A method for modifying prolamin proteins

Abstract

The application provides a modification method of millet prolamin, uniformly mixing the millet prolamin with a solvent to obtain a millet prolamin solution; uniformly mixing a cationic surfactant with the millet prolamin solution to obtain a spinning solution; and electrospinning the spinning solution to obtain a millet prolamin nanofiber product. The cationic surfactant is added in the millet prolamin solution for modification, and the millet prolamin nanofiber product is successfully prepared, the mechanical strength and water stability of the nanofiber product are greatly improved, and the application of the millet prolamin is widened.

Description

Technical Field

[0001] This invention belongs to the field of protein processing technology, and in particular relates to a method for modifying millet prolysin. Background Technology

[0002] Millet is rich in protein, making it a good source of plant-based protein. Millet prolysin contains a high proportion of hydrophobic amino acids (such as valine, leucine, and proline), thus exhibiting strong hydrophobicity and attracting widespread interest in the field of electrospinning. However, unlike synthetic polymers, which maintain high stability over a long period, electrospun fiber materials made directly using millet prolysin as a matrix suffer from limitations in mechanical properties and water stability.

[0003] Protein-based nanofibers have attracted widespread attention due to their superior biodegradability, biocompatibility, in vivo safety, and excellent functional properties compared to other materials. Electrospinning technology widely utilizes various synthetic and natural polymers and their mixtures to produce nanofiber materials. Employing natural bio-based polymers such as polysaccharides and proteins as matrices holds promise for developing nanofiber materials with both biodegradability and biocompatibility.

[0004] Therefore, modifying millet protein to prepare improved fiber materials is of great significance for broadening the application of bio-based polymer electrospun fiber materials. Summary of the Invention

[0005] In view of this, the present invention aims to propose a method for modifying millet prolysin, and to obtain bio-based polymer nanofiber products with strong water stability and excellent mechanical properties using millet prolysin.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0007] In a first aspect, the present invention provides a method for modifying millet prolysin, and also a method for preparing millet prolysin nanofiber products, comprising the following steps:

[0008] Mix millet protein with a solvent until homogeneous to obtain a millet protein solution;

[0009] The cationic surfactant was mixed evenly with the millet alcohol protein solution to obtain the spinning solution;

[0010] Electrospinning was performed on the spinning solution to obtain millet alcohol-soluble protein nanofiber products.

[0011] Furthermore, the solvent is an organic acid; preferably acetic acid.

[0012] Furthermore, the mass concentration of millet prolysin in the millet prolysin solution is 25%~30%.

[0013] Furthermore, the extraction method of millet prolysin includes the following steps:

[0014] Millet is crushed into millet powder, the millet powder is mixed with water and then homogenized by a colloid mill, centrifuged to obtain precipitate;

[0015] The precipitate was mixed with ethanol and centrifuged. Sodium chloride solution was added to the supernatant, and the mixture was centrifuged, washed with water, and freeze-dried to obtain millet alcohol-soluble protein.

[0016] Furthermore, the cationic surfactant is TEBAC.

[0017] Furthermore, the amount of the cationic surfactant added is 1% to 20% of the amount of millet alcohol-soluble protein.

[0018] Furthermore, the electrospinning parameters are: voltage 16-24kV, receiving distance 8-12 cm, roller speed 200-1500 rpm, and flow rate 0.5-1.2 mL / h.

[0019] Secondly, the present invention provides a millet prolysin nanofiber product prepared according to the above-described modification method.

[0020] Thirdly, the present invention provides a millet prolysin nanofiber product prepared according to the above-described modification method or the application of the millet prolysin nanofiber product according to the above-described method in product packaging.

[0021] Fourthly, the present invention provides the application of TEBAC in improving the performance of millet prolysin nanofiber products, the application including at least one of the following:

[0022] (1) Application in reducing the fiber diameter of millet prolysin nanofiber products;

[0023] (2) Application in increasing the tensile strength of millet prolysin nanofiber products;

[0024] (3) Application in increasing the elongation at break of millet prolysin nanofiber products;

[0025] (4) Application in increasing the Young's modulus of millet prolysin nanofiber products;

[0026] (5) Application in improving the water stability of millet prolysin nanofiber products.

[0027] Preferably, the millet prolysin nanofiber product is a millet prolysin nanofiber membrane.

[0028] Compared with existing technologies, the method for modifying millet prolysin according to the present invention has the following advantages:

[0029] (1) The present invention uses millet prolysin as raw material to successfully prepare millet prolysin nanofiber products. Compared with zein, millet prolysin has a more ordered structure and a smaller molecular weight. The prepared millet prolysin nanofiber products have better mechanical strength and water stability compared with zein nanofiber products.

[0030] (2) The modification method of millet prolysin of the present invention further improves the mechanical properties of millet prolysin nanofiber products by adding cationic surfactants. The tensile strength, elongation at break and Young's modulus are all increased, and the water stability of nanofiber products is significantly improved. The millet prolysin nanofiber products prepared by the present invention have broad application prospects. Attached Figure Description

[0031] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0032] Figure 1 This is an SDS-PAGE electrophoresis image of millet gliadin and zein from Example 1; where lane M is the marker; lane 1 is zein; and lane 2 is millet gliadin.

[0033] Figure 2 These are scanning electron microscope images of millet gliadin (MG) and zein from Example 1;

[0034] Figure 3 This is a transmission electron microscope image of millet gliadin (MG) and zein in an ethanol aqueous solution, as shown in Example 1.

[0035] Figure 4 The graphs show the molecular structure results of millet gliadin and corn gliadin from Example 1; where A is the molecular weight distribution curve and B is a circular dichroism chromatogram.

[0036] Figure 5 These are scanning electron microscope images of the samples in Example 2 and Comparative Examples 1-3;

[0037] Figure 6 These are scanning electron microscope images of the MG nanofiber membrane in Example 2 and the zein nanofiber membrane in Comparative Example 1 after immersion in water for 24 h. Detailed Implementation

[0038] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0039] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0040] The main reagents and experimental instruments used in the following examples are shown in Tables 1 and 2.

[0041] Table 1 Main test reagents

[0042]

[0043] Table 2 Main Instruments and Equipment

[0044]

[0045] Example 1: Extraction of millet prolysin

[0046] This embodiment provides a method for extracting millet prolysin (MG), comprising the following steps: Millet grains are pulverized into millet powder using a grinder. Water is added to the millet powder at a ratio of 1:10, and the mixture is homogenized by a colloid mill for 15 min. Then, the mixture is centrifuged at 25°C and 3500×g for 10 min. The supernatant is discarded, and the precipitate is collected. The precipitate is mixed with 80% (v / v) ethanol solution at a ratio of 1:8 (w / v) at 37°C with continuous stirring for 3 h, followed by centrifugation (3500×g for 10 min). Three volumes of cold sodium chloride solution are added to the obtained supernatant until the final sodium chloride concentration is 0.3% (w / v). The mixture is then allowed to stand at 4°C for 24 h. After centrifugation and washing with water, the precipitate is finally freeze-dried to obtain MG.

[0047] Characterization comparison of millet gliadin (MG) and zein:

[0048] 1. Molecular weight distribution

[0049] The molecular weight of MG was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein samples were dissolved in distilled water (pH=12) at a concentration of 2 mg / mL, then mixed with 5X loading buffer in a boiling water bath for 5 min. After cooling, 10 μL was added to each well. SDS-PAGE was performed using a 5% stacking gel and a 12% separating gel. The voltage for the stacking gel was set to 120 V, and the voltage for the separating gel was set to 80 V. Electrophoresis was stopped when bromophenol blue was approximately 1 cm from the bottom of the gel. Finally, the gel was stained with Coomassie Brilliant Blue-R250.

[0050] from Figure 1As can be seen, zein mainly consists of two bands with molecular weights of 21 kDa and 23 kDa (lane 1), indicating that the commercially available zein used in this invention is mainly α-zein. MG consists of four bands with molecular weights of 23 kDa, 19 kDa, 13 kDa, and below 11 kDa. Furthermore, the molecular weights of zein and MG were determined by high-performance liquid chromatography using a Shodex KD-806M gel chromatography column. According to Table 3, the weight-average molecular weight of zein is 13330, significantly greater than the weight-average molecular weight of MG (7623).

[0051] Table 3 Molecular weights of zein and MG

[0052]

[0053] 2. Amino acid analysis

[0054] A suitable amount of protein sample was weighed and dissolved in 6 mol / L hydrochloric acid solution (containing 0.1% phenol). The sample (3 mL) was transferred to an ampoule, purged with nitrogen, sealed, and hydrolyzed in an oven at 110 °C for 24 h. After drying the sample under nitrogen, 3 mL of distilled water was added to reconstitute it. After derivatization, the reconstituted solution was analyzed for amino acids by high performance liquid chromatography (HPLC) using a C18 column (5 μm, 4.6 × 250 mm).

[0055] Table 4 shows the amino acid composition of zein and MG powders. Compared with zein, MG contains more aspartic acid and cysteine, and less leucine, glutamic acid and proline.

[0056] Table 4. Amino acid composition of zein and MG

[0057]

[0058] 3. Microscopic morphological observation

[0059] MG was dispersed in an 80% aqueous ethanol solution (0.1 mg / mL, v / v), and a drop of the solution was placed on a 200-mesh carbon-coated copper grid. After 20 seconds, the droplet was removed with filter paper, allowed to air dry, and used for imaging. The morphology of the MG aggregates in the solution was observed using a transmission electron microscope.

[0060] The microstructure of MG powder was observed using a scanning electron microscope. Prior to observation, the sample was coated with a fine gold layer by sputtering under high vacuum conditions.

[0061] from Figure 2Scanning electron microscopy (SEM) results showed that zein particles were large and irregularly shaped, while MG particles were regular spherical, but mostly existed in clusters. Transmission electron microscopy (TEM) was used to observe the morphology of zein and MG in an ethanol-water solution. Figure 3 As shown, both zein and MG are dense spherical particles. The diameter of the MG spherical particles is 598.03 nm, which is significantly larger than the diameter of the zein spherical aggregates (140.27 nm).

[0062] 4. Molecular structure

[0063] The molecular structure of MG prepared in this embodiment was determined using circular dichroism spectroscopy, fluorescence spectroscopy, and Fourier transform infrared spectroscopy.

[0064] Figure 4 Figure (A) shows the circular dichroism (PD) spectra of zein and MG dissolved in 80% aqueous ethanol solution. It can be clearly seen that both zein and MG exhibit two broad negative peaks at approximately 208 and 222 nm, and a positive peak at approximately 192 nm. The negative peak at 208 nm in the PD spectra is attributed to the π–π* transition, while the negative peak at 222 nm is likely caused by the n–π* transition between the α-helix and the random helix. This indicates that both zein and MG possess typical α+β structures, with the α-helix band being stronger than the β-sheet structure.

[0065] Figure 4 Figure (B) shows the intrinsic fluorescence spectra of zein and MG. Zein has a high concentration of tyrosine residues but a low concentration of tryptophan residues, hence its typical maximum fluorescence peak is around 300 nm. However, MG exhibits the strongest fluorescence intensity at 333 nm, indicating that tryptophan residues contribute the most among the three chromophores. Typically, if λ... max Below 330 nm, tryptophan is considered buried and exists in a "nonpolar" environment; if λ max At wavelengths greater than 330 nm, tryptophan is considered to be in a "polar" environment. In this example, the wavelengths at which zein and MG exhibit maximum fluorescence emission are 300 nm and 333 nm, respectively, indicating that the tryptophan residues in MG are in a polar environment, while the tryptophan residues in zein are in a nonpolar environment. Furthermore, the fluorescence intensity of the tryptophan residues in MG ( FI The value is higher than zein.

[0066] Changes in protein secondary structure content are mainly detected by infrared spectroscopy at 1700-1600 cm⁻¹. -1 The relationship between the peak area and the secondary structure content, calculated from the change in the amide I band, is as follows: α-helix (1659–1648 cm⁻¹) -1 ), β-fold (1700–1680 cm)-1 1640-1610 cm -1 ), β-turn (1679–1660 cm) -1 ), irregular curls (1648–1640 cm) -1 According to Table 5, the relative contents of α-helices, β-sheets, β-turns, and random coils in zein were 25.40%, 32.63%, 18.56%, and 23.40%, respectively. Compared with zein, MG had a higher content of β-sheet structures and a lower content of random coil structures.

[0067] Table 5. Secondary structure content of zein and MG

[0068]

[0069] Example 2: Preparation of millet prolysin nanofiber membrane

[0070] This embodiment provides a millet prolysin nanofiber membrane, the preparation method of which includes the following steps:

[0071] First, using acetic acid as a solvent, millet prolysin extracted in Example 1 was added and magnetically stirred overnight at 25°C to ensure complete dissolution, yielding an MG solution. The amount of millet prolysin added was either 25 g / 100 mL acetic acid or 30 g / 100 mL acetic acid.

[0072] Then, the cationic surfactant TEBAC was added to the MG solution, and the mixture was stirred at room temperature to allow the surfactant to dissolve completely, thus obtaining the spinning solution. The amount of TEBAC added was 2% of the millet alcohol-soluble protein content.

[0073] Finally, the spinning solution was allowed to stand for 10 minutes to remove air bubbles. Then, a certain amount of the spinning solution was drawn into a 10 mL syringe for electrospinning. The spinning parameters were: voltage 16 kV, receiving distance 8 cm, roller speed 1000 rpm, and flow rate 1 mL / h. Millet prolysin nanofiber membranes were prepared and labeled MG1 (millet prolysin added at 25 g / 100 mL acetic acid) and MG2 (millet prolysin added at 30 g / 100 mL acetic acid), respectively.

[0074] Comparative Example 1

[0075] This comparative example provides a zein nanofiber membrane, the preparation method of which includes the following steps:

[0076] First, zein was added to acetic acid as a solvent and magnetically stirred overnight at 25°C to ensure complete dissolution, thus obtaining a zein solution. The amount of zein added was 25 g / 100 mL of acetic acid.

[0077] Then, the cationic surfactant TEBAC is added to the zein solution, and the mixture is stirred at room temperature to allow the surfactant to dissolve completely, thus obtaining the spinning solution. The amount of TEBAC added is 2% of the zein mass.

[0078] Finally, the spinning solution was allowed to stand for 10 minutes to remove air bubbles. Then, a certain amount of the spinning solution was drawn into a 10 mL syringe for electrospinning. The spinning parameters were: voltage 16 kV, receiving distance 8 cm, roller speed 1000 rpm, and flow rate 1 mL / h. A zein nanofiber membrane was prepared and labeled Z1.

[0079] Comparative Example 2

[0080] This comparative example provides a millet prolysin nanofiber membrane, which differs from Example 2 in that the surfactant TEBAC is not added to the MG solution. The specific preparation method is as follows:

[0081] First, using acetic acid as a solvent, millet prolysin extracted in Example 1 was added and magnetically stirred overnight at 25°C to ensure complete dissolution, yielding an MG solution. The amount of millet prolysin added was 25 g / 100 mL of acetic acid.

[0082] Then, using the MG solution as the spinning solution, electrospinning was performed according to the parameters described in Example 1 to prepare a millet alcohol-soluble protein nanofiber membrane, labeled as MG3.

[0083] Comparative Example 3

[0084] This comparative example provides a millet prolysin nanofiber membrane, which differs from Example 2 in that an anionic surfactant SDS is added to the MG solution. The specific preparation method is as follows:

[0085] First, using acetic acid as a solvent, millet prolysin extracted in Example 1 was added and magnetically stirred overnight at 25°C to ensure complete dissolution, yielding an MG solution. The amount of millet prolysin added was 25 g / 100 mL of acetic acid.

[0086] Then, the anionic surfactant SDS was added to the MG solution, and the mixture was stirred at room temperature to allow the surfactant to dissolve completely, thus obtaining the spinning solution. The amount of SDS added was 2% of the millet alcohol-soluble protein content.

[0087] Finally, the spinning solution was allowed to stand for 10 minutes to remove air bubbles. Then, a certain amount of the spinning solution was drawn into a 10 mL syringe for electrospinning. The spinning parameters were: voltage 16 kV, receiving distance 8 cm, roller speed 1000 rpm, and flow rate 1 mL / h. A millet prolysin nanofiber membrane was prepared and labeled MG4.

[0088] Example 3 Performance testing methods and results

[0089] 1. Microscopic morphological observation

[0090] The surface morphology of millet prolysin nanofiber membranes and corn prolysin nanofiber membranes was observed using scanning electron microscopy. All samples underwent gold sputtering under vacuum before observation. The average fiber diameter and diameter distribution were obtained by statistically analyzing 200 randomly selected fibers from each sample using ImageJ image analysis software.

[0091] 2. Determination of water stability

[0092] Millet prolysin nanofiber membranes and corn prolysin nanofiber membranes were cut into 3 cm × 3 cm pieces and then immersed in 40 mL of deionized water in a 90 mm diameter petri dish for 24 h. Images of the samples before and after immersion in deionized water were taken to observe the microstructure of the millet prolysin nanofiber membranes.

[0093] 3. Determination of mechanical properties

[0094] Millet prolysin nanofiber membranes and corn prolysin nanofiber membranes were cut into 30 mm × 3 mm pieces, and their static mechanical properties were analyzed using a universal testing machine at a tensile speed of 1 mm / s. Five measurements were performed for each type of nanofiber membrane. Tensile strength (… TS ), elongation at break ( EB ) and Young's modulus ( YM The calculation is as follows:

[0095]

[0096] in, F m It is the maximum recorded load (N). S It is the cross-sectional area of ​​the sample. L b It is the length (mm) at the break point. L 0 It is the initial length of the sample. L m It is the test length (mm) corresponding to the maximum load.

[0097] result:

[0098] The morphological evaluation of zein nanofiber membranes (Z1) and MG nanofiber membranes (MG1, MG2, MG3, MG4) was performed using scanning electron microscopy. Figure 5 As shown, all nanofiber membranes exhibit randomly oriented, smooth, and uniform fibers, and are free of beads. Compared to MG1, MG2 shows a significantly larger diameter (551.07 ± 149.43 nm). The average fiber diameters of Z1 and MG1 show no significant difference, being 184.79 ± 33.70 nm and 230.99 ± 47.75 nm, respectively. At the same mass concentration, MG1 exhibits a larger fiber diameter than Z1, indicating stronger entanglement between millet prolysin molecular chains. Unlike MG1, MG3 shows an increased diameter (315.32 ± 33.70 nm), primarily attributed to the addition of the cationic surfactant TEBAC. Compared to MG3, the diameter of MG4 decreases to 203.28 ± 42.79 nm after the addition of the anionic surfactant SDS.

[0099] Figure 6 The swelling behavior of zein nanofiber membranes (Z1) and MG nanofiber membranes (MG1, MG2) after immersion in deionized water for 24 h was demonstrated. The results showed that, compared to the original fiber morphology, Z1 lost its original fiber structure after immersion in deionized water for 24 h, with fibers adhering to form a membrane, and pores formed after dissolution were observed; indicating the poor water stability of the zein nanofiber membrane. Unlike the zein nanofiber membrane, MG1 and MG2 maintained their fiber morphology well after immersion in deionized water for 24 h, with only an increase in fiber diameter and a decrease in inter-fiber porosity observed after swelling; and MG2 exhibited better water stability than MG1.

[0100] The tensile strength, elongation at break, and Young's modulus of zein nanofiber membranes and MG nanofiber membranes are shown in Table 6. Compared with zein nanofiber membrane (Z1), MG nanofiber membranes (MG1, MG2) exhibit higher tensile strength, elongation at break, and lower Young's modulus, indicating that MG nanofiber membranes have better mechanical properties. Compared with the fiber membrane without surfactant (MG3), the addition of cationic surfactant increases the tensile strength, elongation at break, and Young's modulus of MG1, indicating that TEBAC can significantly improve the strength and toughness of millet prolysin fiber membranes. However, the addition of anionic surfactant significantly decreases the tensile strength and Young's modulus of MG4, while increasing the elongation at break, indicating that SDS can improve the toughness of millet prolysin fiber membranes and reduce their strength.

[0101] Table 6 Tensile strength, elongation at break, and Young's modulus of fiber membrane samples

[0102]

[0103] In summary, this application prepared millet prolysin nanofiber membranes by electrospinning extracted millet prolysin. The composition and structure of zein and millet prolysin were compared and analyzed. The microstructure, water stability, mechanical properties, and molecular structure of the millet prolysin nanofiber membranes were determined, leading to the following conclusions:

[0104] 1. Millet prolysin and zein have different compositions and conformations. Compared to zein, millet prolysin has a significantly smaller molecular weight; in solution, millet prolysin molecules are more extended. Although the secondary structures of millet prolysin and zein are similar, the structure of millet prolysin is more ordered.

[0105] 2. Compared with zein nanofiber membrane, millet prolysin nanofiber membrane exhibits higher water stability upon initial contact with deionized water.

[0106] 3. Compared with zein nanofiber membrane, millet prolysin nanofiber membrane has significantly improved mechanical properties.

[0107] 4. The addition of cationic surfactant TEBAC (2%wt) can significantly improve the strength and toughness of millet prolysin fiber membrane.

[0108] Finally, it should be noted that the above description is only used to illustrate the technical solutions of the present invention and is not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Claims

1. A method of preparing a prolamin nanoparticle product, characterized by: The preparation method includes the following steps: Mix millet protein with a solvent until homogeneous to obtain a millet protein solution; The cationic surfactant was mixed evenly with the millet alcohol protein solution to obtain the spinning solution; Electrospinning was performed on the spinning solution to obtain millet prolysin nanofibers. The solvent is an organic acid, the mass concentration of millet prolysin in the millet prolysin solution is 25%~30%, the cationic surfactant is TEBAC, the amount of the cationic surfactant added is 1%~20% of the mass of millet prolysin, and the electrospinning parameters are voltage 16-24kV, receiving distance 8-15 cm, roller speed 200-1500 rpm, and flow rate 0.5-1.2 mL / h.

2. The method of claim 1, wherein: The extraction method of millet prolysin includes the following steps: Millet is crushed into millet powder, the millet powder is mixed with water and then homogenized by a colloid mill, centrifuged to obtain precipitate; The precipitate was mixed with ethanol and centrifuged. Sodium chloride solution was added to the supernatant, and the mixture was centrifuged, washed with water, and freeze-dried to obtain millet alcohol-soluble protein.

3. The millet prolysin nanofiber product prepared by the preparation method according to claim 1 or 2.

4. The application of the millet prolysin nanofiber product prepared by the preparation method according to claim 1 or 2 in product packaging.

5. Use of TEBAC to improve the performance of the prolamine nanofiber product according to claim 3, characterized in that, The application includes at least one of the following: (1) Application in reducing the fiber diameter of millet prolysin nanofiber products; (2) Application in increasing the tensile strength of millet prolysin nanofiber products; (3) Application in increasing the elongation at break of millet prolysin nanofiber products; (4) Application in increasing the Young's modulus of millet prolysin nanofiber products; (5) Application in improving the water stability of millet prolysin nanofiber products.

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