Optimal buffer solution extraction test method for optimizing germinated walnut seed protein by response surface method

Abstract

The invention discloses an optimal buffer solution extraction test method for optimizing germinated walnut seed protein by a response surface method.The method comprises the steps that after seeds germinate, the amino acid composition and content of the walnut seeds in different germination periods are measured, and the optimal germination time is determined; then measuring the protein content and the antioxidant activity; and optimizing the optimal extraction condition by using a response surface method. According to the method, the current situation of walnut protein resource utilization and bean and cereal seed germination application research are combined, the protein content and amino acid composition and content change of germinated walnut seeds are analyzed, and the optimal germination time node is determined. According to the method, germinated walnut kernels are used as materials, a response surface optimization protein extraction test is utilized, antioxidant activity evaluation is combined, and an extraction and separation process for the antioxidant protein of the germinated walnut kernels is constructed, so that antioxidant protein components with high additional value are prepared, and accurate preparation of specific functional protein of the germinated walnut kernels is facilitated; a process and a scheme are provided for high-value utilization of subsequent walnut protein resources in the fields of food, health care products, daily chemicals and the like.

Description

Technical Field

[0001] This invention relates to the field of walnut buffer extraction test technology, and in particular to a response surface methodology method for optimizing the optimal buffer extraction test for proteins in germinating walnut seeds. Background Technology

[0002] Germination allows for a high level of seed metabolism. During this process, substances stored in the seed are transformed and upgraded into highly active components that can be used for new life, thereby increasing the content of bioactive substances and potentially generating new highly active substances. For example, during soybean germination, the catalytic action of proteases transforms the poorly soluble macromolecules stored in the seed into soluble small molecules. This includes converting soybean protein, which is difficult for the human body to directly absorb, into easily absorbed amino acids or peptides, and converting polysaccharides that the human body cannot absorb into monosaccharides for absorption. These transformations all contribute to improving the absorption and utilization value of nutrients. Germinated seeds, including germinated brown rice, are rich in various physiologically active components, such as antioxidant peptides, functional proteins, γ-aminobutyric acid (GABA), and polysaccharides. Antioxidant peptides and functional proteins have the functions of scavenging free radicals and regulating immune function. GABA is an important neurotransmitter precursor with effects such as regulating nervous system function, lowering blood pressure, and improving sleep quality; active polysaccharides have multiple effects such as immunomodulation, anti-inflammation, and lowering blood sugar. Dietary fiber promotes intestinal peristalsis, improves the gut microbiota, and prevents digestive tract diseases such as constipation and colon cancer. Phosphatidylinositol and oryzanol possess various physiological activities, including regulating blood lipids, antioxidation, and anti-inflammation, which help reduce the incidence of cardiovascular and cerebrovascular diseases, prevent digestive tract cancers, and slow down the aging process. These components exhibit significant physiological activities and potential health benefits, playing an important role in maintaining human health. Therefore, the bioactive substances in germinated seeds have extremely high development and utilization value, providing a rich resource base for the development of novel functional raw materials in the fields of food, biomedicine, and health products.

[0003] Walnut kernels are rich in various nutrients, promoting brain health and delaying aging. The protein in walnuts is rich in active substances such as antioxidant peptides, blood pressure-lowering peptides, and antibacterial peptides. Consuming walnuts can help prevent cardiovascular and cerebrovascular diseases, cancer, Alzheimer's disease, and other chronic diseases. The fat content of walnut kernels is 60-65%, significantly higher than common oilseed crops such as soybeans, rapeseed, peanuts, and sesame, and its oil components have high utilization value. In addition to oil, walnut kernels are rich in high-quality protein, with a protein content as high as 14%-17%. This protein is mainly composed of gluten, globulin, albumin, and proteoprotein, accounting for 70.11%, 17.57%, 6.81%, and 5.33% of the total protein, respectively. Walnut protein contains 18 amino acids, 8 of which are essential for the human body, and the proportions of each amino acid are appropriate, giving it high nutritional value. In addition to its excellent nutritional performance, walnuts also have traditional medicinal effects such as nourishing the kidneys and blood, moistening the lungs and relieving asthma, and lubricating the intestines and promoting bowel movements. Meanwhile, the antioxidants in walnuts can reduce the level of free radicals in the human body, exerting an anti-aging effect and making them a high-quality natural skincare ingredient. Currently, the walnut industry still faces many shortcomings, with low levels of comprehensive utilization and functional component development. The walnut industry currently faces numerous bottlenecks and challenges. The walnut processing chain is relatively short, mainly concentrated in traditional edible areas such as the direct consumption of walnut kernels, extraction of walnut oil, and simple walnut product processing. Its comprehensive utilization and functional component development are still in their early stages, failing to fully explore its potential value. The short industrial chain leads to significant resource waste. Nevertheless, the development potential of walnut resources and their functional components is enormous, especially the development of functional proteins from germinated walnut seeds, which is currently a blank area in my country and requires further exploration and research.

[0004] Currently, research on germinated seed protein resources in my country mainly focuses on crops such as soybeans, peanuts, peas, and brown rice. These studies have made some progress in protein extraction processes, functional properties, and application development, laying a solid foundation for the functional utilization of plant proteins. However, research and development of germinated seed proteins from woody oil crops is still in its early stages. A comprehensive and systematic evaluation and development of the structural characteristics, functional activities, and application potential in food, medicine, and health products of these proteins has not yet been conducted, and in-depth and systematic research is urgently needed. Therefore, the development and utilization of germinated seed proteins from woody oil crops still faces many challenges. It is imperative to integrate multidisciplinary research and technological innovation to construct extraction processes for germinated seed proteins and deeply explore their functional properties, in order to promote the high-value utilization of proteins from woody oil crops such as walnuts, ginkgo, and camellia, and meet market demand for high-value-added protein products and health foods. Summary of the Invention

[0005] This invention, combining current walnut resource utilization with research results on the germination of legumes and cereals, analyzes the changes in amino acid content and composition during walnut seed germination to determine the optimal germination time. Using germinated walnut seeds as material, a protein extraction process is optimized using response surface methodology. By measuring protein yield and antioxidant activity, an extraction process for germinated walnut seed proteins with antioxidant activity is constructed, providing an innovative research scheme and strategy for producing high-value-added antioxidant protein components. The research results contribute to the development and utilization of high-value-added active proteins from germinated walnut seeds, providing an effective solution for enhancing the overall value of the walnut industry and extending its industrial chain. Furthermore, this extraction process and research scheme can be improved and adjusted for the preparation and development of functional proteins from germinated seeds of other woody oil crops, enhancing the overall industrial added value and economic benefits of woody oil crops, and providing a reference for constructing efficient and scalable antioxidant functional protein preparation processes.

[0006] The purpose of this invention is to address the shortcomings in existing technologies for the diversification and low-value utilization of walnut resources. This invention combines germinated walnut seeds with response surface methodology to optimize the optimal buffer extraction method for protein from germinated walnut seeds.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A response surface methodology method for optimizing the optimal buffer extraction assay for protein extraction from germinating walnut seeds includes the following steps:

[0009] S1: Seed germination;

[0010] S2: Determination of amino acid content and composition in walnut seeds sprouting at different germination stages;

[0011] S3: Determination of protein content and antioxidant activity;

[0012] S4: Optimization of optimal extraction experiment for germinating seed protein using response surface methodology;

[0013] Further, step S1 includes the following steps:

[0014] S11: Select 100-200 walnut seeds from the same plant that have a large yield and plump kernels.

[0015] S12: Divide the above walnut seeds into 6 beakers (2L), heat them in a water bath (constant temperature 50℃), soak them for 7 days, change the water every 12 hours, and stir constantly with a glass rod to ensure that the seeds are heated evenly.

[0016] S13: Place 2-3 layers of baffles on the prepared germination machine, and place 25 walnut seeds on each layer (ensuring moderate density and even distribution);

[0017] S14: Adjust the germinator to the spring setting. The germinator can rotate 360° and automatically circulate water to ensure that the walnut seeds remain moist during the germination process and that the water is changed once a day at 12 o'clock.

[0018] S15: During the period of 0-21 days, samples were taken every 3 days at 12 o'clock. 0.5g of fresh sample was weighed, ground with liquid nitrogen, and 4mL of each of 60mM pH 8.0 borate-borax buffer and 60mM pH 9.5 borate-sodium hydroxide buffer were added to 5mL EP centrifuge tubes. The samples were centrifuged at 12000rpm for 10min at 4℃. The supernatant was then collected and stored in an ultra-low temperature freezer at -80℃ for later use.

[0019] Furthermore, step S3 specifically includes:

[0020] S31: The protein concentration of the extract was determined using the Coomassie Brilliant Blue G-250 colorimetric method; the formula for calculating the protein yield is as follows:

[0021] Protein yield / % (Y) = [protein mass of extract (mg) / seed mass (mg)] × 100%;

[0022] The soluble protein was determined using the Coomassie Brilliant Blue G-250 method. 50 μL of the protein stock solution diluted 15 times was placed into test tubes (3 replicates). 2.5 mL of Coomassie Brilliant Blue solution was added to each test tube. After adding the stock solution, the mixture was thoroughly shaken with a mixer and allowed to stand at room temperature for 3 min. The photometric value was then measured at a wavelength of 595 nm using a 756s spectrophotometer based on the standard curve prepared from bovine serum albumin.

[0023] S32: Determination of DPPH free radical scavenging ability;

[0024] S33: Reducing power measurement;

[0025] S34: Determination of ferrous ion chelating ability.

[0026] Further, step S4 includes the following sub-steps:

[0027] S41: Effect of buffer pH on protein yield in germinated walnut seeds;

[0028] S42: Effect of buffer ionic strength on protein yield from germinated walnut seeds;

[0029] S43: Effect of extraction time on protein yield from germinated walnut seeds;

[0030] S44: Effect of material-to-liquid ratio on protein yield of germinated walnut seeds;

[0031] S45: Response surface methodology optimization experiment for optimal extraction of protein from germinated walnut seeds.

[0032] Further, step S31 specifically includes:

[0033] Weigh 0.5g of germinating walnut seed sample from day 21 and place it in an ice-bathed mortar. Quickly pour in 10mL of liquid nitrogen and grind rapidly. Add 10mL of 60mM buffer solution with pH settings of 7.5, 8.0, 8.5, 9.0, 9.5, and 10.0, respectively, and stir continuously on ice for 10 minutes at room temperature. After extraction, centrifuge at 12000rpm for 10 minutes at 4℃, collect the supernatant, and store for later use.

[0034] Further, step S42 specifically includes:

[0035] Weigh 0.5g of fresh germinating walnut seeds from day 21 and place them in a mortar with an ice bath. Quickly pour in 10mL of liquid nitrogen and grind rapidly. Add 10mL of 20, 40, 60, 80, and 100mM borate-borax buffer solution (pH 9.0) with an ice bath, respectively. Stir continuously for 10 minutes at room temperature on ice. After extraction, centrifuge at 12000rpm for 10 minutes at 4℃, collect the clear liquid, and store it in a 4℃ refrigerator for use as needed.

[0036] Further, step S43 specifically includes:

[0037] Weigh 0.5g of fresh walnut seeds that have germinated on day 21, place them in a mortar with an ice bath, quickly pour in 10mL of liquid nitrogen, and grind rapidly. Add 10mL of 60mM pH 9.0 boric acid and borax solution with an ice bath, and set extraction times of 5, 7.5, 10, 12.5, and 15 min. At room temperature, place the mortar on ice and stir continuously for 5, 7.5, 10, 12.5, and 15 min respectively. After the stirring time is up, place the mortar in a centrifuge at 4℃ and centrifuge at 12000rpm for 10 min. Transfer the supernatant and store it at 4℃ for later use.

[0038] Further, step S44 specifically includes:

[0039] Weigh 0.5g of fresh walnut seeds that have germinated on day 21, place them in a mortar with an ice bath, quickly pour in 10mL of liquid nitrogen, and grind rapidly. Add 6, 8, 10, 12, and 14mL of 60mM pH 9.0 borate-borax solution with an ice bath according to material-to-liquid ratios of 1:4, 1:6, 1:8, 1:10, and 1:12 (g / mL), respectively. Stir continuously on ice for 15min at room temperature. After extraction, centrifuge at 12000rpm for 10min at 4℃, collect the supernatant, and store for later use.

[0040] Further, step S45 specifically includes:

[0041] The results of the single-factor experiments were analyzed, and parameters such as buffer pH, buffer ionic strength, solid-liquid ratio, and extraction time were selected. Using JMP software and the Box-Bhnken central composite model, a four-factor, three-level experiment was designed with randomized sorting, totaling 27 groups. The optimal extraction parameters for the yield of germinating walnut seed protein were determined by using the yield of germinating walnut seed protein as the response value (Y / %), and buffer pH (X1), buffer ionic strength (X2), solid-liquid ratio (X3), and extraction time (X4) as experimental factors.

[0042] Furthermore, step S4 specifically includes:

[0043] An experimental method for extracting protein from germinated walnut seeds using buffer solution was employed. The antioxidant activity of the obtained walnut protein solution was assessed, including its DPPH free radical scavenging capacity, reducing power, and ferrous ion chelating capacity. The optimal extraction parameters for buffer solution extraction of walnut seed protein, determined using response surface methodology, were then used for protein extraction, and the antioxidant activity of the resulting walnut protein solution was measured.

[0044] The beneficial effects of this invention are as follows:

[0045] This invention, combining current walnut resource utilization with research progress on legume or cereal seed germination, analyzes the changes in amino acid composition during walnut seed germination to determine the optimal germination time. Using germinated walnut seeds as material, an orthogonal experimental design for protein extraction is employed, along with the determination of antioxidant activity, to construct an extraction process for antioxidant-active proteins during the germination stage of walnut seeds. This provides an optimal research scheme for producing high-value-added antioxidant protein components. The research results contribute to the development and utilization of high-value-added active proteins from germinating walnut seeds, enhancing the practical and economic value of the overall walnut industry chain. Furthermore, this extraction process and research scheme can be improved and adjusted for application in the development and utilization of other protein-rich agricultural and forestry biological resources, increasing their added value and economic benefits. It also provides a reference and scheme for constructing clean, efficient, and continuous protein separation processes with antioxidant activity. Attached Figure Description

[0046] Figure 1 The effect of buffer pH on protein yield in germinated walnut seeds;

[0047] Figure 2 The effect of buffer ionic strength on protein yield from germinated walnut seeds;

[0048] Figure 3 The effect of the material-to-liquid ratio on the protein yield of germinated walnut seeds;

[0049] Figure 4 The effect of extraction time on the yield of protein from germinated walnut seeds;

[0050] Figure 5 The degree of fit between the model's predicted values ​​and the actual experimental values;

[0051] Figure 6 The response surface plot and contour plot are for the interaction between X1 (buffer pH) and X2 (buffer ionic strength);

[0052] Figure 7 Response surface plot and contour plot of the interaction between X1 (buffer pH) and X3 (feed-to-liquid ratio);

[0053] Figure 8 Response surface plot and contour plot of the interaction between X1 (buffer pH) and X4 (extraction time);

[0054] Figure 9 The response surface plot and contour plot are for the interaction between X2 (buffer ionic strength) and X3 (feed-to-liquid ratio);

[0055] Figure 10 Response surface plot and contour plot of the interaction between X2 (ionic strength) and X4 (extraction time);

[0056] Figure 11 Response surface plot and contour plot of the interaction between X3 (feed-to-liquid ratio) and X4 (extraction time);

[0057] Figure 12 A predictive characterizer for the response surface of protein extraction rate in germinating walnut seeds;

[0058] Figure 13 A flowchart illustrating the steps of an experimental method for optimizing the optimal buffer solution extraction of protein from germinating walnut seeds using response surface methodology. Detailed Implementation

[0059] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0060] Germination refers to the dynamic process by which seeds transition from static dormancy to frequent physiological activity. After dormancy, seed respiration and metabolism increase, as do the types and quantities of enzymes, and their activity intensifies. Seed metabolism can proceed at a high level. During metabolism, numerous enzymes stimulate reactions related to basic energy, leading to biotransformation and degradation. Substances stored in the seed are transformed and upgraded into highly active components usable for new life, thereby increasing the content of bioactive substances and potentially generating new highly active substances. According to relevant research reports, during the germination stage, both cereals and legumes can generate nutrients with significant bioactivity. For example, during soybean germination, through enzymatic catalysis, the poorly soluble macromolecules stored in the seed are transformed into soluble small molecules. For instance, soybean protein, which is difficult for the human body to directly absorb and utilize, is transformed into easily absorbed amino acids or polypeptides; polysaccharides that the human body cannot absorb are transformed into monosaccharides for absorption. These transformations all benefit the absorption and utilization of nutrients. Germination treatment can effectively reduce the content of anti-nutritional factors in soybeans, improving the nutritional and edible value of the food. Scientific research shows that black beans have lipid-lowering, antioxidant, and skin-beautifying effects. Black bean germination effectively improves protein utilization. Due to enzymatic degradation during germination, proteins, polysaccharides, and minerals are released, increasing their absorption and utilization by the human body. Seed germination transforms stored substances into readily absorbable bioactive components, representing a novel way to obtain these bioactive ingredients.

[0061] The buffer-based protein extraction method involves grinding germinated walnut seeds with liquid nitrogen and then extracting them with buffer solutions of different pH and ionic strengths. A three-level experiment was conducted with four factors, including extraction time, pH parameters, ionic strength, and material-to-liquid ratio. The results showed that the protein yield was low with the buffer-based extraction method, but the extracted protein components had high antioxidant activity.

[0062] The results of single-factor experiments provided expected central points for response surface methodology (RSM) parameters. Using protein yield as the response value, RSM was used to optimize the extraction conditions of protein from germinated walnut seeds, thereby determining the optimal extraction parameters and process. Based on the above discussion, this study used germinated walnut seeds as material, employed buffer extraction, and used protein yield as the response value to optimize the extraction parameters affecting protein yield using RSM, thus determining the optimal extraction parameters. Combined with the determination of antioxidant activity, a process for preparing antioxidant proteins from germinated walnut seeds was constructed to produce high-value-added antioxidant protein components.

[0063] A response surface methodology method for optimizing the optimal buffer extraction assay for protein extraction from germinating walnut seeds includes the following steps:

[0064] (1) Seed germination

[0065] Select 100-200 seeds from the same plant, with a large yield and plump kernels. Divide these seeds into 6 beakers (2L each) and soak them in a water bath (50℃) for 7 days, changing the water every 12 hours and stirring constantly with a glass rod to ensure even heating. Place 2-3 layers of baffles on a prepared Roewe bean sprout machine, with 25 walnut seeds in each layer (ensuring moderate density and even distribution). Set the sprouter to the spring setting; the machine features 360° rotating automatic water circulation spraying to keep the seeds moist during germination. Change the water daily at 12 PM. During the 0-21 day period, take 0.5g of fresh sample every 3 days at 12 PM, grind it with liquid nitrogen, and store it in a -80℃ ultra-low temperature freezer for later use.

[0066] (2) Determination of amino acid composition and content in walnut seeds germinated at different germination stages

[0067] Chromatographic conditions: Column: ACQUITYUPLC® BEHC18 column (2.1×100mm, 1.7μm, Waters Corporation, USA); injection volume: 5μL; column temperature: 40℃; mobile phase: A - 10% methanol-water (containing 0.1% formic acid); B - 50% methanol-water (containing 0.1% formic acid). Gradient elution conditions: 0–6.5 min, 10–30% B; 6.5–7 min, 30–100% B; 7–8 min, 100% B; 8–8.5 min, 100–10% B; 8.5–12.5 min, 10% B. Flow rate: 0–8.5 min, 0.3 mL / min; 8.5–12.5 min, 0.3–0.4 mL / min. Mass spectrometry conditions: Electrospray ionization (ESI) source, positive ionization mode. The ion source temperature was 500℃, the ion source voltage was 5500V, the collision gas was 6psi, the curtain gas was 30psi, and the nebulizing gas and auxiliary gas were both 50psi. Multiple reaction monitoring (MRM) was used for scanning.

[0068] Each working standard solution was analyzed by LC-MS. The linear range was examined and a standard curve was plotted by plotting the concentration of the working standard solution on the x-axis and the ratio of the peak area to the internal standard on the y-axis. The correlation coefficient r of the linear regression equation for each amino acid was >0.99. The quantitative results are shown in the table below.

[0069] A response surface methodology method for optimizing the optimal buffer extraction assay for protein extraction from germinating walnut seeds includes the following steps:

[0070] (3) Protein content and antioxidant activity determination

[0071] (31) The protein concentration of the extract was determined by the Coomassie Brilliant Blue G-250 colorimetric method; among which, the formula for calculating the protein yield of germinated walnut seeds is:

[0072] Protein yield / % (Y) = [protein mass of extract (mg) / seed mass (mg)] × 100%;

[0073] Protein content was determined using the Coomassie Brilliant Blue G-250 method. 50 μL of the 20-fold diluted protein stock solution was placed into test tubes (3 replicates). 2.5 mL of Coomassie Brilliant Blue was added to each tube. After adding the stock solution, the mixture was thoroughly shaken with a mixer to ensure homogeneity. After standing at room temperature for 3 minutes, the photometric value was measured at a wavelength of 595 nm using a 756s spectrophotometer, based on a standard curve prepared from bovine serum albumin.

[0074] (32) DPPH free radical scavenging capacity determination

[0075] Pipette 2 mL of DPPH solution into a 5 mL brown centrifuge tube, add 1 mL of 50 mM pH 7.4 Tris-HCl solution, vortex until homogeneous, and measure the absorbance A0 at 517 nm. Similarly, pipette 2 mL of DPPH solution into a 5 mL brown centrifuge tube, add 100 μL of sample solution, add 1 mL of 50 mM pH 7.4 Tris-HCl, mix thoroughly, store in the dark at room temperature, and after standing for 30 min, measure the absorbance A (517 nm).

[0076] (33) Reducing power test

[0077] Add 2 mL of sample to a 5 mL centrifuge tube, then add 2 mL of 1% potassium ferricyanide and 0.2 M pH 6.6 phosphate buffer, shake well, and place the centrifuge tube in 50 °C warm water for 20 min. Remove the tube and place it in an ice pack for rapid cooling. After a 10 min ice-water bath, add 2 mL of 10% TCA, shake well, and centrifuge for 10 min. Then add 1.5 mL of deionized water and 0.3 mL of 0.1% ferric chloride, mix well, and react for 10 min. Measure the absorbance OD (700 nm).

[0078] (34) Determination of ferrous ion chelating ability

[0079] Add 0.5 mL of sample to a 5 mL centrifuge tube, then add 3.7 mL of methanol and 0.1 mL of 2 mM FeCl2·4H2O. After the solution reacts for 30 seconds, add 0.2 mL of 5 mM Ferrozine to the centrifuge tube. After 10 minutes, measure the absorbance OD (562 nm).

[0080] (4) Single-factor experiment on walnut using buffer extraction method.

[0081] (41) Effect of buffer pH on protein yield in germinated walnut seeds

[0082] Weigh 0.5g of fresh germinating walnut seeds from day 21 and place them in an ice-bathed mortar. Quickly pour in 10mL of liquid nitrogen and grind rapidly. Add 10mL of 60mM buffer solution with pH settings of 7.5, 8.0, 8.5, 9.0, 9.5, and 10.0, respectively, and stir continuously on ice for 10 minutes at room temperature. After extraction, centrifuge at 12000rpm for 10 minutes at 4℃, collect the supernatant, and store for later use.

[0083] (42) Effect of buffer ionic strength on protein yield of germinated walnut seeds

[0084] Weigh 0.5g of fresh walnut seeds from day 21 of germination and place them in an ice-bathed mortar. Quickly pour in 10mL of liquid nitrogen and grind rapidly. Add 10mL of 20, 40, 60, 80, and 100mM borate-borax buffer solution (pH 9.0) in an ice-bathed environment. Stir continuously for 10 minutes at room temperature on ice. After extraction, centrifuge at 12000rpm for 10 minutes at 4℃. Collect the clear liquid and store it in a 4℃ refrigerator for use as needed.

[0085] (43) Effect of extraction time on protein yield of germinated walnut seeds

[0086] Weigh 0.5g of fresh walnut seeds from day 21 of germination, place them in an ice bath mortar, quickly pour in 10mL of liquid nitrogen, and grind rapidly. Add 10mL of 60mM pH 9.0 boric acid-borax solution in an ice bath, and set extraction times of 5, 7.5, 10, 12.5, and 15 min. At room temperature, place the mortar on ice and stir continuously for 5, 7.5, 10, 12.5, and 15 min respectively. After the stirring time is up, place the mortar in a centrifuge at 4℃ and centrifuge at 12000rpm for 10 min. Transfer the supernatant and store it at 4℃ for later use.

[0087] (44) Effect of material-liquid ratio on protein yield of germinated walnut seeds

[0088] Weigh 0.5g of fresh walnut seeds from day 21 of germination, place them in a mortar with an ice bath, quickly pour in 10mL of liquid nitrogen, and grind rapidly. Add 6, 8, 10, 12, and 14mL of 60mM pH 9.0 borate-borax solution with an ice bath according to material-to-liquid ratios of 1:4, 1:6, 1:8, 1:10, and 1:12 (g / mL), respectively. Stir continuously on ice for 15min at room temperature. After extraction, centrifuge at 12000rpm for 10min at 4℃, collect the supernatant, and store for later use.

[0089] (45) Optimization of optimal extraction experiment of protein from germinating seeds using response surface methodology

[0090] The results of the single-factor experiments were analyzed, and parameters such as buffer pH, buffer ionic strength, solid-liquid ratio, and extraction time were selected. Using JMP software and the Box-Bhnken central composite model, a four-factor, three-level experiment was designed with randomized sorting, totaling 27 groups. The protein yield of germinating walnut seeds was used as the response value (Y / %), and buffer pH (X1), buffer ionic strength (X2), solid-liquid ratio (X3), and extraction time (X4) were used as experimental factors to determine the optimal parameter conditions for protein extraction from germinating walnut seeds.

[0091] Table 1. Central composite design factors and level coding for buffer-extracted proteins from germinating walnut seeds.

[0092] .

[0093] (5) Data processing and statistical analysis

[0094] The different data from the single-factor experiments were initially integrated and processed, and then plotted and imaged using Origin 8.5 plotting software. The experiment was designed as a central composite experiment, and analysis of variance was performed. The F-values ​​were compared to observe whether the individual factors and their interactions were significant, and the confidence interval of the experiment was ensured to be above 95%.

[0095] in conclusion:

[0096] (1) Effects of germination on GABA and other amino acid composition in walnut seeds

[0097] As shown in Table 2, the total amino acid content in germinated walnuts increases, which can adjust the seed nutrition of walnuts, promote the absorption of nutrients, and improve the bioavailability of walnuts. In particular, the increase in the content of the eight essential amino acids and the functional amino acid GABA not only endows walnut protein with high nutritional value, but the enrichment of GABA also gives germinated walnut seeds the effects of calming the nerves, lowering blood pressure, and improving memory. Except for glutamic acid, the other amino acids are enriched during germination, with the content of essential amino acids being the highest at 21 days (1911.40 μg / gDW). Although the GABA content of germinated walnut seeds at 21 days is not the highest, the overall nutritional value of germinated walnut seeds at 21 days is considered to be relatively high. The increased levels of umami amino acids (aspartic acid), sweet amino acids (threonine, serine, glycine, alanine, methionine, proline, ornithine), and bitter amino acids (valine, leucine, isoleucine, tyrosine, phenylalanine, arginine, lysine, histidine, tryptophan) enrich the flavor of sprouted walnut seeds. From the perspectives of nutrition, function, and flavor, sprouted walnuts have further development value for the industry. Therefore, 21-day-old sprouted walnut seeds were selected as the material for protein extraction.

[0098] Table 2. Changes in amino acid composition and content during walnut seed germination.

[0099] .

[0100] Note: E / T value is the ratio of essential amino acids to total amino acids.

[0101] (2) Effect of buffer pH on protein yield of germinated walnut seeds

[0102] The effect of buffer pH on the yield of walnut seed protein was as follows: (The extraction conditions were: solid-liquid ratio 1:10, ionic strength 60 mM, and extraction time 10 min.) Figure 1 As shown, the yield of walnut seed protein increases between pH 7.5 and 8.5. The yield decreases slowly at pH 9, but increases again at pH 9.5. The protein color darkens with increasing pH. Considering all factors, a pH of 9.7 was chosen.

[0103] (3) Effect of ionic strength on protein yield of germinated walnut seeds

[0104] The effect of ion concentration on the protein content of germinated walnut seeds was investigated under the following extraction conditions: pH 9.0, solid-liquid ratio 1:10, and extraction time 10 min. Figure 2 As shown, when the ion concentration is between 20 and 60 mM, the yield of germinated walnut seed protein increases with the increase of ionic strength, and reaches a peak at an ionic strength of 60 mM. When the ionic strength is higher than 60 mM, the yield of germinated walnut seed protein fluctuates slowly. Considering all factors, a concentration of 60 mM was selected.

[0105] (4) Effect of material-liquid ratio on protein yield of germinated walnut seeds.

[0106] The effect of the solid-liquid ratio on the protein yield of germinated walnut seeds was investigated under the following extraction conditions: pH 9.0, ionic strength 60 mM, and extraction time 10 min. Figure 3 As shown, when the material-to-liquid ratio is between 1:6 and 1:14, the protein yield of walnut seeds fluctuates gently and increases slowly. The difference in protein yield between 1:12 and 1:14 material-to-liquid ratios is small. Considering all factors, the material-to-liquid ratio condition of 1:12 is selected.

[0107] (5) Effect of extraction time on protein yield of germinated walnut seeds

[0108] The effect of extraction time on the protein content of germinated walnut seeds was investigated under the following extraction conditions: pH 9.0, ionic strength 60 mM, and solid-liquid ratio 1:10. Figure 4As shown. The protein yield of germinated walnut seeds increased between 5 min and 7.5 min, decreased between 7.5 min and 10 min, and then fluctuated. The selected time factor was 12.5 min.

[0109] (6) Establishment and analysis of regression model for protein yield of walnut germinated seeds by buffer extraction method

[0110] By comprehensively comparing the results of single-factor experiments and optimizing the experimental design of the response surface by referring to the central combination design of the response surface, an optimized design scheme for the response surface is obtained.

[0111] Using SAS 13.1.0 statistical analysis software, a quadratic regression model was established for the experimental data in Table 2, and the fitting equation for the three-factor, three-level quadratic multiple regression was obtained as follows:

[0112] Y=-6.7626578-0.00464459X1+0.0038414665X2+1.28536931X3+0.08687325X4+0.000114375X1X2 +0.0003583X1X3+0.004875X2X3+0.000218X1X4-0.000805X2X4-0.006867X3X4-0.00002314575X1 2 -0.002677075X2 2 -0.064778X3 2 -0.000249328X4 2

[0113] Table 3. Design, reaction values, and predicted values ​​of central composition for protein yield extracted from walnut seeds using buffer solution (g / 100 g) (n=3)

[0114] .

[0115] Note: X1: Ionic strength of buffer (mM); X2: Meal-to-solvent ratio (g / ml); X3: pH of buffer; X4: Extraction time (h).

[0116] A Box-Behnken central composite model was used to design a four-factor, three-level response surface methodology experiment. The samples were randomly assigned, with the protein yield of germinating walnut seeds as the response value. The experiment had 27 experimental sites, including 24 factorial sites and 3 zero points (center points of the levels). The zero-point experiment was repeated three times to estimate the experimental error.

[0117] By comparing the predicted values ​​obtained from the quadratic regression model in the SAS 13.1.0 statistical analysis software with the actual values ​​measured in the experiment, the conclusion is drawn from... Figure 5 The results show that the two have a good fit.

[0118] Table 4. Analysis of variance results of the regression equation for the buffer extraction method.

[0119] .

[0120] Note: P<0.001 represents highly significant "***"; P<0.01 represents relatively significant "**"; P<0.05 represents significant "*"; P>0.05 represents not significant.

[0121] The results of the ANOVA for the regression equation are shown in Table 4. A significant linear relationship exists between the dependent variable and the independent variable under investigation in the regression model (R² = 0.9143), and the adjusted coefficient of determination AdjR² = 0.8143, indicating that the model can explain 81.43% of the response value variation, demonstrating a high degree of fit. The lack-of-fit term is not significant (P > 0.05), and it can predict the response value well. The F-value of the central composite design of the response surface is 31.6528, with a P-value less than 0.0001, indicating that the experimental model has a high degree of reliability. The model error lack-of-fit term represents the probability that the model prediction does not fit the actual value. The P-value (P = 0.0310) for this lack-of-fit term is less than 0.05, indicating a high degree of fit between the regression equation and the experiment, and strong reliability. Therefore, the established binomial regression model is valid, and the experimentally obtained process parameters for protein extraction can be well analyzed and predicted for the extraction effect on walnut seed protein.

[0122] Table 5. Significance Tests of Regression Model Coefficients

[0123] .

[0124] Significance tests on the regression equation coefficients of the model revealed the following (Table 5): Linear terms X2 (solid-to-liquid ratio) and X3 (pH value) were highly significant, while X1 (ion concentration) and X4 (extraction time) were not significant; the interaction term X1*X4 was significant, while X1*X2, X1*X3, X2*X3, X2*X4, and X3*X4 were not significant; the quadratic terms X1*X1, X2*X2, and X3*X3 were significant, while X4*X4 was not significant. The order of influence of each factor on the yield of walnut seed protein was: solid-to-liquid ratio X2 > pH value X3 > ion concentration X1 > extraction time X4.

[0125] Based on the analysis of variance results, insignificant terms were removed to simplify the regression equation for this experiment:

[0126] Y=-6.7626578+0.0038414665X2+1.28536931X3+0.000218X1X4-0.00002314575X12-0.002677075X22-0.064778X32;

[0127] (7) Response surface methodology for extracting protein yield from germinating seeds using the buffer method

[0128] like Figures 6 to 11 Response surface methodology analysis of each factor shows that X2 (material-liquid ratio) has the greatest impact on the protein yield of germinating walnut seeds, while X1 and X4 have the greatest interaction. Other factors have relatively small interaction effects, with X1 and X3 having the least impact.

[0129] In this case, the solvent / solid ratio (X2) has the greatest impact on protein yield as an independent variable. The interaction terms have the greatest impact on protein yield as X1 and X4, while the other interactions have a smaller impact and are not significant. Among them, the interactions with the smallest impact are X1 and X3. Figure 6-11 The contour plots depict a 3D graph of protein yield as a response to extraction temperature, time, and ethanol concentration parameters. Each time, the effect of two variables on the response is evaluated while the other variable remains constant. It can be seen that the peaks of all 3D response surfaces are very pronounced. Protein yield increases with increasing ionic strength and feed-to-liquid ratio, but when the variables exceed a certain point, protein yield begins to decline despite further increases in the ratio. Figure 6 This is likely due to the increased solubility of the protein caused by the increased feed-to-liquid ratio. However, with increasing ionic strength, the protein's solubility decreases due to the higher salt concentration. Furthermore, ionic strength and pH also show the same effect. Figure 7 Protein yield increases with increasing ionic strength and pH until an optimal point is reached. After this point, protein yield begins to decline. A pH of 9.7 promotes protein exposure and interaction with the cell towards the solvent, resulting in optimal adhesion and dispersibility of soluble proteins. Figure 9 and Figure 10 , 11 In contrast, the time contour lines changed, indicating that the interaction between ion intensity and time caused the contour lines to become flattened, further proving that the interaction between X1 and X4 is significant.

[0130] (8) Optimal extraction conditions and verification of buffer method for extracting proteins from germinating seeds

[0131] Response surface methodology analysis revealed the following extraction conditions for germinating walnut seeds: pH 9.98, ion concentration 72.20 M, liquid-to-solid ratio 1:13.33, extraction time 13.23 min, and predicted protein yield of 15.73%. Based on practical feasibility, these conditions were adjusted to: pH 10.0, ion concentration 70 mM, solid-to-liquid ratio 1:13, and extraction time 13 min.

[0132] (9) Analysis of antioxidant activity of protein seeds extracted using buffer method

[0133] Analysis of experimental data showed that the DPPH free radical scavenging rate of walnut seed protein extracted with buffer solution ranged from 1.14% to 10.06%, with the scavenging rate decreasing as pH increased. The protein reducing power ranged from 0.180% to 0.409, with the reducing power decreasing as pH increased. The protein ferrous ion chelating capacity ranged from 90.15% to 98.59%. Compared with the alkali extraction method, the chelating capacity of germinating seeds was better than that of untreated seeds.

[0134] Summarize:

[0135] (1) The experiment used the buffer method to extract protein from germinated walnut seeds. Through complete single-factor experiments, central composite design, and response surface analysis, the following conclusions were drawn:

[0136] Using protein yield as the response value, the regression model is as follows:

[0137] Y(%)=427.74204-62.88527X1-11.26609X3+0.064422X1X2+0.05794X2X3+0.141497X3X4+3.107744X12-0.0022801X22+0.040447X42

[0138] The order of factors affecting the extraction process of walnut germination seed protein is as follows: solid-liquid ratio (X2) > pH value (X3) > ion concentration (X1) > extraction time (X4). Analysis revealed the following optimal extraction conditions for walnut seeds using buffer solution: pH 9.98, ion concentration 72.20 M, liquid-liquid ratio 1:13.33, and extraction time 13.23 min. After adjustments for practical application, the protein yield under this process was measured to be 16.11%.

[0139] (2) Proteins from germinated walnut seeds were extracted using a buffer method. The DPPH free radical scavenging rate of the protein extract ranged from 1.14 to 10.06%, the protein reducing power ranged from 0.180 to 0.409, and the protein ferrous ion chelating capacity ranged from 90.15% to 98.59%.

[0140] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Response surface methodology was used to optimize the optimal buffer extraction method for protein from germinating walnut seeds, including: S1: Seed germination; S2: Determination of amino acid composition in walnut seeds at different germination stages; S3: Determination of protein content and antioxidant activity; S4: Response surface methodology optimization experiment for optimal extraction of protein from germinated walnut seeds.

2. The method for optimizing the optimal buffer extraction of protein from germinating walnut seeds using response surface methodology according to claim 1, wherein, Step S1, seed germination, includes: S11: Select 100-200 walnut seeds from the same plant that have a large yield and plump kernels. S12: Divide the above walnut seeds into 6 beakers, heat them in a water bath to a constant temperature of 50°C, soak them for 7 days, change the water every 12 hours, and stir them constantly with a glass rod to ensure that the seeds are heated evenly. S13: Place 2-3 layers of baffles on the prepared germination machine, and place 25 walnut seeds on each layer; S14: Adjust the germination machine to the spring setting. The germination machine will automatically rotate and spray water in a 360° rotation. Change the water once a day at 12 noon. S15: Take samples every 3 days at 12 o'clock within 0-21 days. Weigh 0.5g of fresh sample, grind with liquid nitrogen, and add 4mL of each of 60mM pH 8.0 borate-borax buffer and 60mM pH 9.5 borate-sodium hydroxide buffer to 5mL centrifuge tubes. Centrifuge at 12000rpm for 10min at 4℃, collect the supernatant, and store it in an ultra-low temperature freezer at -80℃ for later use.

3. The method for optimizing the optimal buffer extraction of protein from germinating walnut seeds using response surface methodology according to claim 1, wherein, Step S3, the determination of protein content and antioxidant activity, includes: S31: The protein concentration of the extract was determined using the Coomassie Brilliant Blue G-250 colorimetric method; the formula for calculating the protein yield is as follows: Protein yield / % (Y) = [protein mass of extract (mg) / seed mass (mg)] × 100%; The soluble protein was determined using the Coomassie Brilliant Blue G-250 method. 50 μL of the protein stock solution diluted 15 times was placed in a test tube for three replicates. 2.5 mL of Coomassie Brilliant Blue was added to each test tube. After adding the stock solution, the mixture was thoroughly shaken with a mixer and allowed to stand at room temperature for 3 min. The photometric value was then measured at a wavelength of 595 nm using a 756s spectrophotometer based on the standard curve prepared from bovine serum albumin. S32: Determination of DPPH free radical scavenging ability; S33: Reducing power measurement; S34: Determination of ferrous ion chelating ability.

4. The method for optimizing the optimal buffer extraction of protein from germinating walnut seeds using response surface methodology according to claim 1, wherein, Step S4, the response surface methodology optimization experiment for the optimal extraction of germinating seed protein, includes: S41: Effect of buffer pH on protein yield in germinated walnut seeds; S42: Effect of buffer ionic strength on protein yield from germinated walnut seeds; S43: Effect of extraction time on protein yield from germinated walnut seeds; S44: Effect of material-to-liquid ratio on protein yield of germinated walnut seeds; S45: Response surface methodology optimization experiment for optimal extraction of protein from germinated walnut seeds.

5. The method for optimizing the optimal buffer extraction of germinating seed protein using response surface methodology according to claim 4, wherein, Step S41 specifically includes: Weigh 0.5g of fresh walnut seeds from day 21 of germination, place them in an ice bath mortar, quickly pour in 10mL of liquid nitrogen and grind rapidly. Add 10mL and 60mM of ice bath buffer solutions with pH values ​​of 7.5, 8.0, 8.5, 9.0, 9.5 and 10.0 respectively. At room temperature, place on ice and stir continuously for 10min. After extraction, centrifuge at 12000rpm for 10min at 4℃, collect the supernatant and store for later use.

6. The method for optimizing the optimal buffer extraction of protein from germinating walnut seeds using response surface methodology according to claim 4, wherein, Step S42 specifically includes: Weigh 0.5g of fresh walnut seeds from day 21 of germination, place them in an ice bath mortar, quickly pour in 10mL of liquid nitrogen and grind rapidly. Add 10mL of 20, 40, 60, 80 and 100mM borate-borax buffer solution (pH 9.0) in an ice bath, respectively. At room temperature, place on ice and stir continuously for 10min. After extraction, centrifuge at 12000rpm for 10min at 4℃, collect the clear liquid and place it in a 4℃ refrigerator for use as needed.

7. The method for optimizing the optimal buffer extraction of protein from germinating walnut seeds using response surface methodology according to claim 4, wherein, Step S43 specifically includes: Weigh 0.5g of fresh walnut seeds from day 21 of germination and place them in a mortar with an ice bath. Quickly pour in 10mL of liquid nitrogen and grind rapidly. Add 10mL, 60mM, and pH 9.0 boric acid-borax solutions with an ice bath, respectively. Set extraction times of 5, 7.5, 10, 12.5, and 15 min. At room temperature, place the mortar on ice and stir continuously for 5, 7.5, 10, 12.5, and 15 min. After stirring for the specified time, place the mortar in a centrifuge at 4℃ and centrifuge at 12000rpm for 10 min. Transfer the supernatant and store it at 4℃ for later use.

8. The method for optimizing the optimal buffer extraction of protein from germinating walnut seeds using response surface methodology according to claim 4, wherein, Step S44 specifically includes: Weigh 0.5g of fresh walnut seeds from day 21 of germination, place them in a mortar with an ice bath, quickly pour in 10mL of liquid nitrogen, and grind rapidly. Add 6, 8, 10, 12, and 14mL of 60mM boric acid-borax solution with pH 9.0 in an ice bath according to material-to-liquid ratios (g / mL) of 1:4, 1:6, 1:8, 1:10, and 1:12, respectively. Stir continuously on ice for 15min at room temperature. After extraction, centrifuge at 12000rpm for 10min at 4℃, collect the supernatant, and store for later use.

9. The method for optimizing the optimal buffer extraction of protein from germinating walnut seeds using response surface methodology according to claim 4, wherein: Step S45 specifically includes: The results of the single-factor experiments were analyzed, and parameters such as buffer pH, buffer ionic strength, solid-liquid ratio, and extraction time were selected. Using JMP software and the Box-Bhnken central composite model, a four-factor, three-level experiment was designed with randomized sorting, totaling 27 groups. The protein yield of germinated walnut seeds was used as the response value (Y / %), and buffer pH (X1), buffer ionic strength (X2), solid-liquid ratio (X3), and extraction time (X4) were used as experimental factors to determine the optimal parameter conditions for protein extraction from germinated walnut seeds.

10. The method for optimizing the optimal buffer extraction of protein from germinating walnut seeds using response surface methodology according to claim 1, wherein, Step S4, the response surface methodology optimization experiment for the optimal extraction of germinating seed protein, includes: An experimental method for extracting protein from germinated walnut seeds using buffer solution was used. The antioxidant activity of the obtained germinated walnut seed protein solution was tested, and its DPPH free radical scavenging capacity, reducing power, and ferrous ion chelating capacity were determined. The optimal extraction process parameters for extracting protein from germinated walnut seeds using buffer solution were obtained by response surface methodology, and the antioxidant activity of the obtained protein was measured.

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