Optimization method and comprehensive utilization method for extraction of cruciferous seed polyphenol

Abstract

The invention discloses an optimization method for extraction of cruciferous seed polyphenol and a comprehensive utilization method, the extraction condition of rapeseed meal polyphenol is optimized based on a response surface method.The method comprises the following steps: firstly, pretreating cruciferous seed meal-rapeseed meal, crushing and sieving to obtain rapeseed meal powder; then carrying out a single-factor experiment based on five factors of ethanol concentration, hydrochloric acid concentration, extraction time, extraction temperature and solid-liquid ratio, and then carrying out response surface experiment design based on a single-factor experiment result to obtain an extraction condition when the total phenol content of the rapeseed meal polyphenol extracting solution is maximum. The rapeseed meal polyphenol is extracted under the optimal condition, and then the purified rapeseed meal polyphenol is added into the rapeseed oil, so that the rapeseed oil enriched with the rapeseed meal polyphenol is obtained. According to the method disclosed by the invention, suitable conditions for extracting the rapeseed meal polyphenol are determined, a reference basis is provided for development and utilization of the rapeseed meal polyphenol, and a more-developed and more-basis thought is provided for synergistically improving the quality of rapeseed oil by enriching the rapeseed meal polyphenol.

Description

Technical Field

[0001] This invention relates to the field of polyphenol extraction and application technology in agricultural by-products, specifically to the optimization of extraction conditions for rapeseed meal polyphenols derived from cruciferous seed meal and rapeseed meal, and to a processing method for improving rapeseed oil quality; more specifically, it relates to a method for processing and comprehensively utilizing cruciferous seeds. Background Technology

[0002] Rapeseed, a plant belonging to the Brassicaceae family and the genus Brassica, is my country's largest oilseed crop. Rapeseed meal is a byproduct of oil extraction from rapeseed seeds, and it contains abundant phenolic compounds, widely used as a raw material for polyphenol extraction. Rapeseed meal polyphenols possess various functions, including antioxidant, anti-cardiovascular, antiviral, anti-cancer, anti-inflammatory, anti-obesity, hepatoprotective, and neuroprotective properties. It has broad application prospects in the fields of medicine, health products, and cosmetics.

[0003] In recent years, phenolic content has gradually become an important parameter for evaluating rapeseed oil quality because it plays a role in promoting health and stabilizing the oil. However, during the extraction and processing, only a small amount of phenolic substances are transferred to virgin rapeseed oil. Furthermore, the refining process leads to the further loss of most of the remaining phenolic substances in the final product. Currently, there are two main methods to modify the polyphenol content in rapeseed oil: optimizing processing technology to retain endogenous phenolic substances and supplementing with exogenous antioxidants. Adding homologous extracts to rapeseed oil appears to be a better processing method.

[0004] There are many methods for extracting polyphenols, but the optimal process for extracting polyphenols from rapeseed meal under acidic ethanol conditions still needs to be determined. At the same time, it is necessary to further understand the effects of homologous phenolic substances on the stability of oils. Summary of the Invention

[0005] In view of this, the purpose of this invention is to propose an optimized method for extracting and comprehensively utilizing polyphenols from cruciferous seeds. Based on rapeseed meal (a type of cruciferous seed meal) as raw material, this invention utilizes response surface methodology to optimize the extraction conditions of rapeseed meal polyphenols and its enrichment and synergistic enhancement of rapeseed oil quality. Suitable rapeseed meal raw material is selected and pretreated. Then, single-factor experiments are conducted to investigate the effects of ethanol concentration, hydrochloric acid concentration, extraction time, extraction temperature, and material-to-liquid ratio on the total phenol content of the rapeseed meal polyphenol extract. Next, using the total phenol content of the extract as the response value, based on the results of the single-factor experiments, ethanol concentration, hydrochloric acid concentration, and extraction time are selected for optimization. Statistical analysis software is used to analyze the response surface experiment data, establish a mathematical model, and obtain the optimal combination of each factor. Finally, rapeseed meal polyphenols are extracted using the optimal combination and purified. Then, the purified rapeseed meal polyphenols are added to rapeseed oil to obtain rapeseed oil enriched with rapeseed meal polyphenols, and the effect of rapeseed meal polyphenols on the oxidative stability of rapeseed oil is investigated.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] An optimized method for extracting polyphenols from cruciferous seeds includes the following steps:

[0008] Step 1: Pretreatment of rapeseed meal

[0009] The rapeseed meal was crushed and passed through a 40-mesh sieve to obtain rapeseed meal powder.

[0010] Step 2: Single-factor experiment

[0011] Five factors were determined: ethanol concentration, hydrochloric acid concentration, extraction time, extraction temperature, and solid-liquid ratio. The effect of one of these factors on the total phenol content of the extract was measured by using rapeseed meal powder under constant conditions.

[0012] Step 3: Response Surface Design

[0013] Based on the results of the single-factor experiments, ethanol concentration, hydrochloric acid concentration, and extraction time were selected as independent variables, and the total phenol content of the extract was selected as the response value. The Box-Behnken response surface methodology was used to perform regression analysis on the experimental data, establishing a quadratic polynomial model and obtaining the multiple regression equation:

[0014] Y=1.8100-0.1263A+0.0141B+0.0891C-0.0997AB+0.0686AC+0.0568BC-0.2632A 2 -0.0174B 2 -0.0120C 2 ;

[0015] Where Y is the total phenol content of the rapeseed meal polyphenol extract, A is the ethanol concentration, B is the hydrochloric acid concentration, and C is the extraction time.

[0016] Preferably, the volume concentrations of ethanol in step two are 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%.

[0017] The hydrochloric acid concentrations are 0.01 mol / L, 0.05 mol / L, 0.1 mol / L, 0.5 mol / L, 1 mol / L, and 2 mol / L;

[0018] The extraction times were 15 min, 30 min, 60 min, 90 min and 120 min;

[0019] The extraction temperatures were 25℃, 40℃, 55℃, 70℃ and 85℃;

[0020] The material-to-liquid ratio is 1:5, 1:10, 1:20, 1:30 and 1:40.

[0021] Furthermore, the experimental design described in step two is as follows:

[0022] (2.1) Prepare acidic ethanol solutions with ethanol volume concentrations of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90% and hydrochloric acid concentration of 0.5 mol / L respectively. Weigh the pretreated rapeseed meal powder, mix it with the acidic ethanol solution at a material-to-liquid ratio of 1:20, extract at 25℃ for 120 min, centrifuge, collect the supernatant, and determine the total phenol content of the extract.

[0023] (2.2) Prepare acidic ethanol solutions with ethanol volume concentration of 60%, hydrochloric acid concentrations of 0.01 mol / L, 0.05 mol / L, 0.1 mol / L, 0.5 mol / L, 1 mol / L and 2 mol / L respectively. Weigh the pretreated rapeseed meal powder, mix it with the acidic ethanol solution at a material-to-liquid ratio of 1:20, extract at 25℃ for 120 min, centrifuge, collect the supernatant, and determine the total phenol content of the extract.

[0024] (2.3) Prepare an acidic ethanol solution with an ethanol volume concentration of 60% and a hydrochloric acid concentration of 0.5 mol / L. Weigh the pretreated rapeseed meal powder and mix it with the acidic ethanol solution at a material-to-liquid ratio of 1:20. Extract the solution at 25℃ for 15 min, 30 min, 60 min, 90 min and 120 min respectively. Centrifuge the solution, collect the supernatant, and determine the total phenol content of the extract.

[0025] (2.4) Prepare an acidic ethanol solution with an ethanol volume concentration of 60% and a hydrochloric acid concentration of 0.5 mol / L. Weigh the pretreated rapeseed meal powder and mix it with the acidic ethanol solution at a material-to-liquid ratio of 1:20. Extract the solution at 25℃, 40℃, 55℃, 70℃ and 85℃ for 60 min respectively. Centrifuge the solution, collect the supernatant, and determine the total phenol content of the extract.

[0026] (2.5) Prepare an acidic ethanol solution with an ethanol volume concentration of 60% and a hydrochloric acid concentration of 0.5 mol / L. Weigh the pretreated rapeseed meal powder and mix it with the acidic ethanol solution at material-to-liquid ratios of 1:5, 1:10, 1:20, 1:30 and 1:40, respectively. Extract the solution at 85℃ for 60 min, centrifuge, collect the supernatant, and determine the total phenol content of the extract.

[0027] Furthermore, the centrifugation speed is 8000 rpm and the time is 20 min.

[0028] Preferably, the response surface methodology in step three further includes: evaluating the significance and fit of the model using analysis of variance, analyzing the effect of the interaction between the three factors of ethanol concentration, hydrochloric acid concentration and extraction time on the total phenol content, and determining the optimal combination of levels for each factor.

[0029] Furthermore, the optimal extraction conditions are: ethanol volume concentration of 54%, hydrochloric acid concentration of 1 mol / L, extraction time of 90 min, extraction temperature of 55℃, and solid-liquid ratio of 1:10.

[0030] The present invention also provides a rapeseed meal polyphenol extract obtained by using the optimal extraction conditions determined by the above method.

[0031] The technical solution of the present invention further provides a method for preparing and purifying rapeseed meal polyphenols using rapeseed meal polyphenol extract, comprising the following steps:

[0032] AB-8 macroporous resin was soaked in anhydrous ethanol overnight, then rinsed with ultrapure water until the effluent had no alcohol odor. Subsequently, the resin was soaked in 2 BV of 5% hydrochloric acid for 2 hours, then rinsed with ultrapure water until neutral. Next, the resin was soaked in 2 BV of 5% sodium hydroxide for 2 hours, and then rinsed again with ultrapure water until the pH of the effluent reached neutral. The rapeseed meal polyphenol extract was concentrated by vacuum rotary evaporation. The concentrated crude extract was added to a pretreated AB-8 macroporous resin column (1000 mL, 60*600 mm) at a flow rate of 1-2 BV / h. The column, which had adsorbed phenolic compounds, was washed with 2 BV of ultrapure water at a flow rate of 2-3 BV / h to remove proteins and sugars. Then, elution was performed with 70% ethanol at a flow rate of 1-2 BV / h to obtain a purified rapeseed meal polyphenol solution. This solution was collected, concentrated, and dried. The purified solid rapeseed meal polyphenols were stored at 4°C.

[0033] The present invention also claims protection for the application of the purified rapeseed meal polyphenols prepared by the above scheme, wherein the purified rapeseed meal polyphenols are added to rapeseed oil.

[0034] The technical solution of this invention further provides a method for studying the inhibitory effect of rapeseed meal polyphenols on the formation of primary and secondary oxidation products of rapeseed oil, including the following steps:

[0035] Purified rapeseed meal polyphenols were dissolved in rapeseed oil at concentrations of 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, and 1.0 g / L, respectively. Untreated rapeseed oil was used as a negative control, and rapeseed oil containing 0.2 g / L BHT was used as a positive control. Freshly prepared rapeseed oil samples were poured into glass vials and placed in a dark incubator at 60°C. The peroxide value and thiobarbituric acid value were measured to evaluate the degree of oil oxidation.

[0036] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a method for optimizing the extraction conditions of rapeseed meal polyphenols and the application of rapeseed meal polyphenols, which has the following beneficial effects:

[0037] Based on single-factor experiments, this invention utilizes response surface methodology to optimize the extraction process conditions of rapeseed meal polyphenols and verify the antioxidant capacity of rapeseed meal polyphenols against oil oxidation. By determining the effects of five factors—ethanol concentration, hydrochloric acid concentration, extraction time, extraction temperature, and solid-liquid ratio—on the total phenol content of the rapeseed meal polyphenol extract, and selecting ethanol concentration, hydrochloric acid concentration, and extraction time as three factors based on single-factor experiments, the optimal extraction conditions can be quickly and accurately determined. This improves the total phenol content of rapeseed meal polyphenols, simplifies the extraction process, and reduces production costs, which is beneficial for the industrial production and further application of rapeseed meal polyphenols. The optimal extraction conditions for rapeseed meal polyphenols were ultimately determined as follows: ethanol concentration 54%, hydrochloric acid concentration 1 mol / L, extraction time 90 min, extraction temperature 55℃, and solid-liquid ratio 1:10. The rapeseed meal polyphenols were then purified and added to rapeseed oil, and their inhibitory effect on the formation of primary and secondary oxidation products was verified, providing a reference for the development and utilization of rapeseed meal polyphenols. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0039] Figure 1 The graph shows the effect of ethanol concentration on the total phenol content of rapeseed meal polyphenol extract in Example 1.

[0040] Figure 2 The graph shows the effect of hydrochloric acid concentration on the total phenol content of rapeseed meal polyphenol extract in Example 1.

[0041] Figure 3 This is a graph showing the effect of extraction time on the total phenol content of rapeseed meal polyphenol extract in Example 1.

[0042] Figure 4 This is a graph showing the effect of extraction temperature on the total phenol content of rapeseed meal polyphenol extract in Example 1.

[0043] Figure 5 This is a graph showing the effect of the material-to-liquid ratio on the total phenol content of rapeseed meal polyphenol extract in Example 1.

[0044] Figure 6The graph shows the effect of ethanol concentration and hydrochloric acid concentration on the total phenol content of rapeseed meal polyphenol extract in Example 1.

[0045] Figure 7 The graph shows the effect of extraction time and ethanol concentration on the total phenol content of rapeseed meal polyphenol extract in Example 1.

[0046] Figure 8 The graph shows the effect of extraction time and hydrochloric acid concentration on the total phenol content of rapeseed meal polyphenol extract in Example 1.

[0047] Figure 9 This is a graph showing the effect of different concentrations of rapeseed meal polyphenols on the peroxide value of rapeseed oil in Example 3;

[0048] Figure 10 This is a graph showing the effect of different concentrations of rapeseed meal polyphenols on the thiobarbituric acid value of rapeseed oil in Example 3.

[0049] Figure 11 The liquid chromatograms of rapeseed meal polyphenols, sine, and sinapic acid in Example 3 are shown. Detailed Implementation

[0050] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0051] A method for optimizing the extraction conditions of rapeseed meal polyphenols using response surface methodology and a processing method for synergistically improving rapeseed oil quality through enrichment, including...

[0052] S1. Pretreatment of rapeseed meal: After rapeseed is pressed for oil, the cruciferous seed meal - rapeseed meal - is collected, crushed and sieved to obtain rapeseed meal powder;

[0053] S2. Single-factor experiment: Five factors, namely ethanol concentration, hydrochloric acid concentration, extraction time, extraction temperature and material-liquid ratio, were determined. The effect of one factor on the total phenol content of the extract was measured by using rapeseed meal powder under the condition that other conditions remained unchanged.

[0054] S3. Response surface experimental design: Based on the results of the single-factor experiment, ethanol concentration, hydrochloric acid concentration and extraction time were selected as independent variables, and the total phenol content of the extract was selected as the response value. The Box-Behnken response surface design method was used to perform regression analysis on the experimental data, establish a quadratic polynomial model, and obtain a multiple regression equation. The variables were calculated through the regression equation to obtain the extraction conditions when the total phenol content of the rapeseed meal polyphenol extract was maximized.

[0055] S4. Purify rapeseed meal polyphenols using AB-8 macroporous resin;

[0056] S5. Add purified rapeseed meal polyphenols to rapeseed oil to obtain rapeseed oil enriched with rapeseed meal polyphenols.

[0057] The following specific examples further verify and illustrate this point.

[0058] Example 1

[0059] I. Optimization of Extraction Parameters for Rapeseed Meal Polyphenols through Single-Factor Experiments

[0060] An acidic ethanol solution with an ethanol volume concentration of 0–90% and a hydrochloric acid concentration of 0.01–2 mol / L was prepared. Pretreated rapeseed meal powder was weighed and mixed with the acidic ethanol solution at a material-to-liquid ratio of 1:5–1:40. The mixture was extracted at 25–85℃ for 15–120 min. Under the condition that other conditions remain unchanged, the effect of a certain factor on the total phenol content of the rapeseed meal polyphenol extract was determined.

[0061] Determination of total phenol content: The total phenol content of rapeseed meal polyphenol extract was determined by the Folin-Ciocalteu method.

[0062] (1) Effect of ethanol concentration on the total phenol content of rapeseed meal polyphenol extract: Acidic ethanol solutions with ethanol concentrations of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% rapeseed meal powder and a hydrochloric acid concentration of 0.5 mol / L were prepared. Pretreated rapeseed meal polyphenols were weighed and mixed with the acidic ethanol solutions at a material-to-liquid ratio of 1:20. The mixtures were extracted at 25℃ for 120 min, centrifuged, and the supernatant was collected. The total phenol content of the extract was determined. The results are as follows: Figure 1 As shown in Table 1.

[0063] Table 1

[0064]

[0065] The total phenol content of rapeseed meal polyphenol extract increased with increasing ethanol concentration from 0% to 60%, reaching its maximum at 60% ethanol concentration. The total phenol content decreased after the ethanol concentration exceeded 60%. This is because the mixture of ethanol and a small amount of water exhibits stronger polarity and higher efficiency in the extraction of phenolic substances. Therefore, the total phenol content of rapeseed meal polyphenol extract reached its maximum at an ethanol concentration of 60%.

[0066] (2) Effect of hydrochloric acid concentration on the total phenol content of rapeseed meal polyphenol extract: Acidic ethanol solutions with ethanol volume concentration of 60% and hydrochloric acid concentrations of 0.01 mol / L, 0.05 mol / L, 0.1 mol / L, 0.5 mol / L, 1 mol / L and 2 mol / L were prepared. Pretreated rapeseed meal polyphenols were weighed and mixed with the acidic ethanol solutions at a material-to-liquid ratio of 1:20. The mixture was extracted at 25℃ for 120 min, centrifuged, and the supernatant was collected. The total phenol content of the extract was determined. The results are as follows: Figure 2 As shown in Table 2.

[0067] Table 2

[0068]

[0069] As the hydrochloric acid concentration increased from 0.01 mol / L to 0.5 mol / L, the total phenolic content of the rapeseed meal polyphenol extract also increased, reaching its maximum at a hydrochloric acid concentration of 0.5 mol / L. This is because phenolic substances can bind to certain components in plants (such as polysaccharides and proteins) through hydrogen bonds and hydrophobic bonds. Acidic and alkaline hydrolysis can break the tight binding with cells, releasing the bound polyphenols. Therefore, a hydrochloric acid concentration of 0.5 mol / L was chosen to study the relationship between total phenolic content and hydrochloric acid concentration.

[0070] (3) Effect of extraction time on the total phenol content of rapeseed meal polyphenol extract: An acidic ethanol solution with an ethanol volume concentration of 60% and a hydrochloric acid concentration of 0.5 mol / L was prepared. Pretreated rapeseed meal polyphenols were weighed and mixed with the acidic ethanol solution at a material-to-liquid ratio of 1:20. The extraction times were 15 min, 30 min, 60 min, 90 min, and 120 min at 25℃. After centrifugation, the supernatant was collected, and the total phenol content of the extract was determined. The results are as follows: Figure 3 As shown in Table 3.

[0071] Table 3

[0072]

[0073] The total phenol content of rapeseed meal polyphenol extract is affected by the extraction time. After extraction times exceeding 30 minutes, the total phenol content of the rapeseed meal polyphenol extract did not increase significantly with prolonged extraction. Furthermore, excessively long extraction times may lead to the oxidation and polymerization of polyphenols into insoluble compounds. Therefore, an extraction time of 60 minutes is more suitable.

[0074] (4) Effect of extraction temperature on the total phenol content of rapeseed meal polyphenol extract: An acidic ethanol solution with an ethanol volume concentration of 60% and a hydrochloric acid concentration of 0.5 mol / L was prepared. Pretreated rapeseed meal polyphenols were weighed and mixed with the acidic ethanol solution at a material-to-liquid ratio of 1:20. The mixture was extracted at 25℃, 40℃, 55℃, 70℃ and 85℃ for 60 min, respectively. After centrifugation, the supernatant was collected, and the total phenol content of the extract was determined. The results are as follows: Figure 4 As shown in Table 4.

[0075] Table 4

[0076]

[0077] As the extraction temperature increases, the total phenol content of the rapeseed meal polyphenol extract continuously increases. Higher temperatures increase the diffusion rate and solubility of phenolic substances, but some polyphenolic compounds are oxidized and destroyed at high temperatures, undergoing hydroxyl hydrolysis. Therefore, a relatively mild temperature of 55℃ was chosen as the optimal extraction temperature.

[0078] (5) Effect of material-to-liquid ratio on the total phenol content of rapeseed meal polyphenol extract: An acidic ethanol solution with a volume concentration of 60% ethanol and a hydrochloric acid concentration of 0.5 mol / L was prepared. Pretreated rapeseed meal polyphenols were weighed and mixed with the acidic ethanol solution at material-to-liquid ratios of 1:5, 1:10, 1:20, 1:30, and 1:40, respectively. The mixtures were extracted at 85℃ for 60 min, centrifuged, and the supernatant was collected. The total phenol content of the extract was determined. The results are as follows: Figure 5 As shown in Table 5.

[0079] Table 5

[0080]

[0081] The relationship between the feed-to-liquid ratio and the total phenol content is approximately linear, increasing with the increase of the feed-to-liquid ratio. However, due to the strong biosorption capacity of rapeseed meal polyphenols, excess rapeseed meal polyphenols can adsorb free polyphenols. Therefore, this needs to be considered in actual experiments and production, and a feed-to-liquid ratio of 1:10 is selected.

[0082] II. Parameter Extraction through Response Surface Optimization

[0083] (1) Response surface methodology

[0084] Based on the results of the single-factor experiments above, the total phenol content of rapeseed meal polyphenol extract was used as the response value. Three factors that had a significant impact on it were selected: ethanol concentration, hydrochloric acid concentration, and extraction time. Response surface methodology experiments were designed (as shown in Table 6). Corresponding optimization experiments and variance analyses of rapeseed meal polyphenol extraction were conducted. The optimal extraction parameters obtained by fitting the equation were verified (as shown in Table 7).

[0085] Table 6 Response Surface Factors and Coding Levels

[0086]

[0087] Table 7 Response Surface Experimental Design and Total Phenolic Content

[0088]

[0089]

[0090] (2) Regression model establishment and analysis of variance

[0091] Using Design-Expert software to perform regression fitting on the data in the model, the regression equations between the three factors (A, B, C) and the total phenol content (Y) of the rapeseed meal polyphenol extract can be obtained:

[0092] Y=1.8100-0.1263A+0.0141B+0.0891C-0.0997AB+0.0686AC+

[0093] 0.0568BC-0.2632A 2 -0.0174B 2 -0.0120C 2 ;

[0094] Where Y represents the total phenol content of the rapeseed meal polyphenol extract, and the variable parameters A are the ethanol concentration, B are the hydrochloric acid concentration, and C is the extraction time.

[0095] As shown in the analysis of variance results (Table 8), in the significance test of the model, F = 19.53, p = 0.0004, which is statistically significant; while in the lack-of-fit term, F = 1.17, p > 0.05, indicating that the lack-of-fit term is not statistically significant. The corrected coefficient of determination R0 of the model is... 2 Adj =0.9125, indicating that the experimental model fits the experiment well and can effectively reflect the effects of ethanol concentration, hydrochloric acid concentration, and extraction time on the total phenolic content of rapeseed meal polyphenol extract. The F-value shows that A>C>B, indicating that the order of influence of each factor on the experimental results is: ethanol concentration > extraction time > hydrochloric acid concentration. The 3D response surface plot of rapeseed meal polyphenols is shown below. Figure 6 , Figure 7 , Figure 8 As shown.

[0096] Table 8. ANOVA results of the response surface quadratic model

[0097]

[0098] (3) Model Validation

[0099] Analysis using Design Expert software revealed the optimal extraction parameters for rapeseed meal polyphenols to be "ethanol concentration 54.01%, hydrochloric acid concentration 1 mol / L, and extraction time 90 min," with a predicted value of 1.97 mg SAE / mL. Considering actual operation (instrument settings limited accuracy), the optimal extraction process was selected as "ethanol concentration 54%, hydrochloric acid concentration 1 mol / L, and extraction time 90 min."

[0100] Based on the above analysis results, optimized parameters were obtained for the extraction of rapeseed meal polyphenols. Through three parallel experiments, the average total phenol content of the rapeseed meal polyphenol extract was determined to be 1.95 ± 0.04 mg SAE / mL, which is close to the simulated predicted value. This indicates that the extraction process optimized by the model is reliable and has practical significance.

[0101] Example 2

[0102] AB-8 macroporous resin was soaked in anhydrous ethanol overnight, then rinsed with ultrapure water until the effluent had no alcohol odor. Subsequently, the resin was soaked in 2 BV of 5% hydrochloric acid for 2 hours, then rinsed with ultrapure water until neutral. The resin was then soaked in 2 BV of 5% sodium hydroxide for 2 hours, followed by another rinse with ultrapure water until the pH of the effluent was neutral. The rapeseed meal polyphenol extract was concentrated by vacuum rotary evaporation. The concentrated crude extract was added to a pretreated AB-8 macroporous resin column (1000 mL, 60*600 mm) at a flow rate of 1-2 BV / h. The column, which had adsorbed phenolic compounds, was washed with 2 BV of ultrapure water at a flow rate of 2-3 BV / h to remove proteins and sugars. Then, elution was performed with 70% ethanol at a flow rate of 1-2 BV / h to obtain a purified rapeseed meal polyphenol solution. The solution was collected, concentrated, and dried. The purified solid rapeseed meal polyphenols were stored at 4°C.

[0103] The total phenol content of the purified solid rapeseed meal polyphenols obtained was 406.63 mg SAE / g.

[0104] Example 3

[0105] To verify the inhibitory effect of different concentrations of rapeseed meal polyphenols on the formation of primary and secondary oxidation products in rapeseed oil.

[0106] Dried and purified rapeseed meal polyphenols were dissolved in methanol and added to rapeseed oil at concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 g / L, respectively. After vortexing homogenization, the organic solvent components were removed by nitrogen purging. Untreated rapeseed oil served as a negative control, and rapeseed oil containing 0.2 g / L BHT served as a positive control. Freshly prepared rapeseed oil samples were poured into glass bottles and placed in a dark incubator at 60°C to accelerate oxidative rancidity. The degree of oil oxidation was assessed by measuring primary oxidation products (peroxide value) and secondary oxidation products (thiobarbituric acid value).

[0107] The results are as follows Figure 9 , Figure 10 As shown, under accelerated oxidation conditions, the peroxide value and thiobarbituric acid value of rapeseed oil increased with prolonged storage time, indicating that the rapeseed oil samples gradually oxidized. During accelerated oxidation, the peroxide value and thiobarbituric acid value of samples containing rapeseed meal polyphenols were observed to be lower than those of the negative control group. This indicates that rapeseed meal polyphenols can significantly delay the process of lipid oxidation, and the inhibitory effect on primary and secondary oxidation products is enhanced with increasing rapeseed meal polyphenol concentration. Compared with the positive control group containing rapeseed oil (0.2 g / L BHT), when the added concentration of rapeseed meal polyphenols exceeded 0.6 g / L, the inhibitory effect of rapeseed meal polyphenols on primary and secondary oxidation products was more significant.

[0108] Example 3

[0109] Identify the main phenolic components in rapeseed meal polyphenols.

[0110] UPLC chromatographic conditions: Mobile phase A: methanol; Mobile phase B: 2% acetic acid. Gradient elution conditions: 5–25% A (7.40 min), 25–29% A (2.67 min), 29–36% A (6.66 min), 36–45% A (6.67 min), 45–65% A (2.00 min), 65–5% A (2.00 min), 5% A (3.00 min). Equilibrate for 3 min before each injection.

[0111] The results are as follows Figure 11 As shown, the main phenolic components in rapeseed meal polyphenols are sinapic acid and sinapic alkaloids, with contents as high as 13.80% and 7.46%, respectively.

[0112] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0113] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An optimized method for extracting polyphenols from cruciferous seeds, characterized in that, Includes the following steps: Step 1: Pretreatment of rapeseed meal The rapeseed meal was crushed and passed through a 40-mesh sieve to obtain rapeseed meal powder. Step 2: Single-factor experiment Five factors were determined: ethanol concentration, hydrochloric acid concentration, extraction time, extraction temperature, and solid-liquid ratio. The effect of one of these factors on the total phenol content of the extract was measured by using rapeseed meal powder under constant conditions. Step 3: Response Surface Design Based on the results of the single-factor experiments, ethanol concentration, hydrochloric acid concentration, and extraction time were selected as independent variables, and the total phenol content of the extract was selected as the response value. The Box-Behnken response surface methodology was used to perform regression analysis on the experimental data, establishing a quadratic polynomial model and obtaining the multiple regression equation: Y=1.8100-0.1263A+0.0141B+0.0891C-0.0997AB+0.0686AC+0.0568BC-0.2632A 2 -0.0174B 2 -0.0120C 2 ; Where Y is the total phenol content of the rapeseed meal polyphenol extract, A is the ethanol concentration, B is the hydrochloric acid concentration, and C is the extraction time.

2. The optimized method for extracting polyphenols from cruciferous seeds according to claim 1, characterized in that, The volume concentrations of ethanol in step two are: 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%. The hydrochloric acid concentrations are 0.01 mol / L, 0.05 mol / L, 0.1 mol / L, 0.5 mol / L, 1 mol / L, and 2 mol / L; The extraction times were 15 min, 30 min, 60 min, 90 min and 120 min; The extraction temperatures were 25℃, 40℃, 55℃, 70℃ and 85℃; The material-to-liquid ratio is 1:5, 1:10, 1:20, 1:30 and 1:

40.

3. The optimized method for extracting polyphenols from cruciferous seeds according to claim 2, characterized in that, The experimental design described in step two is as follows: (2.1) Prepare acidic ethanol solutions with ethanol volume concentrations of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90% and hydrochloric acid concentration of 0.5 mol / L respectively. Weigh the pretreated rapeseed meal powder, mix it with the acidic ethanol solution at a material-to-liquid ratio of 1:20, extract at 25℃ for 120 min, centrifuge, collect the supernatant, and determine the total phenol content of the extract. (2.2) Prepare acidic ethanol solutions with ethanol volume concentration of 60%, hydrochloric acid concentrations of 0.01 mol / L, 0.05 mol / L, 0.1 mol / L, 0.5 mol / L, 1 mol / L and 2 mol / L respectively. Weigh the pretreated rapeseed meal powder, mix it with the acidic ethanol solution at a material-to-liquid ratio of 1:20, extract at 25℃ for 120 min, centrifuge, collect the supernatant, and determine the total phenol content of the extract. (2.3) Prepare an acidic ethanol solution with an ethanol volume concentration of 60% and a hydrochloric acid concentration of 0.5 mol / L. Weigh the pretreated rapeseed meal powder and mix it with the acidic ethanol solution at a material-to-liquid ratio of 1:

20. Extract the solution at 25℃ for 15 min, 30 min, 60 min, 90 min and 120 min respectively. Centrifuge the solution, collect the supernatant, and determine the total phenol content of the extract. (2.4) Prepare an acidic ethanol solution with an ethanol volume concentration of 60% and a hydrochloric acid concentration of 0.5 mol / L. Weigh the pretreated rapeseed meal powder and mix it with the acidic ethanol solution at a material-to-liquid ratio of 1:

20. Extract the solution at 25℃, 40℃, 55℃, 70℃ and 85℃ for 60 min respectively. Centrifuge the solution, collect the supernatant, and determine the total phenol content of the extract. (2.5) Prepare an acidic ethanol solution with an ethanol volume concentration of 60% and a hydrochloric acid concentration of 0.5 mol / L. Weigh the pretreated rapeseed meal powder and mix it with the acidic ethanol solution at material-to-liquid ratios of 1:5, 1:10, 1:20, 1:30 and 1:40, respectively. Extract the solution at 85℃ for 60 min, centrifuge, collect the supernatant, and determine the total phenol content of the extract.

4. The optimized method for extracting polyphenols from cruciferous seeds according to claim 3, characterized in that, The centrifugation speed was 8000 rpm and the time was 20 min.

5. An optimized method for extracting polyphenols from cruciferous seeds according to claim 1, characterized in that, Step three of the response surface methodology also includes: evaluating the significance and fit of the model using analysis of variance, analyzing the interaction between the three factors of ethanol concentration, hydrochloric acid concentration and extraction time on the total phenol content, and determining the optimal combination of levels for each factor.

6. An optimized method for extracting polyphenols from cruciferous seeds according to claim 5, characterized in that, The optimal extraction conditions are: ethanol volume concentration of 54%, hydrochloric acid concentration of 1 mol / L, extraction time of 90 min, extraction temperature of 55℃, and material-to-liquid ratio of 1:

10.

7. A rapeseed meal polyphenol extract obtained using the optimal extraction conditions described in claim 6.

8. A method for preparing and purifying rapeseed meal polyphenols using the rapeseed meal polyphenol extract according to claim 7, characterized in that, Purified rapeseed meal polyphenols were obtained by purification, concentration and drying using AB-8 resin.

9. The application of purified rapeseed meal polyphenols prepared by the method of claim 8, characterized in that, The purified rapeseed meal polyphenols were added to rapeseed oil.

10. A method for studying the inhibitory effect of rapeseed meal polyphenols on the formation of primary and secondary oxidation products of rapeseed oil, characterized in that, Includes the following steps: Purified rapeseed meal polyphenols were dissolved in rapeseed oil at concentrations of 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, and 1.0 g / L, respectively. Untreated rapeseed oil was used as a negative control, and rapeseed oil containing 0.2 g / L BHT was used as a positive control. Freshly prepared rapeseed oil samples were poured into glass vials and placed in a dark incubator at 60°C. The peroxide value and thiobarbituric acid value were measured to evaluate the degree of oil oxidation.

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