Preparation method of beta-lactoglobulin amyloid fiber

Abstract

The invention belongs to the technical field of biological materials, and particularly relates to a preparation method of beta-lactoglobulin amyloid fibers, which comprises the following steps: dissolving beta-lactoglobulin in distilled water to prepare a 2% beta-lactoglobulin solution, filling the 2% beta-lactoglobulin solution into a vacuum bottle, vacuumizing the vacuum bottle, and carrying out vacuum ultrasonic-assisted pretreatment; and keeping the pretreated protein solution in a vacuum environment, stirring at a rotating speed of 300rpm, heating for 10 hours, and quenching reaction by using an ice-water bath to obtain the product. The beta-lactoglobulin amyloid fiber prepared by the method disclosed by the invention is better in quality and higher in conversion rate; processing in a low-pH environment is not needed, introduction of toxic substances such as TCEP and DTT is avoided, and the restrictive problem of application of an existing preparation method in the fields of food and biological medicine is solved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, and particularly relates to a method for preparing beta-lactoglobulin amyloid fibers. Background Art

[0002] As a new type of biomaterial, amyloid fibrils have attracted much attention in the field of functional materials due to their unique nanofiber structure, high aspect ratio, large specific surface area, and excellent mechanical toughness. Currently, this material system has been successfully applied in various fields such as the food industry, advanced materials, and environmental engineering. In particular, it has demonstrated the following core advantages in the field of food science: (1) optimizing food rheological properties through interfacial stabilization effects; (2) improving bioavailability as a nutrient delivery system; and (3) constructing gel networks to improve product texture characteristics.

[0003] The current mainstream amyloid fiber preparation technologies mainly include acid-heat method, reducing agent method, and ultrasonic method. Although the acid-heat method is mature and has a high conversion rate (>70%), the reaction process requires a strongly acidic environment (pH <3), resulting in an excessively high H+ concentration in the system, which limits its practical application in fields such as food and biomedicine. The reducing agent method can achieve rapid fibrillation within 10-30 minutes by breaking disulfide bonds. However, this method requires the use of highly toxic chemical reducing agents such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and dithiothreitol (DTT), which poses biosafety risks and seriously restricts its application in nutrient delivery systems. Although the ultrasonic method has the advantage of simple operation, it is generally associated with problems such as short and coarse fibers and low conversion rate. In addition, the product has defects such as insufficient viscoelasticity and is currently limited to laboratory-scale research. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for preparing β-lactoglobulin amyloid fibers. The technology uses a vacuum environment and ultrasound assistance to pretreat β-lactoglobulin to unfold its structure, and then heat treats it to promote fiber formation.

[0005] In order to achieve the above object, the present invention provides the following technical solutions: A method for preparing β-lactoglobulin amyloid fibers comprises the following steps: (1) Vacuum ultrasonic assisted pretreatment: β-lactoglobulin was dissolved in distilled water to prepare a 2% β-lactoglobulin solution, which was placed in a vacuum bottle and evacuated for vacuum ultrasonic assisted pretreatment. (2) Fibrillation: The protein solution after pretreatment in step (1) was kept in a vacuum environment, stirred at 300 rpm, heated for 10 h, and then quenched in an ice-water bath to obtain the product.

[0006] Furthermore, in step (1), the vacuum bottle is evacuated to -0.08 MPa.

[0007] Furthermore, the parameters of vacuum ultrasound were as follows: vacuum ultrasound-assisted pretreatment at 36 kHz and 225 W for 3 h.

[0008] Furthermore, the heating temperature is 60°C.

[0009] The beneficial effects of the present invention are as follows: (1) High biosafety, suitable for food and biopharmaceutical fields; the method of the present invention does not require processing in a low pH environment and avoids the introduction of toxic substances such as TCEP and DTT, thus solving the problem of limitations of existing preparation methods in the application of food and biopharmaceutical fields.

[0010] (2) The prepared β-lactoglobulin amyloid fibers have better quality and higher conversion rate; the fibers prepared by the method of the present invention have a slender straight chain structure, a diameter of about 6-8nm, a length of up to micrometer level, can form a three-dimensional network structure, and have highly ordered fibrillation behavior. The fiber conversion rate is significantly higher than that of single heating or single vacuum ultrasonic treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Schematic diagram of the process of the present invention; Figure 2 1 is a comparison of the fluorescence intensity of amyloid protein fiber thioflavin T (ThT) in the examples of the present invention and the comparative examples; Figure 3 This is a comparison chart of protein fiber conversion rates in Examples of the present invention and Comparative Examples; Figure 4 Transmission electron microscope (TEM) photos of amyloid fibers in Example 1 of the present invention and Comparative Examples 1-2; Figure 5 This is a transmission electron microscope (TEM) photograph of the amyloid fiber in Comparative Example 3-4 of the present invention. DETAILED DESCRIPTION

[0012] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be derived from these drawings without inventive effort.

[0013] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The embodiments cannot be described one by one here, but the embodiments of the present invention are not limited to the following embodiments.

[0014] Example 1: A method for preparing β-lactoglobulin amyloid fibers. Figure 1 As shown, the following steps are included: (1) Weigh a certain amount of β-lactoglobulin and dissolve it in water at a concentration of 2%. Transfer the protein solution to a vacuum bottle, evacuate to -0.08 MPa, and vacuum ultrasonicate at 36 kHz and 225 W for 3 h.

[0015] (2) Transfer the pretreated β-lactoglobulin solution from step (1) to a 60°C water bath, stir at 300 rpm, and continue heating for 10 h. After the heat treatment, place in an ice water bath to quench the reaction.

[0016] Comparative Example 1 A method for preparing β-lactoglobulin amyloid fibers comprises the following steps: (1) Weigh a certain amount of β-lactoglobulin and dissolve it in water at a concentration of 2%. Transfer the protein solution to a vacuum bottle, evacuate to -0.06 MPa, and vacuum ultrasonicate at 32 kHz and 200 W for 2 h.

[0017] (2) Transfer the pretreated β-lactoglobulin solution from step (1) to a 60°C water bath, stir at 300 rpm, and continue heating for 10 h. After the heat treatment, place in an ice water bath to quench the reaction.

[0018] Comparative Example 2 A method for preparing β-lactoglobulin amyloid fibers comprises the following steps: (1) Weigh a certain amount of β-lactoglobulin and dissolve it in water at a concentration of 2%. Transfer the protein solution to a vacuum bottle, evacuate to -0.09 MPa, and perform vacuum ultrasonic treatment at 40 kHz and 250 W for 3 h.

[0019] (2) Transfer the pretreated β-lactoglobulin solution from step (1) to an 80°C water bath, stir at 300 rpm, and continue heating for 10 h. After the heat treatment, place in an ice water bath to quench the reaction.

[0020] Comparative Example 3 A method for preparing β-lactoglobulin amyloid fibers, comprising the following steps: A certain amount of β-lactoglobulin was weighed and dissolved in water at a concentration of 2%. The mixture was stirred at 300 rpm and heated at 80°C for 10 h. After the heat treatment, the mixture was placed in an ice-water bath to quench the reaction.

[0021] Comparative Example 4 A method for preparing β-lactoglobulin amyloid fibers, comprising the following steps: A certain amount of β-lactoglobulin was weighed and dissolved in water at a concentration of 2%. The protein solution was transferred to a vacuum bottle, evacuated to -0.08 MPa, and vacuum ultrasonicated at 36 kHz and 225 W for 3 h.

[0022] Test Example The products in Example 1 and Comparative Examples 1-4 of the present invention were tested. Thioflavin T (ThT) fluorescence detection is an important means to evaluate the formation of amyloid fibrils. ThT molecules can specifically embed into the typical β-pleated structure groove of amyloid proteins, and the fixed conformation leads to enhanced fluorescence. ThT solution (30 mg / L) is mixed with the test protein solution at a ratio of 1:1 (v:v), and the fluorescence value is detected under the conditions of 440 nm excitation light and 486 nm emission light. The results are as follows: Figure 2 As shown, Example 1 has the highest fluorescence intensity, significantly superior to Comparative Examples 1 and 2, indicating that it has the best amyloid fibril formation effect. In contrast, the treatment methods of Comparative Examples 3 and 4 only induced partial unfolding of the β-lactoglobulin structure and did not effectively promote the fibrillization process, indicating that such treatments are insufficient to drive the typical amyloid self-assembly process.

[0023] β-lactoglobulin and its amyloid fibrils were separated by isoelectric point separation. The protein solution to be tested was mixed with phosphate-citrate buffer (pH = 4.8) at a ratio of 1:9 (v:v). After standing at 4°C overnight, the mixture was centrifuged at 10,000 rpm for 15 minutes. 200 μL of the supernatant was taken and its absorbance was measured at 278 nm. Figure 3 The fiber conversion rate shown further confirms the above trend. The composite treatment method (vacuum ultrasound combined with heating) adopted in Example 1 significantly improved the fiber conversion efficiency and showed a better structural induction effect compared with other comparative examples.

[0024] (3) Transmission electron microscopy (TEM) images (e.g. Figure 4 and Figure 5 (shown) provides intuitive morphological evidence. Figure 4 It can be seen that the amyloid fibers obtained in Example 1 have good morphology, presenting an elongated straight chain structure with a diameter of about 6-8nm and a length of up to micrometers, and form a certain degree of three-dimensional network structure, showing highly ordered fibrillation behavior. The processing power and vacuum degree used in Comparative Example 1 are relatively low, resulting in incomplete protein unfolding, thereby limiting the number of fibers and the formation of network structure; while the power and vacuum degree in Comparative Example 2 are relatively high, causing the protein to rapidly aggregate hydrophobic residues after rapid unfolding, resulting in a fast fiber formation rate, but it is difficult to effectively capture free residues for continued growth, and ultimately forming fibers with a shorter structure and a thicker diameter. Figure 5It can be seen that the vacuum ultrasonic treatment used in Comparative Example 3 can only unfold the structure of β-lactoglobulin and form amorphous protein aggregates; the heating treatment used in Comparative Example 4 can induce the assembly of β-lactoglobulin to form worm-like amyloid fibers with a length of less than 100 nm, but the fiber conversion rate is low.

[0025] In summary, the synergistic treatment method adopted in the present invention has significant advantages in promoting the unfolding of β-lactoglobulin structure, inducing ordered assembly and improving the quality of fiber formation, and provides an effective process for preparing high-quality amyloid protein fibers.

[0026] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for preparing β-lactoglobulin amyloid fibers, characterized in that: The following steps are involved: (1) Vacuum ultrasonic assisted pretreatment: β-lactoglobulin was dissolved in distilled water to prepare a 2% β-lactoglobulin solution, which was placed in a vacuum bottle and evacuated for vacuum ultrasonic assisted pretreatment. (2) Fibrillation: The protein solution after pretreatment in step (1) was kept in a vacuum environment, stirred at 300 rpm, heated for 10 h, and then quenched in an ice-water bath to obtain the product.

2. The method for preparing β-lactoglobulin amyloid fibers according to claim 1, characterized in that: In step (1), the vacuum bottle is evacuated to -0.08 MPa.

3. The method for preparing β-lactoglobulin amyloid fibers according to claim 1, characterized in that: The parameters of vacuum ultrasound were as follows: vacuum ultrasound-assisted pretreatment at 36 kHz and 225 W for 3 h.

4. The method for preparing β-lactoglobulin amyloid fibers according to claim 1, characterized in that: The heating temperature is 60°C.

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