Ultrasound-regulated sodium caseinate amyloid fiber and preparation method thereof

Abstract

The invention provides the ultrasonically regulated sodium caseinate amyloid fiber and the preparation method thereof, and the fibrosis self-assembly rate and the protein conversion efficiency are improved. The preparation method comprises the following steps: firstly, dispersing sodium caseinate in deionized water, stirring and dissolving, regulating pH with a hydrochloric acid solution, and refrigerating; centrifuging the refrigerated solution, taking supernate, and putting the supernate into an ultrasonic cell crusher for ultrasonic pretreatment; and finally, heating the supernate subjected to ultrasonic pretreatment to carry out fibrosis treatment, and immediately carrying out ice-water bath after the heating is finished, so as to obtain the sodium caseinate amyloid fiber. According to the method, an ultrasonic pretreatment technology is introduced, the initial aggregation state of sodium caseinate is regulated and controlled by utilizing an ultrasonic cavitation effect, uniform dispersion and conformation optimization of protein molecules are promoted, the conversion rate, the fibrosis rate and the fibrosis level of the amyloid fiber are remarkably improved, the length of the formed fiber is increased, branches are fewer, and the size of the fiber is reduced. The method has the advantages of simple process, easily available raw materials, large-scale production and the like.

Description

Technical Field

[0001] This invention relates to the field of amyloid fiber preparation, specifically to ultrasonically controlled sodium caseinate amyloid fibers and their preparation method. Background Technology

[0002] Amyloid fibers are a class of highly ordered nanostructures formed by the partial folding, nucleation, and orderly stacking of protein molecules under specific conditions. Their basic characteristic is a β-sheet structure arranged regularly along the fiber axis. Compared with ordinary protein aggregates, amyloid fibers typically have extremely high aspect ratios, excellent mechanical strength, and good thermal and chemical stability, exhibiting structural characteristics similar to synthetic polymer fibers at the nanoscale. Therefore, achieving efficient and controllable construction of amyloid fibers has become one of the important research directions in related fields.

[0003] There is currently a lack of systematic and in-depth research on the self-assembly behavior of sodium caseinate in amyloid fibril formation under acid-heat conditions. Due to the high flexibility and complex initial aggregation state of sodium caseinate molecules, whether it can form stable and controllable amyloid fiber structures during acid-heat induction, as well as its nucleation behavior and fibrilation kinetics, still need further clarification. In the absence of clear structural evolution laws and process control methods, the fibrilation self-assembly process of sodium caseinate is difficult to achieve in a high-efficiency and controllable manner, which objectively restricts its development and application as a potential amyloid fiber material.

[0004] From the perspective of structural regulation, this study aims to develop a method for preparing amyloid fibers by rationally introducing physical regulation methods such as ultrasonic pretreatment to improve the self-assembly rate of fibrosis and the protein conversion efficiency of sodium caseinate, thereby achieving efficient and controllable preparation of sodium caseinate amyloid fibers. Summary of the Invention

[0005] To address the technical problems mentioned in the background, and considering that amyloid fibers have important structural material value in food protein systems, but the self-assembly behavior of sodium caseinate under acid-heat conditions for amyloid fiber formation lacks systematic research and the means of controlling the fiberization process are unclear, this invention aims to solve the technical problems of slow formation rate, low conversion efficiency, and insufficient controllability of fiber structure in the preparation of amyloid fibers using sodium caseinate. It proposes an ultrasonically controlled sodium caseinate amyloid fiber and its preparation method.

[0006] Therefore, the technical solution adopted by the present invention is as follows:

[0007] Ultrasound-controlled sodium caseinate amyloid fibers and their preparation method, the method comprising:

[0008] S1: Disperse sodium caseinate in deionized water, stir and dissolve at room temperature to prepare sodium caseinate solution; adjust the pH of sodium caseinate solution with hydrochloric acid solution, and refrigerate the adjusted sodium caseinate solution;

[0009] S2: After centrifuging the refrigerated sodium caseinate solution to obtain the supernatant, the sodium caseinate supernatant is placed in an ultrasonic cell disruptor for ultrasonic pretreatment.

[0010] S3: After the sodium caseinate supernatant pretreated by ultrasound is subjected to fiberization treatment, sodium caseinate amyloid fibers are obtained.

[0011] Furthermore, the sodium caseinate is prepared using deionized water as a solvent and has a concentration of 40 to 60 mg / mL;

[0012] The stirring speed is 400 to 800 rpm, and the stirring time is 1 to 4 hours; the room temperature is 15 to 35 degrees Celsius.

[0013] Furthermore, the adjusted pH is 1.8 to 2.5, the refrigeration temperature is 4 degrees Celsius, and the refrigeration time is 8 to 12 hours.

[0014] Furthermore, the centrifugation speed is 4000 to 8000 rpm, the centrifugation time is 10 to 20 min, and the centrifugation temperature is 4 degrees Celsius.

[0015] Furthermore, the ultrasonic power of the ultrasonic cell disruptor is set to 200 to 800 W, the ultrasonic pretreatment time is 0 to 40 min, and the sodium caseinate supernatant is placed in an ice bath during ultrasonic pretreatment.

[0016] Furthermore, the ultrasonically pretreated sodium caseinate supernatant was placed in a borosilicate glass bottle and heated in a constant temperature water bath at 75 to 95 degrees Celsius for 0 to 48 hours, with stirring during the heating process at a speed of 200 to 400 rpm.

[0017] After heating, immediately remove and place in an ice water bath for 20 minutes to obtain sodium caseinate amyloid fibers.

[0018] Compared with the prior art, the advantages of the present invention are as follows:

[0019] 1. This invention is the first to perform acid-heat modification on sodium caseinate and adjust the preparation process through ultrasonic pretreatment, ultimately obtaining a highly efficient and structurally controllable sodium caseinate amyloid fiber. The preparation process of sodium caseinate amyloid fiber is simple, the raw materials are widely available, and the operation is convenient. It does not require the use of various complex chemical reagents and instruments, and can be easily and quickly mass-produced on a large scale.

[0020] 2. This invention introduces a specific ultrasonic pretreatment step to regulate the initial aggregation state of sodium caseinate through the cavitation effect of ultrasound, significantly accelerating the self-assembly rate of fibrillation and improving the conversion efficiency of sodium caseinate to amyloid fibers. In the preparation method of this invention, the conversion rate of sodium caseinate to amyloid fibers is increased by 37.78% to 101.88%, the fibrillation rate and level are also greatly improved, and the formed fibers are longer and have fewer branches. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a fitting diagram of the Th T fluorescence kinetics of the present invention;

[0023] Figure 2 This is a transmission electron microscope image of the present invention;

[0024] Figure 3 This is a comparison graph of the fiber conversion rates of Comparative Example 1 and Example 2 of the present invention after heating for 8 hours;

[0025] Figure 4 This is a comparison graph of the potentials of Comparative Example 1 and Example 2 of the present invention under different heating times;

[0026] Figure 5 This is a comparison chart of the average particle size and PDI value of Comparative Example 1 and Example 2 of the present invention under different heating times;

[0027] Figure 6 This is a comparison graph of turbidity in Comparative Example 1 and Example 2 of the present invention at different heating times;

[0028] Figure 7 These are SDS-PAGE electrophoresis images of Comparative Example 1 and Example 2 of the present invention under different heating times;

[0029] Figure 8 This is a comparison chart of the secondary structure content of Comparative Example 1 and Example 2 of the present invention under different heating times. Detailed Implementation

[0030] To achieve the above objectives, the present invention provides ultrasonically regulated sodium caseinate amyloid fibers and a method for their preparation, the method comprising:

[0031] S1: Disperse sodium caseinate in deionized water, stir and dissolve at room temperature to prepare a sodium caseinate solution; adjust the pH of the sodium caseinate solution with hydrochloric acid solution, and refrigerate the adjusted sodium caseinate solution.

[0032] This step is the basic step in the ultrasonic-controlled sodium caseinate amyloid fiber preparation method. The purpose is to fully dissolve sodium caseinate powder in deionized water to form a homogeneous and stable protein solution, and to create optimal conditions for the subsequent fiberization process through pH adjustment and low temperature treatment. This process includes three key steps: solution preparation, pH adjustment and cold storage treatment.

[0033] In the solution preparation stage, sodium caseinate powder must first be accurately weighed and dispersed in deionized water. The concentration of sodium caseinate has a significant impact on the final fiber formation. Deionized water is used as the solvent for sodium caseinate, and the concentration is set to 40 to 60 mg / mL. The concentration selection is based on the solubility characteristics of sodium caseinate in water and the optimal protein concentration range for fiber formation. When the concentration is below 40 mg / mL, the intermolecular interactions of protein molecules are insufficient, making it difficult to form a stable fiber structure. When the concentration is above 60 mg / mL, the solution viscosity is too high, which is not conducive to uniform mixing and subsequent processing.

[0034] The sodium caseinate solution was mechanically stirred at room temperature to ensure complete dissolution of the protein powder and the formation of a homogeneous solution. A constant-speed stirrer was used, with a stirring speed of 400 to 800 rpm and a stirring time of 1 to 4 hours. The choice of stirring speed needed to balance dissolution efficiency and avoid protein denaturation. Too low a speed (below 400 rpm) would result in incomplete dissolution and protein particle residue; too high a speed (above 800 rpm) might introduce excessive air bubbles and cause partial destruction of the protein structure through shear force. The stirring time depended on the initial state and concentration of the sodium caseinate. Generally, a 40 mg / mL solution required 1 hour of stirring to dissolve completely, while a 60 mg / mL solution required up to 4 hours to ensure complete dissolution. The room temperature was controlled within the range of 15 to 35 degrees Celsius; too low a temperature would reduce the dissolution rate, while too high a temperature might affect the stability of the protein.

[0035] pH adjustment is a crucial step in this process, decisively influencing the formation mechanism of amyloid fibers. Sodium caseinate maintains good solubility and micellar structure under neutral or alkaline conditions, but this structure is unfavorable for amyloid fiber formation. The pH of the sodium caseinate solution is adjusted to an acidic range of 1.8 to 2.5 by adding hydrochloric acid solution (usually 0.1 M or 1 M). The pH value is chosen based on the isoelectric point of sodium caseinate (approximately pH 4.6) and the optimal pH conditions for amyloid fiber formation. In a strongly acidic environment of pH 1.8 to 2.5, the micellar structure of sodium caseinate dissociates, protein molecules unfold, exposing hydrophobic regions and the amino acid sequence required for β-sheet formation. Below pH 1.8, the excessively acidic environment may lead to excessive protein hydrolysis and degradation; above pH 2.5, the degree of protein unfolding is insufficient, making it difficult to form an ordered amyloid fiber structure.

[0036] During pH adjustment, hydrochloric acid solution should be added dropwise while continuously stirring the solution to ensure pH uniformity and avoid excessively high local acid concentrations. After pH adjustment, use a pH meter to accurately measure the final pH value of the solution to ensure it is within the target range.

[0037] The adjusted sodium caseinate solution was refrigerated at 4 degrees Celsius for 8 to 12 hours. The purpose of low-temperature refrigeration was to further stabilize the unfolded conformation of the protein, slow down the protein aggregation rate, and allow protein molecules sufficient time for conformational adjustment and initial intermolecular interactions. At 4 degrees Celsius, the thermal motion of the protein is reduced, which is conducive to the formation of a more ordered prefibrillary structure, while inhibiting non-specific aggregation and precipitation. If the refrigeration time is less than 8 hours, the conformational adjustment of the protein is insufficient, resulting in poor subsequent fibrillation. Although the refrigeration time exceeds 12 hours, it will not have a negative impact on the results, but it will prolong the overall preparation cycle and reduce the process efficiency.

[0038] S2: After centrifuging the refrigerated sodium caseinate solution and collecting the supernatant, the sodium caseinate supernatant is placed in an ultrasonic cell disruptor for ultrasonic pretreatment.

[0039] This step further optimizes the state of the protein solution through centrifugation and ultrasonic pretreatment, providing a homogeneous protein precursor with appropriate molecular conformation for the formation of amyloid fibrils.

[0040] The purpose of centrifugation is to remove insoluble aggregates, incompletely dissolved protein particles, and other impurities that may form during refrigeration, obtaining a clear and transparent supernatant. In the refrigerated sodium caseinate solution, some proteins may precipitate or become turbid due to factors such as excessively high local concentrations, uneven pH adjustment, or spontaneous aggregation. If these insoluble components enter subsequent processing stages, they will affect the uniformity and quality of amyloid fibers.

[0041] Centrifugation was performed using a high-speed centrifuge at a speed of 4000 to 8000 rpm for 10 to 20 minutes, with the centrifugation temperature maintained at 4 degrees Celsius. The selection of centrifugation speed and time was crucial to ensure sufficient separation of insoluble components without disrupting the protein's solubility. Speeds below 4000 rpm were insufficient to effectively settle small aggregates; speeds above 8000 rpm could cause excessive centrifugal force to precipitate some soluble proteins, reducing the protein concentration in the supernatant; centrifugation times less than 10 minutes resulted in incomplete separation, leaving the supernatant turbid; centrifugation times exceeding 20 minutes offered limited improvement in separation but increased processing time. Throughout the centrifugation process, the temperature was maintained at 4 degrees Celsius to preserve the protein's low temperature and prevent conformational changes or accelerated aggregation caused by temperature increases.

[0042] After centrifugation, carefully aspirate the supernatant to avoid aspirating the bottom precipitate; the obtained sodium caseinate supernatant should be a uniform, transparent or slightly yellow liquid with the pH value still maintained within the adjusted range; at this point, the sodium caseinate molecules in the protein solution are in an expanded state, meeting the basic conformational requirements for forming amyloid fibers.

[0043] Ultrasonic pretreatment is the core of this step. Through the mechanical vibration of ultrasound, additional physical energy is applied to protein molecules, promoting further unfolding, depolymerization, and conformational rearrangement of proteins, creating conditions for the efficient formation of amyloid fibrils. The sodium caseinate supernatant is transferred to the processing container of the ultrasonic cell disruptor, and the container is placed in an ice bath to prevent the temperature from rising during the ultrasound process.

[0044] The ultrasonic power of the ultrasonic cell disruptor is set to 200 to 800 W, and the ultrasonic pretreatment time is 0 to 40 minutes. Ultrasonic power and treatment time are two key parameters controlling the ultrasonic effect, jointly determining the total energy input received by protein molecules. Ultrasonic power reflects the intensity of ultrasonic vibration; the higher the power, the stronger the effect of the ultrasound on the protein solution. When the ultrasonic power is below 200 W, the energy density of the ultrasound is insufficient to effectively promote conformational changes and aggregate disruption of proteins. When the ultrasonic power is above 800 W... At time W, excessive ultrasound may cause the breakage and degradation of protein peptide chains, destroying the intact protein structure required for fiber formation. Ultrasound treatment time of 0 minutes represents no ultrasound treatment and serves as a control group to study the effect of ultrasound on fiber formation. Ultrasound treatment time varies from 0 to 40 minutes; the longer the time, the more energy the protein absorbs. Ultrasound time less than 10 minutes has little effect and limited promotion of fiber formation. Ultrasound time between 10 and 30 minutes gradually increases fiber yield and quality with prolonged time. Ultrasound time exceeding 40 minutes has no significant effect on improving fiber formation and may even lead to increased energy consumption and partial protein degradation.

[0045] The mechanism of ultrasonic treatment includes the following aspects: First, the mechanical vibration of ultrasound can break down micro-aggregates or oligomers in protein solution, making protein molecules more uniformly dispersed in the solution. Second, the microjets and shock waves generated by ultrasonic cavitation can apply instantaneous high stress to protein molecules, promoting further unfolding of proteins and exposing more hydrophobic regions, thus enhancing the interaction sites between protein molecules. Third, ultrasound can promote microscopic convection and mixing in the solution, improving the homogeneity of the system and providing more consistent reaction conditions for the subsequent heating and fiberization process. Finally, moderate ultrasonic treatment can induce proteins to form β-sheet structures with characteristics of amyloid fibrous precursors, laying the foundation for rapid fiber growth and assembly during heating.

[0046] After sufficient sonication, the conformation and aggregation state of protein molecules in the sodium caseinate supernatant are optimized, providing an ideal reaction precursor for the next step of heat-induced fibrillation. During the ultrasonic pretreatment, the solution is always placed in an ice bath environment. The cooling effect of the ice bath counteracts the heat generated by ultrasound, keeping the solution temperature below 5 degrees Celsius. This avoids premature aggregation or fibrillation caused by temperature rise, ensuring the singularity and controllability of the ultrasonic treatment.

[0047] S3: After ultrasonic pretreatment of the sodium caseinate supernatant, fiberization was performed to obtain sodium caseinate amyloid fibers.

[0048] This step is a key stage in the formation of amyloid fibers. Through thermal induction, sodium caseinate molecules that have undergone ultrasonic pretreatment are induced to aggregate and assemble in an orderly manner, forming fibrous products with typical amyloid structural characteristics.

[0049] The specific operation of the fiberization process is as follows: the supernatant of sodium caseinate after ultrasonic pretreatment is carefully transferred to a borosilicate glass bottle. The borosilicate glass bottle is selected based on its excellent heat resistance, chemical stability and light transmittance. It can withstand long-term high-temperature heating without deformation or release of harmful substances. At the same time, the transparent material makes it easy to observe the changes in the solution during the fiberization process. The glass bottle should be thoroughly cleaned and dried beforehand to avoid residual impurities interfering with the fiberization process. Air bubbles should be avoided during solution transfer, as the presence of air bubbles may affect the homogeneity of the solution and the heat transfer efficiency.

[0050] High borosilicate glass bottles containing protein solutions were placed in a constant-temperature water bath and heated at 75 to 95 degrees Celsius for 0 to 48 hours. Temperature and time are two core parameters controlling the fibrosis process, jointly determining the formation kinetics, yield, and morphological characteristics of amyloid fibers.

[0051] The choice of heating temperature is based on the thermal stability of proteins and the temperature dependence of amyloid fiber formation. Below 75 degrees Celsius, the thermal energy of protein molecules is insufficient to overcome the energy barrier of aggregation, resulting in a very slow fibrillation rate and requiring an excessively long reaction time to obtain a considerable fiber yield. In the temperature range of 75 to 90 degrees Celsius, as the temperature increases, the collision frequency and interaction strength between protein molecules increase, significantly improving the nucleation and growth rate of amyloid fibers, and consequently increasing the fiber yield and length. Above 95 degrees Celsius, excessively high temperatures may lead to excessive aggregation, disordered precipitation, or even thermal degradation of proteins, which is not conducive to the formation of regular fiber structures.

[0052] The duration of heating directly affects the progress of the fibrosis reaction and the characteristics of the final product. Within the heating time range of 0 to 6 hours, the fibrosis reaction is in the nucleation and early growth stage, with slight changes in turbidity in the solution and short fibrous structures visible under a microscope. Within the heating time range of 6 to 24 hours, the fibrosis reaction enters a rapid growth stage, with a rapid increase in the number and length of fibers and a significant increase in solution viscosity. Within the heating time range of 24 to 48 hours, the fibrosis reaction gradually reaches equilibrium, and the fiber yield and morphology tend to stabilize. After heating for more than 48 hours, further extending the heating time has no significant effect on improving fiber yield and morphology; instead, it may lead to secondary aggregation of some fibers or the formation of larger aggregates.

[0053] Throughout the heating process, the protein solution needs to be continuously mechanically stirred at a speed of 200 to 400 rpm. The purpose of stirring is to maintain the homogeneity of the solution, promote uniform heat transfer, prevent local overheating or the formation of temperature gradients, and avoid the deposition and aggregation of fibers at the bottom or walls of the container. The choice of stirring speed needs to balance the mixing effect and interference with fiber growth. If the speed is too low (below 200 rpm), the stirring effect is insufficient, the homogeneity of the solution is poor, and it may lead to inconsistency in the fiberization reaction. If the speed is too high (above 400 rpm), the excessive shear force may damage the growing fibers, resulting in reduced fiber length or structural damage.

[0054] After heating, the borosilicate glass bottle was immediately removed from the constant temperature water bath and placed in an ice-water bath for rapid cooling for 20 minutes. The purpose of rapid cooling was to quickly lower the reaction temperature, terminate the fiberization reaction, fix the structural state of the fibers, and prevent further aggregation or deagglomeration during the cooling process. The temperature of the ice-water bath was close to 0 degrees Celsius, which could quickly lower the sample temperature from the heating temperature to below room temperature. The cooling time was set to 20 minutes, which was sufficient for the overall temperature of the sample to drop to a stable low temperature, ensuring the stability of the fiber structure.

[0055] After cooling in an ice-water bath, the final product obtained is sodium caseinate amyloid cellulose. The cellulose solution is a uniform milky white or slightly yellow gel with a certain viscosity and fluidity. Through further separation, washing and drying, purified amyloid cellulose solids can be obtained. The morphology and structural characteristics of the fibers can be characterized by transmission electron microscopy (TEM), atomic force microscopy (AFM), circular dichroism spectroscopy (CD), Fourier transform infrared spectroscopy (FTIR), and thiosulfate T (ThT) fluorescence. Typical sodium caseinate amyloid cellulose exhibits a slender fibrous structure with a diameter of 5 to 15 nanometers and a length of hundreds of nanometers to several micrometers, with typical β-sheet secondary structure and ThT positive staining characteristics, proving that it has true amyloid cellulose properties.

[0056] Experiments and Analysis

[0057] Comparative Example 1: 40 mg / mL sodium caseinate (SC) was dissolved in deionized water, and the pH of the SC solution was adjusted to 2.0 using 1M HCl. The solution was then allowed to stand at 4 ℃ for 12 h for hydration and filtered. Subsequently, the sodium caseinate supernatant was subjected to constant temperature water bath heating at 90 ℃ for 0-48 h to allow the protein to self-assemble in an acidic environment and gradually form an amyloid fibrous structure. After heating, the solution was immediately removed and placed in an ice water bath for 30 min to obtain a sodium caseinate amyloid fibrous solution, which was stored in a refrigerator at 4 ℃ or freeze-dried for storage.

[0058] Example 1: 40 mg / mL sodium caseinate (SC) was dissolved in distilled water, and the pH of the SC solution was adjusted to 2.0 using 1M HCl. The solution was then allowed to stand at 4 ℃ for 12 h for hydration and filtered. The supernatant of sodium caseinate was placed in an ultrasonic cell disruptor and treated at 600 W for 10 min, followed by constant temperature water bath heating at 90 ℃ for 0-48 h, allowing the protein to self-assemble in an acidic environment and gradually form amyloid fibrous structures. After heating, the solution was immediately removed and placed in an ice water bath for 30 min to obtain sodium caseinate amyloid fibrous solution, which was stored in a refrigerator at 4 ℃ or freeze-dried for storage.

[0059] Example 2: 40 mg / mL sodium caseinate (SC) was dissolved in distilled water, and the pH of the SC solution was adjusted to 2.0 using 1M HCl. The solution was then allowed to stand at 4 ℃ for 12 h for hydration and filtered. The supernatant of sodium caseinate was placed in an ultrasonic cell disruptor and treated at 600 W for 20 min, followed by constant temperature water bath heating at 90 ℃ for 0-48 h, allowing the protein to self-assemble in an acidic environment and gradually form amyloid fibrous structures. After heating, the solution was immediately removed and placed in an ice water bath for 30 min to obtain sodium caseinate amyloid fibrous solution, which was stored in a refrigerator at 4 ℃ or freeze-dried for storage.

[0060] Example 3: 40 mg / mL sodium caseinate (SC) was dissolved in distilled water, and the pH of the SC solution was adjusted to 2.0 using 1M HCl. The solution was then allowed to stand at 4 ℃ for 12 h for hydration and filtered. The sodium caseinate supernatant was placed in an ultrasonic cell disruptor and treated at 600 W for 30 min, followed by constant temperature water bath heating at 90 ℃ for 0-48 h, allowing the protein to self-assemble in an acidic environment and gradually form amyloid fibrous structures. After heating, the solution was immediately removed and placed in an ice water bath for 30 min to obtain sodium caseinate amyloid fibrous solution, which was stored in a refrigerator at 4 ℃ or freeze-dried for storage.

[0061] Experimental Example 1: The Th-T fluorescence intensity kinetic fitting and fiber conversion rate of sodium caseinate amyloid fibers prepared in Comparative Example 1 and Examples 1-3 were tested; the microstructure of sodium caseinate amyloid fibers prepared in Comparative Example 1 and Examples 1-3 was analyzed using transmission electron microscopy.

[0062] Experimental Example 2: The physicochemical and structural properties of the ultrasonically regulated sodium caseinate amyloid fibers prepared in Comparative Example 1 and Example 2 were analyzed, including the determination of average particle size, potential, PDI value, turbidity, SDS-PAGE gel electrophoresis, and Fourier transform infrared spectroscopy.

[0063] In each experimental example, the relevant detection and characterization are as follows:

[0064] 1) Fitting ThT fluorescence spectra and formation kinetics: 8 mg of ThT was dissolved in 10 mL of PBS buffer (10 mM, 150 mmol / L NaCl, pH 7.2), filtered through a 0.22 μm membrane, and then diluted 50 times to obtain the ThT working solution. 3 mL of the ThT working solution was mixed uniformly with 80 μL of the sample solution. The fluorescence spectrometer parameters were set as follows: excitation wavelength 440 nm, emission wavelength 460–600 nm. The fluorescence intensity of the sample at 486 nm was recorded, and the background fluorescence intensity of the ThT working solution was subtracted to obtain the relative fluorescence intensity value. To further analyze the effect of ultrasonic pretreatment on solution formation kinetics, the relative fluorescence intensity value of ThT was fitted using the following formula:

[0065]

[0066] in, The expression represents the total increase in fluorescence during the exponential phase; k represents the rate constant. Indicates heating time; Indicates the fluorescence intensity at equilibrium; The base of the natural logarithm;

[0067] 2) Fiber conversion rate: The fiber sample was diluted to 10 mg / mL with deionized water pre-adjusted to pH 2.0; then 1 mL of the diluted sample was placed in a 100 kDa ultrafiltration centrifuge tube, 19 mL of pH 2.0 water was added, and the mixture was centrifuged at 8000 rpm at 4°C for 20 min, and the filtrate was collected; after each centrifugation, pH 2.0 deionized water was added to the tube as a washing and retention buffer until no protein was detected in the washing and retention buffer; all filtrates were mixed, and the total volume was measured using a measuring cylinder. The protein concentration and fiber conversion rate were determined using the Bradford method. The calculation formula is:

[0068]

[0069] in, Indicates the mass of total protein; and These represent the protein concentration and volume of the filtrate, respectively.

[0070] 3) Transmission electron microscopy: Dilute the sample solution to 0.5 mg / mL with deionized water at pH 2.0, drop it onto a copper grid, let it stand for 15 seconds, remove excess solution with filter paper, and observe the microstructure of the sample using a transmission electron microscope.

[0071] 4) Zeta potential and particle size distribution: The solution was diluted to 1 mg / mL with deionized water at pH 2.0, and the zeta potential of the sample was measured using a nanoparticle size analyzer; the average particle size and PDI value of the sample were measured using dynamic laser scattering mode at a laser scattering angle of 173°.

[0072] 5) Turbidity determination: The absorbance of the sample solution at 500 nm is measured using an ELISA reader, which is the turbidity value of the sample.

[0073] 6) SDS-PAGE gel electrophoresis: First, prepare a 15% separating gel (pH 8.8) and a 5% stacking gel (pH 6.8); centrifuge the sample solution at 5000 g for 10 min, collect the supernatant and add 5× loading buffer, then heat in a 100°C metal bath for 10 min; inject 10 μL of the prepared sample solution into the wells of the electrophoresis gel and perform electrophoresis at a constant voltage of 110 V; after electrophoresis, stain with Coomassie Brilliant Blue R250 for 40 min, then destain with destaining solution (distilled water containing 10% glacial acetic acid and 5% anhydrous ethanol) and scan for imaging;

[0074] 7) Fourier transform infrared spectroscopy: 1 mg of freeze-dried sample was mixed with KBr at a ratio of 1:100 (w / w), ground evenly, and then compressed into tablets; Fourier transform infrared spectrometer was used to scan the infrared spectrum in the wavelength range of 4000 to 400 cm-1, and the number of scans was set to 64; Peak Fit software was used to fit the amide I band of the sample and analyze the secondary structure content of each sample.

[0075] Results and Analysis

[0076] Experimental Example 1:

[0077] Figure 1 The figure shows the ThT fluorescence kinetics fitting diagram. ThT, as a fluorescent marker, can specifically bind to the β-sheet structure of protein fibers, so the ThT fluorescence intensity can be used as an important indicator to judge the degree of protein fibrillation. Among them, the k value reflects the fibrillation rate, and the A value represents the fiber formation ability. Compared with Comparative Example 1, Examples 1 to 3 all showed an increase in k and A values. In Example 2, the k value increased by 1.89 times and the A value increased by 0.36 times. This indicates that ultrasonic pretreatment can effectively shorten the fiber nucleation lag period and accelerate the formation process, thereby improving the fiber formation ability.

[0078] Figure 2 This is a transmission electron microscope image, by Figure 2It can be seen that the microstructure of sodium caseinate amyloid fibers prepared by Comparative Example 1, Example 1, Example 2, and Example 3 at different heating times; in Examples 1-3, ultrasonic pretreatment can depolymerize the initial aggregated particles of sodium caseinate, promoting their dissociation into monomers, and to a certain extent accelerating the hydrolysis process in the early stage of fiberization; when heated for 2 h, Comparative Example 1 still mainly consists of particles, while short fibers have appeared in Examples 1-3; compared with Comparative Example 1, the fiber length in Examples 1-3 increases rapidly with the extension of heating time, indicating that ultrasonic pretreatment promotes the nucleation of sodium caseinate and significantly improves the fiber formation rate and formation ability.

[0079] Figure 3 This is a graph showing the fiber conversion rate. Figure 3 It can be seen that the conversion rates of sodium caseinate amyloid fibers prepared in Comparative Example 1, Example 1, Example 2, and Example 3 differed after heating for 8 h. The conversion rates of Examples 1-3 were 37.78% to 101.88% higher than those of Comparative Example 1, indicating that ultrasonic pretreatment effectively promoted the formation of amyloid fibers. Meanwhile, compared with Example 1, the conversion rates of Examples 2 and 3 were further increased, but showed a trend of increasing and then decreasing. This indicates that appropriate ultrasonic pretreatment time can significantly improve the fiber conversion rate, but excessive treatment will reduce it.

[0080] Experimental Example 2:

[0081] Figure 4 This is a zeta potential diagram; an increase in potential value is a typical characteristic of protein fibrillation. Figure 4 It can be seen that the potential value of Comparative Example 1 decreased during heating for 0-4 h, which reflects the process of sodium caseinate agglomeration through nucleation in the early stage of fiber formation; in contrast, the potential value of Example 2 continued to rise, which indicates that ultrasonic treatment significantly shortened the hysteresis period of the fiber and improved the fiber formation rate.

[0082] Figure 5 The graph shows the average particle size and PDI value. Figure 5 It can be seen that the average particle size of Comparative Example 1 first decreased and then increased, confirming that the fibrosis process of sodium caseinate follows the typical path of hydrolysis, nucleation, and aggregation. With the extension of heating time, the average particle size of Comparative Example 1 increased significantly after heating for 24 hours, while that of Example 2 increased significantly after heating for 12 hours. This indicates that ultrasonic pretreatment promotes sodium caseinate to quickly enter the fiber growth stage and accelerates its fibrosis process.

[0083] Figure 6The turbidity graph reflects the particle size and aggregation degree of sodium caseinate fibers. Lower turbidity generally indicates smaller particle size and lower aggregation degree. With prolonged heating time, the turbidity of sodium caseinate amyloid fibers initially decreased and then increased. The initial decrease in turbidity may be due to the acid heat treatment causing sodium caseinate to gradually depolymerize into monomers. The resulting fibers have a higher aspect ratio and better light transmittance, thus reducing the system turbidity. Compared to Comparative Example 1, the turbidity of Example 2 was significantly lower. Figure 5 and Figure 2 In Example 2, the sodium caseinate amyloid fibers prepared after heating for 2 hours had larger particle size but longer length. This indicates that ultrasonic pretreatment promotes fiber formation and fiber length extension, reduces fiber branching, and further reduces inter-fiber aggregation, thereby reducing turbidity.

[0084] Figure 7 The SDS-PAGE gel electrophoresis image shows that the intensity of the major subunit bands of sodium caseinate gradually decreases with increasing heating time. At this time, the low molecular weight bands of sodium caseinate (10-30 kDa) gradually increase, indicating that sodium caseinate undergoes hydrolysis during fibrosis, and the generated small molecule peptides participate in fibrosis self-assembly as basic building blocks. Compared with Comparative Example 1, in Example 2, band L is significantly enhanced and the overall lane color deepens after heating for 0 h. This indicates that ultrasound improves the solubility of sodium caseinate and promotes its hydrolysis to generate more fibrosis building blocks. Further comparison of band changes at different heating times shows that bands N and M appear earlier in Example 2 and are darker in color at the same heating time compared with Comparative Example 1, indicating that ultrasonic pretreatment significantly accelerates the fibrosis process of sodium caseinate.

[0085] Figure 8 The diagram shows the content of secondary structure. The formation of protein fibers is usually accompanied by the orderly accumulation of β-sheet structures. Therefore, the increase in β-sheet content is an important indicator of the fibrosis process of sodium caseinate. Compared with Comparative Example 1, the β-sheet content of Example 2 increased significantly after heating for 2 hours, and entered the fiber formation stage more quickly. This indicates that ultrasonic pretreatment effectively accelerates the fibrosis rate of sodium caseinate.

[0086] The present invention proposes an ultrasonically regulated sodium caseinate amyloid fibrous preparation method to solve the problems of long reaction time, low yield, and non-uniform morphology in traditional amyloid fibrous preparation techniques. The method includes three main stages: solution preparation and pH adjustment, centrifugation and ultrasonic pretreatment, and heat-induced fibrillation. The introduction of the ultrasonic pretreatment step is the core. By controlling the ultrasonic power and treatment time, the uniform dispersion and conformation optimization of the protein can be promoted without destroying the protein integrity, providing a more ideal reaction precursor for subsequent fibrillation, thereby significantly shortening the fibrillation time and improving the fiber yield and structural uniformity.

[0087] In summary, this invention, by introducing ultrasonic pretreatment technology and combining it with traditional protein treatment methods such as acidification, low-temperature refrigeration, and temperature-controlled heating, not only solves the efficiency and quality problems in the preparation of amyloid fibers, but also provides a new experimental platform for in-depth research on the regulatory mechanisms of physical fields such as ultrasound on protein aggregation and fibrillation processes. This enables the method to meet the needs of large-scale production of protein-based functional materials in the food industry and provides technical support for the development of new amyloid fiber materials with specific structures and functions.

[0088] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. Ultrasonic-controlled sodium caseinate amyloid fibers and their preparation method, characterized in that, The method includes: S1: Disperse sodium caseinate in deionized water, stir and dissolve at room temperature to prepare sodium caseinate solution; adjust the pH of sodium caseinate solution with hydrochloric acid solution, and refrigerate the adjusted sodium caseinate solution; S2: After centrifuging the refrigerated sodium caseinate solution to obtain the supernatant, the sodium caseinate supernatant is placed in an ultrasonic cell disruptor for ultrasonic pretreatment. S3: After the sodium caseinate supernatant pretreated by ultrasound is subjected to fiberization treatment, sodium caseinate amyloid fibers are obtained.

2. The ultrasound-controlled sodium caseinate amyloid fibers and their preparation method according to claim 1, characterized in that, The sodium caseinate is used in deionized water as a solvent and has a concentration of 40 to 60 mg / mL. The stirring speed is 400 to 800 rpm, and the stirring time is 1 to 4 hours; the room temperature is 15 to 35 degrees Celsius.

3. The ultrasound-controlled sodium caseinate amyloid fibers and their preparation method according to claim 1, characterized in that, The adjusted pH is 1.8 to 2.5, the refrigeration temperature is 4 degrees Celsius, and the refrigeration time is 8 to 12 hours.

4. The ultrasound-controlled sodium caseinate amyloid fibers and their preparation method according to claim 1, characterized in that, The centrifugation speed is 4000 to 8000 rpm, the centrifugation time is 10 to 20 min, and the centrifugation temperature is 4 degrees Celsius.

5. The ultrasound-controlled sodium caseinate amyloid fibers and its preparation method according to claim 1, characterized in that, The ultrasonic power of the ultrasonic cell disruptor is set to 200 to 800 W, the ultrasonic pretreatment time is 0 to 40 min, and the sodium caseinate supernatant is placed in an ice bath during ultrasonic pretreatment.

6. The ultrasound-controlled sodium caseinate amyloid fibers and its preparation method according to claim 1, characterized in that, The ultrasonically pretreated sodium caseinate supernatant was placed in a borosilicate glass bottle and heated in a constant temperature water bath at 75 to 95 degrees Celsius for 0 to 48 hours, with stirring during the heating process at a speed of 200 to 400 rpm. After heating, immediately remove and place in an ice water bath for 20 minutes to obtain sodium caseinate amyloid fibers.

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