer solution with pH of 3-5, such as citric acid-sodium citrate buffer solution, disodium hydrogen phosphate-citric acid buffer solution, citric acid-sodium hydroxide-hydrochloric acid buffer solution, acetic acid-sodium acetate buffer solution, etc., and the last 12 hours of the buffer solution is water, and is carried out at a temperature below 37 deg.C, and the rotation speed is kept constant;

(3) carrying out vacuum freeze drying on the dialyzed lactoferrin solution to obtain iron-free lactoferrin;

(4) dissolving iron-free lactoferrin in a Tris-HCl buffer solution containing NaCl to prepare a 1% solution, and adjusting the pH value to 7.0-7.8 by using HCl;

(5) fresh iron nitrilotriacetic acid (FeNTA) solution was prepared by dissolving 9.9mM iron nitrate and 8.5mM nitrilotriacetic acid or nitrilotriacetic acid disodium salt with ultrapure water, and the pH was adjusted to 7.0 with solid sodium bicarbonate. Adding FeNTA into the iron-free lactoferrin solution in proportion, and reacting for 1 hour at room temperature;

(6) and (4) carrying out vacuum freeze drying on the reacted solution to obtain the lactoferrin with high iron saturation.

The method takes lactoferrin such as natural lactoferrin as raw material to prepare the iron-free lactoferrin, and then prepares the lactoferrin with high iron saturation from the iron-free lactoferrin to improve the heat stability of the lactoferrin.

The invention has the advantages and positive effects that:

the method takes natural lactoferrin as a raw material, prepares the iron-free lactoferrin by dialyzing the natural lactoferrin in an acidic buffer solution, then adds a freshly prepared iron-nitrogen-based triethyl acid solution into the iron-free lactoferrin in proportion to prepare the lactoferrin with high iron saturation, and the stability of the lactoferrin with high iron saturation after being heated is improved by measuring the stability.

Drawings

Figure 1 shows the stability factor of the sample as a whole.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described more clearly and completely in conjunction with the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. In acidic solutions, lactoferrin more readily releases iron ions, so native lactoferrin is dialyzed into iron-free lactoferrin using an acidic buffer. Lactoferrin binds iron ions in the presence of carbonate ions, so sodium bicarbonate is used to adjust the PH, and iron ions are added in the presence of carbonate ions to cause the lactoferrin to bind iron to form lactoferrin of high iron saturation.

Examples

The technical solutions of the present invention will be illustrated below in the form of examples, but the scope of protection of the present invention is not limited to these examples.

The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.

The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.

In the examples, natural lactoferrin with 11% iron saturation is used as a raw material to prepare iron-free lactoferrin, and then lactoferrin with high iron saturation is prepared from the iron-free lactoferrin to improve the heat stability of the lactoferrin. The method comprises the following specific steps:

dissolving natural lactoferrin in ultrapure water to prepare a solution with the concentration of 50mg/ml, fully dissolving the solution on a magnetic stirrer, and putting the solution in a dialysis bag with the interception range of 8-14 kDa;

putting a dialysis bag filled with a natural lactoferrin solution into a buffer solution for dialysis for 24 hours, wherein the buffer solution in the first 12 hours is an acidic buffer solution with the pH value of 3-5, such as a citric acid-sodium citrate buffer solution, a disodium hydrogen phosphate-citric acid buffer solution, a citric acid-sodium hydroxide-hydrochloric acid buffer solution, an acetic acid-sodium acetate buffer solution and the like, and the buffer solution in the last 12 hours is water, and is carried out at room temperature while keeping a constant rotating speed;

thirdly, performing vacuum freeze drying on the dialyzed lactoferrin solution to obtain iron-free lactoferrin, and determining that the saturation of the iron-free lactoferrin is 4%;

fourthly, dissolving the iron-free lactoferrin in a Tris-Hcl buffer solution containing Nacl to prepare a 1% solution, and adjusting the pH value to 7.0-7.8 by using Hcl;

fife, 9.9mM ferric nitrate and 8.5mM nitrilotriacetic acid disodium salt or nitrilotriacetic acid are dissolved by ultrapure water to prepare a fresh solution of iron nitrilotriacetic acid (FeNTA), and the pH is adjusted to 7.0 by using solid sodium bicarbonate. The FeNTA was added to the lactoferrin solution and the amount needed was calculated to achieve iron: the molar ratio of lactoferrin is 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.8:1, respectively. The mixture was reacted at room temperature for 1 hour;

sixthly, performing vacuum freeze drying on the reacted solution to obtain lactoferrin with high iron saturation, wherein the iron saturation is 16%, 22%, 25%, 30% and 40% respectively.

(7) Dissolving the prepared lactoferrin with different iron saturation degrees in ultrapure water to prepare a solution of 1mg/ml, heating at 85 ℃ for 10min, and immediately cooling for later use.

(8) Stability determination

The stability of the heated lactoferrin was determined by a dispersion stability analyzer, and the heated solution was added to a cylindrical glass bottle dedicated to a Turbiscan Lab stability analyzer for measurement. The detection principle of the instrument is as follows: the method takes pulse near infrared as a light source, uses two optical detectors (a transmission light detector and a back scattering light detector) to synchronously scan from the bottom to the top of a glass bottle, and performs data acquisition once at a certain interval height to obtain a function image of transmission light and back scattering light signals to the height of a sample, and can also obtain a change curve of stable dynamic parameters (TSI for short) calculated according to back scattering light along with scanning time. The scanning parameters of the sample were: the sample temperature was set at 25 ℃ and the scan was continued 8 times every 30min and the data was recorded. The TSI value of the stability coefficient reflects the change of particulate matters in the sample along with time, the larger the change amplitude is, the larger the TSI value is, and the more unstable the system is, namely the TSI value is in negative correlation with the stability of the sample.

Figure 1 shows the stability factors of lactoferrin after heating, showing that the stability factors of lactoferrin at 4% and 11% iron saturation are the highest, indicating their worst stability after heating, whereas the stability factor of lactoferrin at a higher iron saturation obtained by the process of the invention is lower, indicating better stability of lactoferrin at a higher iron saturation after heating, and the stability factor of lactoferrin after heating decreases with increasing iron saturation, indicating better stability of lactoferrin at a higher iron saturation than lactoferrin with a lower iron saturation.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.