Method for extracting total phenols from rapeseed oil and method for testing the content thereof

Abstract

This invention discloses a method for extracting total phenols from rapeseed oil and a method for testing its content. The extraction method includes the following steps: Step 1: Weigh rapeseed oil into a glass test tube, add methanol, vortex to mix, sonicate, and freeze; Step 2: Transfer the upper layer liquid in the glass test tube to another clean test tube, dry it under weak nitrogen gas in a metal bath, redissolve the residue in a methanol-water solution, vortex to mix, and store for later use. The extraction method for total phenols from rapeseed oil in this invention uses methanol dissolution, ultrasonic-assisted extraction, and freezing to effectively separate the rapeseed oil and the extract. This method combines organic solvent extraction and ultrasonic-assisted extraction, significantly saving extraction time and yielding rapeseed oil with a high total phenol content. Furthermore, by replacing the standard (gallic acid) in the industry standard for determining the total phenol content of vegetable oils with canolol, this invention is more suitable for determining the total phenol content in rapeseed oil.

Description

Technical Field

[0001] This invention relates to a method for extracting polyphenols from vegetable oils and a method for detecting their content, specifically a method for extracting total phenols from rapeseed oil and a method for testing their content. Background Technology

[0002] There are many methods for extracting total phenols from rapeseed oil, with organic solvent extraction, ultrasound-assisted extraction, and solid-phase extraction being the most reported. Organic solvent extraction is simple, low-cost, and yields high purity, but its extraction rate is low and time-consuming. Ultrasound-assisted extraction has a high extraction rate and the extract is not easily damaged, but the product purity is not high. Solid-phase extraction has a high sample recovery rate and good precision, but solid-phase extraction columns are generally expensive.

[0003] Studies have found that total phenols in rapeseed oil are divided into phenolic acids and tannins. Phenolic acids include free phenolic acids and conjugated phenolic acids. Free phenolic acids account for 6.5-9.0% of total phenols, mainly sinapic acid, accounting for 70-85% of the total. Other components include small amounts of free p-salicylic acid, vanillic acid, gentianic acid, protocatechuic acid, syringic acid, p-coumaric acid, ferulic acid (cis and trans), caffeic acid, and chlorogenic acid. Conjugated phenolic acids account for 80% of total phenols, mainly sinapicine (also known as sinapic acid choline ester), which accounts for approximately 0.4-1.0% of rapeseed oil. Alkaline hydrolysis of conjugated phenolic acids produces small amounts of p-salicylic acid, vanillic acid, protocatechuic acid, syringic acid, p-coumaric acid, ferulic acid, and caffeic acid. Therefore, the polyphenols in cold-pressed and extracted rapeseed oils are mainly sinapic acid, sinapicine, sinapic acid glucoside, and cinnamic acid derivatives. Heat treatment can hydrolyze and decarboxylate sinapic acid and sinapic alkaloids into canolol (2,6-dimethoxy-4-vinylphenol). Therefore, the polyphenols in hot-pressed rapeseed oil are mainly canolol and small amounts of sinapic acid, sinapic alkaloids, sinapic acid glucoside, and cinnamic acid derivatives.

[0004] Currently, the determination of total phenol content in vegetable oils in my country mainly refers to the Folin-Ciocalteu method in the standard LS / T 6119—2017. This method uses gallic acid as a standard, and its detection principle is as follows: under alkaline conditions, Folin-Ciocalteu reagent oxidizes the phenolic hydroxyl groups (-OH groups) in polyphenols, producing a blue color. The intensity of the blue color is directly proportional to the number of phenolic groups. Gallic acid has three -OH groups in its molecular structure, while canolol, a representative polyphenol of rapeseed oil, has only one -OH group in its molecular structure, indicating a discrepancy in the structure and number of functional groups.

[0005] Literature shows that gallic acid is the most common standard for determining total phenols in vegetable oils such as soybean oil, olive oil, and camellia seed oil. However, the standards for determining total phenols in rapeseed or rapeseed oil vary, including sinapic acid, caffeic acid, tannic acid, and tannins. Sinapic acid is the most frequently used standard, and results are expressed as sinapic acid equivalents. However, no research or application of canolol as a standard has been found. Summary of the Invention

[0006] Given that current industry standards for the determination of total phenol content in vegetable oils are not very applicable to the determination of total phenol content in rapeseed oil, this invention provides a method for extracting total phenols from rapeseed oil and a method for testing its content.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A method for extracting total phenols from rapeseed oil, comprising the following steps:

[0009] Step 1: Weigh 0.5~1.5g of rapeseed oil into a glass test tube, add 5~15mL of methanol, vortex and mix for 1~5min, sonicate for 10~30min, and then freeze in a refrigerator at -90~-8℃ for 0.5~12h.

[0010] Step 2: Transfer the upper layer of liquid in the glass test tube to another clean test tube, dry it with weak nitrogen gas in a metal bath at 35~45℃, redissolve the residue in 5~15mL of methanol-water solution (methanol volume concentration 30~100%), vortex for 0.5~1.5min to mix well, and obtain rapeseed oil total phenol extract, which is stored at -18℃ for later use.

[0011] A method for testing the total phenol content of rapeseed oil includes the following steps:

[0012] Step 1: Transfer 0.25-1.25 mL each of the canolol standard working solution, methanol-water solution, and rapeseed oil total phenol extract into 10 mL colorimetric tubes;

[0013] Step 2: First add 4-6 mL of water, then add 0.5-1.5 mL of Folin-Ciocalteu reagent and 1-3 mL of sodium carbonate solution. Vortex for 0.5-1.5 min to mix well, then add water to bring the volume to the mark.

[0014] Step 3: React in a water bath at 60~80℃ for 20~40 minutes, then remove and cool to room temperature;

[0015] Step 4: Measure the absorbance at a wavelength of 760~780 nm;

[0016] Step 5: Plot a standard curve based on the absorbance of the working solution of each canolol standard and the canolol concentration of each working solution.

[0017] Step Six: Based on the standard curve drawn in Step Five, quantitatively determine the total phenol content in rapeseed oil using canolol as a calibration standard. The formula for calculating the total phenol content in rapeseed oil is as follows:

[0018]

[0019] In the formula, X is the total phenol content in rapeseed oil; C is the canolol concentration obtained from the standard curve; D is the dilution factor after the sample is brought to volume; if no dilution is performed, then D=1; V is the reconstituted volume of the extract after drying; and m is the mass of the rapeseed oil total phenol extract sample.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] 1. The extraction method of total phenols from rapeseed oil in this invention uses methanol dissolution and ultrasonic-assisted extraction. At the same time, freezing is used to better separate rapeseed oil and extract. Combining organic solvent extraction and ultrasonic-assisted extraction greatly saves extraction time and the extracted rapeseed oil has a high total phenol content.

[0022] 2. This invention replaces gallic acid (the standard in the industry standard for the determination of total phenol content in vegetable oil) with canolol, making it more suitable for the determination of total phenol content in rapeseed oil. Attached Figure Description

[0023] Figure 1 The chromatogram of polyphenols in rapeseed oil is as follows, from left to right: gallic acid, protocatechuic acid, methyl gallate, p-hydroxybenzoic acid, vanillic acid, syringic acid, vanillin, coniferyl alcohol, syringaldehyde, sinapic acid, ferulic acid, coniferyl aldehyde, salicylic acid, ellagic acid, cinnamic acid, and canolol.

[0024] Figure 2 Examples of liquid chromatograms for different samples;

[0025] Figure 3 For gallic acid standard curve;

[0026] Figure 4 This is the standard curve for Canolol;

[0027] Figure 5 The results are from a full-wavelength scan of the reaction solution. Detailed Implementation

[0028] The technical solution of the present invention will be further described below with reference to the accompanying drawings, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention that do not depart from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention.

[0029] This invention provides a method for testing the total phenol content of rapeseed oil, specifically including the following:

[0030] 1. Reagents and materials

[0031] Methanol (chromatographic grade), UP water, sodium carbonate, Folin-Ciocalteu reagent (2 mol / L), canolol standard (CAS No.: 28343-22-8, molecular formula: C 10 H 12 O3, relative molecular mass: 180.2005, purity: ≥95%.

[0032] 2. Instruments and equipment

[0033] Microplate reader, balance, vortex mixer, nitrogen blower, water bath, stoppered test tubes, 10mL colorimetric tubes.

[0034] 3. Solution preparation

[0035] 50% methanol-water solution: Accurately transfer 50 mL of methanol into a 100 mL volumetric flask, and add water to bring the volume to the mark.

[0036] 7.5% sodium carbonate solution (mass concentration): Weigh 7.5g ± 0.01g sodium carbonate, dissolve in an appropriate amount of water, transfer to a 100mL volumetric flask, dilute to the mark with water, and shake well.

[0037] 4. Preparation of standard solutions:

[0038] Canolol standard stock solution (1 mg / mL): Weigh 0.010 g ± 0.0001 g of canolol standard, dissolve it in a small amount of 50% methanol-water solution, transfer it to a 10 mL volumetric flask, dilute to the mark with 50% methanol-water solution, and shake well.

[0039] Canolol standard working solutions: Pipette 2.0, 1.0, 0.5, 0.2, and 0.1 mL of canolol standard stock solution into 10 mL volumetric flasks, respectively, and dilute to the mark with 50% methanol-water solution. Shake well to prepare canolol standard working solutions with concentrations of 200 µg / mL, 100 µg / mL, 50 µg / mL, 20 µg / mL, and 10 µg / mL.

[0040] 5. Extraction method of total phenols from rapeseed oil

[0041] Weigh 1.00g of rapeseed oil into a glass test tube, add 10mL of methanol, vortex and mix for 2 minutes, sonicate for 20 minutes, and then freeze at -18℃ for 12 hours. Transfer the upper layer of liquid in the glass test tube to another clean test tube, dry it under weak nitrogen gas in a metal bath at 40℃, redissolve the residue in 10mL of 50% methanol-water solution, vortex and mix for 1 minute, and store at -18℃ for later use. This invention combines organic solvent extraction and ultrasonic-assisted extraction, and uses freezing technology to separate rapeseed oil and total phenolic extract, greatly saving extraction time and increasing the polyphenol content of rapeseed oil.

[0042] 6. Detection Methods

[0043] Transfer 1 mL each of the canolol standard working solution, 50% methanol-water solution (blank), and the sample solution to a 10 mL colorimetric tube. First, add 5 mL of water, then add 1 mL of Folin-Ciocalteu reagent and 2 mL of 7.5% sodium carbonate solution. Vortex for 1 min to mix thoroughly. Add water to the mark and incubate in a 70°C water bath for 30 min. Remove from the water bath, cool to room temperature, and measure the absorbance (A) at a wavelength of 765 nm. If the absorbance is outside the range of the standard curve, adjust the sample weight or dilute the test solution appropriately, and then repeat the measurement.

[0044] A standard curve was plotted based on the absorbance (A) of the working solution of each canolol standard and the canolol concentration of each working solution.

[0045] 7. Result Presentation

[0046]

[0047] In the formula: X is the total phenol content in rapeseed oil, in milligrams per kilogram (mg / kg); C is the canolol concentration obtained from the standard curve, in micrograms per milliliter (μg / mL); D is the dilution factor after the sample is brought to volume; if no dilution is performed, D=1; V is the reconstituted volume of the extract after drying, in milliliters (mL); m is the mass of the rapeseed oil total phenol extract sample, in grams (g).

[0048] Parallel determination results are expressed as an arithmetic mean, and the results are rounded to two decimal places. The absolute difference between two independent determinations obtained under repeatability conditions must not exceed 10% of the arithmetic mean.

[0049] 8. Measurement Results

[0050] This invention is an improvement upon the standard LS / T 6119-2017, "Determination of Polyphenols in Vegetable Oils by Spectrophotometric Method for Grain and Oil Inspection." Experiments revealed a deficiency in using the LS / T 6119-2017 industry standard method for detecting total phenol content in rapeseed oil: the standard uses gallic acid as a standard to plot a standard curve and perform calculations, leading to an underestimation of the total phenol content in rapeseed oil. Therefore, this invention uses canolol as the standard, which is more scientifically sound and reasonable.

[0051] To verify the hypothesis that "using canolol as a standard is more scientifically reasonable," the present invention has undertaken the following work:

[0052] (a) Prove that canolol is the most abundant polyphenol component in rapeseed oil.

[0053] Table 1 presents the polyphenol content detection data of rapeseed oil from 13 rapeseed varieties. Figure 1 The table shows the liquid chromatogram of polyphenols in rapeseed oil as a mixed standard. Table 1 and... Figure 2 This indicates that canolol accounts for over 82% of the total phenolic content, verifying that canolol is the most abundant polyphenol in rapeseed oil. Therefore, using gallic acid as a standard to calculate the total phenolic content of rapeseed oil is not consistent with the actual situation of rapeseed oil, while using canolol as a standard to calculate the total phenolic content is more scientific and reasonable.

[0054] (b) Compare the differences in total phenol content between the two standards.

[0055] Gallic acid and canolol standard curves are as follows Figure 3 and Figure 4 As shown in Tables 2-4, the total phenol content of rapeseed oil was detected using gallic acid and canolol standards (detection wavelengths were 750 nm and 765 nm, respectively), and their comparisons are shown.

[0056] The data presented in Tables 2-4 show that when calculating the total phenol content of rapeseed oil using different standards, the calculated value using gallic acid as the standard, as specified in the existing industry standard LS / T 6119-2017, is lower than that using canolol as the standard.

[0057] Among the rapeseed oils from nine different rapeseed varieties, the value calculated using gallic acid was 73% (average) of the value calculated using canolol. This may be related to the molecular structures of the two standards. The structural formulas of gallic acid and canolol are as follows:

[0058]

[0059] Canolol (gallic acid)

[0060] As can be seen from the above structural formulas, gallic acid has three phenolic hydroxyl groups, while canolol has only one. This results in a significant difference when using the Folin-Ciocalteu method to determine the total phenol content; that is, all three phenolic hydroxyl groups in gallic acid participate in the colorimetric reaction, while only one phenolic hydroxyl group in canolol participates.

[0061] (c) Comparison of the detection effects of different extraction methods, reconstituted solutions and standards on rapeseed oil samples.

[0062] Table 5 shows the detection effects of different extraction methods, reconstitution solutions, and standards on rapeseed oil samples. From Table 5, it can be seen that: (1) Under the same reconstitution solution conditions, the detection values ​​of the three groups of samples by column chromatography were all lower than those by direct methanol extraction, so direct methanol extraction had a better detection effect. (2) Under the premise of direct methanol extraction, different reconstitution solutions had different effects on the detection effects of different rapeseed oil samples. For oil 10, the detection effect was better by reconstitution with 50% methanol-water; for oil 11, there was no significant difference in the detection effects of the two reconstitution solutions; for oil 12, the detection effect was better by reconstitution with methanol. (3) The calculated value using canolol as a standard was significantly higher than the calculated value using gallic acid as a standard.

[0063] (d) Results of full-wavelength scan of the reaction solution

[0064] The standard participated in the Folin-Ciocalteu reaction, and the results of a full-wavelength scan of the reaction solution were obtained by... Figure 5 As shown. By Figure 5 It can be seen that the maximum absorption wavelength of canolol after participating in the reaction with Folin-Ciocalteu is between 760 and 770 nm.

[0065] Table 1

[0066]

[0067] Table 2

[0068]

[0069] Table 3

[0070]

[0071] Table 4

[0072]

[0073] Table 5

[0074]

Claims

1. A method for testing the total phenol content of rapeseed oil, characterized in that, The method includes the following steps: Step 1: Weigh rapeseed oil into a glass test tube, add methanol, vortex to mix, sonicate, and freeze; Step 2: Transfer the upper liquid in the glass test tube to another clean test tube, blow it dry with weak nitrogen gas in a metal bath, redissolve the residue in methanol-water solution, vortex and mix well to obtain rapeseed oil total phenol extract. Step 3: Transfer the canolol standard working solution, methanol-water solution, and rapeseed oil total phenol extract into colorimetric tubes respectively; Step 4: Add water, Folin-Ciocalteu reagent, and sodium carbonate solution; vortex to mix; and add water to bring the volume to the mark. Step 5: After the water bath reaction, remove the product and cool it to room temperature; Step 6: Measure the absorbance at a wavelength of 760~780 nm; Step 7: Plot a standard curve based on the absorbance of the working solution of each canolol standard and the canolol concentration of each working solution. Step 8: Based on the standard curve drawn in Step 7, quantitatively determine the total phenol content in rapeseed oil using canolol as a calibration standard.

2. The method for testing the total phenol content of rapeseed oil according to claim 1, characterized in that, In step one, the vortex oscillation mixing time is 1~5 min, the ultrasonic time is 10~30 min, the freezing temperature is -90~-8℃, and the freezing time is 0.5~12 h.

3. The method for testing the total phenol content of rapeseed oil according to claim 1, characterized in that, In step two, the metal bath temperature is 35~45℃, and the vortex oscillation mixing time is 0.5~1.5min.

4. The method for testing the total phenol content of rapeseed oil according to claim 1, characterized in that, The ratio of rapeseed oil, methanol, and methanol-water solution is 0.5~1.5g:5~15mL:5~15mL, and the volume concentration of methanol in the methanol-water solution is 30~100%.

5. The method for testing the total phenol content of rapeseed oil according to claim 1, characterized in that, In step one, the concentrations of the canolol standard working solution are 200µg / mL, 100µg / mL, 50µg / mL, 20µg / mL, and 10µg / mL.

6. The method for testing the total phenol content of rapeseed oil according to claim 1, characterized in that, In step two, the vortex oscillation time is 0.5~1.5 min.

7. The method for testing the total phenol content of rapeseed oil according to claim 1, characterized in that, The volume ratio of the canolol standard working solution / methanol-water solution / rapeseed oil total phenol extract, Folin-Ciocalteu reagent, and sodium carbonate solution is 0.25~1.25mL:0.5~1.5mL:1~3mL, and the mass concentration of the sodium carbonate solution is 7.5%.

8. The method for testing the total phenol content of rapeseed oil according to claim 1, characterized in that, In step three, the water bath reaction temperature is 60~80℃ and the time is 20~40min.

9. The method for testing the total phenol content of rapeseed oil according to claim 1, characterized in that, In step six, the formula for calculating the total phenol content in rapeseed oil is as follows: In the formula, X is the total phenol content in rapeseed oil; C is the canolol concentration obtained from the standard curve; D is the dilution factor after the sample is brought to volume; if no dilution is performed, then D=1; V is the reconstituted volume of the extract after drying; and m is the mass of the rapeseed oil total phenol extract sample.

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