Rapid extraction and component determination method for fatty acid in flax seeds

Abstract

The invention provides a flax seed fatty acid rapid extraction and component determination method, relates to the technical field of agricultural chemical analysis, and has the technical key points that the extraction method comprises the following steps: taking flax seeds and crushing; saponification and methyl esterification reaction: weighing the crushed flax seeds in the step S1, placing the flax seeds in a centrifugal tube with a plug, adding a 1% methanol-sodium hydroxide solution, shaking to fully and uniformly mix the sample and the solution, and standing to complete the reaction; neutralizing the alkali in the solution; n-heptane is added into the solution, and generated fatty acid methyl ester is fully extracted. By optimizing an extraction solvent system, innovatively combining extraction and derivatization steps and adopting an efficient instantaneous extraction / reaction technology, the total extraction time is shortened from tens of hours of a traditional method to tens of minutes on the premise of ensuring the extraction efficiency and the methyl esterification conversion rate, meanwhile, the solvent dosage and the operation steps are reduced, and the production cost is reduced. Powerful technical support is provided for quality control, functional food development and genetic breeding of the flaxseeds.

Description

Technical Field

[0001] This invention relates to the field of agricultural chemical analysis technology, and in particular to a method for rapid extraction and component determination of fatty acids from sesame seeds. Background Technology

[0002] Flax, also known as oil flax, is an important oilseed crop. Its seeds are rich in oil and have a unique fatty acid composition, making them highly nutritious. A prominent feature is its high content of alpha-linolenic acid, an essential omega-3 fatty acid with various physiological functions, including lowering blood lipids, anti-inflammation, improving cognitive function, and preventing cardiovascular diseases. Therefore, accurate and rapid analysis of the fatty acid composition of flax seeds is crucial for its quality identification, nutritional value assessment, variety selection, and processing utilization.

[0003] Currently, the standard procedure for fatty acid analysis in flaxseed is: fat extraction → methyl esterification derivatization → gas chromatography analysis. Among these steps, sample pretreatment (fat extraction) is the main bottleneck and source of error. Traditional Soxhlet extraction is considered the "gold standard," but it is time-consuming (6-24 hours), consumes a large amount of solvent, and is cumbersome and inefficient, failing to meet the demands of high-throughput rapid detection. While some improved methods (such as shaking and ultrasonic extraction) have shortened the time, their extraction efficiency is easily affected by operating parameters, resulting in poor reproducibility. Furthermore, they still require multiple steps such as filtration, transfer, and concentration, making the process cumbersome and increasing the risk of sample loss and contamination.

[0004] Furthermore, the existing technical process has an inherent flaw: extraction and derivatization are two separate, disconnected steps. This not only prolongs the overall analysis time but also introduces additional reagents and operational errors. For flaxseed analysis, its high content of polyunsaturated fatty acids (especially α-linolenic acid) is sensitive to light, heat, and oxygen. The lengthy and complex processing can easily lead to fatty acid oxidation and degradation, distorting the analytical results and severely limiting the efficiency of rapid screening of large-scale samples in modern agricultural breeding. Summary of the Invention

[0005] The purpose of this invention is to solve the problems of unstable extraction efficiency and disconnection from derivation steps in existing technologies.

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

[0007] A method for rapid extraction of fatty acids from sesame seeds, characterized by comprising the following steps:

[0008] S1: Sample preparation: Take sesame seeds and crush them.

[0009] S2: Saponification and methyl esterification reaction: Weigh the crushed sesame seeds from S1, place them in a stoppered centrifuge tube, add 1% methanol-sodium hydroxide solution, shake to mix the sample and solution thoroughly, and let stand to complete the reaction;

[0010] S3: Neutralizes the alkali in the solution;

[0011] S4: Extraction: Add n-heptane to the solution to fully extract the generated fatty acid methyl esters.

[0012] Preferably, the pulverized sesame seeds in step S1 are sieved through a 60-mesh sieve.

[0013] Preferably, the addition ratio of sesame seeds to 1% methanol-sodium hydroxide solution in S2 is 0.5g:5mL, and the standing time in S2 is 30min.

[0014] Preferably, in step S3, an acetic acid solution is added dropwise to a centrifuge tube, and excess alkali is neutralized by gentle shaking.

[0015] Preferably, after adding n-heptane in step S4, the tube is sealed tightly, shaken vigorously for 5 minutes, and then left to stand for 5 minutes. After the solution has fully separated into layers, the upper n-heptane extract is taken and filtered to obtain the desired fatty acid.

[0016] The S4 uses a 0.22 μm organic needle filter for filtration.

[0017] This application also provides a method for detecting fatty acids from sesame seeds, the method being used to detect fatty acids obtained by the rapid extraction method for sesame seeds described above, and the method using gas chromatography.

[0018] Preferably, the detection conditions of the detection method are set as follows:

[0019] Chromatographic column: FFAP flexible quartz capillary column (30 m × 0.125 mm × 0.13 μm);

[0020] The temperature program is as follows: initially 100℃ and hold for 2 minutes, then increase to 220℃ at a rate of 5℃ / min and hold for 13 minutes;

[0021] Inlet temperature: 250℃;

[0022] FID detector temperature: 250℃;

[0023] Airflow rate: 400 mL / min;

[0024] Hydrogen flow rate: 40 mL / min;

[0025] Nitrogen pressure: 11620 kPa;

[0026] Flow split ratio: 1:50;

[0027] Injection volume: 1 μL.

[0028] Compared with the prior art, this application has the following beneficial effects:

[0029] This application optimizes the extraction solvent system, innovatively combines extraction and derivatization steps, and adopts efficient instantaneous extraction / reaction technology. While ensuring extraction efficiency and methyl esterification conversion rate, the total extraction time is shortened from tens of hours in traditional methods to tens of minutes. At the same time, the amount of solvent used and the number of operation steps are reduced, providing strong technical support for the quality control of sesame seeds, the development of functional foods, and genetic breeding. Attached Figure Description

[0030] Figure 1 This is a flowchart illustrating a method for rapid extraction of fatty acids from sesame seeds according to one embodiment of this application.

[0031] Figure 2 This is a typical gas chromatogram according to one embodiment of the present invention;

[0032] Figure 3 This is a comparison chart of the extraction efficiency of the extraction method of the present invention (Method 1) and the traditional Soxhlet extraction-boron trifluoride methyl esterification method (Method 2);

[0033] Figure 4 This is a comparison chart of the extraction efficiency of the method of the present invention and the ultrasonic-assisted extraction method on two different sesame varieties (variety 1 and variety 5). Detailed Implementation

[0034] The present invention will be further described in detail below with reference to specific embodiments.

[0035] Please see Figure 1 This application also provides a method for rapid extraction of fatty acids from sesame seeds, comprising the following steps:

[0036] S1: Sample pretreatment

[0037] Take sesame seeds, crush them, and pass them through a 60-mesh sieve.

[0038] S2: Saponification and methyl esterification reaction:

[0039] Weigh out a sample of sesame seed powder, place it in a stoppered centrifuge tube, add 1% methanol-sodium hydroxide solution, shake to mix the sample and solution thoroughly, let stand for 30 min, and carry out saponification and methyl esterification reactions.

[0040] In one embodiment, 0.5 g of sesame seed powder sample was weighed, accurate to 0.0001 g, and 5 mL of 1% methanol-sodium hydroxide solution was added.

[0041] S3: Neutralizes the alkali in the solution.

[0042] In one embodiment, after the reaction in S2 is complete, 0.2 mL of acetic acid solution is added dropwise to the centrifuge tube, and the excess alkali is neutralized by gentle shaking.

[0043] S4: Extraction:

[0044] Add 5 mL of n-heptane to the S3 mixture, tighten the stopper, and shake vigorously for 5 minutes to fully extract the generated fatty acid methyl esters. Let stand for 5 minutes until the solution has fully separated into layers. Take the upper n-heptane extract and filter it through a 0.22 μm organic syringe filter. The resulting filtrate is the sample for gas chromatography analysis.

[0045] This application also provides a method for detecting fatty acids in sesame seeds as described above, wherein the detection method uses gas chromatography (GC), and the detection conditions are set as follows:

[0046] Chromatographic column: FFAP flexible quartz capillary column (30 m × 0.125 mm × 0.13 μm);

[0047] The temperature program is as follows: initially 100℃ and hold for 2 minutes, then increase to 220℃ at a rate of 5℃ / min and hold for 13 minutes;

[0048] Inlet temperature: 250℃;

[0049] FID detector temperature: 250℃;

[0050] Airflow rate: 400 mL / min;

[0051] Hydrogen flow rate: 40 mL / min;

[0052] Nitrogen pressure: 11620 kPa;

[0053] Flow split ratio: 1:50;

[0054] Injection volume: 1 μL.

[0055] The above content will be elaborated below with specific verification experiments:

[0056] 1. Experimental materials and their sources

[0057] Samples: Different varieties of flaxseed, crushed and passed through a 60-mesh sieve, for later use.

[0058] Reagents: Methanol, sodium hydroxide, acetic acid, and n-heptane, all of analytical grade.

[0059] Reference standards: methyl palmitate, methyl stearate, methyl oleate, methyl linoleate, and methyl alpha-linolenic acid (purity ≥99%).

[0060] Instruments and equipment:

[0061] Analyze the balance scale (1 / 10,000).

[0062] 10 mL stoppered centrifuge tube

[0063] Vortex oscillator

[0064] timer

[0065] 0.22 μm organic needle filter

[0066] Gas chromatograph (equipped with FID detector), FFAP flexible quartz capillary column (30m × 0.125mm × 0.13μm)

[0067] 2. Preparation of standard solutions:

[0068] Accurately weigh 100 mg each of methyl palmitate, methyl stearate, methyl oleate, methyl linoleate and methyl α-linolenic acid reference standards, place them in 10 mL brown volumetric flasks, dissolve them in n-heptane and dilute to the mark, shake well, and prepare stock solutions of each standard with a concentration of 10 mg / mL.

[0069] Accurately measure 3.5 mL of each of the above single standard stock solutions and place them in the same 25 mL brown volumetric flask. Dilute to the mark with n-heptane and shake well to obtain a mixed standard working solution with a concentration of 1.4 mg / mL for methyl palmitate, methyl stearate, methyl oleate, methyl linoleate, and methyl α-linolenic acid.

[0070] 3. Rapid extraction and esterification of sesame seed samples:

[0071] Weigh 0.5 g of sesame seed powder sample (accurate to 0.0001 g) and place it in a 10 mL stoppered centrifuge tube.

[0072] Add 5 mL of 1% (w / v) methanol-sodium hydroxide solution to the centrifuge tube, immediately tighten the stopper, and vortex vigorously for 2 minutes to ensure that the sample powder and solution are thoroughly mixed and free of lumps. Let the centrifuge tube stand at room temperature for 30 minutes to carry out the saponification and methyl esterification reactions.

[0073] After the reaction is complete, add about 0.2 mL (about 5 drops) of 10% acetic acid solution to the centrifuge tube and gently shake to neutralize the excess alkali.

[0074] Subsequently, accurately add 5 mL of n-heptane to the system, tighten the stopper, and shake vigorously again for 5 minutes to fully extract the generated fatty acid methyl esters. Let stand for 5 minutes until the solution has fully separated into layers. Take the upper n-heptane extract and filter it through a 0.22 μm organic syringe filter. The resulting filtrate is the sample for gas chromatography analysis (extraction process as follows). Figure 1 (As shown).

[0075] 4. Gas chromatography analysis:

[0076] The following chromatographic conditions were used to determine the above samples in this application:

[0077] Chromatographic column: FFAP flexible quartz capillary column (30 m × 0.125 mm × 0.13 μm)

[0078] Column oven program: 220 degrees Celsius (hold temperature for 13 minutes)

[0079] Inlet temperature: 250 degrees Celsius

[0080] Detector temperature: 250 degrees Celsius (FID)

[0081] Carrier gas: High-purity nitrogen, column inlet pressure 116.20 kPa

[0082] Airflow rate: 400 mL / min

[0083] Hydrogen flow rate: 40 mL / min

[0084] Flow split ratio: 1:50

[0085] Injection volume: 1 μL

[0086] 5. Results and Discussion:

[0087] The same batch of flaxseed samples was subjected to three parallel replicate measurements (n=3) following the steps described above.

[0088] 5.1 Speed ​​and Ease of Use:

[0089] This method takes only about 40 minutes from sample processing to completion, which is more than 85% shorter than traditional Soxhlet extraction (6-8 hours) and subsequent derivatization (30-60 minutes). The entire process is completed in a single centrifuge tube, eliminating the need for complex operations such as filtration, transfer, and concentration, thus greatly simplifying the process.

[0090] 5.2 Validation of the method:

[0091] Chromatographic resolution: Under the above chromatographic conditions, baseline separation was achieved for the five major fatty acid methyl esters (methyl palmitate, methyl stearate, methyl oleate, methyl linoleate, and methyl α-linolenic acid) in flaxseed, with symmetrical and sharp peaks and no tailing phenomenon. Typical chromatographic results are shown in [Figure number missing]. Figure 2 .

[0092] Repeatability: The relative standard deviation (SD) of the relative content of each fatty acid measured in three parallel experiments was less than 1%, indicating that the method has good repeatability (Table 1).

[0093] Experimental Materials: Five representative sesame varieties were selected for method verification in this experiment, including:

[0094] Variety 1: A common high-oleic sesame variety, with an oleic acid content as high as 27%;

[0095] Variety 5: This is a high-linolenic acid variety, with its α-linolenic acid content significantly higher than other varieties, reaching 52%.

[0096] Other varieties (2-4) also have unique characteristics in their fatty acid composition, which are used to comprehensively evaluate the applicability of the method. (Table 1)

[0097] Table 1. Relative content and relative standard deviation (SD) of fatty acids in different varieties obtained from three parallel experiments.

[0098] name Palmitic acid (%) SD Stearic acid (%) SD Oleic acid (%) SD Linoleic acid (%) SD Linolenic acid (%) SD 6.3471 5.2773 27.4485 15.1963 45.2958 No. 1 6.3280 0.010 5.4203 0.072 27.4682 0.034 15.1761 0.011 45.1232 0.113 6.3378 5.3380 27.5145 15.1947 45.0826 6.7228 5.9029 25.5134 14.1068 47.1448 No. 2 6.7107 0.007 5.9332 0.020 25.5134 0.018 14.1087 0.006 47.1396 0.015 6.7114 5.9417 25.5440 14.0982 47.1165 7.0808 5.9681 24.4683 16.0112 45.9206 No. 3 7.0899 0.009 5.9696 0.024 24.4010 0.071 15.9407 0.043 46.0906 0.086 7.0725 5.9276 24.3269 16.0173 45.9866 6.4136 4.7897 23.5211 14.0748 50.6350 No. 4 6.4150 0.009 4.8229 0.017 23.6107 0.075 14.1864 0.064 50.5477 0.087 6.3992 4.8097 23.6706 14.1864 50.4614 6.8283 4.2055 24.0108 12.2603 52.4295 No. 5 6.8763 0.026 4.1454 0.038 24.0365 0.060 12.1624 0.054 52.5212 0.091 6.8331 4.2163 24.1247 12.1715 52.3394

[0099] Comparison with standard methods: To verify the extraction and esterification efficiency of this method, the same flaxseed sample was used, and both this method (Method 1) and the traditional Soxhlet extraction-boron trifluoride methyl esterification method (Method 2) were employed for processing and determination. The results showed that the relative contents of each fatty acid determined by this method were not significantly different from those of the classical method (P>0.05), and the determination results for unsaturated fatty acids (such as α-linolenic acid) were more stable, avoiding oxidation losses that may be caused by prolonged heating. Specific comparison results are shown in [link to comparison]. Figure 3 .

[0100] In summary, this embodiment provides a rapid extraction and component determination method for fatty acids from flax seeds. It successfully integrates alkali-catalyzed methyl esterification and liquid-liquid extraction steps into a single centrifuge tube, achieving rapid, efficient, and accurate determination of fatty acids in flax seeds. This method significantly improves analytical efficiency, reduces solvent consumption and operational complexity, and is particularly suitable for rapid evaluation of flax seed quality, high-throughput screening of breeding materials, and quality control in the production process.

[0101] Example 2: Comparative experiment with ultrasound-assisted extraction method:

[0102] This implementation case aims to demonstrate the significant advantage of the present invention in extraction efficiency by comparing the method of the present invention with another common rapid extraction method—ultrasound-assisted extraction—with objective data.

[0103] 1. Experimental Materials and Instruments

[0104] Sample: Flaxseeds from the same batch, crushed and passed through a 60-mesh sieve, mixed evenly and set aside.

[0105] Reagents: Methanol, sodium hydroxide, acetic acid, n-heptane, and petroleum ether (60-90℃), all of analytical grade.

[0106] Instruments and equipment:

[0107] Analyze the balance scale (1 / 10,000).

[0108] 10 mL stoppered centrifuge tube

[0109] Vortex oscillator

[0110] Ultrasonic cleaner (600W power, 40kHz frequency)

[0111] centrifuge

[0112] 0.22 μm organic needle filter

[0113] Gas chromatograph (configuration as described in the invention)

[0114] 2. Experimental Methods

[0115] 2.1 The method of the present invention (one-step extraction-esterification method)

[0116] Accurately weigh 0.5 g (accurate to 0.0001 g) of sesame seed powder into a 10 mL centrifuge tube. Then, add 5 mL of 1% methanol-sodium hydroxide solution, vortex for 2 minutes, and let stand at room temperature for 30 minutes. Subsequent steps, including adding acetic acid, extracting with 5 mL of n-heptane, and filtration, are exactly the same as described in [Implementation Case 1]. The final volume is approximately 5 mL.

[0117] 2.2 Comparison Method (Ultrasound-Assisted Extraction)

[0118] Accurately weigh 0.5 g (accurate to 0.0001 g) of sesame seed powder into a 10 mL centrifuge tube. Add 5 mL of petroleum ether, vortex to mix, and then place in an ultrasonic cleaner for ultrasonic extraction at 40°C for 30 minutes. After extraction, centrifuge at 4000 rpm for 5 minutes and collect the supernatant. Transfer the supernatant to a new centrifuge tube and dry it under nitrogen gas in a 40°C water bath. Add 5 mL of 1% methanol-sodium hydroxide solution to the dried residue, vortex, and react in an 80°C water bath for 15 minutes for methylation. Subsequent neutralization, n-heptane extraction, and filtration steps are the same as in this invention. The final volume is also 5 mL.

[0119] 3. Gas chromatography analysis

[0120] Samples prepared by both methods were subjected to gas chromatography analysis under identical chromatographic conditions (see the chromatographic conditions in the aforementioned [Implementation Case 1]). Each method was prepared in triplicate (n=3).

[0121] 4. Results and Data Analysis

[0122] In the gas chromatogram, the peak areas of all major fatty acid methyl ester peaks (including methyl palmitate, methyl oleate, methyl linoleate, and methyl α-linolenic acid) obtained by the method of this invention are significantly higher than the peak areas of the corresponding components obtained by the ultrasonic-assisted extraction method. The chromatogram visually demonstrates the advantage of the peak height of each component obtained by the method of this invention. A comparison of the extraction efficiency of the two methods on varieties 1 and 5 is shown in [the figure]. Figure 4 .

[0123] 5. Conclusions and Analysis

[0124] Simpler operation and less loss: Ultrasonic-assisted extraction requires four main steps: extraction, concentration, derivatization, and extraction, involving solvent transfer and nitrogen blowing concentration. This process is prone to loss of volatile or semi-volatile components, as well as operational errors. In contrast, the method of this invention completes saponification, esterification, and extraction in a single centrifuge tube in one step, avoiding losses from intermediate steps. This is the key reason for its higher extraction efficiency.

[0125] More favorable to unsaturated fatty acids: The present invention performs esterification at room temperature, while the comparative method requires reaction at 80 degrees Celsius. The relatively mild conditions are beneficial to protecting highly unsaturated fatty acids such as α-linolenic acid and avoiding their oxidative degradation, which may be one of the reasons for the higher α-linolenic acid determination results.

[0126] In conclusion, this case study strongly demonstrates that the rapid extraction and component determination method for fatty acids from sesame seeds described in this invention not only has advantages in speed, but its core extraction efficiency is also significantly better than other rapid extraction methods, while also exhibiting better repeatability and reliability.

[0127] In summary, this application improves extraction efficiency and reduces toxicity by optimizing the mixed solvent system. The use of a vortex mixer for extraction significantly shortens the extraction time compared to traditional methods; the extraction process can be completed in as little as 40 minutes. Furthermore, the practical use of the vortex mixer effectively avoids high-temperature oxidation. This application also utilizes a one-step methyl esterification method combined with GC detection, resulting in significantly higher fatty acid component extraction efficiency than traditional methods, making it suitable for large-scale oilseed quality evaluation.

Claims

1. A method for rapid extraction of fatty acids from flax seed, the method comprising: The method comprises the following steps: ​ S1: sample preparation: taking the flaxseed and crushing it; S2: saponification and methyl esterification reaction: taking the crushed flaxseed in S1, placing it in a centrifuge tube with a plug, adding 1% methanol-sodium hydroxide solution, shaking to mix the sample and the solution thoroughly, and standing to complete the reaction; S3: neutralizing the alkali in the solution; S4: extraction: adding n-heptane to the solution to extract the generated fatty acid methyl ester thoroughly.

2. A method of rapid extraction of fatty acids from flax seed according to claim 1, characterized in that: The crushed flaxseed in S1 is sieved using a 60-mesh sieve.

3. A method of rapid extraction of fatty acids from flax seed according to claim 2, characterized in that: The addition ratio of the flaxseed to 1% methanol-sodium hydroxide solution in S2 is 0.5 g:5 mL, and the standing time in S2 is 30 min.

4. A method of rapid extraction of fatty acids from flax seed according to claim 3, characterized in that: In S3, the excess alkali is neutralized by adding acetic acid solution dropwise in the centrifuge tube and by gently shaking.

5. A method of rapid extraction of fatty acids from flax seed according to claim 4, characterized in that: After adding n-heptane in S4, the tube plug is tightly covered, and the solution is shaken again for 5 min, and then it is stood for 5 min; after the solution is fully layered, the upper n-heptane extraction liquid is taken and filtered to obtain the required fatty acid.

6. A method of rapid extraction of fatty acids from flax seed according to claim 5, characterized in that: In S4, a 0.22-μm organic needle filter is used for filtering.

7. A method for detecting flax seed fatty acids, characterized by: The detection method is used to detect the fatty acid obtained by the rapid extraction method of flaxseed fatty acid according to any one of claims 1-6, and the detection method uses gas chromatography detection.

8. A method for detecting the fatty acids of flax seed according to claim 7, characterized in that: The detection conditions of the detection method are set as follows: Chromatographic column: FFAP elastic quartz capillary column (30 m×0.125 mm×0.13 μm); Programmed temperature: initial 100℃ for 2 min, increased to 220℃ at 5℃ / min, and maintained for 13 min; Injection port temperature: 250℃; FID detector temperature: 250℃; Air flow rate: 400 mL / min; Hydrogen flow rate: 40 mL / min; Nitrogen pressure: 11620 kPa; Split ratio: 1:50; Injection amount: 1 μL.

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