阅读说明：本技术 一种乳铁蛋白冷冻干燥保护剂及其应用 (Lactoferrin freeze-drying protective agent and application thereof ) 是由 罗述博 闫序东 杨蕾蕾 王彩云 程英 于 2018-06-05 设计创作，主要内容包括：本发明提供了一种乳铁蛋白冷冻干燥保护剂及其应用,所述乳铁蛋白冷冻干燥保护剂包括糖类及氨基酸,以所述乳铁蛋白的总重量为100％计,所述糖类的用量为0.01％-5％,所述氨基酸的用量为0.01％-2％。以乳铁蛋白对大肠杆菌的抑菌活性为评价指标,添加了本发明的冷冻干燥保护剂而冻干的乳铁蛋白的抑菌率明显高于未添加冷冻干燥保护剂冻干的乳铁蛋白,这表明本发明所提供的冷冻干燥保护剂可以有效降低乳铁蛋白在冷冻干燥过程中的活性损失。(The invention provides a lactoferrin freeze-drying protective agent and application thereof, wherein the lactoferrin freeze-drying protective agent comprises saccharides and amino acids, the total weight of the lactoferrin is 100%, the amount of the saccharides is 0.01% -5%, and the amount of the amino acids is 0.01% -2%. The antibacterial activity of the lactoferrin on escherichia coli is taken as an evaluation index, the bacteriostatic rate of the freeze-dried lactoferrin added with the freeze-drying protective agent is obviously higher than that of the lactoferrin freeze-dried without the freeze-drying protective agent, and the freeze-drying protective agent provided by the invention can effectively reduce the activity loss of the lactoferrin in the freeze-drying process.)

1. The freeze-drying protective agent for the lactoferrin is characterized by comprising saccharides and amino acids, wherein the total weight of the lactoferrin is 100%, the saccharides are 0.01-5%, and the amino acids are 0.01-2%.

2. A lactoferrin freeze-drying protector according to claim 1, characterized in that the carbohydrate is used in an amount of 0.1-2% and the amino acid in an amount of 0.05-1%.

3. A lactoferrin freeze-drying protector according to claim 1 or 2, characterized in that the sugar comprises one or a combination of several of sucrose, trehalose, lactose, fructose, lactulose, fructo-oligosaccharide, galacto-oligosaccharide, xylo-oligosaccharide, dextran, maltodextrin and pregelatinized starch.

4. A lactoferrin freeze-drying protector according to claim 1 or 2, characterized in that the amino acids comprise one or a combination of arginine, lysine, tryptophan and glycine;

preferably, when the amino acids include arginine and lysine, the molar ratio of arginine to lysine is 20:1 to 1: 20.

5. use of a lactoferrin freeze-drying protectant according to any one of claims 1 to 4 in lactoferrin freeze-drying.

6. a method for freeze-drying lactoferrin, wherein the method uses the lactoferrin freeze-drying protectant according to any one of claims 1 to 4, the method comprising the steps of:

(1) Adding the lactoferrin freeze-drying protective agent into the lactoferrin aqueous solution, and uniformly mixing at room temperature;

(2) Freeze-drying the mixed solution obtained in the step (1); wherein the freeze drying comprises the following four stages:

the first stage is as follows: the freeze drying temperature is in the range of-50 to-30 deg.C, and the pressure is 0.3bar for 0.5-3 h;

and a second stage: rapidly heating to-20 deg.C to-15 deg.C, maintaining for 3-8h, and keeping pressure at 0.3 bar;

and a third stage: heating to 0 deg.C, maintaining for 1-3h under 0.3 bar;

A fourth stage: heating to 25-35 deg.C, maintaining for 5-10 hr at 0.3 bar;

Preferably, the temperature rise rate of the rapid temperature rise is 1-1.5 ℃/min.

7. Lactoferrin freeze-drying process according to claim 6, characterized in that the mixing time is 1-60 min.

8. the freeze-drying method of lactoferrin according to claim 6, wherein the thickness of the control solution during the freeze-drying process is 1-5 cm.

9. A lactoferrin freeze-drying process according to claim 6, characterised in that the second stage is a rapid warming to a temperature of-15 ℃.

10. A lactoferrin freeze-drying process according to any of claims 6-9, characterized in that the concentration of lactoferrin is 1-10%, preferably 5%, based on 100% total weight of the lactoferrin aqueous solution;

Also preferably, the pH of the lactoferrin aqueous solution is 6.5-7.0;

Also preferably, the conductivity of the lactoferrin aqueous solution is 1.5mS/cm or less.

Technical Field

The invention relates to a lactoferrin freeze-drying protective agent and application thereof, belonging to the technical field of biology.

Background

Lactoferrin (LF) is a glycoprotein naturally present in the external secretions of milk, tears, saliva, etc. with bioactive functions including broad-spectrum antibacterial activity, enhancement of iron transmission and absorption, immunomodulation, regulation of inflammatory responses, and anti-oxidant action. The biological activity of lactoferrin is closely related to the structure of lactoferrin, and the mechanism of action of some lactoferrin structures on the biological activity is researched and confirmed that various excellent biological activities of lactoferrin must depend on the normal maintenance of protein structures at all levels. However, lactoferrin is sensitive to external environments such as heat, pH, ionic strength, and the like, and the structure thereof is easily changed to lose activity.

At present, commercial lactoferrin is mainly extracted from cow milk or whey, the final step of the lactoferrin production process is to dry a liquid lactoferrin solution into a solid state, and due to the high sensitivity of lactoferrin, the main drying methods at present are freeze drying and low-temperature spray drying.

Freeze drying (freeze drying for short) is a drying method in which a water-containing substance is frozen at a low temperature and then ice is sublimated by heating the freeze-dried substance under a vacuum condition, thereby removing water. Because the material is always at a lower temperature in the freeze-drying process, the change of the physical and chemical properties of the material is smaller, the quality of the freeze-dried material is higher, and the freeze-dried material has a loose and porous structure, thereby being very beneficial to the re-dissolution of the material. It is clear that freeze-drying is the most suitable method for drying structurally unstable proteins such as lactoferrin. Compared with drying modes such as roller drying, spray drying and the like, the structural change and activity loss of the lactoferrin caused by freeze drying are minimum. However, the lyophilization process is a complex phase transition process, and there are many factors inducing protein denaturation during freezing, primary drying, secondary drying, and the like. Thus, in fact, lactoferrin still suffers a relatively slight loss of its biological activity after freeze-drying. Besides adjusting the process parameters such as pre-freezing temperature, cooling rate and the like, the freeze-drying protective agent is also a particularly effective method for maintaining the biological activity of the lactoferrin.

The inventor of the present invention has found through practical research that the bioactivity of the lactoferrin reconstituted after conventional freeze-drying is reduced, which indicates that the lactoferrin is denatured to some extent during the freeze-drying process.

Therefore, the technical problem to be solved in the art is to provide a freeze-drying protective agent which is suitable for lactoferrin and can effectively reduce the activity loss of lactoferrin in the freeze-drying process.

Disclosure of Invention

In order to solve the above disadvantages and shortcomings, the present invention aims to provide a freeze-drying protective agent for lactoferrin. The lactoferrin freeze-drying protective agent provided by the invention can greatly reduce the activity loss of lactoferrin in the freeze-drying process.

the invention also aims to provide the application of the lactoferrin freeze-drying protective agent in lactoferrin freeze drying.

The invention also aims to provide a lactoferrin freeze-drying method, which adopts the lactoferrin freeze-drying protective agent.

In order to achieve the above object, in one aspect, the present invention provides a lactoferrin freeze-drying protective agent, wherein the lactoferrin freeze-drying protective agent includes saccharides and amino acids, the saccharides are used in an amount of 0.01% to 5% and the amino acids are used in an amount of 0.01% to 2% based on the total weight of the lactoferrin as 100%.

The lactoferrin freeze-drying protective agent is preferably used in an amount of 0.1% -2% of the saccharides and 0.05% -1% of the amino acids.

The sugar has two main protection mechanisms, one is that the sugar with high viscosity surrounds the protein molecules, limits the diffusion of substances, and reduces the extension degree of the protein molecules, for example, sucrose has a relatively high glass transition temperature, so that the sucrose can be concentrated with the protein in a frozen state to dilute the protein, thereby preventing polymerization and maintaining the structure of the protein molecules; secondly, after the protein loses water, hydrogen bonds originally combined with water molecules are combined with sugar molecules, so that the hydrogen bonds and polar groups on the surface of the protein cannot be denatured due to exposure.

according to the lactoferrin freeze-drying protective agent, preferably, the saccharide comprises one or more of sucrose, trehalose, lactose, fructose, lactulose, fructo-oligosaccharide, galacto-oligosaccharide, xylo-oligosaccharide, glucan, maltodextrin and pregelatinized starch.

When the amino acid is used as a protein freeze-drying protective agent, the glass transition temperature of the amino acid is relatively higher than that of a non-amino acid buffer agent, so that the amino acid plays a certain role in the stability of a freeze-drying preparation; in the freezing and freeze-drying processes, specific interaction occurs between amphoteric amino acid molecules and protein molecules, so that the protein invariance can be protected; in addition, amino acids can also protect proteins by preventing crystallization of buffer salts. Therefore, the freeze-drying protective agent claimed by the invention can significantly reduce the loss of lactoferrin activity in the freeze-drying process by adopting the combination of the saccharides and the amino acids.

According to the lactoferrin freeze-drying protective agent, preferably, the amino acid comprises one or a combination of arginine, lysine, tryptophan and glycine.

According to the lactoferrin freeze-drying protective agent, preferably, when the amino acid comprises arginine and lysine, the molar ratio of the arginine to the lysine is 20:1-1: 20.

According to the application field and relevant regulations of lactoferrin, different combinations of saccharides and amino acids can be selected as a lactoferrin freeze-drying protective agent in the freeze-drying process, for example, for lactoferrin in infant formula milk powder, the freeze-drying protective agent can be selected from fructo-oligosaccharide and/or galacto-oligosaccharide and tryptophan, wherein the fructo-oligosaccharide and the galacto-oligosaccharide are prebiotics which are beneficial to intestinal health of infants, and the tryptophan is important for neural development of infants; and for lactoferrin for energy supplement use, a freeze-drying protective agent used for the lactoferrin can be fructose and various amino acids in combination; the freeze-drying protective agent used for the lactoferrin used for the yoghourt and the liquid milk can be selected from lactose and/or a combination of glucan and glycine with relatively low cost.

On the other hand, the invention also provides the application of the lactoferrin freeze-drying protective agent in lactoferrin freeze drying.

In another aspect, the present invention also provides a freeze-drying method of lactoferrin, which uses the freeze-drying protective agent of lactoferrin, the method comprising the following steps:

(1) Adding the lactoferrin freeze-drying protective agent into the lactoferrin aqueous solution, and uniformly mixing at room temperature;

(2) freeze-drying the mixed solution obtained in the step (1); wherein the freeze drying comprises the following four stages:

The first stage is as follows: the freeze drying temperature is in the range of-50 to-30 deg.C, and the pressure is 0.3bar for 0.5-3 h;

And a second stage: rapidly heating to-20 deg.C to-15 deg.C, maintaining for 3-8h, and keeping pressure at 0.3 bar;

And a third stage: heating to 0 deg.C, maintaining for 1-3h under 0.3 bar;

a fourth stage: heating to 25-35 deg.C, and maintaining at 0.3bar for 5-10 h.

According to the lactoferrin freeze-drying method, the four-stage freeze-drying method can greatly reduce freeze-drying time and freeze-drying cost, wherein the second stage is mainly adopted, and the temperature is increased to be within the range of-20 to-15 ℃ by adopting rapid heating. The main purposes of this operation are: since the water of the freeze-dried product is mainly discharged at a low temperature, i.e. below the eutectic point of the solution, the eutectic point of the lactoferrin is between-14 and-12 ℃, and therefore, the supply of more heat at the eutectic point temperature during freeze-drying is an important factor for shortening the freeze-drying time. The moisture is removed as much as possible in the early stage, and the later drying time can be shortened.

according to the lactoferrin freeze-drying method of the present invention, preferably, the mixing time is 1-60 min.

According to the lactoferrin freeze-drying method, the thickness of the feed liquid is preferably controlled to be 1-5cm in the freeze-drying process. Wherein, in the freeze drying process, if the thickness of the feed liquid is too large, the feed liquid can not be completely freeze-dried, so the thickness of the feed liquid needs to be controlled to be 1-5cm in the lactoferrin freeze drying method provided by the invention.

According to the lactoferrin freeze-drying method of the present invention, preferably, the second stage is to rapidly raise the temperature to-15 ℃.

according to the lactoferrin freeze-drying method, preferably, the temperature rise rate of the rapid temperature rise is 1-1.5 ℃/min.

According to the lactoferrin freeze-drying method of the present invention, preferably, the concentration of lactoferrin is 1 to 10%, more preferably 5%, based on the total weight of the lactoferrin aqueous solution as 100%.

According to the lactoferrin freeze-drying method of the present invention, preferably, the pH of the lactoferrin aqueous solution is 6.5 to 7.0.

According to the lactoferrin freeze-drying method of the present invention, preferably, the conductivity of the lactoferrin aqueous solution is 1.5mS/cm or less. Wherein, in the freeze drying process of the lactoferrin, the amount of salts contained in the lactoferrin solution can be reduced by controlling the conductivity of the lactoferrin solution to be less than 1.5mS/cm, and the finally obtained lactoferrin has higher purity.

The lactoferrin freeze-drying method provided by the invention is characterized in that the lactoferrin aqueous solution is prepared by chromatography and membrane filtration technologies, and specifically, the preparation method comprises the following steps: carrying out ion exchange chromatography on a milk raw material containing lactoferrin, such as skim milk or whey protein, to obtain a lactoferrin crude extract, wherein the concentration of the lactoferrin is about 1% by taking the total weight of the lactoferrin crude extract as 100%;

And carrying out ultrafiltration, concentration and desalination on the lactoferrin crude extract to obtain a lactoferrin water solution with the pH value of 6.5-7.0, the conductivity of less than 1.5mS/cm and the concentration of 1-10%.

If the concentration of the lactoferrin is more than 10%, the lactoferrin aqueous solution can precipitate to block the membrane filter, so that the concentration speed is influenced, and further the concentration cannot be continued.

Adding the lactoferrin freeze-drying protective agent provided by the invention into the lactoferrin aqueous solution, uniformly stirring, and freeze-drying the lactoferrin according to the freeze-drying program. The antibacterial activity of the lactoferrin on escherichia coli is taken as an evaluation index, and the antibacterial rate of the freeze-dried lactoferrin added with the freeze-drying protective agent is obviously higher than that of the lactoferrin freeze-dried without the freeze-drying protective agent, which shows that the freeze-drying protective agent provided by the invention can effectively reduce the activity loss of the lactoferrin in the freeze-drying process, namely the freeze-drying protective agent can effectively reduce the damage of the lactoferrin in the freeze-drying process, and the biological activity preservation rate of the lactoferrin in the production and processing is improved to a certain extent.

Detailed Description

In order to clearly understand the technical features, objects and advantages of the present invention, the following detailed description of the technical solutions of the present invention will be made with reference to the following specific examples, which should not be construed as limiting the implementable scope of the present invention.

the procedures and equipment used in the present invention, which are not specifically mentioned in the present invention, can be performed according to the conventional procedures in the art or by referring to the specifications of the equipment used.