Method for extracting peanut meal protein by using natural eutectic solvent
By combining natural eutectic solvents with peanut meal, and using magnetic stirring, heating and centrifugation, the problems of long time and low protein quality in traditional methods are solved, and high-purity peanut protein is achieved. It maintains its good nutritional properties and emulsification properties.
Patent Information
- Application Number
- CN202411244372.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-06
- Publication Date
- 2024-12-13
AI Technical Summary
Traditional methods extract peanut meal protein for a long time and low protein quality, resulting in poor nutritional content loss and emulsification performance.
Using natural eutectic solvents (prepared from mixing choline chloride and glycerol or choline chloride with xylitol) with peanut meal, magnetic stirring, heating and stirring, cooling, centrifugation and ethanol washing, etc., to obtain high quality by freeze-drying Purity peanut protein.
It significantly improves the extraction rate and purity of peanut meal protein, maintains the nutritional composition and emulsification performance of the protein, and provides a more effective peanut protein extraction method.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention relates to a method for extracting peanut meal protein with a natural low eutectic solvent, belonging to the technical field of peanut processing. Background Art
[0002] Peanut meal contains a variety of sugars, flavonoids and protein compounds as well as essential elements for the human body such as potassium, calcium, iron, sodium, zinc, and magnesium. It has high nutritional value, but most peanut meal resources are not fully utilized at present. Peanut meal contains 40% to 50% peanut protein, which has good emulsification, water retention and foaming ability. In addition, compared with soy protein, peanut protein is easier to absorb when the activity and functional properties are similar. Peanut protein is rich in amino acids, of which the essential amino acids for the human body are as high as 38.29%, and the umami amino acids in the non-essential amino acids are relatively high, so it can be used to develop flavored foods.
[0003] The commonly used methods for extracting peanut protein from peanut meal are mainly alkali dissolution and acid precipitation and alcohol washing. The alkali dissolution and acid precipitation method is simple to operate, but the extraction time is long, and it will produce wastewater, which may cause environmental pollution, and the acid-base treatment will cause a certain degree of damage to the structure and properties of the protein; the alcohol washing method has a simple process and ethanol is recyclable, so the extraction cost is low, it is suitable for large-scale production, and will not cause environmental pollution, but the extraction time is long, and ethanol treatment will have an adverse effect on the properties of the protein, reducing the protein's potency nitrogen solubility index and nitrogen solubility index.
[0004] Deep eutectic solvent (DES) is a homogeneous liquid mixture formed by hydrogen bond donors (HBD) and hydrogen bond acceptors (HBA) at a specific molar ratio through intermolecular hydrogen bonds. Natural deep eutectic solvent (NADES) is a subclass of DES. Because its HBA and HBD are both non-toxic and harmless natural derivative compounds, it is called the third solvent in living cells. NADES is simple to prepare and has good biodegradability, low cytotoxicity and reusability. In terms of physicochemical properties, NADE is not volatile, viscosity and polarity can be adjusted, and it has good solubility for a variety of compounds. In addition, due to the "designability" of NADES, it can be freely combined to adapt to different applications by selecting different HBA and HBD. Therefore, the use of NADES to extract natural biological compounds from various natural biological components has an excellent development prospect in the extraction of natural biological compounds, which has great theoretical and practical significance for the extraction of peanut meal protein in the future, and provides ideas for the processing of peanut protein in my country. Summary of the invention
[0005] The purpose of the present invention is to provide a method for extracting peanut meal protein using a natural low eutectic solvent, so as to solve the problem that the traditional method of extracting peanut meal protein has a long extraction time and low protein quality.
[0006] To achieve the above object, the present invention adopts the following technical scheme: a method for extracting peanut meal protein with a natural deep eutectic solvent, characterized in that it comprises the following steps:
[0007] 1) Mixing choline chloride and glycerol is recorded as CG or mixing choline chloride and xylitol is recorded as CX;
[0008] 2) subjecting the CG or CX obtained in step 1) to magnetic stirring and cooling to room temperature; diluting with water to obtain a natural low eutectic solvent CG or a natural low eutectic solvent CX;
[0009] 3) mixing the natural deep eutectic solvent obtained in step 2) and peanut meal in proportion;
[0010] 4) heating and stirring the peanut meal solvent mixture obtained in step 3);
[0011] 5) cooling the peanut meal solvent mixture obtained in step 4) to room temperature;
[0012] 6) centrifuging the peanut meal solvent mixture obtained in step 5);
[0013] 7) adding ethanol to the supernatant obtained in step 6) for regeneration;
[0014] 8) centrifuging the solution obtained in step 7);
[0015] 9) washing the precipitate obtained in step 8) with ethanol;
[0016] 10) freeze-drying the precipitate obtained in step 9).
[0017] The above-mentioned method for extracting peanut meal protein with a natural low eutectic solvent is characterized in that, in step 2), the conditions for magnetic stirring are: temperature of 80° C., rotation speed of 500 r / min, and time of 40 min.
[0018] The above-mentioned method for extracting peanut meal protein with a natural low eutectic solvent is characterized in that, in step 2), water is added to dilute it to a moisture content of 20%.
[0019] The above-mentioned method for extracting peanut meal protein with a natural low eutectic solvent is characterized in that, in step 3), the solid-liquid ratio is 1g:15g.
[0020] The above-mentioned method for extracting peanut meal protein with a natural low eutectic solvent is characterized in that, in step 4), the conditions for heating and stirring are: temperature is 60° C., rotation speed is 500 r / min, and time is 120 min.
[0021] The above-mentioned method for extracting peanut meal protein with a natural low eutectic solvent is characterized in that, in step 6), the centrifugal conditions are: temperature is 25° C., rotation speed is 5000 r / min, and time is 10 min.
[0022] The above-mentioned method for extracting peanut meal protein with a natural low eutectic solvent is characterized in that, in step 8), the centrifugal conditions are: temperature is 4° C., rotation speed is 10000 r / min, and time is 30 min.
[0023] The peanut meal protein extracted by the above method can maintain the nutritional components of peanut meal protein to a large extent, and has better nutritional characteristics and emulsifying properties.
[0024] The beneficial effects of the present invention are as follows: a method for extracting peanut meal protein with a natural deep eutectic solvent is demonstrated, wherein cold-pressed peanut meal is used as a raw material, and a natural deep eutectic solvent is added for extraction, that is, choline chloride is used as a hydrogen bond acceptor, and glycerol and xylitol are respectively hydrogen bond donors. Through this method, the problems of more nutrient loss and long extraction time in the traditional extraction method of peanut meal protein can be solved to a certain extent, the nutrient components of peanut meal protein can be maintained to a large extent, and the extracted peanut meal protein has better nutritional characteristics and emulsifying properties, which provides a new idea for the extraction of peanut meal protein in my country. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 SDS-PAGE image of peanut protein.
[0026] Figure 2 Fourier transform infrared spectrum of peanut protein.
[0027] Figure 3 UV-visible absorption spectrum of peanut protein.
[0028] Figure 4 Fluorescence emission spectrum of peanut protein. DETAILED DESCRIPTION
[0029] Example 1 Method for extracting peanut meal protein using a natural deep eutectic solvent
[0030] (I) Preparation of natural deep eutectic solvents
[0031] 1) 25.87 g of choline chloride and 34.13 g of glycerol were mixed, which was recorded as CG; 28.71 g of choline chloride and 31.29 g of xylitol were mixed, which was recorded as CX;
[0032] 2) the two groups of mixtures obtained in step 1) were subjected to magnetic stirring at 80° C. and a speed of 500 r / min for 40 min;
[0033] 3) cooling the two groups of mixtures obtained in step 2) to room temperature;
[0034] 4) The two groups of mixtures obtained in step 3) are diluted with water to reach a water content of 20%, thereby obtaining natural deep eutectic solvent CG and natural deep eutectic solvent CX.
[0035] (II) Peanut protein extraction
[0036] 5) mixing the peanut meal obtained in step 4) and the natural deep eutectic solvent in a ratio of 1:15 (g / g) and recording them as CG or CX;
[0037] The peanut meal and deionized water were mixed in a ratio of 1:12 (g / g) and recorded as the alkali-soluble and acid-precipitated group;
[0038] 6) heating and stirring the peanut meal solvent mixture of CG or CX obtained in step 5) at 60° C. and 500 r / min for 120 min; adjusting the pH of the alkali-dissolved and acid-precipitated mixture to 9.5 with 1.0 mol / L NaOH solution;
[0039] 7) cooling the CG or CX peanut meal solvent mixture obtained in step 6) to room temperature; heating the alkali-soluble and acid-precipitated mixture at 60° C. for 2 h;
[0040] 8) The CG or CX peanut meal solvent mixture obtained in step 7) was centrifuged at 5000 r / min for 10 min at 25° C.; the alkali-soluble and acid-precipitated mixture was centrifuged at 5000 r / min for 30 min;
[0041] 9) adding ethanol to the CG or CX supernatant obtained in step 8) for regeneration; the supernatant of the alkali-dissolved and acid-precipitated group is used
[0042] 1.0 mol / L HCl solution adjusted pH to 4.5;
[0043] 10) centrifuging the solution obtained in step 9) at 10000 r / min for 30 min at 4°C;
[0044] 11) washing the precipitate obtained in step 10) with ethanol;
[0045] 12) freeze-drying the precipitate obtained in step 11).
[0046] (III) Protein content determination
[0047] The protein content was determined by the Coomassie brilliant blue method, and a protein standard curve was drawn. The absorbance values of the samples were determined and substituted into the regression equation to calculate the peanut protein content. The results are shown in Table 1. The extraction rates of natural deep eutectic solvent CG and natural deep eutectic solvent CX were 46.18% and 45.51%, respectively, and the protein purities were as high as 73.02% and 74.95%, respectively, which were significantly higher than 37.94% and 44.68% of the alkali dissolution and acid precipitation group (A1). This shows that the NADES extraction method has not only a higher extraction rate than the traditional alkali dissolution and acid precipitation method, but also a higher protein purity of the crude protein obtained.
[0048] Table 1 Peanut protein extraction rate and yield
[0049]
[0050] Note: The results are expressed as mean ± standard deviation. Different letters in the same column indicate significant differences among the groups (P < 0.05).
[0051] (IV) Physical and chemical properties of peanut protein
[0052] (1) SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
[0053] Gel Doc XR+ gel imaging was used to evaluate the protein content of peanut meal in different treatment groups. Figure 1 As shown in the figure, the approximate molecular weight of the sample protein subunits can be obtained by calculation. For the electrophoretic band on the left without the addition of β-mercaptoethanol, it is speculated from the literature that the high molecular weight subunit 65.2kDa is conarachidin II, the low molecular weight subunits 34.5, 30.2 and 23.6kDa are conarachidin, and 19.3 and 14.5kDa are conarachidin I. β-mercaptoethanol can reduce the disulfide bonds in protein molecules, so in the presence of β-mercaptoethanol, the polypeptide chains connected by disulfide bonds will dissociate into single peptide chains. Compared with the left side, it can be seen that the electrophoretic bands on the right side after adding β-mercaptoethanol are significantly increased, indicating that the sample protein contains intermolecular disulfide bonds. Regardless of whether β-mercaptoethanol is added, there is no obvious difference in the bands of the three groups of protein samples, but it can be seen that the bands of the crude protein sample extracted using NADES are darker in color, indicating that the protein content in the sample is higher.
[0054] (2) Fourier transform infrared spectroscopy (FT-IR)
[0055] The results of the FTIR-8400S infrared spectrometer are as follows: Figure 2 As shown, it can be seen that at 1000cm -1The infrared spectra of the proteins in each group were basically the same. The characteristic absorption peaks differed only in peak height, and there was almost no change in peak shift and peak shape, indicating that the various extraction methods had almost the same effect on the secondary structure of the protein. -1 The difference in the subsequent absorption peaks may be the result of the residual NADES.
[0056] (2) UV-visible absorption spectrum
[0057] The conformational changes of proteins were determined using a UV-2550 ultraviolet-visible spectrophotometer. Figure 3 The peak positions and absorbances of UV-visible absorption spectra of peanut protein in CG and CX groups are almost the same, and the peak positions of UV-visible absorption spectra of peanut protein in Al group are also the same as those of the first two, but the absorbance is higher than that of the first two, which indicates that the content of aromatic amino acids on the surface of the protein is higher. The acid-base treatment during the alkaline extraction process will unfold the protein molecular structure, exposing the tryptophan and tyrosine residues buried inside the protein molecules. The NADES extraction process does not change the structure of the protein molecule, so there will be differences in absorbance.
[0058] (2) Fluorescence emission spectrum measurement
[0059] The results of the Cary Eclipse fluorescence spectrophotometer are as follows Figure 4 As shown in the figure, there is no obvious difference in the fluorescence emission spectra of peanut proteins in the CG group and the CX group, while the peanut protein in the Al group undergoes a red shift and the fluorescence intensity also increases, indicating that the protein molecules unfold after acid-base treatment and the tyrosine residues inside the molecules are exposed, which is consistent with the results of the UV-visible absorption spectrum.
Claims
1. A method for extracting peanut meal protein using a natural deep eutectic solvent, characterized in that: The steps include: 1) Mixing choline chloride and glycerol is recorded as CG or mixing choline chloride and xylitol is recorded as CX; 2) subjecting the CG or CX obtained in step 1) to magnetic stirring and cooling to room temperature; diluting with water to obtain a natural low eutectic solvent CG or a natural low eutectic solvent CX; 3) mixing the natural deep eutectic solvent obtained in step 2) and peanut meal in proportion; 4) heating and stirring the peanut meal solvent mixture obtained in step 3); 5) cooling the peanut meal solvent mixture obtained in step 4) to room temperature; 6) centrifuging the peanut meal solvent mixture obtained in step 5); 7) adding ethanol to the supernatant obtained in step 6) for regeneration; 8) centrifuging the solution obtained in step 7); 9) washing the precipitate obtained in step 8) with ethanol; 10) freeze-drying the precipitate obtained in step 9).
2. The method for extracting peanut meal protein using a natural deep eutectic solvent according to claim 1, characterized in that: Step 2), the conditions for magnetic stirring are: temperature of 80°C, rotation speed of 500r / min, and time of 40min.
3. The method for extracting peanut meal protein using a natural deep eutectic solvent according to claim 1, characterized in that: Step 2), dilute with water to a moisture content of 20%.
4. The method for extracting peanut meal protein using a natural deep eutectic solvent according to claim 1, characterized in that: Step 3), the material-liquid ratio is 1g:15g.
5. The method for extracting peanut meal protein using a natural deep eutectic solvent according to claim 1, characterized in that: Step 4), the conditions for heating and stirring are: temperature is 60°C, rotation speed is 500r / min, and time is 120min.
6. The method for extracting peanut meal protein using a natural deep eutectic solvent according to claim 1, characterized in that: Step 6), the centrifugal conditions are: temperature is 25°C, rotation speed is 5000r / min, and time is 10min.
7. The method for extracting peanut meal protein using a natural deep eutectic solvent according to claim 1, characterized in that: In step 8), the centrifugal conditions are: temperature is 4°C, rotation speed is 10000r / min, and time is 30min.