Lactoferrin nanofiber and preparation method thereof

Abstract

The invention discloses a lactoferrin nanofiber and a preparation method thereof, and the preparation method comprises the following steps: adjusting the pH value of a lactoferrin aqueous solution with the mass volume concentration of 2.5% (w / v) to 2.0, then heating at 90 DEG C for 4 hours, and finally cooling to terminate the reaction to obtain the lactoferrin nanofiber. The method is simple in process, controllable in condition and high in fiber forming speed. The prepared lactoferrin nanofiber is worm-shaped, rich in beta-folded structure, has positive charges on the surface, has good colloidal stability and functional characteristics, can be used as an excellent carrier material for encapsulating and protecting hydrophobic bioactive substances, and has wide application prospects in the fields of functional foods and medicines.

Description

Technical Field

[0001] This invention relates to the fields of biopolymer materials and food science and technology, and more specifically to a lactoferrin nanofiber and its preparation method. Background Technology

[0002] Protein nanofibers, particularly amyloid fibers formed through self-assembly, are a new class of bionanomaterials. These fibers typically possess high aspect ratios, abundant β-sheet structures, numerous hydrophobic regions, and excellent mechanical properties and stability, showing broad application prospects in the food, pharmaceutical, and materials fields, such as as delivery carriers for active ingredients, food texture modifiers, or building blocks for hydrogels.

[0003] Currently, studies have reported the preparation of protein nanofibers using whey protein, soy protein, and ovalbumin as raw materials. Lactoferrin is a multifunctional globular glycoprotein found in milk, possessing various natural biological activities (such as antibacterial and immunomodulatory effects) and a high isoelectric point (pI ~8-9). Compared to the aforementioned proteins, research on the preparation of nanofibers using lactoferrin is still insufficient. Its fiber-forming kinetics, optimal processing conditions, fine fiber structure, and properties as a functional material lack systematic elucidation. In particular, existing technologies lack a method for rapidly and efficiently preparing lactoferrin nanofibers with uniform morphology, stable structure, and excellent function. Developing such a method is crucial for expanding the application value of lactoferrin in high-end functional foods and nutritional supplements. Summary of the Invention

[0004] In view of this, the present invention provides lactoferrin nanofibers and a method for preparing the same, which is simple, has controllable conditions, and is highly efficient.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for preparing lactoferrin nanofibers includes the following steps:

[0007] (1) Dissolve lactoferrin powder in ultrapure water, stir at room temperature to prepare lactoferrin solution, and store in a refrigerator at 4°C overnight;

[0008] (2) Adjust the pH of the lactoferrin solution obtained in step (1) to 2.0-3.0 with hydrochloric acid, incubate in a water bath at 80-90℃ for 0-24h, and immediately place it in an ice bath to cool after incubation to obtain lactoferrin nanofibers.

[0009] Preferably, the stirring speed in step (1) is 500 rpm and the time is 4 hours.

[0010] Preferably, the mass-volume concentration of the lactoferrin solution in step (1) is 2.5%.

[0011] Preferably, the concentration of hydrochloric acid in step (2) is 2 mol / L.

[0012] The present invention also provides lactoferrin nanofibers prepared by the method described above.

[0013] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a lactoferrin nanofiber and its preparation method, which has the following beneficial effects:

[0014] Rapid fiber formation: This invention promotes the formation of a large number of nanofibers from lactoferrin in just 4 hours by heating at a specific acidic pH, which is faster than many reported plant or animal-derived proteins.

[0015] The process is simple and controllable: This invention can be achieved by adjusting only three key parameters: pH, temperature and time. No complicated equipment or additional reagents are required, making it easy to scale up production.

[0016] Excellent product performance: The prepared lactoferrin nanofibers have a unique worm-like morphology, a stable β-sheet structure and a high surface positive charge, which provides ideal conditions for them to serve as efficient carriers of hydrophobic bioactive substances (such as curcumin and resveratrol), and are expected to exhibit excellent encapsulation ability, protective effect and colloidal stability. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0018] Figure 1 The variation of thioflavin T (ThT) fluorescence intensity in the nanofibers formed by lactoferrin under different heating times in Example 1 of this invention;

[0019] Figure 2 The images shown are SDS-PAGE electrophoresis patterns of lactoferrin at different heating times in Example 1 of this invention.

[0020] Figure 3 This is a transmission electron microscope (TEM) image of the lactoferrin nanofibers prepared in Example 2 of the present invention;

[0021] Figure 4Potential measurement of the lactoferrin nanofibers prepared in Example 2 of this invention. Detailed Implementation

[0022] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1

[0024] Determination of formation kinetics and optimal conditions for lactoferrin nanofibers

[0025] Lactoferrin (LF, PUREnFERRIN™, >95% purity) was purchased from Pactum Dairy Group Pty. Ltd. Thioflavin T (ThT), hydrochloric acid, etc., were all of analytical grade. Ultrapure water was used in the experiments. Major instruments included a pH meter, a constant temperature water bath, and a multi-functional microplate reader (Agilent Technologies, USA).

[0026] Experimental methods:

[0027] 1) Preparation of lactoferrin fibrils (LFF)

[0028] Accurately weigh lactoferrin powder and dissolve it in ultrapure water to prepare a 2.5% (w / v) solution. Stir at 500 rpm for 4 hours at room temperature until fully dissolved, then let stand overnight at 4°C. Adjust the pH of the solution to 2.0 precisely using 2M HCl. Then, aliquot the solution into several centrifuge tubes and heat them in a 90°C water bath for 0, 1, 2, 4, 8, 12, and 24 hours, respectively. After the predetermined time, immediately immerse the sample tubes in an ice-water bath for rapid cooling to terminate the fibrosis reaction.

[0029] 2) Characterization

[0030] 2.1) ThT fluorescence spectroscopy analysis

[0031] Preparation of ThT working solution: In a black 96-well plate, add 10 μL of LFF sample heated for different times and 200 μL of ThT working solution in sequence, mix well and incubate in the dark for 10 minutes.

[0032] Fluorescence intensity was measured using a microplate reader at an excitation wavelength of 440 nm and an emission wavelength of 490 nm. The results are as follows: Figure 1As shown, the fluorescence intensity reaches its peak after 2 hours of heating, indicating that the fiber formation is at its maximum at this time. After 4 hours of heating, the fluorescence intensity still remains at about 90% of the peak level, indicating that the fiber structure is mature and stable.

[0033] 2.2) SDS-PAGE Analysis

[0034] Samples heated for different times were analyzed by electrophoresis using a Tricine-PAGE kit. Results are as follows: Figure 2 As shown, unheated natural LF (0h) exhibits a main band at approximately 80 kDa. With prolonged heating, the 80 kDa band weakens and disappears, while peptide bands at 25 kDa, 13 kDa, 12 kDa, and even smaller appear. After 4 hours of heating, the 13 kDa and 12 kDa peptides become dominant. This indicates that under high-temperature, acidic conditions, lactoferrin undergoes controlled hydrolysis, and the resulting small peptides are the basic building blocks of nanofibers.

[0035] Example 2

[0036] Morphology and structural characterization of lactoferrin nanofibers

[0037] This embodiment characterizes the morphology and structure of lactoferrin nanofibers prepared under the optimal conditions of Example 1 (90°C, pH 2.0, 4 h).

[0038] The main instruments include a dynamic light scattering (DLS) device (NanoBrook 90plus PALS, Brookhaven, USA) and a transmission electron microscope (Talos F200S G2, Thermo Fisher Scientific Co., USA.).

[0039] TEM observation

[0040] Unheated lactoferrin solution and LFF samples heated for 4 hours were diluted to 0.1 mg / mL with ultrapure water at pH 2.0. 10 μL of the diluted solution was dropped onto a copper grid on a carbon-supported membrane, allowed to stand for 5 minutes, and excess liquid was blotted away with filter paper. The membrane was negatively stained with 2% (w / v) phosphotungstic acid solution for 5 minutes, the stain was removed, and the membrane was dried at room temperature. The results were observed using TEM (Thermo Fisher Scientific, USA) at an accelerating voltage of 200 kV. Figure 3 As shown, a large number of discrete, unbranched worm-like nanofibers with a length of about 50 nm were formed in the sample, proving that the method of the present invention can successfully prepare lactoferrin nanofibers with uniform morphology.

[0041] Zeta potential measurement

[0042] The prepared LF and LFF dispersions were appropriately diluted with ultrapure water pre-adjusted to pH 2.0 and measured using a Zeta potential analyzer (Brookhaven, USA). The measured zeta potentials of LF and LFF were 21.69 mV and 29.63 mV, respectively, indicating that lactoferrin fibrils carry more positive charge than natural lactoferrin, their surface chemistry has changed, and they may have stronger surface activity and aggregation stability.

[0043] The various embodiments described in this specification are presented in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing lactoferrin nanofibers, characterized in that, Includes the following steps: (1) Dissolve lactoferrin powder in ultrapure water, stir at room temperature to prepare lactoferrin solution, and store in a refrigerator at 4°C overnight; (2) Adjust the pH of the lactoferrin solution obtained in step (1) to 2.0-3.0 with hydrochloric acid, incubate in a water bath at 80-90℃ for 0-24h, and immediately place it in an ice bath to cool after incubation to obtain lactoferrin nanofibers.

2. The method for preparing lactoferrin nanofibers according to claim 1, characterized in that, In step (1), the stirring speed is 500 rpm and the time is 4 hours.

3. The method for preparing lactoferrin nanofibers according to claim 1, characterized in that, The mass-volume concentration of the lactoferrin solution in step (1) is 2.5%.

4. The method for preparing lactoferrin nanofibers according to claim 1, characterized in that, The concentration of hydrochloric acid in step (2) is 2 mol / L.

5. Lactoferrin nanofibers prepared by the method according to any one of claims 1-4.

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