Method for modifying wheat gluten protein with lactic acid

Abstract

The present invention discloses a method for modifying wheat gluten protein with lactic acid, which belongs to the technical field of wheat protein processing. The present invention involves dispersing wheat gluten protein in a lactic acid solution or an aqueous solution containing sodium lactate, and modifying the protein with lactic acid to change the internal structure of the gluten protein molecule and improve the functional properties of the wheat gluten protein, such as solubility and emulsifying ability.

Description

[Technical Field]

[0001] The present invention relates to the technical field of wheat protein processing, and in particular to a method for modifying wheat gluten proteins with lactic acid. [Background technology]

[0002] Wheat is one of the world's most widely cultivated, most widely planted, and oldest cultivated food crops. In China, wheat is second only to rice in terms of planted area, and plays a vital role in food structure and human consumption. Wheat protein is a by-product of wheat starch production and is also known as gluten flour or wheat gluten protein. In the food industry, wheat gluten protein's uses are primarily reflected in two aspects: as a protein nutritional supplement and as a dough strengthener to improve dough rheological properties and product quality.

[0003] Based on differences in wheat gluten protein solubility, wheat gluten proteins can be divided into four types of proteins: albumin, globulin, gliadin, and glutenin, the latter two of which are the major components of wheat gluten proteins. Glutenin is a naturally occurring heterogeneous polymeric protein that can be divided into two types of glutenin subunits: high-molecular-weight glutenin subunit (HMW-GS) and low-molecular-weight glutenin subunit (LMW-GS). These two subunits are linked via polypeptide bonds and intermolecular disulfide bonds. Gliadin, a low-molecular-weight protein, is non-covalently bound to glutenin and embedded in the three-dimensional network structure of glutenin, which results in undesirably poor processing properties (e.g., solubility, dispersibility) of wheat gluten proteins. Furthermore, due to factors such as genetics, growth environment, and processing techniques, wheat gluten proteins are characterized by diverse amino acid sequences and complex overall structures, which significantly limit their applications. Therefore, there is a need to rationally modify wheat gluten proteins to increase their solubility and improve other functional properties.

[0004] Currently, there are three techniques for modifying wheat gluten protein: physical, enzymatic, and chemical. Physical modification uses physical effects such as heat, pressure, radiation, and sound waves to change the intermolecular aggregation and secondary and tertiary structure of protein molecules. Chemical modification uses chemical methods to partially cleave the peptide chain of a protein or introduce new active groups to modify the protein's structure, hydrophobic groups, charge characteristics, etc., ultimately changing its spatial structure and functional properties. Enzymatic modification uses proteases to selectively hydrolyze the peptide and / or amide bonds of a protein or to induce covalent crosslinking between molecules, thereby changing its functional properties. Compared to other modification methods, chemical modification has the advantages of fast reaction rate, high efficiency, good performance, clear modification effects, and easy crosslinking, making it the main technology for protein modification. Summary of the Invention

[0005] Wheat gluten protein is a high-quality vegetable protein with high nutritional value. However, due to its structural characteristics, wheat gluten protein has low water solubility and its functional properties are not fully exerted, hindering its application in the food and other industries.

[0006] The present invention provides a method for modifying wheat gluten protein with lactic acid, which can change the internal structure of gluten protein molecules and improve the functional properties of wheat gluten protein, such as solubility and emulsifying ability.

[0007] The method comprises: Preparation of protein dispersion: Step (1) of dispersing wheat gluten protein in a lactic acid solution or an aqueous solution containing sodium lactate to obtain a wheat gluten protein suspension; Modification: The wheat gluten protein suspension is stirred for a certain time to allow the lactic acid groups to fully contact with the amino acids of the gluten protein and bond to the amino acids. If sodium lactate is selected in step (1), the pH should be kept alkaline with an alkaline solution during the stirring process. After the stirring is completed, a step (2) of obtaining a lactic acidified wheat gluten protein treated solution; Protein precipitation: adjusting the pH of the lactic acidified wheat gluten protein treatment solution to neutral with a pH adjuster to precipitate the gluten protein, then centrifuging, collecting the precipitated protein, and washing it with water to remove the remaining pH adjuster and some of the free lactic acid and sodium lactate that did not participate in the reaction; Dialysis: Redissolving the washed precipitated protein and dialyzing it against deionized water to remove salt ions, to obtain a wheat gluten protein lactic acid purified solution, the dialysis being for removing hydrogen ions, sodium ions, free lactic acid and sodium lactate; and (5) freeze-drying the purified wheat gluten protein lactic acid solution to obtain lactic acid modified wheat gluten protein.

[0008] Further, in step (1), the mass of wheat gluten protein is 25 g, the lactic acid solution is 250 mL (solid-liquid ratio 1:10), and the concentration of the lactic acid solution is 0.1 mol / L; or the mass of wheat gluten protein is 25 g, and the aqueous solution of sodium lactate is 250 mL, where the mass of sodium lactate is 5 g, that is, the mass ratio of sodium lactate to wheat gluten protein is 1:5.

[0009] Furthermore, in step (2), the reaction time is 3 hours, the reaction temperature is 25° C., and the stirring speed is 100 rpm.

[0010] Furthermore, in step (2), the alkaline solution is 2 mol / L NaOH and has a pH of 10.0.

[0011] Furthermore, in step (3), the neutral pH is 7.0, the pH adjuster used is 2 mol / L NaOH, the centrifugation speed is 4500 r / min, the centrifugation time is 15 minutes, and the precipitation is performed by washing with water three times.

[0012] Furthermore, in step (4), the dialysis time is 72 hours.

[0013] In the present invention, after treating the wheat gluten protein solution with lactic acid and sodium lactate, the wheat gluten protein is modified with lactic acid, and its solubility and emulsifying properties are significantly improved. At a pH of about 7, the solubility of gluten protein increases from the original 0.05 mg / mL to 0.19 mg / mL and 0.21 mg / mL, respectively, and the emulsifying activity increases from the original 16.66 mg / mL. 2 / g to 19.27m respectively 2 / g, 19.58m 2 / g.

[0014] Lactic acid is a natural organic weak acid that can be used as a pH adjuster and has a certain preservative effect, making it a widely used food additive. The present invention effectively improves the solubility and emulsification properties of wheat gluten protein through lactation, expanding the scope of its application to systems such as protein drinks, edible packaging films, non-dairy creamers, ice cream, and vegetable proteins.

[0015] Unlike organic acids, which usually improve the functional properties of proteins by deamidation, the present invention alters the structure of proteins by attaching lactic acid groups to the amino acid side chains of proteins, further improving their functional properties, as evidenced by the fact that lactate, i.e., sodium lactate, can modify gluten proteins. [Brief explanation of the drawings]

[0016] [Figure 1] Western blot analysis is shown. [Figure 2] 1 shows the solubility of lactated wheat gluten proteins. [Figure 3] The water retention capacity (WHC) and oil retention capacity (OAC) of lactated wheat gluten proteins are shown. [Figure 4] 1 shows the emulsifying ability of lactic oxidized wheat gluten proteins. [Figure 5] 1 shows the rheological properties of lactated wheat gluten proteins. [Figure 6] 1 shows the infrared spectrum of lactated wheat gluten protein. [Figure 7] Fluorescence spectra of lactated wheat gluten proteins. [Figure 8] 1 shows the ultraviolet spectrum of lactated wheat gluten protein. [Figure 9] 1 shows an SDS-PAGE spectrum of lactated wheat gluten protein.

[0017] In Figures 2 to 8, Raw refers to untreated wheat gluten protein, Lactate refers to wheat gluten protein treated with lactic acid, and Sodium lactate refers to wheat gluten protein treated with sodium lactate. DETAILED DESCRIPTION OF THE INVENTION

[0018] Example 1 (1) 25 g of wheat gluten protein was weighed into 250 mL of 0.1 mol / L lactic acid solution to give a solid-liquid ratio of 1:10 (m / v) to obtain a wheat gluten protein suspension; (2) The wheat gluten protein suspension is stirred at 100 rpm for 3 hours to allow the lactic acid groups to fully react with the amino acids of the gluten protein and bond to the amino acids of the gluten protein; (3) After the reaction is completed, the pH of the reaction system is adjusted to 7.0 with 2 mol / L NaOH, centrifuged at 4500 rpm for 15 minutes, the supernatant is discarded, and the protein is washed three times with water to precipitate. (4) Dialysis: After washing with water, the precipitated protein was redissolved and dialyzed against deionized water for 72 hours to obtain a purified wheat gluten protein lactic acid solution. (5) The wheat gluten protein lactic acid purified solution was vacuum freeze-dried to obtain lactic acid-modified wheat gluten protein, and the dried wheat gluten protein was stored in a drying dish.

[0019] Example 2 (1) 25 g of wheat gluten protein was weighed into 250 mL of deionized water to give a solid-liquid ratio of 1:10 (m / v), and 5 g of sodium lactate was added thereto to obtain a wheat gluten protein suspension; (2) The wheat gluten protein suspension was stirred at 100 rpm for 3 hours to allow the lactic acid groups to fully react with the amino acids in the gluten protein and bond to the amino acids in the gluten protein. During the stirring process, the pH of the reaction system was adjusted to 10.0 with 2 mol / L NaOH. (3) After the reaction is completed, the pH of the reaction system is adjusted to 7.0 with 2 mol / L NaOH, centrifuged at 4500 rpm for 15 minutes, the supernatant is discarded, and the protein is washed three times with water to precipitate. (4) Dialysis: After washing with water, the precipitated protein was redissolved and dialyzed against deionized water for 72 hours to obtain a purified wheat gluten protein lactic acid solution. (5) The wheat gluten protein lactic acid purified solution was vacuum freeze-dried to obtain lactic acid-modified wheat gluten protein, and the dried wheat gluten protein was stored in a drying dish.

[0020] Test results: 1. Verification of lactate modification Sample preparation: The protein concentrations of the samples stored in the dry dishes in Examples 1 and 2 were measured using the BCA protein analysis reagent. The proteins were then adjusted to the same concentration (1 mg / mL) and added to a protein loading buffer. After mixing, the mixture was heated at 100°C for 10 minutes to denature the proteins. The protein loading buffer consisted of 50 mM Tris-HCl, 0.1% bromophenol blue, 10% glycerol (v / v), and 2% SDS (w / v), with a pH of 6.8. Electrophoresis: A 10% SDS polyacrylamide gel was prepared, loaded with 10 μL of protein per sample, and electrophoresis was performed at a constant voltage of 120 V. The electrophoresis was terminated when the bromophenol blue at the front end disappeared from the bottom of the gel. Transfer: The PVDF membrane was immersed in methanol for 1 minute, then placed in the transfer solution with the gel and allowed to equilibrate for 5 minutes. The membrane was then placed in the following order: anode-sponge-two layers of filter paper-PVDF membrane-gel-two layers of filter paper-sponge-cathode. The transfer was then placed in a wet transfer machine. The transfer machine was then embedded in ice and transferred at a constant current of 300 mA for 2.5 hours. Blocking: The PVDF membrane was removed and blocked with 5% skim milk for 1 hour. Primary antibody incubation: After blocking, a lactate-specific antibody was added and incubated overnight at 4°C on a horizontal shaker. Washing: The primary antibody was removed, and the membrane was washed three times with TBST, for 5–10 minutes each time. Secondary antibody incubation: The corresponding secondary antibody (1:3000) diluted in 5% skim milk was added and incubated at room temperature for 1.5 hours. Washing: The secondary antibody was removed, and the membrane was washed three times with TBST, for 5–10 minutes each time. ECL development: Photographs were taken using a digital chemiluminescence imager.

[0021] The results are shown in Figure 1. Lane 1 is untreated wheat gluten protein, lane 2 is wheat gluten protein treated with lactic acid, and lane 3 is wheat gluten protein treated with sodium lactate. Western blot results show that the bands at 15-20 and 30-50 kDa in lanes 2 and 3 are significantly stronger. Western blot analysis involves staining proteins treated with gel electrophoresis with specific antibodies and analyzing the location and intensity of the staining to determine the expression status of specific proteins. Specifically, the lactic acid pan-antibody used in this experiment is an antibody that can identify specific proteins with lactic acid groups attached to amino acid side chains. Lane 1, which represents untreated gluten protein, shows no distinct bands, while lanes 2 and 3, which represent wheat gluten protein treated with lactic acid and sodium lactate, respectively, produce distinct bands. This indicates that treatment with lactic acid and sodium lactate caused significant lactic acid modification of gluten protein. The molecular weight of wheat gliadin is approximately 22 to 58 kDa, the molecular weight of the high molecular weight glutenin subunit is 80 to 130 kDa, and the molecular weight of the low molecular weight glutenin subunit is 10 to 70 kDa. As can be seen from Figure 1, the band intensities at 15 to 20 and 30 to 50 kDa in lanes 2 and 3 are clearly stronger, and therefore it is inferred that the modification sites are mainly concentrated in gliadin and low molecular weight glutenin subunits.

[0022] II. Measurement of the functional properties of lactated wheat gluten protein (1) Solubility measurement: The protein samples stored in the drying dishes of Examples 1 and 2 were dissolved in deionized water, the pH of the mixture was adjusted to 3.0-10.0 with HCl and NaOH, and the mixture was stirred at room temperature for 120 minutes. The mixture was centrifuged at 10,000 rpm for 15 minutes, and the protein content in the supernatant was measured using a BCA protein assay kit.

[0023] As shown in Figure 2, the solubility of lactic acid-modified wheat gluten protein is significantly improved in the pH range of 3.0 to 10.0. First, the newly introduced lactic acid groups unfold the gluten protein structure and strengthen the interaction between the protein side chains and water, which is consistent with the fluorescence spectrum results shown below. Second, the introduction of lactic acid groups increases the negative charge, which may lead to improved solubility.

[0024] Water retention measurement: 0.4 g of wheat gluten protein was accurately weighed into a 15 mL centrifuge tube. The combined weight of the tube and sample was recorded, and 4 mL of deionized water was added to the tube. The sample was thoroughly mixed using a vortex mixer, then left at room temperature for 60 minutes, centrifuged at 4000 rpm for 20 minutes, the supernatant was carefully removed, and the mass was accurately weighed. The water retention capacity of the sample was expressed as the mass of water absorbed per gram of protein. Oil retention measurement: 0.3 g of wheat gluten protein was accurately weighed into a 15 mL centrifuge tube. The combined weight of the tube and sample was recorded, and 2 mL of sunflower oil was added to the tube. The sample was thoroughly mixed using a vortex mixer, then left at room temperature for 60 minutes, centrifuged at 4000 rpm for 20 minutes, the supernatant was carefully removed, and the mass was accurately weighed. The oil retention capacity of the sample was expressed as the mass of oil absorbed per gram of protein.

[0025] The effect of lactic acid modification on the water and oil retention of wheat gluten proteins is shown in Figure 3. The decrease in water retention may be due to the improved solubility, as it has been reported that highly water-soluble proteins have a low water absorption capacity. The improvement in oil retention may be due to the unfolding of the protein structure by lactic acid modification, which exposes hydrophobic groups that can further form protein-oil complexes with oil.

[0026] (3) Measurement of emulsifying ability: 20 mL of 10 mg / mL wheat gluten protein solution (0.05 mol / L phosphate buffer) was taken, 5 mL of sunflower oil was added, and the mixture was sheared for 1 minute at 10,000 r / min using an Ultra-Turrax 18 emulsifier / disperser. Immediately afterwards, 50 μL of liquid was aspirated from a position approximately 5 mm from the bottom of the container, 4.95 mL of 0.1% SDS solution was added, and the mixture was mixed homogeneously. The absorbance A0 was measured at 500 nm using the 0.1% SDS solution as a blank. After leaving the mixture at room temperature for 10 minutes, the mixture was sampled again, and the absorbance A0 was measured in the A0 measurement step. t The emulsifying ability was calculated according to the following formula: TIFF0007825080000001.tif27170

[0027] A 500 is the absorbance measured at 500 nm (0 min), n is the dilution factor, C is the mass concentration of the protein, φ is the volume fraction of the dispersed phase, L is the light path of the colorimetric cell, A0 and A t is the absorbance measured at 0 and 10 min, and ΔT is 10 min.

[0028] Proteins with good emulsifying ability can effectively reduce surface tension, rapidly absorb at the oil-water interface, and continuously expand, uniting the oil-water system. Protein solubility plays an important role in the emulsification process, and solubility is a prerequisite for proteins to have good emulsifying properties; only highly soluble proteins can easily diffuse at the oil-water interface. The good emulsifying activity and emulsion stability of wheat gluten proteins after lactic oxidation are thought to be due to the improved protein solubility.

[0029] (4) Rheological Properties: The rheological properties of wheat gluten protein were measured using a DHR-3 rheometer with a 40 mm flat plate. After mixing the samples with deionized water, they were allowed to relax for 30 minutes. To prevent moisture loss, silicone oil was coated on the edges of each sample. A strain of 1% was selected in the linear viscoelastic region, and the frequency sweep range was set to 0.1 Hz to 10 Hz. The wheat gluten protein was allowed to equilibrate for 300 s before testing, and the test temperature was set to 25°C.

[0030] As shown in Figure 5, all samples exhibited typical viscoelastic and solid-like behavior, i.e., the storage modulus was greater than the loss modulus. Both lactic acid and sodium lactate treatments could significantly improve the storage modulus and loss modulus of wheat gluten protein, which means that lactic oxidation is an effective method for improving the viscoelasticity of gluten protein, because the solubility of wheat gluten protein after lactic oxidation treatment was significantly improved and the protein was more fully hydrated.

[0031] 3. Measurement of the structural characteristics of lactated wheat gluten protein (1) Measurement of secondary structure: The secondary structure of wheat gluten protein was measured using the Fourier transform infrared method. 1 mg of wheat gluten protein and 100 mg of potassium bromide were weighed and ground into powder, then pressed into a press to form semitransparent, uniform flakes. The sample was then placed in a Fourier transform infrared spectrometer and scanned using air as the background. The scanning frequency was 4000-400 cm. -1 The number of scans was 32, and the resolution was 4cm. -1 and finally Peakfit was used to analyze the secondary structure content of the samples.

[0032] TIFF0007825080000002.tif81170

[0033] As shown in Table 1, lactic acid modification converts the β-turn portion of the secondary structure of wheat gluten protein to β-sheet. β-sheet is a relatively stable secondary structure, while α-helix, β-turn, and random coil are relatively flexible. The significant increase in β-sheet structure indicates the improved stability of lactic acid-treated wheat gluten protein. At the same time, the increase in β-sheet structure significantly changes the protein-protein interactions, favoring the viscoelasticity of gluten protein, which is consistent with the previous rheological results. Furthermore, the arrangement of peptide chains is reversed by the β-turns on the protein surface, which causes the proteins to bind tightly. The β-turn content of wheat gluten protein after lactic acid modification significantly decreases, indicating further unfolding of the protein molecules.

[0034] (2) Intrinsic fluorescence spectra: Fluorescence spectra of wheat gluten proteins were measured using an F-7000 fluorescence spectrometer. Wheat gluten proteins were dissolved in phosphate buffer (0.05 mol / L, pH 7.0), and the protein concentration was measured using a BCA kit to adjust the final sample concentration to 0.4 mg / mL. The fluorescence spectrometer parameters were: excitation wavelength 280 nm, emission wavelength range 300–500 nm, scan speed 1200 nm / min, reaction time 0.5 s, voltage 600 V, and excitation and emission gaps 5 nm.

[0035] Amino acids in gluten proteins, such as tryptophan, can emit fluorescence. Measuring protein fluorescence spectra can reflect the microenvironment of amino acids within proteins and even infer changes in the protein's tertiary structure. As shown in Figure 7, the maximum absorption fluorescence peak of wheat gluten protein after lactic acid modification is red-shifted, indicating that lactic acid modification causes the structure of wheat gluten protein to unfold, exposing amino acid side groups to a more hydrophilic environment. However, the exposed amino acids do not increase in fluorescence intensity, which may be due to tryptophan fluorescence quenching.

[0036] (3) UV Spectrum: UV absorption spectra were recorded using a UV-1800 UV Spectrophotometer. Wheat gluten protein was dissolved in phosphate buffer (0.05 mol / L, pH 7.0), and the protein concentration was measured using a BCA kit. The final sample concentration was adjusted to 0.4 mg / mL. The scan range of the UV spectrophotometer was 200–400 nm, the optical path length was 1 cm, and the response time was 0.1 s.

[0037] UV absorption spectra are also commonly used to predict changes in the tertiary structure of proteins. As shown in Figure 8, the maximum absorption peak of wheat gluten protein after lactation exhibits a blue shift, indicating that the microenvironment of tryptophan and tyrosine in wheat gluten protein changes after treatment. The maximum UV absorption peak also increases significantly after treatment, further demonstrating that lactation has a significant effect on the tertiary structure of wheat gluten protein.

[0038] (4) SDS-PAGE analysis: The sample solution (1 mg / mL) was added to the protein loading buffer, mixed, and heated at 100°C for 10 minutes. The protein loading buffer consisted of 50 mM Tris-HCl, 0.1% bromophenol blue, 10% glycerol (v / v), and 2% SDS (w / v), with a pH of 6.8. After cooling to room temperature, 10 μL of the sample was loaded onto the gel and maintained at 120 V for 60 minutes. The gel was stained with Coomassie Brilliant Blue solution for 30 minutes and destained overnight at 37°C using a 10% acetic acid and 5.5% ethanol destaining solution. Lane 1 was untreated wheat gluten protein, lane 2 was lactic acid-treated wheat gluten protein, and lane 3 was sodium lactate-treated wheat gluten protein.

[0039] Based on the changes in the secondary and tertiary structures of gluten proteins, the effect of lactation on the molecular weight distribution of gluten proteins can be further analyzed by SDS-PAGE. As shown in Figure 9, after lactation, the bands at 40-70 kDa were clearly intensified. This may be due to the increase in molecular weight of the protein molecules after binding to lactic acid groups, or it may be due to the aggregation of lactated proteins, which leads to the intensification of the bands.

[0040] Although the present invention has been disclosed above with preferred embodiments, these embodiments are not intended to limit the present invention, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, and therefore the scope of protection of the present invention should be defined by the claims.

Claims

1. 1. A method for preparing lactic acid modified wheat gluten protein, comprising: Preparation of protein dispersion: Step (1) of dispersing wheat gluten protein in a lactic acid solution or an aqueous solution containing sodium lactate to obtain a wheat gluten protein suspension, wherein the solid-liquid ratio of wheat gluten protein to lactic acid solution is 1 g:10 mL, the concentration of the lactic acid solution is 0.1 mol / L, the solid-liquid ratio of wheat gluten protein to sodium lactate solution is 1 g:10 mL, and the mass ratio of sodium lactate to wheat gluten protein is 1:5; Modification: Step (2) is to stir the wheat gluten protein suspension to obtain a lactic acid wheat gluten protein treatment liquid, the stirring time is 3 hours, the temperature is 25°C, the stirring speed is 100 rpm, and when sodium lactate aqueous solution is used in step (1), the pH is adjusted to alkaline with an alkaline solution in the stirring process, the alkaline solution is 2 mol / L NaOH, and the pH is 10.0; Protein precipitation: adjusting the pH of the lactic acidified wheat gluten protein treatment solution to neutral with a pH adjuster to precipitate gluten protein, then centrifuging, collecting the precipitated protein and washing it with water (3); Dialysis: Redissolving the precipitated protein after washing with water and dialysis it against deionized water to obtain a wheat gluten protein lactic acid purified solution (4); Vacuum freeze-drying: Vacuum freeze-drying the wheat gluten protein lactic acid purified solution to obtain lactic acid modified wheat gluten protein (5); A method for preparing lactic acid modified wheat gluten protein, comprising:

2. 2. The method for preparing lactic acid modified wheat gluten protein according to claim 1, wherein the neutral pH in step (3) is 7.

0.

3. 2. The method for preparing lactic acid modified wheat gluten protein according to claim 1, characterized in that in step (3), the centrifugation speed is 4500 r / min and the centrifugation time is 15 minutes.

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