Preparation method of peanut-egg white coprecipitation protein

Abstract

The invention belongs to the technical field of protein modification, and particularly relates to a preparation method of peanut-egg white coprecipitation protein. The peanut-egg white coprecipitation protein is prepared by using alkali dissolution and an isoelectric precipitation method. The result shows that when the ratio of the peanut protein to the egg white protein is 2: 1, the property of the prepared coprecipitation protein is optimal. The solubility and the free sulfydryl content of the hydrogel are respectively increased to 55.7% and 15.7 [mu] mol / g, and the gel strength and the water-holding capacity of the hydrogel are respectively increased to 911.9 g and 85.5%. The interaction force between gel molecules shows that the hydrophobic interaction is dominant and the disulfide bond is secondary, but the property of the gel is obviously influenced. According to the method, the defects that the peanut protein is poor in solubility and relatively low in free sulfydryl content can be overcome by adding the egg white protein, so that the strength, the water binding capacity and the like of the peanut protein gel are improved, and the industrial application range of the peanut protein is expected to be expanded.

Description

Technical Field

[0001] This invention belongs to the field of protein modification technology, specifically relating to a method for preparing peanut-egg white co-precipitated protein. Background Technology

[0002] Peanuts have significant potential for development and utilization. Peanut kernels are high in protein, and peanut protein possesses excellent nutritional properties. However, natural peanut protein has drawbacks such as low methionine content and significant intermolecular hydrophobic interactions, which limit its application in the food industry.

[0003] Protein modification methods can improve the functional properties of proteins and broaden the processing and application range of natural proteins. Plant protein modification technology mainly utilizes physical, chemical, and biological methods to treat plant proteins, thereby altering their molecular structure or chemical groups and enhancing their functional properties. Coprecipitation modification technology is an emerging protein modification method that enables simultaneous protein co-assembly and modification during protein extraction. The core principle of this technology is to precisely adjust the pH value to near the isoelectric point of the protein, thereby weakening the protein's surface charge, reducing its hydrophilicity, and promoting the unfolding and interaction of protein molecules from different sources, ultimately leading to refolding and the formation of co-assembled protein complexes.

[0004] Researchers Tian et al. successfully prepared a soluble composite protein of soybean protein isolate (SPI) and wheat protein (WP) using co-precipitation technology, and found that its gel properties were significantly improved (Tian T, Tong X, Ren K, et al. Influence of protein ratios on the structure and gel properties of soybean-wheat co-precipitated proteins[J]. Lwt, 2022, 170: 114045.). Zhou Xiaohu prepared a pea-grass carp co-precipitated dual protein using isoelectric solubilization / precipitation technology. The results showed that the composite protein not only had a more reasonable amino acid composition and higher nutritional value, but its gel properties were also significantly improved (Zhou Xiaohu. Structural characterization and functional properties of pea-grass carp co-precipitated dual protein[D]. Guangdong: Guangdong Ocean University, 2022.). Summary of the Invention

[0005] Based on the principle of amino acid complementarity and functional synergy, this invention co-assembles egg white protein rich in sulfur-containing amino acids with peanut protein, and prepares peanut-egg white coprecipitated protein by alkaline dissolution-isoelectric point precipitation technology. This regulates the interaction between the two proteins, improves the amino acid balance and functional properties of peanut protein, enhances the gel properties of peanut protein, and expands the application range of peanut protein.

[0006] The present invention specifically adopts the following technical solution:

[0007] A method for preparing peanut-egg white coprecipitated protein, using peanut protein and egg white protein as raw materials, employs alkali dissolution and isoelectric point precipitation methods to prepare peanut-egg white coprecipitated protein. Specifically, it includes the following steps:

[0008] Step 1: Dissolve the mixture of peanut protein and egg white protein under alkaline conditions; further, the mass ratio of peanut protein to egg white protein is 6:1 to 2:1; the alkaline conditions refer to a pH value of 10 to 12, which can be adjusted by using NaOH; even further, the mass ratio of peanut protein to egg white protein is 2:1, the peanut protein content is 45%, and the egg white protein content is 80.2%.

[0009] Step 2: Then, in a 50°C water bath, centrifuge to collect the supernatant, adjust the pH to acidic, and centrifuge to collect the protein precipitate. Further, the water bath time is 30–90 minutes, where the water bath is to further promote the dissolution of peanut protein and egg white protein under alkaline conditions. The acidic conditions refer to a pH of 4–5.5, which can be adjusted using HCl. pH 4–5.5 is the isoelectric point of the protein precipitate (i.e., peanut-egg white co-precipitated protein). In this step, water is added and allowed to stand naturally before centrifuging to collect the protein precipitate. Adding water shifts the pH of the solution towards the isoelectric point of the protein, causing natural precipitation. Without external force, the resulting protein has better properties.

[0010] Step 3: Disperse the protein precipitate in distilled water, adjust the pH to 7.0, and freeze-dry to obtain protein powder. Further, adjust the pH to 7.0 using NaOH.

[0011] This invention explores the differences in solubility, fluorescence, free thiol content, and surface hydrophobicity of peanut-egg white coprecipitated proteins prepared at different ratios, clarifying the influence of raw material ratios on the physicochemical properties of peanut-egg white coprecipitated proteins. It also investigates the differences in gel strength, water retention, and intermolecular forces of gels formed from peanut-egg white coprecipitated proteins prepared at different ratios, clarifying the influence of raw material ratios on the gel properties of peanut-egg white coprecipitated proteins.

[0012] The beneficial effects of this invention are as follows:

[0013] This invention systematically reveals the improved functional properties of peanut-egg white co-precipitated proteins. By comparing the physicochemical properties of the co-precipitated proteins with those of single peanut protein, it was found that key indicators such as solubility, structural stability, active thiol content, and surface hydrophobicity of the co-precipitated protein system all showed an optimization trend, with higher egg white protein ratios resulting in better optimization. When the peanut protein:egg white protein ratio was 2:1, the solubility, free thiol content, structural stability, and surface hydrophobicity of the co-precipitated protein were significantly better than those of PP. The strength and water-holding capacity of the composite gel also showed an optimization trend with increasing egg white protein ratio. The Co 2:1 gel exhibited significantly higher strength and water-holding capacity than the pure peanut protein gel, and was also the best among all samples. Hydrophobic interactions played a major role in the formation of the gel structure, followed by disulfide bonds, while ionic bonds and hydrogen bonds contributed much less. In conclusion, co-assembling egg white protein and peanut protein can significantly improve the gel properties of peanut protein, providing theoretical and technical support for expanding the application of peanut protein in the food industry. Attached Figure Description

[0014] Figure 1 : Solubility of peanut-egg white coprecipitated protein prepared under different raw material ratios; Note: Different letters indicate significant differences between samples (P < 0.05).

[0015] Figure 2 Fluorescence intensity of peanut-egg white coprecipitated proteins prepared under different raw material ratios.

[0016] Figure 3 : Free sulfhydryl content of peanut-egg white coprecipitated protein prepared under different raw material ratios; Note: Different letters indicate significant differences between samples (P < 0.05).

[0017] Figure 4 : Surface hydrophobicity of peanut-egg white coprecipitated protein prepared under different raw material ratios; Note: Different letters indicate significant differences between samples (P < 0.05).

[0018] Figure 5 : Gel strength of peanut-egg white coprecipitated protein prepared under different raw material ratios; Note: Different letters indicate significant differences between samples (P < 0.05).

[0019] Figure 6 : Gel water-holding capacity of peanut-egg white coprecipitated protein prepared under different raw material ratios; Note: Different letters indicate significant differences between samples (P<0.05).

[0020] Figure 7Intermolecular forces of peanut-egg white coprecipitated proteins prepared under different raw material ratios; Note: Different letters indicate significant differences between samples (P < 0.05). Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of the embodiments of this invention will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0022] The peanut protein (45% protein content) used in this embodiment was purchased from Anyang Tianxiangrui Food Technology Co., Ltd., and the egg white protein (80.2% protein content) was purchased from Anhui Rongda Food Co., Ltd. 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and 8-aniline-1-naphthalenesulfonic acid (ANS) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0023] Example 1

[0024] 1. Preparation of co-precipitated proteins

[0025] Weigh out 60g of peanut protein (denoted as PP group), and then weigh out 60g of peanut protein and egg white protein in weight ratios of 6:1, 4:1, and 2:1 respectively to prepare co-precipitated proteins (denoted as Co 6:1, Co 4:1, and Co 2:1 groups).

[0026] Dissolve peanut protein or a mixture of peanut protein and egg white protein in 600 mL of distilled water, adjust the pH to 10 with 2 mol / L NaOH, then incubate in a water bath at 50 °C for 30 min, centrifuge at 4000 r / min for 15 min, take the supernatant, add 0.5 mol / L HCl to adjust the pH to 4.5, add water and let stand to settle naturally, then centrifuge (5000 r / min, 10 min) to collect the protein precipitate.

[0027] Disperse the protein precipitate in an appropriate amount of distilled water, adjust the pH to 7.0 with 2 mol / L NaOH, pour the pH-adjusted sample into an aluminum box, seal it with plastic wrap, freeze it completely in a refrigerator, and then freeze-dry it for 48 hours. Place the resulting protein powder in a sealed bag.

[0028] 2. Solubility determination

[0029] Calculate the solubility using the following formula:

[0030]

[0031] In the formula: C is the protein content (mg / mL) in the sample after centrifugation; C0 is the protein content (mg / mL) in the sample before centrifugation.

[0032] Protein solubility is an important indicator of the functional value of protein-based foods and a crucial method for assessing the potential applications of proteins in food. It can be used to predict a protein's foaming, emulsifying, and gel-forming abilities. Figure 1 It is evident that the ratio of peanut protein to egg white protein significantly affects the solubility of the coprecipitated protein during the preparation of peanut-egg white coprecipitated protein using the alkali dissolution-isoelectric point precipitation method. As the proportion of egg white protein in the coprecipitated protein increases, the solubility also gradually increases. This is determined by the inherent properties of the two protein components: natural egg white protein has a better hydrophilic structure, and the surface of egg white protein molecules has a large number of polar groups, enabling egg white protein molecules to disperse into a stable hydration film, while peanut protein itself has strong hydrophobicity. The combination of the two improves the hydrophilicity of the composite protein product.

[0033] 3. Intrinsic fluorescence spectroscopy determination

[0034] The protein powders prepared in the "Preparation of Co-precipitated Proteins" section (PP group, Co 6:1 group, Co 4:1 group, and Co 2:1 group) were each prepared into 0.1 mg / mL protein solutions, and protein fluorescence was detected using a fluorescence spectrophotometer.

[0035] The aromatic amino acids in peanut-egg white co-precipitated proteins, such as tryptophan, tyrosine, and phenylalanine, are intrinsic fluorescent chromophores of the protein. When excited at 290 nm, they emit intrinsic fluorescence, with tryptophan being the main contributor. When tryptophan is exposed from its internal hydrophobic region to the external hydrophilic environment, the quenching effect of the polar environment (water) reduces the fluorescence intensity of the characteristic group and causes a red shift in the maximum emission peak. These changes characterize the degree of conformational unfolding of the protein molecule, thus reflecting the trend of changes in the protein's tertiary structure. Figure 2 The changes in fluorescence intensity of peanut-egg white coprecipitated proteins under different protein ratios were shown, confirming that the conformational state of the composite protein is affected by the component ratio. All four proteins exhibited maximum emission spectra at 325 nm, with the Co2:1 group showing the highest intensity. Comparison revealed that a higher proportion of egg white protein in the four coprecipitated proteins resulted in a greater fluorescence intensity at the maximum emission peak. This may be because the hydrophilic egg white protein can shield some hydrophobic groups, effectively reducing the exposure probability of tryptophan, thus increasing the fluorescence intensity. This indicates that within a certain range, a higher proportion of egg white protein leads to a more stable coprecipitated protein composite structure, suggesting that coprecipitated proteins with this ratio may possess superior functional properties.

[0036] 4. Determination of free sulfhydryl content

[0037] Weigh 100 mg of the protein powders prepared in the "Preparation of Co-precipitated Proteins" section, namely the PP group, Co 6:1 group, Co 4:1 group, and Co 2:1 group. Dissolve them in 10 mL of Tris-Gly buffer (0.086 mol / L tris(hydroxymethyl)aminomethane, 0.09 mol / L glycine, 0.04 mol / L ethylenediaminetetraacetic acid, pH 8.0) containing 8 mol / L urea. Let stand overnight with slow stirring, and then determine the free thiol content.

[0038] The formation and stability of protein gel networks are accomplished through intermolecular synergistic interactions, with disulfide bonds serving as crucial covalent cross-linking forces maintaining the three-dimensional network structure of the gel. Free thiol groups are precursors to disulfide bond formation; variations in their content reflect the degree of thiol oxidation during gelation and also indicate the potential for constructing intermolecular cross-linked networks within proteins. The free thiol content of four proteins is shown below. Figure 3 As shown, the PP group had the lowest thiol content. With the increase of the egg white protein ratio, the thiol content in the co-assembled proteins gradually increased. This is because egg white protein is rich in thiol groups, which can compensate for the lack of thiol groups in peanut protein and provide a large number of disulfide bond precursors. Therefore, the degree of cross-linking of the composite system can be controlled by using an appropriate protein blending ratio to obtain gel products with better performance, which is also beneficial to the development and application of peanut protein. Generally, the disulfide bonds generated by thiol oxidation are positively correlated with gel hardness, indicating the quality of the gel. Therefore, it is speculated that the gel formed by Co 2:1 may have better performance.

[0039] 5. Surface hydrophobicity determination

[0040] The surface hydrophobicity (H0) of proteins (PP group, Co 6:1 group, Co 4:1 group, and Co 2:1 group protein powders prepared in the "Preparation of Co-precipitated Proteins" section) was determined using the ANS fluorescent probe method.

[0041] The formation and stability of protein gel networks require the participation of multiple intermolecular forces, among which hydrophobic interactions are the fundamental driving force for gel formation and play a crucial role in the formation of the three-dimensional network structure of protein molecules. Surface hydrophobicity, as an important physical quantity reflecting the hydrophobic interactions of proteins, indicates the degree of binding between hydrophobic groups and water. It can be used to predict the spatial distribution and intensity of hydrophobic interactions during gelation and can also reflect the functional properties of proteins.

[0042] Figure 4This indicates that the surface hydrophobicity of the co-precipitated protein system is affected by the component ratio. Within a certain range, the higher the proportion of egg white protein in the co-precipitated protein, the lower the surface hydrophobicity. This is because egg white protein molecules contain more hydrophilic amino acids, which can cover the hydrophobic regions of peanut protein and induce conformational changes in peanut protein, bringing the hydrophobic core of peanut protein closer to the molecular interior and reducing the possibility of exposed hydrophobic groups. The magnitude of protein surface hydrophobicity has a significant impact on functional properties: a certain level of hydrophobic interaction can form a dense and ordered gel network, but if the protein surface hydrophobicity is too high, it will lead to excessive protein aggregation, which will reduce the solubility of the protein. For example, PP histidine powder has the strongest hydrophobic interaction and the lowest solubility. Figure 1 In summary, the surface hydrophobicity of proteins can be adjusted by modifying the ratio of egg white protein to achieve a balance between hydrophobic interactions and protein solubility, providing a reference for developing composite protein materials with excellent processing properties and functional stability.

[0043] 6. Gel strength test

[0044] The protein powders prepared in the "Preparation of Co-precipitated Proteins" section (PP group, Co 6:1 group, Co 4:1 group, and Co 2:1 group) were prepared into 15% (w / w) solutions. The protein samples were placed in a water bath and the temperature was maintained at 90°C for 30 minutes. The samples were then rapidly cooled and stored at 4°C.

[0045] The gel samples were removed from a 4°C freezer and allowed to equilibrate at room temperature for 1 hour. Subsequently, the gel properties of the samples were evaluated using a texture analyzer equipped with a P / 0.5 probe. During the test, the speeds before, during, and after the test were set to 2 mm / s, 1 mm / s, and 2 mm / s, respectively. The test distance was set to 10 mm, and the trigger force to 1 g.

[0046] The aggregation ability of a gel network can be represented by gel strength. From Figure 5 It can be seen that the strength of the coprecipitated protein gel increases with the increase of the proportion of egg white protein. When the ratio of peanut protein to egg white protein is 2:1, the gel strength is significantly higher than that of using peanut protein alone. This is mainly because egg white protein can provide a large number of sulfhydryl groups to participate in the formation of disulfide bonds between peptide chains, thereby promoting the covalent cross-linking between protein molecules to form a denser network structure, which significantly improves the mechanical strength, water retention, and thermal stability of the coprecipitated protein gel. The experimental results demonstrate the feasibility and rationality of using protein co-assembly to improve plant-based gels, and it is expected to be applied in vegetarian meat products or dairy substitutes.

[0047] 7. Determination of gel water-holding capacity

[0048] Weigh approximately 5g of each of the four gel samples prepared in the "Gel Strength Determination" section. Wrap each sample in two layers of filter paper and place them in centrifuge tubes. Weigh the total mass. Centrifuge at 4℃ and 5000 rpm for 10 minutes, remove the gels, and weigh them again. The formula for calculating water-holding capacity (WHC) is as follows:

[0049] WHC(%)=(m2-m0) / (m1-m0)

[0050] In the formula, m0 is the mass of the centrifuge tube (g); m1 is the total mass of the gel and centrifuge tube before centrifugation (g); and m2 is the total mass of the gel and centrifuge tube after centrifugation (g).

[0051] The water-holding capacity of protein gels is one of the main parameters characterizing their functional properties, determining the texture and sensory quality of food. This stems from the dual effect of the gel network on water molecules: physical retention and chemical adsorption. The strength of this interaction determines the perfection of the three-dimensional network structure, and its water-holding capacity directly affects the gel's texture. Figure 6 It can be seen that the water-holding capacity of the complex protein system increases systematically with the increase of the proportion of egg white protein. This is because egg white protein has a large number of hydrophilic amino acid residues, which can enhance the interaction between protein and water molecules and improve the hydration capacity of the co-precipitated protein.

[0052] The water-holding capacity of a gel depends on its three-dimensional network structure and the role of chemical bonds. A dense and uniform three-dimensional network can achieve physical water retention by hindering the diffusion of water molecules; abundant disulfide cross-linking increases the rigidity of the network to resist deformation caused by external forces. The combination of these two factors can maintain the stable retention of water in the gel. By controlling the proportion of raw protein, the water-holding capacity of the gel can be optimized, providing a reference for the formation of plant-based protein products with high-quality texture.

[0053] 8. Measurement of intermolecular forces in gels

[0054] Prepare extraction solutions A1, A2, A3, and A4 according to the following compositions: A1 (10 mL 0.6 mol / L NaCl), A2 (10 mL 1.5 mol / L urea, 0.6 mol / L NaCl), A3 (10 mL 8 mol / L urea, 0.6 mol / L NaCl), and A4 (10 mL 0.5 mol / L β-mercaptoethanol, 0.6 mol / L NaCl, 8 mol / L urea, pH 7.0). Add 2 g of each of the four gel samples prepared in the "Gel Strength Measurement" section to extraction solution A1, homogenize at 5000 rpm for 3 min, and then let stand at 4°C for 30 min. Next, centrifuge at 5000 rpm for 30 min at 4°C. Store the supernatant at 4°C; this supernatant is labeled S1. The precipitate is used for subsequent operations. Collect the precipitate and repeat the above operation (replacing A1 extract with A2 extract) to obtain S2 supernatant and precipitate. Similarly, repeat the operation (replacing A1 extract with A3 or A4 extract) to obtain S3 and S4 extracts. Mix S1, S2, S3, and S4 extracts with equal volumes of 20% trichloroacetic acid solution, then centrifuge at 4℃ and 5000 r / min for 15 min. Discard the supernatant, dissolve the precipitate in 5 mL of 1 mol / L NaOH solution, and determine the protein content using the Coomassie Brilliant Blue method. The protein mass fractions dissolved in S1, S2, S3, and S4 extracts represent the contributions of ionic bonds, hydrogen bonds, hydrophobic interactions, and disulfide bonds in the gel system, respectively.

[0055] The formation and stability of protein gel networks depend on the coordination of different types of intermolecular forces, and the proportional distribution of each type of force determines the properties exhibited by the gel. Figure 7 It is known that hydrophobic interactions dominate the thermal gelation process of proteins. This is because heating denatures protein molecules, exposing a large number of hydrophobic groups within the molecules. Hydrophobic interactions then become the dominant force in protein gel formation. Furthermore, because peanut protein has a low sulfur amino acid content and is a major component of co-precipitated proteins, hydrophobic interactions remain dominant even after the gel has stabilized. After the initial aggregation of a large number of protein molecules, disulfide bonds act as covalent cross-linking agents, providing strong cross-linking strength to support the gel structure and significantly contributing to the gel's strength and stability. The Co 2:1 gel has significantly higher strength than other gels. Figure 5 Correspondingly, its disulfide bond ratio is also the highest among all samples. In contrast, non-covalent bonds such as hydrogen bonds and ionic bonds have limited contribution to the stability of gel strength due to their weaker interactions.

[0056] The intermolecular forces of coprecipitated proteins differ. With increasing egg white protein ratio, the contribution of hydrophobic interactions decreases. This may be because the abundant hydrophilic amino acid residues within egg white protein molecules can cover the hydrophobic sites within peanut protein molecules, causing the hydrophobic core of peanut protein to fold inward, reducing the exposed hydrophobic groups. Pure peanut protein exhibits stronger hydrophobic interactions, which is a significant reason for its low solubility. Disulfide bond forming ability increases with increasing egg white protein ratio. When the peanut protein:egg white protein ratio is 2:1, the coprecipitated proteins show the highest disulfide bond contribution rate, possibly because egg white protein contains abundant free sulfhydryl groups, providing ample raw materials for disulfide bond formation. Conversely, pure peanut protein, due to the lack of sulfur-containing amino acids, has a lower disulfide bond content in the gel system, resulting in a looser gel structure. The gel strength and water-holding capacity are significantly lower than those of the Co 2:1 ratio.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing peanut-egg white co-precipitated protein, characterized in that, Peanut protein and egg white protein were used as raw materials to prepare peanut-egg white coprecipitated protein by alkali dissolution and isoelectric point precipitation method.

2. The method for preparing peanut-egg white co-precipitated protein according to claim 1, characterized in that, The mass ratio of peanut protein to egg white protein is 6:1 to 2:

1.

3. The method for preparing peanut-egg white co-precipitated protein according to claim 1, characterized in that, The mass ratio of peanut protein to egg white protein is 2:

1.

4. The method for preparing peanut-egg white co-precipitated protein according to claim 1, characterized in that, The peanut protein has a protein content of 45%, and the egg white protein has a protein content of 80.2%.

5. The method for preparing peanut-egg white co-precipitated protein according to claim 1, characterized in that, include: Step 1: Dissolve the mixture of peanut protein and egg white protein under alkaline conditions; Step 2: Then, in a water bath at 50°C, centrifuge to collect the supernatant, adjust the pH to acidic, and centrifuge to collect the protein precipitate; Step 3: Disperse the protein precipitate in distilled water, adjust the pH to 7.0, and freeze-dry to obtain protein powder.

6. The method for preparing peanut-egg white co-precipitated protein according to claim 5, characterized in that, In step 1, the alkaline conditions refer to a pH value of 10 to 12.

7. The method for preparing peanut-egg white co-precipitated protein according to claim 5, characterized in that, In step 2, the water bath time is 30 to 90 minutes.

8. The method for preparing peanut-egg white co-precipitated protein according to claim 5, characterized in that, In step 2, the acidic conditions refer to conditions with a pH value of 4 to 5.

5.

9. The method for preparing peanut-egg white co-precipitated protein according to claim 5, characterized in that, In step 2, water is added and the mixture is allowed to stand and settle naturally before centrifugation to collect the protein precipitate.

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