Preparation method of mixed protein nanofiber
By adding lactoglobulin to plasma proteins and using precision pH regulation and heat induction treatment technology, the problem of low preparation efficiency of plasma protein nanofibers was solved, and cross-beta-structured mixed protein nanofibers suitable for industrialization were prepared, which were used in high-value foods and bioplastics.
Patent Information
- Application Number
- CN202510877165.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-27
- Publication Date
- 2025-08-12
AI Technical Summary
The prior art is difficult to efficiently prepare plasma protein nanofibers, especially due to the lack of β-sheet structure of BSA, which leads to low preparation efficiency, long reaction time, high energy consumption, and low blood utilization rate of livestock and poultry, which pollutes the environment.
Lactoglobulin rich in β-sheet structure was added to plasma proteins, and the protein conformation transformation was induced by precision pH regulation, dialysis purification, centrifugal clarification and microporous membrane fine filtration technology, combined with acidic pH conditions, and continuous thermal induction treatment was performed using a constant temperature dynamic incubation system to control reaction uniformity.
It has achieved shortening the reaction time, improving the fiber yield and fluorescence intensity, and prepared mixed protein nanofibers rich in cross β-structures, which are suitable for industrial production and are used in high-value food, water treatment materials and bioplastics.
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Figure CN120465126A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of protein nanofibers, and particularly relates to a method for preparing mixed protein nanofibers. Background Art
[0002] Livestock blood is a major byproduct of livestock and poultry slaughter and processing. Statistics show that my country produces approximately 5 million tons of livestock blood annually, containing the equivalent of 1.25 million tons of meat or 1.56 million tons of whole eggs. Due to regional, religious, cultural, and technological differences, while some blood is utilized, a considerable amount is discarded. Livestock blood utilization in my country is still primarily focused on primary processing, with backward deep-processing technology and equipment, resulting in a low blood utilization rate. This not only wastes resources but also pollutes the environment. As a byproduct of livestock and poultry slaughter and processing, blood accounts for approximately 3%-4% of the mass of a live animal or 6%-7% of lean carcass meat. Blood is composed of cells and plasma (red blood cells, white blood cells, and platelets), containing a variety of biologically active substances such as immunoglobulins, bioactive peptides, hemoglobin, and superoxide dismutase, offering significant application potential and commercial value. Plasma proteins are the golden component of blood, abundant in abundance and possessing excellent nutritional properties. However, plasma proteins are spherical and have small molecular weight, limited functional properties, and their processing methods are extensive and unsustainable, which seriously restricts their comprehensive utilization and the creation of high-end products.
[0003] To address the challenges of traditional protein monomers in meeting the demands of creating new healthy foods, numerous approaches have been proposed and studied, including enzymatic modification of proteins, compounding of different protein types, and assembly of protein nanofibers. Protein nanofibers stand out due to their exceptional physical properties (high aspect ratio, specific surface area, and mechanical properties), chemical properties (high acid and heat stability, and multiple surface-active functional groups), and biological properties (hypoallergenicity, edibility, and antioxidant properties). Protein nanofibers, also known as amyloid fibrils, are widely found in nature. Amyloid fibrils were initially thought to be closely associated with neurodegenerative diseases. Subsequent studies have demonstrated that amyloid fibrils can be synthesized in vitro through acid and heat treatment. The formation process of protein nanofibers involves hydrogen bonding of β-strands to form β-sheets, which then stack in parallel and along the main fiber axis to form protofilaments. These protofilaments then intertwine to form different types of protein nanofibers. With the convergence of materials science, nanoscience, and food science, protein nanofibers are now being widely researched and applied in the construction of high-value foods, water treatment materials, bioplastics, and renewable energy materials.
[0004] Plasma proteins are a collective term for the over a thousand proteins found in plasma. In descending order of abundance, the most important proteins include: serum albumin (50%-60%), globulins (40%-50%), and a small amount of fibrinogen. Plasma albumin has a molecular weight of 66 kDa, while plasma globulins are composed of three isoforms: α, β, and γ. In reality, the composition of plasma proteins is not fixed and is influenced by factors such as species, variety, and environmental factors. BSA, among others, is often used as a biochemical model due to its low cost, widespread availability, and low allergenicity. It is widely used in biochemical research, pharmaceuticals, and food science. Studies have shown that BSA has a molecular weight of approximately 66 kDa and an isoelectric point of approximately 4.7. The secondary structure of BSA is primarily α-helical, with a low content of β-sheets. Due to the lack of β-sheets, the traditional acid-heat method for the fibrillization of BSA suffers from low efficiency, long reaction times, and high energy consumption. Consequently, research on the preparation of BSA and plasma protein nanofibers is relatively limited. Improving preparation efficiency, shortening reaction time, and realizing high-value applications of livestock and poultry plasma proteins are currently urgent issues to be addressed. Current studies have shown that plasma proteins, as mixed proteins, can be generated into nanofibers under acidic and hot conditions, but the conditions are relatively harsh. Summary of the Invention
[0005] To address the above-mentioned issues, the present invention provides a method for preparing hybrid protein nanofibers. Lactoglobulin, rich in β-sheet structure, is added to plasma proteins to shorten the heating time of the protein nanofibers, increase fiber yield, and enhance fluorescence intensity. This enables diversified fiber production, meeting diverse application requirements. This method features environmentally friendly, efficient, highly operable, and safe processes, making it suitable for industrial production. The method utilizes an anticoagulated whole blood sample and centrifuges to separate the plasma supernatant. The target precipitate is then purified through multi-stage centrifugation. The precipitate buffer is adjusted to a neutral pH using a precision pH control system. Dialysis is then used to remove low-molecular-weight impurities, and the plasma protein powder is prepared through freeze-drying. The resulting product is then compounded with the model protein lactoglobulin at a specific mass ratio, dispersed in ultrapure water, and subjected to acidic pH conditions to induce protein conformational transitions. Centrifugal clarification combined with microporous membrane fine filtration is used to obtain a stable sol system. Finally, continuous heat induction treatment is performed in a constant-temperature dynamic incubation system. Magnetic stirring maintains reaction uniformity, promoting the exposure of protein functional groups and conformational reorganization.
[0006] The present invention provides a method for preparing mixed protein nanofibers, comprising the following steps:
[0007] Plasma protein powder and β-lactoglobulin were mixed and dispersed in ultrapure water to prepare a 5% mixed protein solution. The pH value was adjusted to 2 and centrifuged. The resulting supernatant was filtered through a filter membrane and then incubated at 80-90°C with a magnetic stirrer at 300 rpm for 12 hours.
[0008] In one embodiment of the present invention, the mass ratio of plasma protein powder to β-lactoglobulin is 1-3:1-3.
[0009] In one embodiment of the present invention, the mass ratio of plasma protein powder to β-lactoglobulin is 1:1.
[0010] In one embodiment of the present invention, the mass ratio of plasma protein powder to β-lactoglobulin is 1:3.
[0011] In one embodiment of the present invention, the mass ratio of plasma protein powder to β-lactoglobulin must be 3:1.
[0012] In one embodiment of the present invention, a method for preparing plasma protein powder includes: collecting fresh bovine blood into a clean container, adding 3.8g / 100mL sodium citrate solution for anticoagulation at a volume ratio of 1:10 to obtain anticoagulated whole blood, centrifuging the anticoagulated whole blood at 4°C and 5,000g for 15 minutes, collecting the supernatant, and continuing to centrifuge three times. The collected precipitate is first adjusted to a pH of 7.0, then dialyzed in ultrapure water for two days using a 5,000Da dialysis bag to remove residual electrolytes. Finally, the dialyzed liquid is freeze-dried for two days to obtain plasma protein powder.
[0013] The present invention provides mixed protein nanofibers prepared by the above method.
[0014] The present invention provides applications of the above-mentioned mixed protein nanofibers in food, water treatment materials, and bioplastics.
[0015] The present invention provides the use of the above-mentioned mixed protein nanofibers in the preparation of nanofiber plastic films.
[0016] [Beneficial Effects]
[0017] (1) The present invention scientifically and rationally reveals the assembly pattern of the mixed protein nanofiber system, realizes the directional regulation of plasma protein and lactoglobulin nanofibers, and finally produces mixed protein nanofibers rich in cross-β-structure, which can be used for the development of high-value food, water treatment materials, and bioplastics, and recycles waste materials from the livestock and poultry slaughtering industry, with good application prospects and social and economic benefits.
[0018] (2) The present invention increases the yield and fluorescence intensity of nanofibers by adding β-lactoglobulin to plasma protein. ThT is a specific fluorescent dye that specifically binds to protein nanofiber molecules in the grooves formed by the side chain ladders, parallel to the long axis of the fiber surface. Higher fluorescence intensity indicates the formation of more cross-β-structures. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 The fluorescence intensity of the pure protein or mixed protein solution of Examples 1 to 3 and Comparative Examples 1 to 2 of the present invention after acid-heat treatment and then reacting with ThT fluorescent dye is measured by a fluorescence microplate reader.
[0020] Figure 2 Comparison of electrophoresis patterns of protein monomers and protein nanofiber solutions before and after acid-heat treatment of pure protein or mixed protein solutions in Examples 1 to 3 and Comparative Examples 1 to 2 of the present invention.
[0021] Figure 3 The mesostructure of the protein nanofibers was characterized by AFM after acid-heat treatment of the purified or mixed proteins from Examples 1-3 of the present invention. A and B represent plasma protein nanofibers and lactoglobulin nanofibers, respectively. C and D represent the mixed protein nanofibers prepared in Example 1.
[0022] Figure 4 The present invention provides a flow chart for preparing mixed protein nanofibers.
[0023] Figure 5 The present invention provides a prospective application of the protein nanofibers of comparative example 1 in preparing bioplastic films for food packaging. DETAILED DESCRIPTION
[0024] Collect fresh bovine blood in a clean container and add 3.8g / 100mL sodium citrate solution for anticoagulation at a volume ratio of 1:10.
[0025] β-Lactoglobulin was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.
[0026] The test method used in the present invention is:
[0027] Yield: Fiber yield was estimated by centrifugation. The protein nanofiber solution was diluted fivefold with pH 2 hydrochloric acid. 2 mL of the diluted sample was transferred to a 100 kDa centrifugal filter and centrifuged at 2500 g for 20 minutes. The precipitate was washed twice and then dissolved in 1 mL of pH 2 hydrochloric acid. The precipitate was centrifuged to remove any remaining non-fiber proteins. The net weight was obtained by subtracting the weight of the filtrate from the total weight. The protein concentration was determined using a BCA assay. The yield was calculated using the following formula:
[0028]
[0029] Where X is the concentration of protein nanofibers (mg / mL), V X is the volume of protein nanofibers (mL), Y is the concentration of the filtrate, V Y is the volume of the filtrate (mL).
[0030] Fluorescence intensity test method: Prepare 0.08% (w / v) ThT stock solution. First, dissolve ThT powder in phosphate buffer (pH 7.0, containing 150mM sodium chloride) and filter through a 0.22μm filter membrane to remove any insoluble precipitates. The ThT working solution is prepared by diluting the ThT stock solution 10 times with Milli-Q ultrapure water. Take 10μL of the fibrillation reaction product and 200μL of ThT working solution in a 96-well plate for mixing and incubation. Use a fluorescence microplate reader to measure the fluorescence intensity associated with amyloid fibers, with the excitation wavelength set to 440nm and the emission wavelength range of 460-550nm. The slit width is set to 9nm.
[0031] Test method for fiber mesoscopic properties (contour length, persistence length): A sample of plasma protein nanofiber solution was diluted to 0.01% (w / v) with Milli-Q water at pH 2. Subsequently, 20 μL of the diluted solution was added dropwise to the surface of freshly cut mica. After incubation for 5 minutes, the mica substrate was gently rinsed with Milli-Q water adjusted to pH 2 and dried with clean air. The microscopic morphology of the amyloid fiber sample was observed using AFM in tapping mode at room temperature. Finally, the collected images were analyzed using NanoScope analysis software (NanoScope version 2.0). The acquired images were statistically analyzed using the open source software FiberApp. First, the surface potential was cleared, and more than 500 protein nanofibers were selected within the largest possible range to statistically analyze the average length (contour length) and persistence length characteristics of the plasma protein nanofibers.
[0032] The average length distribution can reflect the nucleation rate of protein nanofibers. The distribution of protein nanofibers can be well described by the lognormal probability density function, which is expressed as:
[0033]
[0034] In this function, L is the total length, μ and σ are the mean and standard deviation of the natural logarithm of the length, and A is a constant.
[0035] Persistence length is an important metric for quantifying polymer stiffness. In this example, the mean square end-to-end distance method was used to calculate the length distribution. The degree of match between the mean square end-to-end distance and the contour length can be described by the following formula:
[0036]
[0037] Where lp is the persistence length of the protein nanofiber. Fast Fourier transform (FFT) is a method for efficiently calculating discrete Fourier transform (DFT), and its specific form is:
[0038] The flexibility and semi-flexibility of protein nanofibers are relative. When the persistence length is less than the contour length, it is a flexible fiber; when the persistence length is close to the contour length, it is a semi-flexible fiber; when the persistence length is greater than the contour length, it is a rigid fiber.
[0039] The method for preparing anticoagulated whole blood of the present invention specifically comprises: collecting fresh bovine blood into a clean container, adding 3.8g / 100mL sodium citrate solution for anticoagulation, with a volume ratio of 1:10, to obtain anticoagulated whole blood.
[0040] Example 1
[0041] A method for preparing cross-β-structure protein-rich nanofibers using plasma protein and β-lactoglobulin as raw materials through acid-heat treatment, wherein the specific process steps are as follows:
[0042] Anticoagulated whole blood was centrifuged at 4°C and 5,000g for 15 minutes. The supernatant was collected and centrifuged three more times. The pH of the collected precipitate was first adjusted to 7.0, then dialyzed in Milli-Q water using a 5,000Da dialysis bag for two days to remove residual electrolytes. Finally, the dialyzed liquid was freeze-dried for two days to obtain plasma protein powder. Plasma protein and β-lactoglobulin were mixed and dispersed in Milli-Q water at a mass fraction of 1:1 to prepare a 5% mixed protein solution. The pH of the dispersion was adjusted to pH 2 using 6M hydrochloric acid. After centrifugation at 4°C and 5,000g for 30 minutes, the supernatant was filtered through a 0.22μm filter membrane and incubated at 90°C for 12 hours with a magnetic stirrer at 300 rpm / min.
[0043] The fluorescence intensity of the mixed protein nanofibers prepared in this example was enhanced, and the addition of lactoglobulin significantly increased the yield of the mixed protein nanofibers, reaching 50% to 54%, with average fiber lengths of 419.31 nm and 2928.37 nm, respectively. Electrophoresis results indicated that protein hydrolysis occurred during the nanofiber formation process. The yield of the protein nanofibers prepared in this example was significantly higher than that of Comparative Example 1, and a significant shift in flexibility was observed, from rigid to semi-flexible, making them advantageous for the preparation of protein nanofiber bioplastics.
[0044] Example 2
[0045] A method for preparing cross-β-structure protein-rich nanofibers using plasma protein and β-lactoglobulin as raw materials through acid-heat treatment, wherein the specific process steps are as follows:
[0046] Anticoagulated whole blood was centrifuged at 4°C and 5,000g for 15 minutes. The supernatant was collected and centrifuged three more times. The collected precipitate was first adjusted to pH 7.0 and then dialyzed in Milli-Q water using a 5,000Da dialysis bag for two days to remove residual electrolytes. Finally, the dialyzed liquid was freeze-dried for two days to obtain plasma protein powder. β-lactoglobulin and plasma protein were dispersed in Milli-Q water at a mass fraction of 3:1 to prepare a 5% mixed protein solution. The pH of the dispersion was adjusted to pH 2 using 6M hydrochloric acid. After centrifugation at 4°C and 5,000g for 30 minutes, the supernatant was filtered through a 0.22μm filter membrane and incubated at 90°C for 12 hours with a magnetic stirrer at 300 rpm / min.
[0047] The fluorescence intensity of the mixed protein nanofibers prepared in this embodiment is moderate, and the yield is 61% to 67%. The electrophoresis results show that protein hydrolysis occurs during the nanofiber formation process.
[0048] Example 3
[0049] A method for preparing cross-β-structure protein-rich nanofibers using plasma protein and β-lactoglobulin as raw materials through acid-heat treatment, wherein the specific process steps are as follows:
[0050] Anticoagulated whole blood was centrifuged at 4°C and 5,000g for 15 minutes. The supernatant was collected and centrifuged three more times. The pH of the collected precipitate was first adjusted to 7.0, then dialyzed in Milli-Q water using a 5,000Da dialysis bag for two days to remove residual electrolytes. Finally, the dialyzed liquid was freeze-dried for two days to obtain plasma protein powder. β-lactoglobulin and plasma protein were dispersed in Milli-Q water at a mass fraction of 1:3 to prepare a 5% mixed protein solution. The pH of the dispersion was adjusted to pH 2 using 6M hydrochloric acid. After centrifugation at 4°C and 5,000g for 30 minutes, the supernatant was filtered through a 0.22μm filter membrane and incubated at 90°C for 12 hours with a magnetic stirrer at 300 rpm / min.
[0051] The fluorescence intensity of the mixed protein nanofibers prepared in this embodiment is moderate, and the yield is 45% to 47%. The electrophoresis results show that hydrolysis occurs during the process of forming the protein nanofibers.
[0052] Comparative Example 1
[0053] A method for preparing cross-β-structure protein nanofibers by acid-heat treatment using plasma protein as raw material, wherein the specific process steps are as follows:
[0054] The anticoagulated whole blood was centrifuged at 4°C and 5,000g for 15 minutes. The supernatant was taken and centrifuged three more times. The pH of the collected precipitate was first adjusted to 7.0, and then dialyzed in Milli-Q water for two days using a 5,000Da dialysis bag to remove residual electrolytes. Finally, the dialyzed liquid was freeze-dried for two days to obtain plasma protein powder, which was dispersed in Milli-Q water to prepare a 5% plasma protein solution by mass, and the pH of the dispersion was adjusted to pH 2 using 6M hydrochloric acid. Centrifuged at 4°C and 5000g for 30 minutes, the resulting supernatant was filtered through a 0.22μm filter membrane, and then incubated at 90°C with a magnetic stirrer at 300rpm for 12 hours.
[0055] The fluorescence intensity of the pure plasma protein nanofibers prepared in this comparative example was the lowest, with a yield of 32% to 36%. Hydrolysis occurred during the nanofiber formation process, and the average fiber length was 471.83 nm. Electrophoresis results showed that protein hydrolysis occurred during the nanofiber formation process.
[0056] Comparative Example 2
[0057] A method for preparing cross-β-structure protein nanofibers by acid-heat treatment using β-lg as raw material, wherein the specific process steps are as follows:
[0058] β-lg was dispersed in Milli-Q water to prepare a 5% protein solution, and the pH of the dispersion was adjusted to pH 2 using 6 M hydrochloric acid. The solution was centrifuged at 5000 g for 30 min at 4°C, and the supernatant was filtered through a 0.22 μm filter membrane and incubated at 90°C with a magnetic stirrer at 300 rpm / min for 12 h.
[0059] The lactoglobulin nanofibers prepared in this comparative example had the highest fluorescence intensity, with a yield of 70% to 76%. This high yield of pure lactoglobulin nanofibers is attributed to the fact that lactoglobulin's secondary structure contains fewer helical structures and more folded structures. Hydrolysis occurs during the nanofiber formation process, resulting in an average fiber length of 1628.25 nm. Electrophoresis results indicate that protein hydrolysis occurs during the nanofiber formation process.
[0060] Table 1 Properties of the mixed protein nanofibers prepared by the present invention
[0061]
[0062] The embodiments provided above are not intended to limit the scope of the present invention, nor are the steps described to limit their execution order. Any obvious improvements to the present invention made by those skilled in the art in combination with existing common knowledge shall fall within the scope of protection defined by the claims of the present invention.
Claims
1. A method for preparing mixed protein nanofibers, characterized in that: Plasma protein powder and beta-lactoglobulin are mixed and dispersed in ultrapure water to obtain a mixed protein solution, the pH of which is adjusted to 2, and the solution is centrifuged, filtered through a microporous membrane, and finally stirred and incubated at 80-90° C. to obtain the mixed protein nanofibers.
2. The method according to claim 1, characterized in that The mass fraction of the mixed protein solution was 5%; and the incubation time was 12 h.
3. The method according to claim 1, characterized in that The mass ratio of plasma protein powder to β-lactoglobulin is 1-3:1-3.
4. The method according to claim 1, wherein The mass ratio of plasma protein powder and β-lactoglobulin is 1:
1.
5. The method according to claim 1, wherein The mass ratio of plasma protein powder to β-lactoglobulin is 1:
3.
6. The method according to claim 1, characterized in that The mass ratio of plasma protein powder to β-lactoglobulin is 3:
1.
7. The method according to claim 1, characterized in that The preparation method of plasma protein powder includes: centrifuging anticoagulated whole blood at 4°C and 5,000g for 15 minutes, taking the supernatant and continuing to centrifuge three times, adjusting the pH of the collected precipitate to 7.0, dialyzing it in an ultrapure medium with a 5,000Da dialysis bag for two days to remove residual electrolytes, and finally, freeze-drying the dialyzed liquid for two days to obtain plasma protein powder; the anticoagulated whole blood is prepared by adding 3.8g / 100mL sodium citrate solution to bovine blood for anticoagulation, with a volume ratio of 1:10, to obtain anticoagulated whole blood.
8. The mixed protein nanofiber prepared by the method according to any one of claims 1 to 7.
9. Use of the mixed protein nanofiber according to claim 8 in food, water treatment materials, and bioplastics.
10. Use of the mixed protein nanofiber according to claim 8 in the preparation of nanofiber plastic film.