Extraction method of oat bran protein

Abstract

The invention relates to an extraction method of oat bran protein, which comprises the following steps: mixing HBD and HBA according to different molar ratios, and carrying out magnetic stirring until a clear and transparent liquid is formed; the preparation method comprises the following steps: cleaning oat with deionized water, drying at 40 DEG C until the water content is 10%, crushing, sieving with a 60-mesh sieve, and taking an oversize product of the 60-mesh sieve as oat bran; the preparation method comprises the following steps: mixing oat bran and n-hexane (g / ml) according to a ratio of 1: 5, magnetically stirring for 3 hours at room temperature at 600 rpm, centrifuging for 10 minutes at 4000 rpm, degreasing for 3 times, and naturally volatilizing and airing to obtain degreased oat bran; mixing the degreased oat bran and the DES solution in a vortex manner according to a solid-to-liquid ratio of 1: 10, stirring for 60 minutes at the temperature of 50 DEG C and at the speed of 600rpm, cooling to room temperature, and centrifuging for 10 minutes at the speed of 3500rpm; taking the supernatant, extracting the precipitate twice by using the fresh DES in the same extraction process, merging the obtained supernatant, filling the merged supernatant into a dialysis bag, dialyzing at the temperature of 3,500 D4 DEG C for about 3 days by molecular weight closure, and freeze-drying the obtained suspension to obtain crude protein powder.

Description

Technical Field

[0001] The invention belongs to the field of extraction of cereal processing by-products, and particularly relates to a method for extracting oat bran protein. Background Art

[0002] According to United Nations statistics, the world population reached 8 billion in 2022 and is expected to reach about 9.7 billion by 2050. The demand for food is expected to grow by 30-50% in the next 30 years. Due to the concerns about global population growth, climate change, environmental pollution and animal welfare, healthy and sustainable development are required, and eating habits are gradually changing. More and more people choose foods rich in plant-based protein and reduce the demand for animal-based products. Plant protein is becoming more and more popular. It is reported that the production of traditional animal proteins such as meat, seafood, eggs and milk has a negative impact on climate change, such as increased greenhouse gas emissions, land desertification, severe pollution and loss of biodiversity. It is reported that excessive intake of animal-based protein will have adverse effects on human health, such as causing high blood pressure, cardiovascular disease, cancer, etc. Therefore, people have been looking for affordable, sustainable and healthy sources of plant-based protein to replace animal protein in the human diet.

[0003] Plant protein comes from a wide range of sources, including almonds, nuts, green leaves, cereals, beans, and plant seeds. About 160.3 million tons of grain bran are produced each year worldwide, which is a source of high-quality protein, thereby converting this waste into a functional ingredient with nutritional value. Among them, oats are annual grass plants, generally including hulled oats (Avena sativa L.) and naked oats (Avena nuda L.), belonging to the genus Avena of the Poaceae family. Oats have the characteristics of strong stress resistance, high nutritional value, and good processing performance, and have been widely planted around the world. According to the 2023 statistics of the Food and Agriculture Organization database (FAO), the global oat planting area is 95.08 million hectares and the output is 18.16 million tons, an increase of 4.5% and 11.1% respectively compared with 2022. Hulled oats are mainly used to feed livestock and poultry, and only a small part is processed into oatmeal for consumption, while all naked oat kernels are edible. A large amount of by-product bran is produced during the processing of oats, accounting for about 30-50% of the dry weight of oats. The protein content in oat bran is 14-20%, mainly including four types: globulin (50-80%), albumin (1-12%), prolamin (4-15%) and glutenin (<10%).

[0004] Alkali extraction and acid precipitation is a common and traditional method for protein extraction. This method is easy to operate for protein extraction, does not produce harmful substances, has little pollution to the environment, is low in cost, and is suitable for industrial production. However, high concentrations of alkali will change the nutritional properties of proteins, and high concentrations of alkali will also promote the Maillard reaction between carbohydrates and proteins. Deep eutectic solvents (DES) have recently been considered as environmentally friendly extraction solvents. They are mixed in a specific molar ratio and heated and stirred to reduce the melting point, forming hydrogen bonds from solid to liquid. They are usually composed of choline chloride as a hydrogen bond donor (HBD) and glycerol, diols, urea, etc. as hydrogen bond acceptors (HBA). The extraction and separation of organic substances are achieved through the interaction between hydrogen bonds and coordination components in the system. DES is easy to prepare, and is green, biodegradable, non-volatile, non-toxic, and highly stable. It is safer than traditional extraction solvents with concentrated alkali. In addition, it is not only used to extract proteins from plants and animals, but also enzymes and some essential amino acids. Therefore, establishing an efficient and green method for DES to extract protein from oat by-product bran is very necessary to supplement plant protein sources. Summary of the invention

[0005] In order to solve the problems existing in the above-mentioned prior art, the object of the present invention is to provide a method for extracting oat bran protein, which is suitable for a DES extraction solvent for oat bran. Based on DES extraction, single factor optimization and response surface optimization are used to explore the optimal extraction conditions. By comparing the physicochemical properties of proteins extracted by two different extraction methods, an extraction method suitable for oat bran protein is screened.

[0006] To achieve the above object, the technical solution of the present invention is:

[0007] A method for extracting oat bran protein comprises the following steps:

[0008] Step 1: Preparation of 15 types of DES

[0009] Choline chloride was selected as HBA; HBD and HBA were mixed in different molar ratios as follows:

[0010]

[0011] Incubate at 80°C with magnetic stirring at 600 rpm until a clear liquid is formed; after cooling to room temperature, the liquid still does not solidify, indicating the formation of DES; to enhance the formation of DES and reduce the viscosity, 20% water (w / w) is added to DES to reduce the viscosity; DESs are stored in sealed glass bottles at room temperature until use;

[0012] Step 2: DES extraction of oat bran protein:

[0013] (1) The oats are cleaned with deionized water to remove dust and impurities on the surface, dried at 40°C to a moisture content of 10%, and ground in a grinder. The ground powder is passed through a 60-mesh sieve, and the material on the 60-mesh sieve is the oat bran.

[0014] (2) Oat bran: n-hexane (g / ml) 1:5 mixture, magnetic stirring at 600 rpm for 3 h at room temperature, centrifugation at 4000 rpm for 10 min, defatting was repeated 3 times, and naturally evaporated and air-dried to obtain defatted oat bran.

[0015] (3) Defatted oat bran and DES solution were vortex mixed at a solid-liquid ratio of 1:10, stirred at 600 rpm for 60 min at 50°C, cooled to room temperature and centrifuged at 3500 rpm for 10 min. The supernatant was taken and the precipitate was extracted twice with fresh DES using the same extraction process. The obtained supernatants were combined and placed in a dialysis bag with a molecular weight cutoff of 3500. The suspension was dialyzed at 4°C for about 3 days. The obtained suspension was freeze-dried to obtain crude protein powder.

[0016] Furthermore, in the step 1, choline chloride is used as a hydrogen bond acceptor, glycerol, 1,4-butanediol, 2,3-butanediol, 1,2-butanediol, and urea are used as hydrogen bond donors, the molar ratios are 1:1, 1:2, and 1:3, respectively, and the water content is 20%, to prepare DES.

[0017] Furthermore, in the step 1, formulas DES3, DES7 and DES14 are selected to extract oat bran protein.

[0018] Furthermore, in the step 1, the DES7 formula is selected to extract oat bran protein.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The new process improves the extraction rate and the technical functions and nutritional characteristics of the protein, shortens the protein extraction time, and improves the protein extraction rate. It overcomes the disadvantages of the traditional extraction method: large solvent loss, long extraction time, decreased protein quality, low recovery rate and the like. Oat bran protein is more difficult to extract than oat flour protein, and a higher extraction rate can only be obtained under strong alkaline conditions. However, alkaline conditions may cause adverse reactions, resulting in the loss of protein nutritional value and the formation of potential toxic substances such as lysine and alanine. 2. The DES solution used in this technology can be recycled and reused, and is safe and non-toxic, with high cost performance and environmental friendliness. In addition, the DES solution is a solvent that has been screened for the extraction of oat bran protein. 3. The protein purity of DES extracted protein is as high as 81%, and the appearance is milky white powder. The overall water solubility is higher than that of alkali extraction, the average particle size is reduced by more than half, the globulin structure remains intact, and the gelling properties are enhanced. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the extraction rate of different DES proteins.

[0021] Figure 2 SDS-PAGE electrophoresis and solubility of proteins extracted from different DES (A), total protein content (B), water-soluble proteins (C, E) and salt-soluble proteins (D, F).

[0022] Figure 3 Scanning electron microscopy (A) and particle size distribution (B) of proteins extracted from different DES.

[0023] Figure 4 FT-IR spectra (A) and secondary structure analysis (B) of proteins extracted for different DES.

[0024] Figure 5 To characterize the viscosity and rheological properties of proteins extracted by different DES.

[0025] Figure 6 These are the single factor optimization results of extraction temperature (A and B), solid-liquid ratio (C), and extraction time (D).

[0026] Figure 7 3D surface plot and contour plot for response surface optimization results. Extraction temperature and solid-liquid ratio (A and D), solid-liquid ratio and extraction time (B and E), extraction temperature and extraction time (C and F). DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments:

[0028] like Figure 1-7 As shown, a method for extracting oat bran protein,

[0029] As shown in Table 1, 15 different DES compositions, with choline chloride as hydrogen bond acceptor, glycerol, 1,4-butanediol, 2,3-butanediol, 1,2-butanediol, urea as hydrogen bond donors, the molar ratios were 1:1, 1:2, 1:3, respectively, and the water content was 20%. It was found that DES1-14 could be stably formed, but DES15 could not be formed. The extraction rates of 14 different DES proteins were screened, and it was found that DES3, DES7, and DES14 had higher extraction rates, and they could represent different hydrogen bond donors ( Figure 1 ).

[0030] Table 1: DES composition

[0031]

[0032]

[0033] Weigh 5g of defatted oat bran, add 95g of DES3, 7, and 14 respectively, mix in a conical flask, and then incubate in a water bath shaker at 50°C and 200rpm for 1h. After cooling to room temperature, centrifuge at 4000rpm for 10min. Transfer the supernatant to a beaker, extract the precipitate twice with fresh DES under the same conditions, and combine the obtained supernatants. Pour the supernatant into a dialysis membrane with a pore size of 3.5kDa molecular weight cutoff (MWCO) and dialyze, change the water every 4 hours, and dialyze at 4°C for 5 days to ensure the removal of the solvent. The resulting protein solution was freeze-dried to obtain DES-extracted oat bran protein (DES3, DES7, DES14).

[0034] Extraction of oat bran protein by alkali dissolution and acid precipitation. Mix defatted oat bran with deionized water (g / ml) at 1:20, adjust pH to 9.0 with 1M NaOH solution, stir with a magnetic stirrer at room temperature for 1h, speed 300rpm, and keep pH = 9.0 during this period to obtain maximum extraction. Centrifuge at 4000rpm for 10min and take the supernatant. Stir the supernatant with a magnetic stirrer at a speed of 100rpm, adjust pH to 5 with 1M hydrochloric acid, and place in a 4℃ refrigerator for precipitation overnight. Centrifuge at 10,000rpm for 20min, pour off the supernatant, disperse the protein precipitate in deionized water, adjust pH to 7.0, pour into a dialysis membrane with a pore size of 3.5kDa molecular weight cutoff (MWCO) for dialyzing, change water every 4 hours, and dialyze at 4℃ for 5 days to ensure the removal of salt ions. Freeze-dry to obtain alkali-soluble acid-precipitated oat bran protein powder (JRSC). The above four kinds of dried protein samples were ground into fine powder using a mortar and pestle and stored in a refrigerator at 4°C until testing.

[0035] The properties of DES solution will affect the protein extraction rate. The characterization results of the properties of 14 different DES solutions are shown in Table 2. The density of the 14 different DES is greater than that of water, among which DES1-3 in the glycerol group and DES13-14 in the urea group are larger, and the isomer DES4-12 of butanediol is smaller. The pH values ​​of the 14 different DES are quite different, with the highest pH of DES13-14 in the urea group at around 8.4, the lowest pH of DES1-3 in the glycerol group at around 3.4, and the isomer DES4-12 of butanediol in the middle, 5.3-5.5, 3.6-3.8. The conductivity results show that as the molar ratio increases, the conductivity decreases, and the urea group DES13-14 has the highest pH, reaching 18.56-19.80μs / cm. The viscosity results of the 14 different DES show little difference.

[0036] Table 2: Physicochemical properties of 14 synthesized deep eutectic solvents

[0037]

[0038] Table 3: Oat bran protein particle size distribution

[0039]

[0040] The total protein content of the extracted 4 protein sample powders was determined by the Kjeldahl method. Weigh 0.5-2g of protein content, put it into the digestion tube, add 0.3g of copper sulfate, 6g of potassium sulfate, and 12mL of concentrated sulfuric acid, and make a blank control at the same time. Put the digestion tube into the digestion furnace, heat it slowly at the beginning, and raise the temperature of the digestion furnace after the bubbling stops and the contents are uniform, and keep the liquid surface slightly boiling. When the solution is blue-green and transparent, continue to heat for 0.5-1h. After cooling, distillation and absorption are carried out. The digestion tube is fixed on the Kjeldahl nitrogen analyzer, and the receiver is prepared (add 1-2 drops of indicator). The determination conditions are: dilution water volume 10mL, boric acid volume 25mL, alkali volume 50mL, distillation time 5min, elution water volume 10mL. The distillate is titrated with a hydrochloric acid standard solution (0.1mol / L), and the color changes from blue-green to slightly red, which is the end point. The results show that the total protein content of DES3 extracted protein is the highest, which is 80.19%. JRSC and DES7 were 70.40% and 72.03% respectively, while DES14 was the lowest at 55.30%.

[0041] The water solubility and salt solubility of proteins were extracted by JRSC, DES3, DES7 and DES14, and SDS-PAGE gel electrophoresis was performed. It was found that DES3 can extract water-soluble proteins. For salt-soluble proteins, DES3 has the highest salt solubility; the molecular weights of JRSC and DES3 proteins are similar. The surface microstructure of the four protein samples was observed by scanning electron microscopy, and SEM photos were taken at an accelerating voltage of 10kV and magnifications of 5000 and 20,000. Before observation, the samples were glued with conductive glue and gold was sputtered on the samples for 120 seconds using an ion sputtering coating machine. The results are as follows: Figure 3 As shown in A, the JRSC protein structure undergoes microstructural changes due to pH changes, while the DES3 and DES7 protein globulin structures are intact, and DES14 is partially intact. The particle size of the protein is closely related to the functional properties of the protein. The particle size distribution and aggregation of the protein are represented by various parameters of the particle size determination, among which D 4,3 Ability to monitor changes in aggregates, D 3,2 Represents the size of most particles. The particle size results of 4 different oat bran proteins are as follows Figure 3 As shown in Figure B and Table 3, the particle size of proteins extracted by JRSC is significantly larger than that of proteins extracted by low eutectic solvents DES3, DES7 and DES14. It is worth noting that proteins extracted by DES3 have a small amount of distribution at 10 μm. This is closely related to the strong water solubility of proteins extracted by DES3.

[0042] Fourier transform mid-infrared spectroscopy (400~4000cm -1) is recognized as a powerful tool for analyzing food chemical composition and is widely used in the determination of protein secondary structure. FTIR spectra of proteins extracted from JRSC, DES3, DES7 and DES14 are shown in Figure 2. Figure 4 As shown, 1660-1656cm -1 Corresponding to the vibration of amide I, 1542-1534 cm -1 The absorption peaks of JRSC and DES14 proteins at amide I and amide II were significantly weakened, but no significant shift occurred. The characteristic peaks in amide I are mainly caused by C=O and NH stretching vibrations, while amide II can indicate that the intermolecular or intramolecular hydrogen bonds are broken. -1 The absorption peak near the hydroxyl group (-OH) corresponds to the absorption peak of the hydroxyl group, which represents the strength of the intermolecular hydrogen bond in the sample. Compared with DES3 and DES9, the absorption peaks of JRSC and DES14 proteins are significantly weakened, and the absorption peaks shift to the short-wave infrared region, indicating that the strength of the intermolecular hydrogen bond in the protein is weakened. Since both DES14 and JRSC are extracted with alkaline solutions, it indicates that extraction under conditions of pH>8 will change the properties of the protein.

[0043] The secondary structure of a protein is a local folded structure that is usually determined by the interaction between the amino acid sequence and the main chain atoms; the secondary structure of a protein is determined by the ratio of α-helix, β-sheet, β-turn, and random coil. -1 ) peak area in each region, the peptide bond in the α-helix (1650-1660cm -1 ), β-folding (1600~1640cm -1 ), β-angle (1661~1700cm -1 ) and irregular curl (1640~1650cm -1 ) to study the folding, unfolding and aggregation of proteins. The secondary contents of the four oat bran proteins are as follows Figure 4 B and Table 4 show that compared with JRSC, the β-fold content of DES3 and DES7 is reduced and the α-helix content is increased. It has been reported in the literature that the β-fold content in the protein structure is negatively correlated with the water solubility and digestibility of the protein. The β-fold content in DES3 and DES7 proteins is reduced, indicating that the protein extracted by DES has higher water solubility and better digestibility. The increase in α-helix content means the contraction of protein molecules, which is consistent with the results of scanning electron microscopy, and the globulin structure is relatively complete. The β-turn has a higher content in the protein extracted by DES, indicating that the protein tends to be disordered.

[0044]

[0045] Table 4: Secondary structure content of oat bran protein

[0046] Viscosity and rheology of proteins extracted from JRSC, DES3, DES7 and DES14 Figure 5 As shown, in terms of viscosity, DES7 protein has the largest viscosity, followed by DES3, JRSC and DES14. The storage modulus G' represents the elastic property, and the loss modulus G" represents the viscosity property. The rheological properties of oat bran protein, both the storage modulus and the loss modulus, increase with increasing frequency, and the modulus is highly dependent on frequency. The loss coefficient = G" / G', and the G" of oat protein is significantly greater than G', showing viscoelastic solid properties.

[0047] Based on the above results, DES7 solution was selected as the extraction solvent to perform single factor optimization of the effects of extraction temperature, solid-liquid ratio, and extraction time on the extraction rate of oat bran protein. First, the solid-liquid ratio of 1:20 and the extraction time of 1h were selected to screen the effects of different extraction temperatures on the protein extraction rate. Low temperature gradient: room temperature (RT), 30℃, 40℃, 50℃, 60℃ and higher temperature gradient: 50℃, 60℃, 70℃, 80℃, 90℃. At an extraction temperature of 50℃ and an extraction time of 1h, the effects of different solid-liquid ratios: 1:10, 1:15, 1:20, 1:30, and 1:40 on the extraction rate of oat bran protein were screened. Under the conditions of extraction temperature of 50℃ and solid-liquid ratio of 1:20, the effects of different extraction times: 30min, 60min, 120min, 180min, and 240min on the extraction rate of oat bran protein were screened. The results are as follows Figure 6 As shown, the maximum extraction rates were achieved at extraction temperature of 80°C, solid-liquid ratio of 1:30, and extraction time of 180 min.

[0048] In order to explore the effects of extraction temperature, solid-liquid ratio, and extraction time on the extraction rate of oat bran protein, a response surface optimization experiment was conducted to obtain the optimal extraction conditions with the maximum extraction rate as the response value. According to the single factor optimization results, a three-factor, three-level response surface optimization was performed for temperature, solid-liquid ratio, and extraction time, and the setting range is shown in Table 5.

[0049] Table 5: Response surface optimization extraction condition parameter settings

[0050]

[0051] Table 6: Response surface experimental design and results

[0052]

[0053] The data in Table 2 were fitted with a quadratic polynomial regression using Design-Expert 13.0 software, and the regression model equation of protein extraction rate on extraction temperature (A), solid-liquid ratio (B), and extraction time (C) was obtained as follows: Protein extraction rate (%) = 67.3602-4.28689A-0.519137B+2.28355C+0.38784AB+0.50114AC-0.935998BC-8.05372A 2 -2.52033B 2 -2.9288C 2 The established quadratic regression model was subjected to variance analysis (Table 3). The results showed that the regression model had significant differences, the lack-of-fit test was not significant, and R 2 =0.9955, AdjR 2 =0.9898, indicating that the equation fits the experiment well and can also better reflect the relationship between each factor and the response value. It is feasible to use this equation model to analyze and predict the process parameters of oat bran protein extraction.

[0054] Table 7: Analysis of variance of regression equation

[0055]

[0056] Note: P value < 0.05 indicates that the index is significant*; P value < 0.01 indicates that the index is extremely significant**

[0057] The response surface 3D diagram and contour map results of temperature A and material-liquid ratio B are as follows: Figure 7 As shown, when the material-liquid ratio is constant, with the increase of temperature, the protein extraction rate first increases and then decreases. When the temperature is constant, the change of protein extraction rate shows a relatively gentle trend. The interaction between AB is significant. The response surface 3D map and contour map results of material-liquid ratio B and extraction time C show that when the material-liquid ratio is constant, with the extension of extraction time, the protein extraction rate gradually increases. When the extraction time is constant, with the increase of material-liquid ratio, the protein extraction rate first increases slowly and then decreases slowly. The interaction between BC is significant. The response surface 3D map and contour map results of temperature A and extraction time C show that when the temperature is constant, with the extension of time, the protein extraction rate gradually increases. When the extraction time is constant, with the increase of temperature, the protein extraction rate first increases and then decreases. The interaction between AC is significant. The optimal extraction conditions for extracting oat bran protein by DES are as follows: extraction temperature: 78.10℃, material-liquid ratio: 1:22.52, extraction time: 1.71h, and the predicted value of protein extraction rate is: 68.29%. Taking into account the actual operating conditions, the final extraction conditions were determined as follows: extraction temperature: 78°C, solid-liquid ratio: 1:23, extraction time: 1.7h, and the actual value of protein extraction rate was: 63.06%±2.95. The relative error with the theoretical value was 3.69%.

[0058] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that are not conceived through creative work should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope defined in the claims.

Claims

1. A method for extracting oat bran protein, characterized in that, It consists of the following steps: Step 1: Preparation of 15 types of DES Choline chloride was selected as HBA; HBD and HBA were mixed in different molar ratios as follows: Incubate at 80°C with magnetic stirring at 600 rpm until a clear liquid is formed; after cooling to room temperature, the liquid still does not solidify, indicating the formation of DES; to enhance the formation of DES and reduce the viscosity, 20% water (w / w) is added to DES to reduce the viscosity; DESs are stored in sealed glass bottles at room temperature until use; Step 2: DES extraction of oat bran protein: (1) Wash oats with deionized water to remove dust and impurities on the surface, dry them at 40° C. to a moisture content of 10%, grind them in a grinder, pass the ground powder through a 60-mesh sieve, and take the oat bran on the 60-mesh sieve; (2) Oat bran: n-hexane (g / ml) 1:5 mixture, magnetic stirring at 600 rpm for 3 h at room temperature, centrifugation at 4000 rpm for 10 min, defatting repeated 3 times, natural evaporation and air drying to obtain defatted oat bran; (3) Defatted oat bran and DES solution were vortex mixed at a solid-liquid ratio of 1:10, stirred at 600 rpm for 60 min at 50°C, cooled to room temperature and centrifuged at 3500 rpm for 10 min; the supernatant was taken and the precipitate was extracted twice with fresh DES using the same extraction process. The obtained supernatants were combined and placed in a dialysis bag with a molecular weight cutoff of 3500D. Dialysis was performed at 4°C for about 3 days. The obtained suspension was freeze-dried to obtain a crude protein powder.

2. The method according to claim 1, characterized in that: In the step 1, choline chloride is used as a hydrogen bond acceptor, glycerol, 1,4-butanediol, 2,3-butanediol, 1,2-butanediol and urea are used as hydrogen bond donors, the molar ratios are 1:1, 1:2 and 1:3 respectively, and the water content is 20%, to prepare DES.

3. The method according to claim 2, characterized in that: In the step 1, DES3, DES7 and DES14 formulas are selected to extract oat bran protein.

4. The method according to claim 2, characterized in that: In the step 1, the DES7 formula is selected to extract oat bran protein.

Images