Modification method of millet protein and modified millet protein

Abstract

The invention is suitable for the technical field of food processing, and provides a modification method of millet protein and modified millet protein.The modification method of the millet protein comprises the steps that the millet protein and water are stirred to be uniform, then the mixture is placed in a boiling water bath to be subjected to incubation pretreatment, cooling is conducted, and protein suspension liquid is obtained; and adding lactobacillus plantarum into the protein suspension, carrying out shaking table incubation treatment, and carrying out vacuum freeze drying treatment to obtain the modified millet protein. According to the present invention, the millet protein is subjected to incubation treatment through the lactobacillus plantarum, such that the soluble protein content and the peptide content of the millet protein are significantly improved, the essential amino acid ratio in the millet protein is optimized, and the nutritional value of the millet protein is significantly enhanced. Besides, the modification method can improve the physical properties of the millet protein and optimize the gel network structure of the millet protein, is simple and convenient to operate, environment-friendly, reasonable in cost control and suitable for large-scale industrial production, and does not need to depend on complex equipment or expensive raw materials.

Description

Technical Field

[0001] The present application belongs to the field of food processing technology, and in particular relates to a method for modifying millet protein and modified millet protein. Background Art

[0002] Millet originated more than 10,000 years ago and is still an important food crop in arid and semi-arid regions of Asia, Africa, and Europe. Millet is rich in starch, protein, fat, phenolic compounds, carotenoids, and other nutrients. The protein content ranges from 7.9% to 21.9%, making it one of the most important sources of plant protein. Millet protein has been reported to have bioactive functions such as hypoglycemic, hypotensive, anti-inflammatory, and antioxidant. In addition, according to our previous research, the higher the protein content in millet, the worse its cooking and sensory quality is generally, and therefore the less favor it has by consumers. Therefore, the rational use of protein in this type of millet is of great significance to increase the added value of millet. However, the solubility and digestibility of natural millet protein are poor, so some processing technologies are urgently needed to improve it in order to improve its application in the food industry.

[0003] In recent years, physical technologies such as extrusion, microwave, cold plasma, high static pressure, chemical technologies such as acid / base hydrolysis, glycosylation, succinylation, and biotechnology such as enzyme cross-linking have been widely used in the modification of plant proteins to change the structure and biological properties of proteins and improve their utilization. Fermentation, as an ancient and emerging biotechnology, is also considered to be an effective means of protein modification. Studies have shown that fermentation can reduce the content of allergenic peptides in plant proteins and increase the antihypertensive and antioxidant activities of proteins. Lactic acid bacteria, as a probiotic with both acidification and enzymatic hydrolysis, have been widely used to change the physicochemical and functional properties of bean proteins. However, research on the effects of lactic acid bacteria on cereal proteins is still very limited, and the understanding of the effect of lactic acid bacteria on the modification of millet proteins is even more lacking. Therefore, based on the above situation, this field urgently needs to clarify the effects of fermentation on the structure and properties of millet proteins, and provide a theoretical basis for the application of millet proteins in the food industry, in order to enhance the added value of millet. Summary of the invention

[0004] The purpose of the embodiments of the present application is to provide a method for modifying millet protein and modified millet protein, aiming to solve the problems of limited application of millet protein in the prior art and insufficient research on cereal protein by lactic acid bacteria.

[0005] The embodiment of the present application is implemented as follows: a method for modifying millet protein, comprising:

[0006] After the millet protein and water are uniformly stirred, the mixture is placed in a boiling water bath for incubation pretreatment, and then cooled to obtain a protein suspension;

[0007] The plant lactobacillus is added into the protein suspension, and the suspension is incubated on a shaking table, and then subjected to vacuum freeze-drying to obtain the modified millet protein.

[0008] The embodiment of the present application also provides a modified millet protein, which is prepared by the above-mentioned millet protein modification method.

[0009] The embodiments of the present application have the following advantages:

[0010] (1) The present invention significantly improves the soluble protein and peptide content of millet protein by incubating millet protein with Lactobacillus plantarum, optimizes the ratio of essential amino acids in millet protein, and thus significantly enhances the nutritional value of millet protein. In addition, the fermentation and metabolic activity of Lactobacillus plantarum can produce a large amount of organic acids, which not only further improve the taste and digestibility of millet protein, but also give it more biological activity and functional properties.

[0011] (2) The modification method of the embodiment of the present application can improve the physical properties of millet protein and optimize the network structure of millet protein gel. Specifically, incubation allows the protein to form intermolecular and intramolecular interactions through covalent (forming new disulfide bonds or cross-linking through exchange reactions of thiol groups and disulfide bonds) and non-covalent reactions (forming hydrogen bonds, hydrophobic forces), promoting the formation of millet protein gel structure. This modification method makes millet protein a promising delivery carrier for bioactive substances, providing a broader prospect for the application of millet protein in the food industry.

[0012] (3) The modification method of the embodiment of the present application is not only simple to operate, but also has good potential for industrial application. The method is suitable for large-scale industrial production, has reasonable cost control, does not rely on complex equipment or expensive raw materials, and has low production costs. At the same time, the process is green and environmentally friendly and meets the requirements of sustainable development.

[0013] In summary, the millet protein modified by the embodiments of the present application can be widely used in multiple fields such as food processing, health food and nutritional supplements, etc., can meet consumers' demand for high-quality healthy food, has important application value and promotion prospects, and provides a new technical option for the field of food processing. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The results of the effects of different fermentation times on the microstructure of millet protein provided in the examples of the present application;

[0015] Figure 2 The results of the effects of different fermentation times on the circular dichroism spectrum (A), secondary structure (B), FTIR spectrum (C) and intrinsic fluorescence spectrum (D) of millet protein provided in the examples of the present application;

[0016] Figure 3 The results of the effects of different fermentation times provided by the embodiments of the present application on the surface hydrophobicity (A), free sulfhydryl group content (B), and Zeta potential (C) of millet protein;

[0017] Figure 4 The results of the effects of different fermentation times provided by the embodiments of the present application on SDS-PAGE of millet protein under non-reducing and reducing conditions.

[0018] Figure 5 The results of the effects of different fermentation times provided by the embodiments of the present application on the emulsifying activity, emulsion stability (A), and in vitro digestion characteristics (B) of millet protein. Detailed implementation manners

[0019] In order to make the objectives, technical solutions, and advantages of the present application clearer, the present application will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0020] The embodiments of the present application provide a method for modifying millet protein, including:

[0021] After stirring millet protein and water evenly, incubate and pretreat it in a boiling water bath, and after cooling, obtain a protein suspension;

[0022] Add Lactobacillus plantarum to the protein suspension, perform shaking incubation treatment, and perform vacuum freeze-drying treatment to obtain modified millet protein.

[0023] Among them, the Lactobacillus plantarum used in the embodiments of the present application is freeze-dried powder of Lactobacillus plantarum, which is derived from Shaanxi Yijun Biotechnology Co., Ltd. In addition, in the previous R & D process of the present application, other strains were also used for experiments, such as Lactobacillus bulgaricus, and the effect was that the change in the pH value of the fermentation broth was not significant, and the pH value of the fermentation decreased from 5.72 to 4.92 after 96 h of fermentation.

[0024] In the embodiment of the present application, the step of uniformly stirring millet protein and water, then incubating and pretreating in a boiling water bath, and obtaining a protein suspension after cooling includes: mixing millet protein and water, stirring at room temperature for 30 min, then incubating and pretreating in a boiling water bath for 10 min, and obtaining a protein suspension after cooling. Before adding Lactobacillus plantarum for fermentation, in the present application, millet protein and water are first mixed and stirred at room temperature for 30 min. This pretreatment method helps to fully hydrate millet protein, making it more easily acted on by Lactobacillus plantarum; incubating in a boiling water bath for 10 min helps to kill most of the microorganisms in millet protein and water, and at the same time changes the structure of millet protein, thereby improving the fermentation effect and protein digestibility.

[0025] In the embodiment of the present application, the weight ratio of millet protein to water is 1:4 - 6, preferably 1:6.

[0026] In the embodiment of the present application, the added mass ratio of Lactobacillus plantarum in the protein suspension is 1 - 2%, preferably 2%.

[0027] In the embodiment of the present application, in the shaking incubator treatment, the incubation temperature is 35 - 37 °C, the rotation speed is 100 rmp, and the incubation time is 0 - 60 h.

[0028] In the embodiment of the present application, the step of adding Lactobacillus plantarum into the protein suspension, performing shaking incubator treatment, and obtaining modified millet protein after vacuum freeze-drying treatment can be: incubating the Lactobacillus plantarum - millet protein mixture in a shaking incubator for a certain time, the shaking incubator temperature is 35 °C, the rotation speed is 100 rmp, and the incubation time is 0 - 60 h; vacuum freeze-drying, grinding the incubated Lactobacillus plantarum - millet protein, and passing through an 80-mesh sieve to obtain Lactobacillus plantarum-fermented and modified millet protein. In the present application, after fermentation is completed, the methods of vacuum freeze-drying, grinding, and passing through an 80-mesh sieve can better maintain the nutritional components and activity of the fermentation product, and at the same time make the particle size of the product uniform, which is beneficial to improving its stability and solubility in applications. Compared with ordinary drying and pulverization methods, it is more helpful to improve the quality of the product.

[0029] It should be noted that by controlling the fermentation time, the present application effectively changes the structure of millet protein, increases the content of small molecule proteins and peptides, makes up for the problem of unbalanced amino acid ratio in millet protein, and thus significantly improves the digestibility of millet protein, which is of great significance for improving the nutritional absorption of millet protein. Compared with traditional millet protein processing methods, there is an obvious breakthrough in improving digestibility.

[0030] In addition, this application not only improves the digestibility, but also enhances the gel properties of millet protein, broadening the application scope of millet protein in the food industry, such as its application in gel-like foods, dairy products, etc., making it have better texture and taste. This is the unique innovation of this method in improving the functional properties of millet protein.

[0031] Furthermore, the method of this application is easy to operate, does not require complex equipment or expensive raw materials, has low production costs, and the process is green and environmentally friendly. It has good potential for industrial application, is suitable for large-scale industrial production, can effectively reduce production costs, improve production efficiency, and meet the market's demand for high-quality and low-cost millet protein products. Compared with some complex biotechnological or chemical modification methods, it has obvious advantages in industrial production.

[0032] The following uses specific examples to describe in detail the modification method of millet protein, as shown below. For those not specifying specific conditions in the following examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0033] All experiments in this example were repeated three times, and the data were expressed as the mean ± standard deviation (SD) of three replicates. The data were analyzed using one-way analysis of variance (ANOVA) and SPSS, and the Duncan multiple range test was used to compare the means and significance (P value is 0.05). The figures drawn in this application were drawn by Origin software.

[0034] Example 1: Modification method of millet protein

[0035] Disperse 25.0 g of millet protein in 150 mL of distilled water and stir at room temperature for 30 min. The suspension is sterilized in a boiling water bath for 10 min and then quickly cooled to 35°C in an ice bath. Add 2% freeze-dried powder of Lactobacillus plantarum, incubate in a shaker at 35°C and 100 rmp, and take samples at 0, 12, 24, 36, 48, and 60 h respectively. After freeze-drying, grinding, and sieving, the fermented millet protein samples are obtained, named FMP, FFMP-12, FFMP-24, FFMP-36, FFMP-48, and FFMP-60 respectively.

[0036] Example 2: Determination of microbial quantity, pH value, soluble protein content, and peptide content

[0037] The number of viable bacteria in the fermentation broth is calculated by culturing on MRS medium at 35°C for 48 h. The pH value of the fermentation broth is measured using a pH meter. The soluble protein content and peptide content in the supernatant of the fermentation broth are determined using the Bradford protein detection kit and the OPA method respectively.

[0038] The microbial count, pH value, content of soluble proteins and peptides during the entire fermentation process are shown in Table 1. During the fermentation process, the viable cell count increased sharply first and then entered the stationary growth phase, increasing from 8.46 log CFU / mL at the beginning to 9.03 log CFU / mL at the end. The organic acids produced by Lactobacillus plantarum caused the pH value of the fermentation broth to gradually decrease from 5.71 to 3.78. During the fermentation process, the content of soluble proteins and peptides gradually increased, and this increase was attributed to the continuous decrease in pH value, which in turn triggered the activation of endogenous proteases in Lactobacillus plantarum, ultimately leading to the degradation of millet proteins into smaller proteins or short peptides. In addition, as the starch degraded during the fermentation process, the interaction between starch and proteins weakened, which might also lead to an increase in the content of soluble proteins.

[0039] Table 1 Microbial count, pH, soluble protein content, and polypeptide content during the fermentation process

[0040] Sample Number of microorganisms during fermentation (log CFU / mL) pH Soluble protein content (mg / mL) Polypeptide content (mg / mL) FMP 8.46±0.06e 5.71±0.01a 0.16±0.00e 0.20±0.00f FFMP-12 8.76±0.03d 4.61±0.00b 0.28±0.02d 0.22±0.00e FFMP-24 8.87±0.04c 4.15±0.01c 0.31±0.01c 0.24±0.01d FFMP-36 8.93±0.03b 3.93±0.02d 0.33±0.02c 0.28±0.01c FFMP-48 8.98±0.02ab 3.86±0.01e 0.38±0.00b 0.32±0.01b FFMP-60 9.03±0.01a 3.78±0.01f 0.41±0.02a 0.39±0.00a

[0041] Example 3: Scanning electron microscopy (SEM) test

[0042] The millet protein samples were sputter-coated with gold and then observed using a scanning electron microscope (Hitachi Regulus 8230, Japan) at an acceleration voltage of 5.0 kV. As Figure 1 shown, the original millet protein particles presented an irregular spherical or oval shape, with relatively uniform sizes and a relatively smooth surface; the protein particles of FFMP-12 and FFMP-24 showed greater irregularity and more obvious surface textures; the FFMP-36 and FFMP-48 samples had various morphologies, including oval, flat, and granular agglomerate shapes, and the concavities and convexities and textures on the particle surfaces were more obvious and complex; FFMP-60 showed unique structural and textural characteristics. In summary, as the fermentation time extended, the protein particles gradually changed from a relatively compact and smooth morphology to a loose and porous complex morphology. This might be because during the fermentation process of millet proteins, under the combined action of proteases and organic acids secreted by microorganisms, they were partially decomposed, resulting in larger pores and smaller particle sizes. As the fermentation continued, the small particles were rearranged and agglomerated under the action of intermolecular forces to form particle clusters with various morphologies.

[0043] Example 4: Circular dichroism (CD) spectroscopy analysis

[0044] The sample was dissolved in 0.01 M phosphate-buffered saline (PBS; pH 7.0) at a final concentration of 0.1 mg / mL. The protein solution was scanned using a circular dichroism spectrometer (Chirascan, Applied Photophysics Ltd, UK) in the wavelength range of 190 - 260 nm with a path length of 1 mm.

[0045] Circular dichroism (CD) spectra, especially in the far-ultraviolet region (190 - 260 nm), are commonly used to evaluate changes in protein secondary structure. As Figure 2 shown in A, the CD spectrum of FMP shows a positive peak at 192 nm and a strong negative peak at 205 nm, which are typical features of α-helix-rich proteins. As the fermentation time extended, the positive peak of the protein sample at 192 nm decreased (FFMP - 12) or even disappeared (FFMP - 24, FFMP - 36, FFMP - 48, FFMP - 60); in addition, the negative peak at 205 nm significantly blue-shifted to 200 - 202 nm and became narrower, indicating a change in the protein structure.

[0046] The proportions of the secondary structure of millet protein were calculated using CDNN software. As can be seen from Figure 2 B, the contents of α-helix, β-turn, and random coil gradually decreased from 33.22%, 19.57%, and 21.01% to 21.30%, 18.51%, and 16.34%, respectively. While the proportion of β-sheet gradually increased from 26.11% to 33.67%, 39.53%, 42.29%, 43.00%, and 43.58%. The stability of the α-helix mainly depends on the hydrogen bonds formed between adjacent amino acid residues. All peptide bonds in the peptide chain can form hydrogen bonds, so the α-helix is very stable. As fermentation proceeds, the α-helix structure is destroyed as the hydrogen bonds gradually break, and this result confirms that the microstructure of millet protein becomes loose and porous after fermentation. The β-sheet structure is mainly maintained by the hydrogen bonds formed between the N-H and C=O of adjacent peptide chains. Fermentation greatly increased the content of β-sheet, which may be due to the self-assembly behavior of small peptides produced by fermentation reconnecting through hydrogen bonds. The decrease in the content of α-helix and the increase in the content of β-sheet in millet protein corresponded to the extension of fermentation time, indicating the formation of a more ordered structure. In addition, the ratio of the content of α-helix to β-sheet decreased rapidly first and then slowly, which means that fermentation reduced the rigidity and enhanced the flexibility of the protein.

[0047] Example 5: Fourier transform infrared spectroscopy (FTIR) analysis

[0048] A 2 mg protein sample was mixed with 200 mg KBr and pressed into a thin film. The Fourier transform infrared spectrum of the protein sample was measured in the range of 4000 - 400 cm -1 using a Fourier transform infrared spectrometer (VERTEX 80v, BRUKER, Germany) with a scanning frequency of 64 times and a resolution of 4 cm -1 . Air was used as a control.

[0049] Fourier transform infrared spectroscopy is an effective method for detecting conformational changes in proteins. Figure 2 Figure C shows the Fourier transform infrared spectra of millet proteins between 4000 and 400 cm -1 after fermentation treatment. All spectra showed characteristic peaks of proteins, and no new absorption peaks were observed, indicating that the functional groups of the proteins did not change. However, the positions of the absorption peaks shifted slightly, indicating that the fermentation treatment changed the secondary structure of the proteins. The amide A band (3600 - 3200 cm -1 ) is caused by the stretching vibrations of N - H and O - H, and the amide B band (3000 - 2800 cm -1 ) represents the stretching and bending vibrations of C - H. The shift of these two peaks towards lower wavelengths indicates an enhancement of hydrogen bond interactions. In our study, compared with FMP, the peak of FFMP in the amide A band shifted significantly towards lower wavelengths (from 3294 to 3273, 3283, 3283, 3292, and 3276 cm -1 ). The peak of the amide B band tended to shift towards higher wavelengths, but the deviation was small (from 2921 to 2923, 2920, 2924, 2925, and 2922 cm -1 ). Therefore, this application concludes that fermentation enhances the hydrogen bonds of proteins, which may be due to the promotion of intermolecular interactions by hydrophobic aggregation during the fermentation process. The amide I band (1700 - 1600 cm -1 ) is generated by the stretching vibration of the C = O bond. Fermentation caused a slight shift in the amide I peak of the protein (from 1628 to 1635, 1627, 1629, 1628, and 1635 cm -1 ), indicating that the C = O stretching changed, which may be due to the electrostatic and hydrogen bond interactions between protein molecules. The amide II band (1550 - 1500 cm -1 ) is caused by the stretching vibration of the C - N bond and the in - plane bending vibration of the N - H bond, while the amide III band (1300 - 1200 cm -1 ) is attributed to the stretching vibration of the C - N bond and the deformation vibration of the N - H bond, and the peaks of the amide II and amide III bands changed slightly. These results indicate that different fermentation times lead to different protein conformations.

[0050] Example 6: Intrinsic fluorescence spectroscopy analysis

[0051] Prepare a 0.1 mg / mL protein solution using PBS buffer (0.01 M, pH 7.0). Perform intrinsic fluorescence spectroscopy measurements using a fluorescence spectrometer (FL970, Techcomp, China). The excitation wavelength is 280 nm, and the emission wavelength range is 300 - 450 nm.

[0052] Intrinsic fluorescence spectroscopy is an effective tool for characterizing the tertiary structure of proteins. The principle is that the endogenous fluorescence of aromatic amino acid residues (tryptophan and tyrosine) exists in the hydrophobic core region of proteins and can be excited at specific wavelengths to produce a certain fluorescence intensity. Generally, the emission intensity of tyrosine residues is weak, and tryptophan plays a major role. When tryptophan residues are buried in the hydrophobic core of the protein structure, λ max is usually less than 330 nm; while in a polar environment, λ max will undergo a red shift, indicating the loss of the tertiary or quaternary structure of the protein. In this study ( Figure 2 D), the λ max of both FMP and FFMP samples is greater than 330 nm, indicating that the tryptophan residues of foxtail millet proteins are in a polar environment before and after fermentation. In addition, changes in fluorescence intensity also reflect the spatial changes in the tertiary structure of proteins. Fluorescence intensity is affected by energy transfer from Tyr to Trp and fluorescence quenching by neighboring groups. In this study, with the extension of fermentation time, the fluorescence intensity of foxtail millet proteins gradually increased. This may be due to fermentation increasing the energy transfer from Tyr to Trp or reducing the accessibility of Trp to fluorescence.

[0053] Example 7: Surface hydrophobicity (H 0 ) analysis

[0054] Prepare a protein solution with a concentration of 0.01 - 0.1 mg / mL using PBS buffer (0.01 M, pH 7.0). Then, mix 2 mL of the protein solution with 20 μL of ANS solution (8 mM) and react for 15 min in a dark room. Measure the fluorescence intensity of the complex using a fluorescence spectrometer. The excitation wavelength is 390 nm, the emission wavelength range is 410 - 650 nm, the slit width is 5 nm, and the scanning speed is 10 nm / s. Through linear regression analysis, the slope of the fluorescence intensity versus the corresponding protein concentration is the H 0 value of the sample.

[0055] The surface hydrophobicity (H 0 ) of proteins reflects the degree of protein aggregation / folding and the exposure of surface hydrophobic amino acids. The H 0 values of foxtail millet proteins before and after fermentation are shown inFigure 3 A. The H of the protein after fermentation 0 value first decreases and then increases, and the H of FFMP-24 0 is the lowest. The decrease in the H value of FFMP-12 and FFMP-24 0 may be due to the relatively small degree of damage to the protein by enzymes and acids, failing to fully expose the hydrophobic groups originally hidden inside the protein; at the same time, the hydrophobic groups originally exposed in the polar environment may aggregate due to interactions. As fermentation progresses, the amino acid residues originally buried in the hydrophobic core of the protein are gradually exposed as the protein molecule unfolds, resulting in a gradual increase in the H 0 value of the protein. The surface hydrophobicity of FFMP-60 remains at a relatively high level, which may be because the exposure effect of enzymes and acids on hydrophobic amino acids exceeds the burial effect of millet protein aggregation on hydrophobic amino acids.

[0056] Example 8: Determination of Free SH Content

[0057] Disperse 20 mg of the sample in 4 mL of Tris-glycine-SDS buffer solution [0.086 M Tris, 0.09 M Glycine, 0.004 M disodium ethylenediaminetetraacetate (EDTA-2Na), and 2.5% SDS, pH 8.0]. Then, centrifuge the above solution at 5000 g for 10 min. Mix the supernatant (4 mL) with 0.04 mL of Ellman's reagent [5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) dissolved in Tris-glycine buffer, 4 mg / mL, pH 8.0], and incubate in the dark at 25 °C for 30 min. Taking the reagent blank as the reference, measure the absorbance at a wavelength of 412 nm. The Free SH content is calculated according to formula (1):

[0058] (1)

[0059] where A 412 represents the absorbance at a wavelength of 412 nm; C and D represent the protein concentration (mg / mL) and dilution factor, respectively.

[0060] The change in the free SH content can reflect the three-dimensional conformation of the protein. As can be seen from Figure 3 B, the free SH content in FMP is 1.66 μmol / g. The free SH content after fermentation is significantly lower than that in FMP. On the one hand, the decrease in the free SH content can be attributed to the aggregation of protein molecules; on the other hand, the exposed sulfhydryl groups are oxidized to disulfide bonds under the action of temperature or peroxides, which also leads to a decrease in the sulfhydryl content.

[0061] Example 9: Zeta potential analysis

[0062] The surface charge of the protein sample was measured using a Zeta potential analyzer (BeNano, 90 Zeta, Dandong Better Instrument Co., China).

[0063] Zeta potential is a reliable indicator of the dispersion stability of colloidal suspensions or solutions. Generally speaking, the smaller the absolute value of the Zeta potential, the lower the level of electrostatic repulsion, which leads to a decrease in the dispersibility of proteins and an increased likelihood of aggregation. As fermentation progresses, the absolute value of the Zeta potential of millet protein gradually decreases from 12.41 mV to 5.72 mV( Figure 3 C), indicating that fermentation reduces the electrostatic repulsion between millet proteins. Compared with FMP, the reason for the decrease in electrostatic repulsion may be the increased number of protons produced by Lactobacillus plantarum during fermentation, which offsets the negative charge on the surface of protein particles. In addition, the decrease in Zeta potential also means that proteins are more likely to aggregate, which is consistent with the results of SEM and SDS-PAGE.

[0064] Example 10: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis

[0065] A protein solution with a dry matter content of 10 μg / μL was prepared in PBS buffer. For non-reducing condition gel electrophoresis, the protein solution was mixed with a denaturing and non-reducing gel sample loading buffer (Beijing Solarbio Science & Technology Co., Ltd.) at a ratio of 4:1 and then centrifuged. For reducing condition gel electrophoresis, the protein solution was mixed with an SDS-PAGE sample loading buffer (containing DTT) (Beijing LABLEAD Commerce Co., Ltd.) at a ratio of 4:1, and the sample was centrifuged after heating for 10 min. During electrophoresis, 10 μL of the sample mixture was added to a LabPAGE 4-12% protein precast gel, and a LABLEAD prestained protein standard (Beijing LABLEAD Commerce Co., Ltd.) was used as the molecular weight marker. The gel was run for 40 min, stained with Coomassie Brilliant Blue G-250 (Beijing LABLEAD Commerce Co., Ltd.), and then decolorized with distilled water.

[0066] SDS-PAGE analysis was used to evaluate the molecular weight of proteins. The SDS-PAGE patterns of millet proteins under non-reducing and reducing conditions are shown in Figure 4。The electrophoretic band patterns and intensities of the protein samples were significantly different under reducing and non-reducing conditions, indicating the presence of disulfide bonds in the millet protein subunits. Under reducing conditions, distinct characteristic bands appeared with molecular weights of 1-15 kDa, 21 kDa, 34 kDa, 53 kDa, and 63 kDa for FMP. Meanwhile, the entire gel showed uniform blue staining because of the wide molecular weight range of albumin, globulin, and glutelin components in millet protein. In the protein electrophoretograms of FFMP-12 and FFMP-24, a significant decrease in the intensity of bands above 34 kDa was observed, which was attributed to the degradation of macromolecules by fermentation. However, as fermentation proceeded, an increase in the intensity of bands above 34 kDa was observed, along with the appearance of proteins with molecular weights below 10 kDa, indicating that protein aggregation and decomposition occurred simultaneously.

[0067] Example 11: Determination of Emulsifying Activity (EA) and Emulsifying Stability (ES)

[0068] Homogenize 3 mL of a protein dispersion (5 mg / mL) and 1 mL of soybean oil for 2 min at 15000 rpm using a homogenizer (THF500-G, China Tuohe Electromechanical Technology Co., Ltd.). Then, take 40 μL of the emulsion from the bottom of the mixture and mix it with 4 mL of SDS solution (0.1 %). Using the SDS solution as a blank control, measure the absorbance at a wavelength of 500 nm. Calculate EA and ES according to formulas (6) and (7):

[0069] (6)

[0070] where T is 2.303, D is the dilution factor (100), C is the protein concentration (0.005 g / mL), and φ is the volume fraction of oil (0.25).

[0071] (7)

[0072] where A 0 and A 10 represent the absorbances at 0 min and 10 min, respectively; Δt is 10 min.

[0073] The results of the emulsifying properties are shown in Figure 5 A. The emulsifying ability of FMP was the highest, at 1.67 m 2 / g. As fermentation proceeded, the emulsifying capacity of the protein decreased by 13.31%, 21.48%, 23.79%, 35.67% and 49.26% respectively. In addition, the emulsifying stability of FFMP-60 was the highest (50.58 min), while the emulsifying stability of other fermented samples was slightly lower than that of FMP (43.83 min). This indicates that fermentation reduces the amphiphilicity of the protein, which is consistent with the result that the interfacial tension increases with fermentation. Research shows that low molecular weight peptides can quickly enter the oil-water interface and reposition, so they have good emulsifying properties. In the research of this application, some low molecular weight proteins and peptides were produced during fermentation, but the emulsifying properties of millet protein still decreased. It has been reported that fermentation reduces the emulsifying properties of pea protein, probably due to protein aggregation and microbial interaction.

[0074] Example 12: Determination of in vitro digestibility (IVD) of protein

[0075] In the gastric juice stage, 0.5 g of the protein sample was mixed with 8 mL of simulated gastric juice, and the pH value was adjusted to 3.0 with 1 M HCl. 5.0 μL of 0.3 M CaCl 2 and 1 mL of pepsin solution (40000 U / mL distilled water) were added, and the volume was made up to 20.0 mL with distilled water. The mixture was stirred at 37°C for 2 h. 2.0 mL of bile (160 mmol / L simulated intestinal juice), 40 μL of 0.3 M CaCl 2 and 5 mL of trypsin solution (80 U / mL simulated intestinal juice) were added, and the volume was made up to 40.0 mL with distilled water. The mixture was stirred at 37°C for 2 h. After each digestion stage, the sample was heated in a boiling water bath for 10 min to completely inactivate the enzyme. Finally, the sample was cooled to room temperature and centrifuged at 5000 g for 15 min to obtain the supernatant.

[0076] The IVD of the protein was calculated using formula (8):

[0077] (8)

[0078] where C is the concentration (mg / mL) of the peptides released after subtracting the blank (buffer and digestive enzymes), V is the digestion volume (mL), and M is the mass (mg) of the protein.

[0079] Protein digestibility is one of the most basic indicators for evaluating the nutritional value of proteins. As Figure 5 shown in B, the gastric digestibility of FMP was 5.49% and the intestinal digestibility was 68.63%. After 60 h of fermentation, the gastric IVD was 6.14% and the intestinal IVD was 78.55%, which were 12% and 14% higher than those of the unfermented sample respectively. Combining with SDS-PAGE (Figure 3 C), during the fermentation of Lactobacillus plantarum, proteins are hydrolyzed into smaller fragments by various intracellular and extracellular peptidases, thus reducing the burden on pepsin and trypsin during the digestion of millet proteins and making it more conducive to hydrolysis. In addition, changes in the secondary structure of proteins also affect the digestibility of proteins. Some studies have shown that the β-sheet structure causes proteins to form a rigid closed structure, thus limiting the accessibility of digestive enzymes and reducing the digestibility of proteins. This is contrary to our research results. However, some studies have also shown that a decrease in the α-helix content and the α-helix / β-sheet ratio also increases the digestibility of proteins.

[0080] The above-described embodiments merely represent several implementation manners of the present application. The description is relatively specific and detailed, but it should not be construed as a limitation on the patent scope of the present application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims.

[0081] The foregoing is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for modifying millet protein, characterized in that: include: After the millet protein and water are uniformly stirred, the mixture is placed in a boiling water bath for incubation pretreatment, and then cooled to obtain a protein suspension; The plant lactobacillus is added into the protein suspension, and the suspension is incubated on a shaking table, and then subjected to vacuum freeze-drying to obtain the modified millet protein.

2. The method for modifying millet protein according to claim 1, characterized in that: The weight ratio of the millet protein to water is 1:4-6.

3. The method for modifying millet protein according to claim 1, characterized in that: The mass ratio of Lactobacillus plantarum added to the protein suspension is 1-2%.

4. The method for modifying millet protein according to claim 1, characterized in that: During the shaking incubation process, the incubation temperature is 35-37°C, the rotation speed is 100 rpm, and the incubation time is 0-60 h.

5. The method for modifying millet protein according to claim 1, characterized in that: The step of uniformly stirring the millet protein and water, placing the millet protein and water in a boiling water bath for incubation pretreatment, and cooling the millet protein suspension comprises: The millet protein was mixed with water, stirred at room temperature for 30 min, placed in a boiling water bath for incubation pretreatment for 10 min, and cooled to obtain a protein suspension.

6. A modified millet protein, characterized in that: The modified millet protein is prepared by the millet protein modification method described in any one of claims 1-5.

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