Method for promoting fibrosis of plant protein by subcritical water technology

Abstract

The invention discloses a method for promoting fibrosis of plant proteins by a subcritical water technology, which comprises the following steps: (1) dispersing plant proteins in water to obtain a uniform protein dispersion liquid; (2) adjusting the pH value of the protein dispersion liquid to 2.0-2.5 to obtain an acidic protein dispersion liquid; and (3) treating the acidic protein dispersion liquid in a subcritical water environment at 105-120 DEG C for 20-60 minutes to obtain the vegetable protein nanofiber. According to the method, protein is placed under an acidic condition, and a protein space structure is rearranged in a subcritical water environment. In a subcritical water environment, a tertiary structure of the protein is partially or completely expanded, so that a hydrophobic group is exposed. The process promotes the conversion of the protein from a spherical structure to a fibrous structure.

Description

Technical Field

[0001] This invention relates to plant protein processing, and more specifically, to a method for efficiently promoting plant protein fibrosis using subcritical water technology. Background Technology

[0002] Amyloid fibrous structures are ordered aggregates of proteins rich in cross-beta structures (EISENBERG DS, SAWAYA M R. Structural Studies of Amyloid Proteins at the Molecular Level[J]. 2017, 86(Volume 86, 2017): 69-95.). Fibrosis typically begins with the complete or partial unfolding of globular proteins and ends with ordered fibrous structures (ADAMCIK J, MEZZENGA R. Proteins Fibrils from a Polymer Physics Perspective[J]. Macromolecules, 2012, 45(3): 1137-50.). Due to their dense and ordered hydrogen bond network and low free energy, protein amyloid fibrils exhibit strong tolerance to extreme environments such as acid, heat, and enzymes, making them one of the toughest protein materials (WU H, NIAN Y, LIU Y, et al. Formation of pea protein amyloid fibrils to stabilize high internal phase emulsions for encapsulation of lutein [J]. Journal of Functional Foods, 2022, 94: 105110.).

[0003] In recent years, the application of amyloid fibers in food has attracted widespread attention, and their unique structural characteristics have provided new solutions for the food industry. Due to their high aspect ratio and rigid structure, amyloid fibers can be used to improve the fibrous texture of plant-based meat products (Zhang H, Lv S, Jin C, Ren F, Wang J. Wheat gluten amyloidfibrils: Conditions, mechanism, characterization, application, and future perspectives. Int J Biol Macromol. 2023 Dec 31;253(Pt 3):126435.). Furthermore, the surface functional groups (such as hydroxyl and amino groups) of amyloid fibers can be used to load bioactive components to achieve sustained release and protection (Yu Z, Li N, Liu Y, Zhang B, Zhang M, Wang X, Wang X. Formation, structure and functional characteristics of amyloid fibrils formed based on soy proteinisolates. Int J Biol Macromol. 2024 Jan;254(Pt 3):127956.). Meanwhile, its biodegradability and mechanical properties make it a potential choice for environmentally friendly packaging materials. In meat alternative production, high-moisture extrusion technology combined with plant protein amyloid fibers can produce plant-based products with a structure similar to animal meat fibers, improving physical stability, enhancing nutrient utilization efficiency, and expanding the applications of plant proteins. Its core advantages lie in its natural biodegradability, extreme stability, functional diversity, low cost, and sustainability.

[0004] Currently, the main methods for preparing protein fibers are hydrothermal and microwave heating. The traditional hydrothermal method (85℃, 12 h) for preparing protein amyloid fibers is time-consuming and energy-intensive, making industrial production difficult (WANG F, LIN L, WANG X, et al. Astaxanthin complexes of five plant protein fibrils: Aggregation behavior, interaction, and delivery properties [J]. Food Hydrocolloids, 2025, 162: 110995.). Furthermore, the reaction conditions and degree of reaction are difficult to control in the microwave heating process. Summary of the Invention

[0005] To address the current problems of high energy consumption and difficulty in controlling the reaction rate of protein fibers, this invention provides a method for promoting plant protein fiberization using subcritical water technology. This method is simple to operate and has low operating costs. Compared with traditional hydrothermal methods, subcritical water technology can efficiently, energy-savingly, and time-savingly prepare protein amyloid fibers, and is easy to industrialize.

[0006] To achieve the above objectives, the present invention provides a method for promoting plant protein fibrosis using subcritical water technology, comprising the following steps: (1) Disperse the plant protein in water to obtain a uniform protein dispersion; (2) Adjust the pH of the protein dispersion to 2.0-2.5 to obtain an acidic protein dispersion; (3) The acidic protein dispersion was treated in a subcritical water environment at 105-120℃ for 20-60 min to obtain plant protein nanofibers.

[0007] The core of protein fibrillation lies in the process of globular protein structures unfolding into ordered aggregates of fibers. First, a homogeneous dispersion is obtained through physical dispersion, then the pH is adjusted to a strongly acidic environment. This step does not directly trigger fibrillation but rather paves the way for subsequent structural changes. Subcritical water (liquid water below the critical temperature and pressure Tc=374℃, Pc=22.1MPa) is a key driving factor for fibrillation; its low viscosity and low dielectric constant physicochemical properties can efficiently disrupt the tertiary structure of proteins. Transmission electron microscopy results show that untreated proteins are all globular, while fibrous structures are observed in the subcritical water-treated group. Particle size data corroborate this, with walnut protein decreasing from 460 nm to 234 nm and flaxseed protein from 890 nm to 344 nm, indicating that after the tertiary structure unfolds, protein molecules change from tightly packed globular structures to loose linear structures. The Congo red spectral absorbance of the subcritical water group is significantly higher than that of the untreated group and the hydrothermal group. Congo red can specifically bind to the cross-β structure of amyloid fibers, indicating that subcritical water promotes the formation of more ordered fibers.

[0008] Preferably, in step (1), the concentration of plant protein in the protein dispersion is 1-6 mg / mL.

[0009] Specifically, in step (1), the dispersion is achieved by stirring for 1-2 hours.

[0010] Preferably, in step (3), before performing subcritical treatment, the acidic protein dispersion is centrifuged at 4000-5000 r / min for 5-10 min.

[0011] The purpose of centrifugation is to ensure that the obtained acidic protein dispersions are completely dissolved, so that they can undergo better fibrotic transformation in the subsequent subcritical water environment.

[0012] Preferably, in step (3), the subcritical water ambient temperature is 110-115℃.

[0013] A second aspect of the present invention provides plant protein fibers obtained by the above-described method.

[0014] Through the above technical solution, the present invention achieves the following beneficial effects: 1. This invention places the protein under acidic conditions, utilizing a subcritical water environment to cause a rearrangement of the protein's spatial structure. In a subcritical water environment, the protein's tertiary structure partially or completely unfolds, leading to the exposure of hydrophobic groups. This process promotes the transformation of the protein from a globular structure to a fibrous structure.

[0015] 2. The plant protein nanofibers prepared by this invention have smaller particle sizes and exhibit a stronger degree of fibrosis compared to plant protein nanofibers prepared by existing hydrothermal methods.

[0016] 3. This invention is simple to operate, has low operating costs, saves energy and time. Subcritical water technology can greatly improve the efficiency of protein transformation from a globular structure to a fibrous structure, reducing it from 12 hours to less than 1 hour, making it easy for industrial production. Attached Figure Description

[0017] Figure 1 Particle size and potential diagrams of walnut protein samples prepared in Comparative Example 1, Comparative Example 4, Example 1, Example 2 and Example 3; Figure 2 Particle size and potential diagrams of flaxseed protein samples prepared in Comparative Example 2, Comparative Example 5, Example 4, Example 5 and Example 6; Figure 3 Particle size and potential diagrams of sunflower seed protein samples prepared in Comparative Examples 3, 6, 7, 8 and 9; Figure 4 This is a schematic diagram showing the Congo red intensity of walnut protein samples prepared in Comparative Example 1, Comparative Example 4, Example 1, Example 2 and Example 3; Figure 5 This is a schematic diagram showing the Congo red intensity of flaxseed protein samples prepared in Comparative Example 2, Comparative Example 5, Example 4, Example 5, and Example 6. Figure 6 A schematic diagram showing the Congo red intensity of sunflower seed protein samples prepared in Comparative Example 3, Comparative Example 6, Example 7, Example 8 and Example 9; Figure 7 Transmission electron microscopy (TEM) schematic diagrams of walnut protein samples prepared in Comparative Example 1, Comparative Example 4, Example 1, Example 2, and Example 3; Figure 8Transmission electron microscopy (TEM) diagrams of flaxseed protein samples prepared in Comparative Examples 2, 5, 4, 5, and 6. Figure 9 This is a transmission electron microscope (TEM) schematic diagram of the sunflower seed protein samples prepared in Comparative Examples 3, 6, 7, 8, and 9. Detailed Implementation

[0018] The specific embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0019] Example 1 Preparation of walnut protein nanofibers via subcritical water method: 2 mg / mL walnut protein was dispersed in water and stirred for 1.5 h until fully hydrated to obtain a homogeneous walnut protein dispersion. The pH of the walnut protein dispersion was adjusted to 2.0 using 3M hydrochloric acid solution, and after thorough stirring, an acidic walnut protein dispersion was obtained. The acidic walnut protein dispersion was centrifuged at 4000 r / min for 10 min and placed in a subcritical water reactor. The treatment temperature and time were adjusted to obtain walnut protein nanofibers. The subcritical water environmental conditions were: temperature 110℃ and time 50 min.

[0020] Example 2 Preparation of walnut protein nanofibers via subcritical water method: 6 mg / mL walnut protein was dispersed in water and stirred for 2 h until fully hydrated to obtain a homogeneous walnut protein dispersion. The pH of the walnut protein dispersion was adjusted to 2.0 using 3M hydrochloric acid solution, and after thorough stirring, an acidic walnut protein dispersion was obtained. The acidic walnut protein dispersion was centrifuged at 5000 r / min for 50 min and placed in a subcritical water reactor. The treatment temperature and time were adjusted to obtain walnut protein nanofibers. The subcritical water environmental conditions were: temperature 105℃ and time 50 min.

[0021] Example 3 Preparation of walnut protein nanofibers via subcritical water method: 3 mg / mL walnut protein was dispersed in water and stirred for 1 h until fully hydrated to obtain a homogeneous walnut protein dispersion. The pH of the walnut protein dispersion was adjusted to 2.0 using 3M hydrochloric acid solution, and after thorough stirring, an acidic walnut protein dispersion was obtained. The acidic walnut protein dispersion was centrifuged at 4000 r / min for 10 min and placed in a subcritical water reactor. The treatment temperature and time were adjusted to obtain walnut protein nanofibers. The subcritical water environmental conditions were: temperature 120℃ and time 50 min.

[0022] Example 4 Preparation of flaxseed protein nanofibers via subcritical water method: 5 mg / mL flaxseed protein was dispersed in water and stirred until fully hydrated to obtain a homogeneous flaxseed protein dispersion. The pH of the flaxseed protein dispersion was adjusted to 2.0 using 3M hydrochloric acid solution, and after thorough stirring, an acidic flaxseed protein dispersion was obtained. The acidic flaxseed protein dispersion was centrifuged at 4000 r / min for 10 min and placed in a subcritical water reactor. The treatment temperature and time were adjusted to obtain flaxseed protein nanofibers. The subcritical water environmental conditions were: temperature 110℃ and time 50 min.

[0023] Example 5 Preparation of flaxseed protein nanofibers via subcritical water method: 5 mg / mL flaxseed protein was dispersed in water and stirred until fully hydrated to obtain a homogeneous flaxseed protein dispersion. The pH of the flaxseed protein dispersion was adjusted to 2.0 using 3M hydrochloric acid solution, and after thorough stirring, an acidic flaxseed protein dispersion was obtained. The acidic flaxseed protein dispersion was centrifuged at 5000 r / min for 5 min and placed in a subcritical water reactor. The treatment temperature and time were adjusted to obtain flaxseed protein nanofibers. The subcritical water environmental conditions were: temperature 115℃ and time 20 min.

[0024] Example 6 Preparation of flaxseed protein nanofibers via subcritical water method: 5 mg / mL flaxseed protein was dispersed in water and stirred until fully hydrated to obtain a homogeneous flaxseed protein dispersion. The pH of the flaxseed protein dispersion was adjusted to 2.0 using 3M hydrochloric acid solution, and after thorough stirring, an acidic flaxseed protein dispersion was obtained. The acidic flaxseed protein dispersion was centrifuged at 4000 r / min for 10 min and placed in a subcritical water reactor. The treatment temperature and time were adjusted to obtain flaxseed protein nanofibers. The subcritical water environmental conditions were: temperature 105℃ and time 60 min.

[0025] Example 7 Preparation of sunflower seed protein nanofibers via subcritical water method: 5 mg / mL sunflower seed protein was dispersed in water and stirred until fully hydrated to obtain a homogeneous sunflower seed protein dispersion. The pH of the sunflower seed protein dispersion was adjusted to 2.0 using 3M hydrochloric acid solution, and after thorough stirring, an acidic sunflower seed protein dispersion was obtained. The acidic sunflower seed protein dispersion was centrifuged at 5000 r / min for 5 min and placed in a subcritical water reactor. The treatment temperature and time were adjusted to obtain sunflower seed protein nanofibers. The subcritical water environmental conditions were: temperature 110℃ and time 40 min.

[0026] Example 8 Preparation of sunflower seed protein nanofibers via subcritical water method: 5 mg / mL sunflower seed protein was dispersed in water and stirred until fully hydrated to obtain a homogeneous sunflower seed protein dispersion. The pH of the sunflower seed protein dispersion was adjusted to 2.5 using 3M hydrochloric acid solution, and after thorough stirring, an acidic sunflower seed protein dispersion was obtained. The acidic sunflower seed protein dispersion was centrifuged at 4000 r / min for 10 min and placed in a subcritical water reactor. The treatment temperature and time were adjusted to obtain sunflower seed protein nanofibers. The subcritical water environmental conditions were: temperature 105℃ and time 40 min.

[0027] Example 9 Preparation of sunflower seed protein nanofibers via subcritical water method: 5 mg / mL sunflower seed protein was dispersed in water and stirred until fully hydrated to obtain a homogeneous sunflower seed protein dispersion. The pH of the sunflower seed protein dispersion was adjusted to 2.0 using 3M hydrochloric acid solution, and after thorough stirring, an acidic sunflower seed protein dispersion was obtained. The acidic sunflower seed protein dispersion was centrifuged at 4000 r / min for 10 min and placed in a subcritical water reactor. The treatment temperature and time were adjusted to obtain sunflower seed protein nanofibers. The subcritical water environmental conditions were: temperature 120℃ and time 40 min.

[0028] Comparative Example 1 Preparation of walnut protein nanofibers by hydrothermal method: 5 mg / mL of walnut protein was dispersed in water and stirred for 1.5 h until fully hydrated to obtain a homogeneous walnut protein dispersion. The pH of the walnut protein dispersion was adjusted to 2.0 using 3M hydrochloric acid solution, and after thorough stirring, an acidic walnut protein dispersion was obtained. The acidic walnut protein dispersion was centrifuged at 4000 r / min for 10 min, and then stirred in an 85℃ water bath for 12 h to obtain hydrothermal walnut protein nanofibers.

[0029] Comparative Example 2 Preparation of flaxseed protein nanofibers by hydrothermal method: 5 mg / mL flaxseed protein was dispersed in water and stirred until fully hydrated to obtain a homogeneous flaxseed protein dispersion. The pH of the flaxseed protein dispersion was adjusted to 2.0 using 3M hydrochloric acid solution, and after thorough stirring, an acidic flaxseed protein dispersion was obtained. The acidic flaxseed protein dispersion was centrifuged at 4000 r / min for 10 min, and then stirred in an 85℃ water bath for 12 h to obtain hydrothermal flaxseed protein nanofibers.

[0030] Comparative Example 3 Preparation of sunflower seed protein nanofibers by hydrothermal method: 5 mg / mL sunflower seed protein was dispersed in water and stirred until fully hydrated to obtain a homogeneous sunflower seed protein dispersion. The pH of the sunflower seed protein dispersion was adjusted to 2.0 using 3M hydrochloric acid solution, and after thorough stirring, an acidic sunflower seed protein dispersion was obtained. The acidic sunflower seed protein dispersion was centrifuged at 5000 r / min for 5 min, and then stirred in an 85℃ water bath for 12 h to obtain hydrothermally prepared sunflower seed protein nanofibers.

[0031] Comparative Example 4 Untreated walnut protein: 5 mg / mL of walnut protein was dispersed in water and stirred until fully hydrated to obtain a homogeneous walnut protein dispersion; the pH of the walnut protein dispersion was adjusted to 2.0 using 3M hydrochloric acid solution, and after thorough stirring, it was centrifuged at 4000 r / min for 10 min to obtain an acidic walnut protein dispersion.

[0032] Comparative Example 5 Untreated flaxseed protein: 5 mg / mL of flaxseed protein was dispersed in water and stirred until fully hydrated to obtain a homogeneous flaxseed protein dispersion; the pH of the flaxseed protein dispersion was adjusted to 2.0 using 3M hydrochloric acid solution, and after thorough stirring, it was centrifuged at 4000 r / min for 10 min to obtain an acidic flaxseed protein dispersion.

[0033] Comparative Example 6 Untreated sunflower seed protein: Disperse 5 mg / mL sunflower seed protein in water and stir until fully hydrated to obtain a homogeneous sunflower seed protein dispersion; adjust the pH of the sunflower seed protein dispersion to 2.0 using 3M hydrochloric acid solution, stir thoroughly, and centrifuge at 4000 r / min for 10 min to obtain an acidic sunflower seed protein dispersion.

[0034] Performance testing: (1) The particle size and potential of protein nanofibers prepared by subcritical water method, protein nanofibers prepared by hydrothermal method and untreated protein were determined by Malvern particle size analyzer (ES90 Nano).

[0035] The results are as follows Figure 1 , Figure 2 and Figure 3As shown, the particle size of plant protein nanofibers treated with subcritical water showed a significant decreasing trend compared to the untreated protein sample group (walnut protein particle size decreased from 460 nm to 234 nm, and flaxseed protein particle size decreased from 890 nm to 344 nm). Furthermore, except for flaxseed protein, the particle size of nanofibers prepared by the subcritical water method differed significantly from that prepared by the hydrothermal method for the other two proteins. However, there was no significant difference in potential between the subcritical water and hydrothermal methods. This indicates that treatment with both subcritical water and hydrothermal methods can significantly reduce the particle size of proteins, with the subcritical water method having a more significant impact on particle size, while the potential showed little difference during the treatment process.

[0036] (2) Mix 500 μL of sample with 5 mL of Congo red solution thoroughly and react in the dark for 30 min. Use a UV spectrophotometer (Cary 5000) to scan the spectral intensity of protein nanofibers prepared by subcritical water method, protein nanofibers prepared by hydrothermal method, and untreated protein in the range of 400-600 nm.

[0037] The results are as follows Figure 4 , Figure 5 and Figure 6 As shown, within the wavelength range of 400-600 nm, the Congo red spectral intensity of protein nanofibers prepared by the subcritical water method was higher than that of the untreated group, indicating that the subcritical water method can promote protein fibrillation. Furthermore, the Congo red spectral absorbance of the three types of protein nanofibers prepared by the subcritical water method was higher than that of the protein nanofibers prepared by the hydrothermal method. This indicates that the conversion efficiency of plant protein nanofibers prepared by the subcritical water method is significantly better than that prepared by the hydrothermal method.

[0038] (3) Protein nanofibers prepared by the subcritical water method, protein nanofibers prepared by the hydrothermal method, and untreated protein solution were adsorbed onto a carbon-coated copper grid and completely dried at room temperature. Images were recorded using a transmission electron microscope (HT7800, Hitachi) with an accelerating voltage of 200 kV and a magnification of 10 k.

[0039] The results are as follows Figure 7 , Figure 8 and Figure 9 As shown in the transmission electron microscope (TEM) diagram, the untreated plant protein samples all exhibited a spherical structure. The plant protein samples treated with subcritical water showed a greater degree of fibrosis compared to those treated with hydrothermal methods. This indicates that subcritical water can more efficiently convert plant proteins into fibrous structures compared to the traditional hydrothermal method.

[0040] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0041] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0042] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A method for promoting plant protein fibrosis using subcritical water technology, characterized in that, Includes the following steps: (1) Disperse the plant protein in water to obtain a uniform protein dispersion; (2) Adjust the pH of the protein dispersion to 2.0-2.5 to obtain an acidic protein dispersion; (3) The acidic protein dispersion was treated in a subcritical water environment at 105-120℃ for 20-60 min to obtain plant protein nanofibers.

2. The method according to claim 1, characterized in that, In step (1), the concentration of plant protein in the protein dispersion is 1-6 mg / mL.

3. The method according to claim 1, characterized in that, In step (1), the dispersion is carried out by stirring for 1-2 hours.

4. The method according to claim 1, characterized in that, In step (3), before performing subcritical treatment, the acidic protein dispersion is centrifuged at 4000-5000 r / min for 5-10 min.

5. The method according to claim 1, characterized in that, In step (3), the subcritical water ambient temperature is 105-120℃.

6. The method according to claim 1, characterized in that, In step (3), the subcritical water environment treatment time is 20-60 min.

7. Plant protein fiber obtained by the method of any one of claims 1 to 5.

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