Method for extracting lutein from wheat
By using a heating extraction method involving a mixture of wheat flour products and n-butanol-water solution with acid, the problem of low extraction efficiency of wheat lutein was solved, achieving efficient and low-cost lutein extraction and improving extraction efficiency and stability.
Patent Information
- Application Number
- CN202610016057.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-07
- Publication Date
- 2026-04-10
AI Technical Summary
Existing methods for extracting lutein from wheat are inefficient and costly, and their efficiency improvement is limited, especially for high-lutein varieties.
The process involves mixing wheat flour products with a mixture of n-butanol and water, then mixing with an acid solution (such as phosphoric acid, hydrochloric acid, or sulfuric acid), heating for extraction, and optimizing process parameters through vortexing and centrifugation to improve extraction efficiency.
It significantly improved the extraction efficiency of wheat lutein, increasing it by 74% in common wheat varieties and by 36% in high-lutein varieties, while reducing solvent usage and operation time costs.
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Figure CN121824384A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of agricultural product processing and natural product extraction technology, specifically relating to a method for extracting wheat lutein. Background Technology
[0002] Lutein is one of the main components of carotenoids and a major pigment in the macular region of the human retina. It can effectively filter blue light and prevent eye diseases such as retinitis pigmentosa. The molecular structure of lutein contains conjugated double bonds and hydroxyl groups, giving it strong antioxidant properties. It can serve as an important natural antioxidant, and the extraction efficiency of lutein directly affects its application value.
[0003] Lutein lacks an effective synthetic pathway in humans and animals and can only be obtained from daily diets. Wheat, as a staple crop, can significantly enrich and increase the content and extraction yield of lutein in its grains through targeted breeding and optimized extraction processes, meeting the demand for fortified nutritional functions of whole grains. Wheat grain lutein exists in two forms: free and esterified. Esterified lutein is formed by lutein and fatty acids linked by ester bonds; its content is usually higher than that of the free form, and it has strong stability, but its solubility in organic solvents is low. Traditional methods for extracting lutein from wheat grains use a water-saturated n-butanol single solvent system, which suffers from technical drawbacks such as long extraction time and large solvent consumption. Existing literature on wheat lutein extraction methods still suffers from low extraction efficiency and high material costs, especially with limited improvement in extraction efficiency for high-lutein varieties. Summary of the Invention
[0004] In view of the deficiencies in the prior art, the purpose of this invention is to provide a method for extracting lutein from wheat, which can significantly improve the extraction efficiency of lutein.
[0005] The objective of this invention is achieved through the following technical solution: This invention provides a method for extracting lutein from wheat, comprising: Wheat flour products are mixed with a n-butanol-water mixture to obtain a wheat solvent system; Wheat lutein was obtained by mixing a wheat solvent system with an acid solution and then heating and extracting it. The acid solution includes any one or more of phosphoric acid solution, hydrochloric acid solution, and sulfuric acid solution.
[0006] Preferably, the molar concentration of hydrogen ions in the acid solution is 0.03~0.1 mol / L; when the wheat solvent system is mixed with the acid solution, the mass-to-volume ratio of the wheat processed flour product to the acid solution is (0.2~0.25) g : (24~30) μL.
[0007] Preferably, the temperature for heating and extraction is 35~55℃; and the time for heating and extraction is 20~22 min.
[0008] Preferably, the n-butanol-water mixture system comprises water-saturated n-butanol.
[0009] Preferably, the mass-to-volume ratio of the wheat flour product to the n-butanol-water mixture is (0.2~0.25) g : (1000~1250) μL.
[0010] Preferably, after mixing the wheat solvent system with the acid solution, the mixture is further subjected to vortex oscillation.
[0011] Preferably, the vortex oscillation time is 14-16 min.
[0012] Preferably, after the heating extraction is completed, centrifugation is also included.
[0013] Preferably, the centrifugation speed is 8000~10000 r / min, and the centrifugation time is 5~10 min.
[0014] This invention provides the application of the extraction method described in the above technical solution in improving the extraction rate of wheat lutein.
[0015] The beneficial effects of this invention are: This invention provides a method for extracting lutein from wheat, comprising: mixing wheat processed flour with a n-butanol-water mixture to obtain a wheat solvent system; mixing the wheat solvent system with an acid solution and then heating for extraction to obtain wheat lutein; wherein the acid solution includes any one or more of phosphoric acid solution, hydrochloric acid solution, and sulfuric acid solution. Esterified lutein is formed by the combination of lutein and fatty acids through ester bonds. The content of esterified lutein in wheat grains is higher than that of free lutein, exhibiting strong stability, but low solubility in organic solvents. Under acid catalysis, the acid can promote the cleavage of ester bonds by releasing protons, effectively promoting ester hydrolysis, promoting the release of bound lutein, and increasing the extraction yield of lutein. Simultaneously, the acid can also inhibit the oxidative decomposition of lutein. The heating extraction method used in this invention enhances the effect of the acid and accelerates solvent penetration, thereby further improving the extraction efficiency of lutein. The results of the embodiments of this invention show that the extraction method significantly improves the extraction efficiency of lutein from different varieties of wheat flour compared to conventional methods. The extraction method described in this invention improves the extraction efficiency by an average of 74% for common wheat varieties (an average increase of 1.2 μg / g in lutein extraction) and by an average of 36% for high-lutein varieties (an average increase of 1.3 μg / g in lutein extraction). The correlation analysis of the lutein content measured by the methods before and after the improvement showed a highly significant positive correlation, with a correlation coefficient of 0.974 (P < 0.001). Attached Figure Description
[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0017] Figure 1 The graph shows the correlation between the lutein content obtained in the comparative examples and the lutein content obtained in comparative examples 1-7. Figure 2 This is a comparison chart of wheat lutein content measured by two different methods. Detailed Implementation
[0018] This invention provides a method for extracting lutein from wheat, comprising: Wheat flour products are mixed with a n-butanol-water mixture to obtain a wheat solvent system; Wheat lutein was obtained by mixing a wheat solvent system with an acid solution and then heating and extracting it. The acid solution includes any one or more of phosphoric acid solution, hydrochloric acid solution, and sulfuric acid solution.
[0019] This invention mixes wheat processed flour products with a n-butanol-water mixture to obtain a wheat solvent system. This invention does not specifically limit the source of the wheat processed flour products; any conventional wheat processed flour products in the art can be used. As an optional embodiment of this invention, the wheat processed flour products can be wheat processed flour products of different wheat varieties; the different wheat varieties include any one or more of Zhoumai 36, Jimai 22, Jimai 24, Jimai 1803, Jimai 8040, Shannong 48, and Luliang 1. As an optional embodiment of this invention, the wheat processed flour products can be any one or more of whole wheat flour, wheat kernel flour, refined flour, and wheat bran. As an optional embodiment of the present invention, the n-butanol-water mixture system includes water-saturated n-butanol; the mass-to-volume ratio of the wheat flour product to the n-butanol-water mixture system can be (0.2~0.25) g:(1000~1250) μL, or 0.2 g:1000 μL, or 0.25 g:1250 μL, preferably 0.2 g:1000 μL. The extraction method provided by the present invention significantly reduces the amount of n-butanol-water mixture system added without adversely affecting the extraction efficiency of lutein. In combination with other reagents and steps, it can also significantly improve the extraction efficiency of lutein, achieving cost reduction and efficiency improvement. The amount of water-saturated n-butanol added is reduced from 1250 μL to 1000 μL, which, in addition to significantly reducing the amount of reagent used, also considers the ease of operation in practical applications. The 1000 μL volumetric pipette is the mainstream volumetric pipette used in laboratories. The 1000 μL volume of water-saturated n-butanol is well-suited to the aspiration volume of a 1000 μL volumetric pipette, allowing for the completion of a single sample addition in one operation without the need for secondary additions. In contrast, the 1250 μL volume requires at least two repeated aspirations to complete the addition of a single reagent. Compared to the 1000 μL volume of water-saturated n-butanol, under the catalysis of trace amounts of acid, not only does not reduce the extraction yield of lutein, but also improves the overall efficiency of the experiment and reduces time costs.
[0020] After obtaining the wheat solvent system, this invention mixes the wheat solvent system with an acid solution and then heats and extracts the mixture to obtain wheat lutein. The acid solution includes any one or more of phosphoric acid solution, hydrochloric acid solution, and sulfuric acid solution. As an optional embodiment of this invention, the molar concentration of hydrogen ions in the acid solution can be 0.03~0.1 mol / L, or it can be 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1 mol / L.
[0021] As an optional embodiment of the present invention, when the acid solution is a phosphoric acid solution, the molar concentration of phosphoric acid can be 0.34 mol / L; when the acid solution is a hydrochloric acid solution, the molar concentration of hydrochloric acid can be 0.05~0.1 mol / L, or 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1 mol / L; when the acid solution is a sulfuric acid solution, the molar concentration of sulfuric acid can be 0.025~0.05 mol / L, or 0.025, 0.03, 0.035, 0.04, 0.045, or 0.05 mol / L. In the present invention, when the wheat solvent system is mixed with the acid solution, the mass-to-volume ratio of the wheat processed flour product to the acid solution can be (0.2~0.25) g:(24~30) μL, or 0.2 g:24 μL, or 0.25 g:30 μL. In the preparation of wheat lutein, this invention involves adding phosphoric acid, hydrochloric acid, or sulfuric acid to the wheat solvent system. The acidic solution acts as a catalyst while simultaneously creating an acidic solvent system and an acidic environment. This environment inhibits the oxidative decomposition of lutein and promotes the release of bound lutein. Further comparative analysis reveals that phosphoric acid significantly and efficiently improves the extraction efficiency of wheat lutein. Based on its weak acidity and complexing properties, phosphoric acid gently disrupts cell structure while efficiently releasing bound lutein through phosphate complexation, simultaneously maximizing the stability of lutein, reducing degradation and side reactions, and thus enhancing chlorophyll extraction efficiency.
[0022] This invention, after mixing the wheat solvent system with an acid solution, further includes vortexing the mixture; the vortexing time can be 14-16 min, or 15 min. The main purpose of vortexing in this invention is to enhance the mass transfer efficiency of the extraction system, promoting the rapid and complete dissolution of lutein from the whole wheat flour matrix, laying the foundation for subsequent efficient extraction and accurate quantification. The shortened vortexing time and the introduction of water bath heating are complementary in efficiency. The main function of vortexing is to promote thorough mixing of the sample solution by increasing the contact area between the sample and the extract. Water bath heating increases the solubility of the organic solvent, accelerating the diffusion efficiency of lutein, effectively improving the solubility of lutein while avoiding degradation of lutein due to high temperatures. Both complement each other, jointly maximizing extraction efficiency. In addition, by introducing a water bath heating step, equipment can be diverted, enabling rapid turnover of equipment for processing multiple batches of samples. The water bath heating step can independently undertake part of the processing tasks without occupying core equipment such as shakers and centrifuges. This allows different equipment to complement each other in terms of time and function, avoiding the problem of "queuing" for single equipment, fundamentally reducing the competition for equipment resources and idleness, and enabling efficient parallel detection of large batches of samples.
[0023] After vortex oscillation, the resulting mixture is subjected to heating extraction. As an optional embodiment of the invention, the heating extraction temperature can be 35℃~55℃, or 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55℃; the heating extraction time can be 20~22 min, or 20, 21, or 22 min. As an optional embodiment of the invention, the heating extraction can be carried out under water bath conditions. The invention employs heating extraction during lutein extraction, which enhances the acid reaction effect and accelerates solvent penetration, thus further improving the extraction efficiency of lutein. Furthermore, by introducing a heating extraction step, the invention achieves equipment diversion, enabling rapid equipment turnover for multiple batches of sample processing. This allows different equipment to complement each other in terms of time and function, avoiding the "queueing" problem of a single device and enabling efficient parallel detection of large batches of samples.
[0024] After heating and extraction, the present invention preferably further includes centrifuging the reaction system. As an optional embodiment of the present invention, the centrifugation speed can be 8000~10000 r / min, or 8000, 9000, or 10000 r / min; the centrifugation time can be 5~10 min, or 5, 6, 7, 8, 9, or 10 min. The present invention primarily removes solid impurities and interfering components from the extraction system through centrifugation, obtaining a clear and pure lutein extract, laying the foundation for accurate absorbance determination by spectrophotometry. In the extraction method provided by the present invention, the centrifugation speed is reduced by 20%, and the centrifugation time is shortened by 50%, reducing equipment wear and extending the centrifuge's service life. Energy consumption is reduced while ensuring extraction efficiency. Furthermore, the factor causing the reduction in centrifugation speed and time is that the addition of trace amounts of acid effectively promotes the extraction efficiency of lutein from wheat seeds, and 8000 rpm for 5 minutes is sufficient to settle impurities and obtain a clear supernatant, eliminating the need to increase the extraction time of the sample solution through centrifugation.
[0025] After centrifugation, the present invention obtains a system containing wheat lutein, that is, wheat lutein.
[0026] After obtaining a system containing wheat lutein, this invention allows for the determination of lutein content in the system using an enzyme-linked immunosorbent assay (ELISA) reader. This invention replaces a spectrophotometer with an ELISA reader, enabling simultaneous detection of multiple samples and reducing sample loading volume. By using an ELISA reader instead of a spectrophotometer to detect the absorbance value of lutein content, this invention transforms the detection of a single sample at a time into the simultaneous detection of multiple samples, reducing the sample loading volume from 1 mL to 200 μL, significantly improving detection efficiency and reducing sample loading volume.
[0027] This invention provides the application of the extraction method described above in improving the extraction rate of lutein from wheat. The results of the embodiments of this invention demonstrate that the extraction method described above can significantly improve the extraction efficiency of lutein from whole wheat flour of different varieties compared to conventional methods.
[0028] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.
[0029] The preparation method of whole wheat flour used in the following technical solutions is as follows: Wheat grain moisture content was determined using a PM-8188-A grain moisture meter, and grain hardness was determined using an SKCS 4100 single-grain grain characteristic tester. The required water content for rinsing was determined based on the measured hardness: 14% water was required for soft wheat with a hardness index ≤ 40; 15% for mixed wheat with a hardness index range of 40–60; and 16% for hard wheat with a hardness index > 60. The wheat was rinsed at room temperature for 16–18 hours. Whole wheat flour was prepared using an FW400A high-speed universal grinder. The whole wheat flour was stored at 4°C.
[0030] The formula for calculating lutein content is: Lutein content (μg / g) = (Sample A - Blank A) × 30.1.
[0031] In the following examples or comparative examples, each experiment was performed in ≥3 parallel trials.
[0032] The preparation method of water-saturated n-butanol is as follows: at room temperature, n-butanol (Chinese medicine) and distilled water are mixed at a volume ratio of 1:1, and the mixture is allowed to stand overnight or sonicated for 10 min and then allowed to stand until the solution is separated into layers. The upper layer solution is water-saturated n-butanol.
[0033] Unless otherwise specified, all the following tests were conducted at room temperature (20~25℃).
[0034] Wheat kernel flour, refined flour, and bran were all prepared using the French CHOPIN experimental mill.
[0035] Example 1 A method for extracting lutein, comprising the following steps: Ingredients: Shannong 48 whole wheat flour.
[0036] (1) Accurately weigh 0.20 g of whole wheat flour into a 2 mL centrifuge tube.
[0037] (2) Add 1000 μL of water-saturated n-butanol to the centrifuge tube and accurately inject 24 μL of H3PO4 (≥85%) solution (phosphoric acid concentration of 0.34 mol / L) using a pipette.
[0038] (3) Immediately vortex oscillate for 15 minutes to ensure sufficient and uniform oscillation.
[0039] (4) Transfer to a constant temperature water bath and heat at 45°C for 20 min.
[0040] (5) Centrifuge at 8000 r / min for 5 min, and transfer 200 μL of the supernatant to the enzyme label strip.
[0041] (6) Using water-saturated n-butanol as a blank control, the absorbance at 436.5 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.
[0042] (7) Calculate the final lutein content according to the formula: lutein content (μg / g) = (A sample - A blank) × 30.1, which is the amount of lutein extracted per gram of whole wheat flour.
[0043] The method for preparing lutein is referred to as the modified method in the following tables, and so on.
[0044] Comparative Example 1 A method for extracting lutein, comprising the following steps: Ingredients: Shannong 48 whole wheat flour.
[0045] (1) Accurately weigh 0.25 g of whole wheat flour into a 2 mL centrifuge tube.
[0046] (2) Add 1250 μL of water-saturated n-butanol.
[0047] (3) Immediately vortex oscillate for 30 minutes to ensure sufficient and uniform oscillation.
[0048] (4) Let stand for 10 minutes to fully extract.
[0049] (5) Centrifuge at 10000 r / min for 10 min.
[0050] (6) Calibrate using water-saturated n-butanol as a blank control and measure the absorbance value A at 436.5 nm using a spectrophotometer.
[0051] (7) Calculate the final lutein content according to the formula: Lutein content (μg / g) = A × 30.1.
[0052] The method for preparing lutein is referred to as the pre-modification method in Table 1 below, and the same applies hereinafter.
[0053] Example 2 A method for extracting lutein, with the same steps as in Example 1, the only difference being that the raw material is Zhoumai 36 whole wheat flour.
[0054] Comparative Example 2 A method for extracting lutein, with the same steps as Comparative Example 1, the only difference being that the raw material is Zhoumai 36 whole wheat flour.
[0055] Example 3 A method for extracting lutein, with the same steps as in Example 1, the only difference being that the raw material is Jimai 22 whole wheat flour.
[0056] Comparative Example 3 A method for extracting lutein, with the same steps as Comparative Example 1, the only difference being that the raw material is Jimai 22 whole wheat flour.
[0057] Example 4 A method for extracting lutein, with the same steps as in Example 1, the only difference being that the raw material is Jimai 24 whole wheat flour.
[0058] Comparative Example 4 A method for extracting lutein, with the same steps as Comparative Example 1, the only difference being that the raw material is Jimai 24 whole wheat flour.
[0059] Example 5 A method for extracting lutein, the steps are the same as in Example 1, the only difference being that the raw material is Jimai 1803 whole wheat flour.
[0060] Comparative Example 5 A method for extracting lutein, with the same steps as Comparative Example 1, the only difference being that the raw material is Jimai 1803 whole wheat flour.
[0061] Example 6 A method for extracting lutein, the steps are the same as in Example 1, the only difference being that the raw material is Jimai 8040 whole wheat flour.
[0062] Comparative Example 6 A method for extracting lutein, with the same steps as Comparative Example 1, the only difference being that the raw material is Jimai 8040 whole wheat flour.
[0063] Example 7 A method for extracting lutein, the steps are the same as in Example 1, the only difference being that the raw material is Luliang No. 1 whole wheat flour.
[0064] Comparative Example 7 A method for extracting lutein, with the same steps as Comparative Example 1, the only difference being that the raw material is Luliang No. 1 whole wheat flour.
[0065] Application Example 1 The correlation between the lutein content obtained in Examples 1-7 and the lutein content obtained in Comparative Examples 1-7 is as follows: Figure 1 As shown in Table 1, the results of lutein content preparation for each wheat variety in Examples 1-7 and Comparative Examples 1-7 are presented in Table 1 and 2. Figure 2As shown in Table 2, the mean values obtained by the methods before and after the improvement are shown in Table 2.
[0066] Table 1. Results of lutein content preparation for each wheat variety in Examples 1-7 and Comparative Examples 1-7
[0067] Table 2. Basic information on the average lutein content of 7 wheat varieties.
[0068] From Tables 1-2 and Figure 1 It can be seen that the lutein content extracted by the method before the improvement is significantly lower than that of the lutein extraction method used in this invention. The lutein extraction method provided by this invention can effectively increase the amount of lutein extracted from wheat grains while reducing costs.
[0069] Example 8 A method for extracting lutein, with the same steps as in Example 1, the only difference being that the raw material is wheat kernel powder. Lutein was extracted from the wheat kernel powder of Shannong 48, Jimai 24, Luliang 1, Jimai 1803, and Jimai 8040, respectively.
[0070] Example 9 A method for extracting lutein, with the same steps as in Example 1, the only difference being that the raw material is refined flour. Lutein was extracted from the refined flour of Shannong 48, Jimai 24, Luliang 1, Jimai 1803, and Jimai 8040, respectively.
[0071] Example 10 A method for extracting lutein, with the same steps as in Example 1, the only difference being that the raw material is wheat bran. Lutein was extracted from the wheat bran of Shannong 48, Jimai 24, Luliang 1, Jimai 1803, and Jimai 8040, respectively.
[0072] Application Example 2 The results of measuring the lutein content obtained from Examples 8-10 are shown in Table 3.
[0073] Table 3. Results of lutein content determination obtained from Examples 8-10
[0074] The lutein content was calculated as the average of three biological replicates and three technical replicates, with the unit being μg / g.
[0075] Example 11 A method for extracting lutein, with the same steps as in Example 1, except that the centrifugation speed and time are 10,000 rpm for 10 min.
[0076] Lutein was extracted from whole wheat flour using the method described in Example 1.
[0077] The results of lutein extraction in Example 11 are shown in Table 4.
[0078] Table 4 Results of lutein extraction in Example 11
[0079] The lutein content was calculated by averaging three biological replicates and three technical replicates, and the results showed no significant differences. The unit for each result is μg / g.
[0080] Comparative Example 8 A method for extracting lutein, with the same steps as Comparative Example 1, except that: 0.2 g of whole wheat flour and 1000 μL of water-saturated n-butanol are used.
[0081] Meanwhile, lutein was extracted from wheat using the method of Comparative Example 1, and the results are shown in Table 5.
[0082] Table 5 Results of lutein extraction in Comparative Example 8
[0083] The lutein content was calculated as the average of three biological replicates and three technical replicates, and there were no significant differences in the results. The units were all µg / g.
[0084] Comparative Example 9 A method for extracting lutein, with the same steps as in Example 1, except that phosphoric acid solution is not added. The specific steps are as follows: Ingredients: Shannong 48 whole wheat flour.
[0085] (1) Accurately weigh 0.20 g of whole wheat flour into a 2 mL centrifuge tube.
[0086] (2) Add 1000 μL of water-saturated n-butanol to the centrifuge tubes.
[0087] (3) Immediately vortex oscillate for 15 minutes to ensure sufficient and uniform oscillation.
[0088] (4) Transfer to a constant temperature water bath and heat at 45°C for 20 min.
[0089] (5) Centrifuge at 8000 r / min for 5 min, and transfer 200 μL of the supernatant to the enzyme label strip.
[0090] (6) Using water-saturated n-butanol as a blank control, the absorbance at 436.5 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.
[0091] (7) Calculate the final lutein content according to the formula: Lutein content (μg / g) = (A sample - A blank) × 30.1.
[0092] Lutein was extracted from whole wheat flour using the method described in Example 1. The results are shown in Table 6. The control group represents the results obtained by conducting the experiment entirely according to the method described in Example 1, while the experimental group did not contain added phosphoric acid.
[0093] Table 6 Results of lutein extraction in Comparative Example 9
[0094] The lutein content results are the average of three biological replicates and three technical replicates, and the unit is μg / g.
[0095] Comparative Example 10 A method for extracting lutein, with the same steps as in Example 1, except that a water bath heating process is not performed. The specific steps are as follows: Ingredients: Shannong 48 whole wheat flour.
[0096] (1) Accurately weigh 0.20 g of whole wheat flour into a 2 mL centrifuge tube.
[0097] (2) Add 1000 μL of water-saturated n-butanol to the centrifuge tube and accurately inject 24 μL of H3PO4 (≥85%) solution (phosphoric acid concentration of 0.34 mol / L) using a pipette.
[0098] (3) Immediately vortex oscillate for 15 minutes to ensure sufficient and uniform oscillation.
[0099] (4) Let stand at room temperature for 20 min.
[0100] (5) Centrifuge at 8000 r / min for 5 min, and transfer 200 μL of the supernatant to the enzyme label strip.
[0101] (6) Using water-saturated n-butanol as a blank control, the absorbance at 436.5 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.
[0102] (7) Calculate the final lutein content according to the formula: Lutein content (μg / g) = (A sample - A blank) × 30.1.
[0103] Lutein was extracted from whole wheat flour simultaneously using the method described in Example 1. The control group consisted of samples obtained entirely according to the method described in Example 1, while the experimental group did not undergo water bath heating. The results of lutein extraction from Comparative Example 10 are shown in Table 7.
[0104] Table 7 Results of lutein extraction in Comparative Example 10
[0105] The lutein content results are the average of three biological replicates and three technical replicates, and the unit is μg / g.
[0106] Comparative Example 11 A method for extracting lutein, with the same steps as in Example 1, except that acetic acid solution is used instead of phosphoric acid solution, the concentration of acetic acid in the acetic acid solution is 0.41 mol / L, and the amount used is 24 μL.
[0107] Lutein was extracted from whole wheat flour simultaneously using the method described in Example 1. The control group consisted entirely of samples obtained by following the method described in Example 1, while the experimental group used acetic acid solution instead of phosphoric acid solution. The results of lutein extraction from Comparative Example 11 are shown in Table 8.
[0108] Table 8 Results of lutein extraction in Comparative Example 11
[0109] The lutein content results are the average of three biological replicates and three technical replicates, and the unit is μg / g.
[0110] Example 12 A method for extracting lutein, with the same steps as in Example 1, except that hydrochloric acid solution of different molar concentrations is used instead of phosphoric acid solution, with the different molar concentrations of hydrochloric acid solution being 0.05 mol / L and 0.1 mol / L, respectively.
[0111] Lutein was extracted from whole wheat flour simultaneously using the method described in Example 1. The control group consisted entirely of samples obtained by following the method described in Example 1. The experimental group used 0.05 mol / L and 0.1 mol / L hydrochloric acid solutions instead of phosphoric acid solution. The results of lutein extraction in Example 12 are shown in Table 9.
[0112] Table 9 Results of lutein extraction in Example 12
[0113] The lutein content results are the average of three biological replicates and three technical replicates, and the unit is μg / g.
[0114] Example 13 A method for extracting lutein, with the same steps as in Example 1, except that sulfuric acid solution of different molar concentrations is used instead of phosphoric acid solution, with the sulfuric acid solutions of different molar concentrations being 0.025 mol / L and 0.05 mol / L, respectively.
[0115] Lutein was extracted from whole wheat flour simultaneously using the method described in Example 1. The control group consisted entirely of samples obtained by following the method described in Example 1. The experimental group used 0.025 mol / L and 0.05 mol / L sulfuric acid solutions instead of phosphoric acid solution. The results of lutein extraction in Example 13 are shown in Table 10.
[0116] Table 10 Results of lutein extraction in Example 13
[0117] The lutein content results are the average of three biological replicates and three technical replicates, and the unit is μg / g.
[0118] Hydrochloric acid and sulfuric acid dilutions were prepared according to the listed gradients. The results showed that, under consistent experimental conditions, the addition of phosphoric acid solution maintained the best extraction effect for lutein from wheat seeds when the acid solution addition volume was 24 μL. The addition of acetic acid, hydrochloric acid, and sulfuric acid, compared to the previous method, had a certain promoting effect on the extraction effect of lutein from wheat seeds. However, due to its strong pungent odor and volatility, and its strong corrosiveness and hygroscopicity, sulfuric acid solution was also excluded from conventional extraction experiments for safety reasons. Therefore, acetic acid and phosphoric acid were determined to be the relatively optimal reagents for preparing lutein.
[0119] Example 14 A method for extracting lutein, with the same steps as in Example 1, the only difference being: heating in a constant temperature water bath at 35°C for 20 minutes.
[0120] Lutein was extracted from whole wheat flour simultaneously using the method described in Example 1. The control group was obtained by conducting the experiment entirely according to the method described in Example 1, while the experimental group was heated in a constant temperature water bath at 35°C for 20 min. The results of lutein extraction from Comparative Example 14 are shown in Table 11.
[0121] Table 11 Results of lutein extraction in Example 14
[0122] The lutein content results are the average of three biological replicates and three technical replicates, and the unit is μg / g.
[0123] Example 15 A method for extracting lutein, with the same steps as in Example 1, except that the constant temperature water bath is heated at 55°C for 20 minutes.
[0124] Lutein was extracted from whole wheat flour simultaneously using the method described in Example 1. The control group consisted of samples obtained entirely according to the method described in Example 1, while the experimental group was heated in a constant temperature water bath at 35°C for 20 min. The results of lutein extraction in Example 15 are shown in Table 12.
[0125] Table 12 Results of lutein extraction in Example 15
[0126] The lutein content results are the average of three biological replicates and three technical replicates, and the unit is μg / g.
[0127] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A method for extracting lutein from wheat, characterized in that, include: Wheat flour products are mixed with a n-butanol-water mixture to obtain a wheat solvent system; Wheat lutein was obtained by mixing a wheat solvent system with an acid solution and then heating and extracting it. The acid solution includes any one or more of phosphoric acid solution, hydrochloric acid solution, and sulfuric acid solution.
2. The extraction method according to claim 1, characterized in that, The molar concentration of hydrogen ions in the acid solution is 0.03~0.1 mol / L; when the wheat solvent system is mixed with the acid solution, the mass-volume ratio of the wheat processed flour product to the acid solution is (0.2~0.25) g : (24~30) µL.
3. The extraction method according to claim 1, characterized in that, The temperature for the heating extraction is 35℃~55℃; the heating extraction time is 20~22 min.
4. The extraction method according to claim 1, characterized in that, The n-butanol-water mixture system includes water-saturated n-butanol.
5. The extraction method according to claim 1 or 4, characterized in that, The mass-to-volume ratio of the wheat flour product to the n-butanol-water mixture is (0.2~0.25) g : (1000~1250) µL.
6. The extraction method according to claim 1, characterized in that, The process of mixing the wheat solvent system with the acid solution also includes vortexing the mixture.
7. The extraction method according to claim 6, characterized in that, The duration of the vortex oscillation is 14-16 minutes.
8. The extraction method according to claim 1, characterized in that, After the heating extraction is complete, centrifugation is also performed.
9. The extraction method according to claim 8, characterized in that, The centrifugation speed is 8000~10000 r / min, and the centrifugation time is 5~10 min.
10. The application of the extraction method according to any one of claims 1 to 9 in improving the extraction rate of wheat lutein.