A method for determining the sn-2 fatty acid distribution in long chain polyunsaturated fatty acid-enriched oils

Abstract

This invention discloses a method for determining the distribution of fatty acids at the sn-2 position in oils rich in long-chain polyunsaturated fatty acids, belonging to the fields of analytical chemistry and food science. The method introduces a quality control oil sample containing a correction fatty acid, which is bound to any acyl position in the triglyceride molecule. A correction factor is calculated from the measured data of the quality control oil sample, and this correction factor is used to correct for enzymatic hydrolysis efficiency and analytical system bias. This invention innovatively introduces a quality control sample to monitor and correct enzymatic hydrolysis efficiency and separation recovery rate, eliminating systematic errors caused by factors such as enzyme activity fluctuations and operational differences, and greatly improving the reproducibility of the method across different times and operators.

Description

Technical Field

[0001] This invention belongs to the fields of analytical chemistry and food science, specifically relating to a method for determining the distribution of fatty acids at the sn-2 position in oils rich in long-chain polyunsaturated fatty acids. Background Technology

[0002] Triglycerides are the main components of lipids and fats, formed by the esterification of one molecule of glycerol and three molecules of fatty acids. The three carbon atoms on the glycerol backbone are stereotyped as sn-1, sn-2, and sn-3. The positional distribution of fatty acids on the glycerol backbone (i.e., Sn-position distribution) directly affects the physical properties, metabolic pathways, and nutritional functions of lipids and fats. In particular, the proportion of long-chain polyunsaturated fatty acids (such as DHA, EPA, and ARA) at the sn-2 position is crucial for assessing the nutritional value of products such as infant formula and certain medical fat emulsions. The underlying mechanism lies in the human digestive and metabolic process: pancreatic lipase secreted by the pancreas has sn-1 and sn-3 position specificity, which hydrolyzes fatty acids at the sn-1 and sn-3 positions to generate sn-2 monoglycerides. Sn-2 monoglyceride molecules are polar and readily form mixed micelles with bile salts, thus being efficiently absorbed by the small intestinal epithelial cells. Therefore, nutrients such as DHA, existing in the form of sn-2 monoglycerides, exhibit significantly higher bioavailability than other forms.

[0003] Currently, the mainstream method for determining the composition of fatty acids at the sn-2 position is enzymatic hydrolysis. This method utilizes pancreatic lipase or other lipases with sn-1 and sn-3 position specificity to enzymatically hydrolyze oils, specifically targeting the sn-1 and sn-3 ester bonds of triglycerides (TAGs), aiming to retain the fatty acid at the sn-2 position to generate sn-2 monoglycerides. The sn-2 monoglycerides are then separated and extracted, and their composition is analyzed using chromatographic techniques.

[0004] However, this classic method has significant technical bottlenecks in practical applications: First, the unavoidable acyl migration during enzymatic hydrolysis severely interferes with the accuracy of assays. In the enzymatic hydrolysis of triglycerides using sn-1,3-specific lipases, the formation rate of diacylglycerols (DAG) is generally higher than that of monoacylglycerols (MAG). However, the hydrolysis products (mainly 1,2-DAG, 2,3-DAG, and 2-MAG) are thermodynamically unstable and readily undergo non-enzymatic acyl migration. This process involves the spontaneous transfer of the acyl group from the sn-2 hydroxyl group to the adjacent sn-1 or sn-3 hydroxyl group, leading to isomerization: i.e., 1,2-DAG or 2,3-DAG rearranges to 1,3-DAG, and 2-MAG rearranges to 1-MAG or 3-MAG. Since the rearranged acyl group reoccupies the sn-1 or sn-3 position, it becomes a substrate again for sn-1,3-specific lipases and is further hydrolyzed. Therefore, as the reaction time increases, the system undergoes cascade enzymatic hydrolysis, and the final product tends to be glycerol that has been completely hydrolyzed, resulting in the loss of fatty acid information at the sn-2 position.

[0005] Secondly, existing technologies face two major challenges in detecting long-chain polyunsaturated fatty acids (LCPUFA): 1. Oxidation loss leads to inaccurate test results: Long-chain polyunsaturated fatty acids are chemically active and are easily oxidized during sample pretreatment, enzymatic hydrolysis, separation and other processes, resulting in lower measured content values ​​and thus inaccurate data on the proportion of fatty acids at the sn-2 position.

[0006] 2. Poor method reproducibility: The extent of the enzymatic hydrolysis reaction is affected by various factors (such as enzyme activity, reaction time, temperature, pH value, etc.), making it difficult to control precisely; at the same time, the recovery rate of subsequent separation steps such as thin-layer chromatography also fluctuates. These factors together lead to poor repeatability of the test results, making it difficult to compare and verify test results between different laboratories or even between different batches of the same laboratory.

[0007] Therefore, there is an urgent need in this field to develop a novel detection method that can effectively protect easily oxidized fatty acids and achieve precise process control of key steps, thereby improving the accuracy and reproducibility of the analysis of the proportion of fatty acids at the sn-2 position in oils rich in long-chain polyunsaturated fatty acids. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for determining the distribution of fatty acids at the sn-2 position in oils rich in long-chain polyunsaturated fatty acids.

[0009] This application provides a method for determining the distribution of fatty acids at the sn-2 position in oils rich in long-chain polyunsaturated fatty acids, comprising any combination of the following steps: (a) Prepare the oil sample to be tested and the quality control oil sample, wherein the quality control oil sample contains a correction fatty acid, which is bound to any acyl position of the triglyceride molecule; (b) The oil sample to be tested is subjected to enzymatic hydrolysis with a specific lipase at position sn-1,3 to obtain the hydrolysis product; (c) The quality control oil sample is subjected to enzymatic hydrolysis with a specific lipase at the sn-1,3 position to obtain the hydrolysis product; (d) Separate the enzymatic hydrolysis products obtained in steps (b) and (c) respectively to obtain the sn-2 position monoglyceride from the oil sample to be tested and the sn-2 position monoglyceride from the quality control oil sample. (e) Determine the content of the target fatty acid in the sn-2 position monoglyceride obtained from the oil sample to be tested in step (d), and the total content of the target fatty acid in the oil sample to be tested; (f) The content of correction fatty acids in the sn-2 position monoglyceride obtained from the quality control oil sample in step (d) and the total content of correction fatty acids in the quality control oil sample are determined respectively. (g) Based on the determination results of steps (e) and (f), the proportion of the target fatty acid in the sn-2 position monoglyceride of the tested oil sample is calculated using the following formula: Sn-2% (X) = [(content of X in sn-2 monoglycerides in the tested oil sample %) / (content of X in total fatty acids of triglycerides in the tested oil sample % × 3)] × (F) × 100%; the content is in molar percentage (mol%) or mass percentage (wt%). Wherein, X represents the target fatty acid to be tested; F is a correction factor, which is calculated using the measured data of the quality control oil samples and is used to correct for enzymatic hydrolysis efficiency and analytical system bias.

[0010] In some embodiments, the quality control oil samples include quality control oil sample 1 and quality control oil sample 2. The quality control oil sample 1 is selected from medium-chain fatty acid oils, including at least one of sunflower seed oil, soybean oil, corn oil, rapeseed oil, tea seed oil, peanut oil, olive oil and avocado oil; the quality control oil sample 2 is prepared by mixing the quality control oil sample 1 and the oil sample to be tested at a mass ratio of 1:10 to 2:1.

[0011] In some embodiments, the medium-chain fatty acid oils selected for the quality control oil sample 1 also include high-oleic sunflower seed oil and high-oleic rapeseed oil. This application selects the aforementioned medium-chain fatty acid oils as quality control samples, as these oils have the characteristics of stable fatty acid composition and relatively simple components.

[0012] In some implementations, the correction factor in step (g) is calculated using the following formula: For quality control oil sample 1: Sn-2%(Y)1 = (content of Y in sn-2 monoglyceride in quality control oil sample 1) / (content of Y in total fatty acids in triglycerides in quality control oil sample 1) × 3) × 100%; For quality control oil sample 2: Sn%(Y)2 = (content of Y in sn-2 monoglyceride in quality control oil sample 2) / (content of Y in total fatty acids of triglycerides in quality control oil sample 2) × 3) × 100%; The content is expressed as a mole percentage (mol%) or a mass percentage (wt%). Correction factor = Sn%(Y)2 ÷ Sn%(Y)1; Y represents the corrective fatty acid, which is one of the long-chain unsaturated fatty acids with a carbon chain length of 16-20, including at least one of oleic acid, linoleic acid, linolenic acid, and eicosatrienoic acid.

[0013] Preferably, Y is one of the long-chain unsaturated fatty acids with a carbon chain length of 18, selected from oleic acid (C18:1), linoleic acid (C18:2) or linolenic acid (C18:3).

[0014] In some implementations, the test sample and the quality control sample may be selectively purified by alumina column chromatography before the detection begins.

[0015] In some embodiments, the sn-1,3-position specific lipase described in steps (b) and (c) includes at least one of pancreatic lipase, pregastric lipase, lipase derived from Candida antarctica, Rhizomucor miehei lipase (or Rhizomucor miehei lipase (RML)), Rhizopus miehei lipase, Aspergillus miehei lipase, Rhizopus dellemarum EU O93 / Rd lipase, Yersinia lipolytica lipase, Thermomyces lanuginosus lipase (or Lipozyme TL IM), Penicillium camembertii lipase, Rhizopus arrhizus lipase, Penicillium roquefortii lipase, and Candida rubra lipase.

[0016] In some embodiments, the reaction temperature of the enzymatic hydrolysis in steps (b) and (c) is ≤40°C and the reaction time is ≤3h.

[0017] In some embodiments, the separation in step (d) includes at least one of thin-layer chromatography or silica gel solid-phase extraction.

[0018] In some embodiments, the determination method described in steps (e) and (f) is gas chromatography.

[0019] In some implementations, all steps that may involve contact with air are treated with antioxidants.

[0020] In some embodiments, the antioxidant protection treatment includes at least one of inert gas protection treatment, light protection treatment, and the addition of antioxidants.

[0021] In some embodiments, the inert gas includes at least one of nitrogen and argon.

[0022] In some embodiments, the antioxidant includes at least one of vitamin E, BHT, and TBHQ.

[0023] In summary, compared with the prior art, the present invention achieves the following technical effects: 1. This invention effectively avoids the oxidative loss of long-chain polyunsaturated fatty acids through antioxidant protection measures throughout the entire process, ensuring the authenticity and reliability of the test data.

[0024] 2. This invention innovatively introduces quality control samples to monitor and correct the enzymatic hydrolysis efficiency and separation recovery rate, eliminating systematic errors caused by factors such as enzyme activity fluctuations and operational differences, and greatly improving the reproducibility of the method of this invention at different times and by different operators.

[0025] 3. The method of this invention has a clear process, simple and effective quality control measures, and is easy to promote and apply in conventional laboratories, providing reliable technical support for the quality control and nutritional evaluation of oil products.

[0026] 4. The method of the present invention is particularly suitable for the analysis of the distribution of fatty acids at the sn-2 position in oils and fats containing long-chain (C>20) polyunsaturated fatty acids and with complex fatty acid composition. Detailed Implementation

[0027] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described. 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 skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0028] This application systematically addresses the accuracy and reproducibility issues in the analysis of the fatty acid composition at the sn-2 position in oils rich in long-chain polyunsaturated fatty acids by combining whole-process antioxidant protection with process calibration using quality control samples.

[0029] Throughout the entire sample processing, enzymatic hydrolysis, and subsequent separation process, physical or chemical measures are taken to protect the long-chain polyunsaturated fatty acids in the oil samples and prevent them from being oxidized. For example, inert gases (such as nitrogen or argon) are introduced into the reaction system, or antioxidants (such as vitamin E, BHT, or TBHQ) are added during the pretreatment and enzymatic hydrolysis of the oils to be tested, and light-protected operation is also employed.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used are commercially available.

[0031] Example 1 The entire sample processing, enzymatic digestion, and subsequent separation process was carried out under nitrogen protection. Simultaneously, all organic solvents used were pre-purged with nitrogen to remove oxygen.

[0032] Step 1. Prepare samples and buffer solutions, including (1) Take the DHA algal oil to be tested and add 1.5% mixed tocopherols to it. Vortex until completely dissolved to obtain the sample to be tested. (2) High oleic sunflower seed oil was selected as quality control oil sample 1 (hereinafter referred to as quality control sample 1); (3) The test sample and quality control sample 1 are mixed at a mass ratio of 1:1 to obtain quality control oil sample 2 (hereinafter referred to as quality control sample 2).

[0033] (4) Buffer system: 1M Tris buffer pH 8.0, 2.2% calcium chloride solution, bile salt solution (0.05%), 6M hydrochloric acid solution.

[0034] In subsequent experiments, quality control sample 1 and quality control sample 2 were subjected to the same enzymatic digestion, thin-layer chromatography separation and detection process as the test sample under exactly the same conditions.

[0035] The technical solution of this application is based on the results of the proportion of medium-chain fatty acids (correction fatty acids) at the sn-2 position in quality control sample 1 and quality control sample 2 obtained by the detection and analysis method, to infer the enzymatic hydrolysis of long-chain polyunsaturated fatty acids in quality control sample 2.

[0036] Step 2. Enzymatic hydrolysis of triglycerides Weigh 5 mg of the sample to be tested into a test tube. Add 1 mL of Tris buffer, 0.1 mL of calcium chloride solution, and 0.25 mL of bile salt solution to the tube sequentially. Preheat the test tube in a 38°C water bath for 2 minutes. Then add approximately 4 mg of porcine pancreatic lipase (which has regioselectivity and directionally hydrolyzes the sn-1 and sn-3 fatty acids of triglycerides). Mix the sample, lipase, and buffer system thoroughly to ensure that the lipase does not clump or stick to the walls. Under constant temperature of 38°C, vigorously shake the test tube for 5 minutes to disperse the triglycerides and directionally hydrolyze the fatty acids at the sn-1 and sn-3 positions of the triglycerides, generating sn-2 monoglycerides and free fatty acids, thus obtaining the enzymatic reaction product. Add 1 mL of ethanol to the reaction mixture, and then add 1 mL of 6M hydrochloric acid to terminate the reaction.

[0037] Extraction with ether solution three times (3 × 3 ml) to form an emulsion was followed by centrifugation. The upper solvent layer was collected separately in a test tube and washed once with distilled water (3 ml). The organic phase was then passed through a pre-washed sodium sulfate Pasteur column, and the filtrate was collected in a 20 ml round-bottom flask. The sodium sulfate column was then washed with ether (2 ml), and the washing solution was also collected in a round-bottom flask. The solvent was removed using a rotary evaporator in a hot water bath at <40°C. The resulting product was dissolved in a small amount of ether (150-200 μl) for later use.

[0038] Following the method described in step 2, quality control samples 1 and 2 were treated under the same conditions. During the testing process, two parallel samples were set up for parallel determination of both the sample to be tested and quality control sample 2.

[0039] Step 3. Thin-layer chromatography separation and purification of sn-2 monoglyceride: Preparation of thin-layer chromatography developing solvent (isohexane: ether: formic acid equals 70:30:2, V:V): Add 70 ml isohexane, 30 ml ether and 2 ml formic acid to a round-bottom flask, mix well to obtain the thin-layer chromatography developing solvent. Preparation of 0.01% Primulin colorimetric solution: Weigh 10 mg of primulin and place it in a spray bottle. Add 100 ml of a mixed solvent prepared by acetone and distilled water in a volume ratio of 60:40 to the bottle and shake well to dissolve the solute.

[0040] Preparation of TLC standards: Prepare 20 mg / ml TLC mix 34 and 20 mg / ml TLC mix 40 solutions, respectively. These TLC standard solutions serve as qualitative analysis references, indicating the migration positions (Rf values) of the substrate (triglycerides) and enzymatic hydrolysis products (monoglycerides and diglycerides) on the thin-layer chromatography plate.

[0041] The separation of the above products by thin-layer chromatography includes the following steps: (1) Thin-layer chromatography spotting: Take a thin-layer chromatography plate and draw a baseline (horizontal line) 2.0 cm from the bottom edge of the plate. Spot TLL mix 34 standard 0.5 cm from the left edge of the baseline and TLC mix 40 standard 0.5 cm from the right edge of the plate. Take 150-200 μL of the enzymatic hydrolysis product ether solution prepared in step 2 and spot it in the baseline area between the two standards.

[0042] (2) Thin-layer chromatography separation and product recovery: Add developing solvent to the developing tank, seal and let stand for 10 min to saturate the atmosphere; place the spotted thin-layer chromatographic plate into the developing tank for development until the solvent front reaches 1.0 cm from the edge of the thin-layer chromatographic plate, then remove it. Place the chromatographic plate in a nitrogen atmosphere and dry it at about 20°C to remove the solvent. After drying, spray 0.01% primrose extract evenly onto the plate surface. Observe the chromatographic plate under ultraviolet light, identify and mark the monoglyceride bands, scrape the silica containing the monoglyceride bands into a test tube with a scraper, and elute and extract with an organic solvent to achieve effective separation of monoglycerides from free fatty acids and byproducts such as diglycerides (1,2-DAG / 2,3-DAG).

[0043] The same method was used to process quality control samples 1 and 2.

[0044] Step 4. Fatty acid composition analysis: The sn-2 monoglycerides obtained in step 3 (corresponding to the test sample, quality control sample 1, and quality control sample 2, respectively) were subjected to methyl esterification according to the method described in the National Food Safety Standard GB 5009.168 to prepare the corresponding fatty acid methyl ester derivatives. Subsequently, the fatty acid composition at the sn-2 position was analyzed by gas chromatography normalization as described in GB 5009.168.

[0045] Simultaneously, the raw oil samples (without enzymatic hydrolysis and thin-layer chromatography separation) of the test sample, quality control sample 1, and quality control sample 2 were directly subjected to methyl esterification treatment according to the method described in GB 5009.168. The total fatty acid composition of the triglycerides was analyzed using gas chromatography normalization.

[0046] In this invention, process calibration is performed in each batch of testing by setting up quality control samples. The quality control samples undergo enzymatic digestion, thin-layer chromatography separation, and subsequent detection procedures under identical conditions as the test samples. By comparing the actual test results of the quality control samples, the overall recovery rate of the experimental system (covering both enzymatic digestion efficiency and thin-layer chromatography recovery rate) is calculated, and a correction factor is obtained accordingly. This correction factor is then used to calibrate and correct the final calculation results for the test samples, thereby eliminating systematic errors caused by batch-to-batch operational differences.

[0047] Step 5. Data Calculation: Based on the theory of random distribution of fatty acids, use the following formulas to calculate the proportions of oleic acid, linoleic acid, linolenic acid, DPA, and DHA, as well as the target fatty acid, at the sn-2 position: The percentage of oleic acid or linoleic acid at the sn-2 position in quality control sample 1 = (the content of oleic acid or linoleic acid in sn-2 monoglycerides in quality control sample 1%) / (the content of oleic acid or linoleic acid in total fatty acids of triglycerides in quality control sample 1%) × 100%; The percentage of oleic acid or linoleic acid at the sn-2 position in quality control sample 2 = (the content of oleic acid or linoleic acid in sn-2 monoglycerides in quality control sample 2) / (the content of oleic acid or linoleic acid in total fatty acids of triglycerides in quality control sample 2) × 100%; The content is a mass percentage (wt%). Correction factor = percentage of oleic acid at the sn-2 position in quality control sample 2 / percentage of oleic acid at the sn-2 position in quality control sample 1.

[0048] Sn-2% (DHA) = [(DHA content in sn-2 monoglycerides in the sample) % / (DHA content in total fatty acids of triglycerides in the sample) % × 3) ] × (correction factor) × 100%. The results are summarized in Table 1.

[0049] Typically, at least two parallel samples are used in the test setup, and the average value is taken as the final result.

[0050] Comparative Example 1 According to the standard method of GB / T24894-2010, the test sample, quality control sample 1 and quality control sample 2 in Example 1 were tested respectively, and the proportion of the target fatty acid at the sn-2 position was calculated according to the following formula.

[0051] Sn-2% (X) = [ (X content in sn-2 monoglyceride %) / (X content in total fatty acids of triglycerides % × 3) ] × 100%.

[0052] The distribution ratio (occupancy ratio) of each polyunsaturated fatty acid at the sn-2 position of sn-2 monoglyceride in Example 1 and Comparative Example 1 is summarized in Table 1.

[0053] Table 1 The fatty acid used for correction is oleic acid or linoleic acid.

[0054] Results Analysis: For quality control sample 1, the ratio of oleic acid and linoleic acid at the sn-2 position was similar. However, for quality control sample 2, which was a mixture of high-oleic sunflower seed oil and DHA algal oil, the ratio of oleic acid and linoleic acid at the sn-2 position differed significantly. This demonstrates that when the fatty acid composition of the oil is complex, the detection results can be distorted. The technical solution of this invention effectively overcomes matrix interference, thereby obtaining objective and accurate distribution data. Furthermore, the repeated detection results of the embodiments were similar, further verifying the stability and reliability of the detection system when handling complex samples.

[0055] Example 2 The entire sample processing, enzymatic digestion, and subsequent separation process was carried out under nitrogen protection. Simultaneously, all organic solvents used were pre-purged with nitrogen to remove oxygen.

[0056] Step 1. Prepare the sample, including (1) Take the ARA oil sample to be tested and add 1% vitamin E to it. Vortex until completely dissolved to obtain the sample to be tested. (2) High oleic acid rapeseed oil was selected as quality control oil sample 1 (hereinafter referred to as quality control sample 1); (3) The test sample and quality control sample 1 are mixed at a mass ratio of 1:1 to obtain quality control oil sample 2 (hereinafter referred to as quality control sample 2).

[0057] In subsequent experiments, quality control sample 1 and quality control sample 2 were subjected to the same enzymatic digestion, thin-layer chromatography separation and detection process as the test sample under exactly the same conditions.

[0058] Step 2. Enzymatic hydrolysis reaction Weigh approximately 100 mg of the oil sample to be tested into a centrifuge tube, preheat it in a water bath at 35°C for 2 minutes, add 2 g of ethanol and 0.2 g of Novozym 435, cover the tube, mix the sample thoroughly, and react in a constant temperature shaking water bath or constant temperature shaker at 32°C and 180 rpm for 2 hours. Then filter to remove lipase and obtain the enzymatic reaction product.

[0059] Following the method in step 2, quality control samples 1 and 2 were processed under the same conditions, with two parallel samples set up for the test sample and quality control sample 2 respectively.

[0060] Step 3. Thin-layer chromatography separation and purification of sn-2 monoglyceride Using silica gel G thin-layer plates, the developing solvent system was a mixed solvent of petroleum ether: diethyl ether: formic acid (volume ratio 70:30:1). After spotting and development, the thin-layer plate was dried in a nitrogen atmosphere at 20°C. A slightly alkaline dichlorofluorescein ethanol solution was sprayed from a spray bottle, and the positions of monoglycerides were observed and marked under ultraviolet light. The silica gel strip corresponding to the Rf value of the sn-2 monoglyceride standard was scraped off.

[0061] The same method was used to process quality control samples 1 and 2.

[0062] Step 4. Fatty acid methyl esterification and GC analysis The scraped silica gel was extracted with an organic solvent (such as chloroform). After the extract was concentrated by nitrogen blowing, it was subjected to methyl esterification according to the method described in GB 5009.168 to prepare the corresponding fatty acid methyl ester derivatized products. Subsequently, the fatty acid composition at the sn-2 position was analyzed by gas chromatography normalization as described in GB 5009.168. Quality control samples 1 and 2 were treated in the same way.

[0063] Simultaneously, the raw oil samples (without enzymatic hydrolysis and thin-layer chromatography separation) of the test sample, quality control sample 1, and quality control sample 2 were directly subjected to methyl esterification treatment according to the method described in GB 5009.168. The total fatty acid composition of the triglycerides was analyzed using gas chromatography normalization.

[0064] Step 5. Data Calculation: Sn% (Y) in quality control sample 1 = (content of Y in sn-2 monoglyceride in quality control sample 1) / (content of Y in total fatty acids in triglycerides in quality control sample 1) × 3) × 100%; Sn% (Y) in quality control sample 2 = (content of Y in sn-2 monoglyceride in quality control sample 2) / (content of Y in total fatty acids of triglycerides in quality control sample 2) × 3) × 100%; The content is a mass percentage (wt%). Correction factor = Sn% (Y) in quality control sample 2 / Sn% (Y) in quality control sample 1.

[0065] Sn-2% (X) = [(content of X in sn-2 monoglycerides in the sample) % / (content of X in total fatty acids of triglycerides in the sample) % × 3) ] × (correction factor) × 100%; The content is expressed as a mass percentage (wt).

[0066] Where X represents the target fatty acid to be tested, and Y represents the calibration fatty acid, which is one of the long-chain unsaturated fatty acids with a carbon chain length of 16-20 in quality control sample 1, including oleic acid, linoleic acid, linolenic acid and eicosatrienoic acid.

[0067] Comparative Example 2 According to the method of GB / T24894-2010 standard, the test sample, quality control sample 1 and quality control sample 2 in Example 2 were tested respectively, and the sn-2 position ratio of the target fatty acid was calculated according to the following formula.

[0068] Sn-2% (X) = [ (X content in sn-2 monoglyceride %) / (X content in total fatty acids of triglycerides % × 3) ] × 100%.

[0069] The distribution ratio (occupancy ratio) of each polyunsaturated fatty acid at the sn-2 position of sn-2 monoglyceride in Example 2 and Comparative Example 2 is summarized in Table 2, where the corrected fatty acid is oleic acid or linoleic acid.

[0070] Table 2 Example 3 The entire sample processing, enzymatic digestion, and subsequent separation process was carried out under nitrogen protection. Simultaneously, all organic solvents used were pre-purged with nitrogen to remove oxygen.

[0071] Step 1. Prepare the sample, including (1) Take a mixture of DHA and ARA oils as the sample to be tested; (2) Soybean oil was selected as the quality control oil sample 1 (hereinafter referred to as quality control sample 1); (3) The test sample and quality control sample 1 are mixed at a mass ratio of 1:1 to obtain quality control oil sample 2 (hereinafter referred to as quality control sample 2).

[0072] In subsequent experiments, control samples 1 and 2 were subjected to the same enzymatic digestion, Sep-Pak silica column separation, and detection procedures as the test samples under identical conditions.

[0073] Step 2. Enzymatic hydrolysis of triglycerides Weigh 5 mg of the sample to be tested into a test tube, then add 1 mL of Tris buffer, 0.1 mL of calcium chloride solution, and 0.25 mL of bile salt solution. Preheat the test tube in a 38°C water bath for 2 minutes. Then add approximately 4 mg of porcine pancreatic lipase (which has regioselectivity and directs the enzymatic hydrolysis of fatty acids at the sn-1 and sn-3 positions of triglycerides). Mix the sample, lipase, and buffer system thoroughly to ensure that the lipase does not clump or stick to the walls. Under constant temperature of 38°C, vigorously shake the test tube for 5 minutes to disperse the triglycerides. The lipase then directionally hydrolyzes the fatty acids at the sn-1 and sn-3 positions of the triglycerides, generating sn-2 monoglycerides and free fatty acids, yielding the enzymatic hydrolysis product. Add 1 mL of ethanol to the mixture, and then add 1 mL of 6M hydrochloric acid to terminate the reaction.

[0074] Extraction with ether solution three times (3 × 3 ml) to form an emulsion was followed by centrifugation. The upper solvent layer was collected separately in a test tube, and the collected organic phase was washed once with distilled water (3 ml). The organic phase was then passed through a pre-washed sodium sulfate Pasteur column, and the filtrate was collected in a 20 ml round-bottom flask. The sodium sulfate column was then washed with ether (2 ml), and the washing solution was also collected in a round-bottom flask. The solvent was removed using a rotary evaporator in a hot water bath at <40°C. The resulting product was dissolved in a small amount of ether (150-200 μl) for later use.

[0075] Following the method described in step 2, quality control samples 1 and 2 were treated under the same conditions. During the testing process, two parallel samples were set up for parallel determination of both the sample to be tested and quality control sample 2.

[0076] Step 3. Separation and purification of monoglycerides 0.1 mL of the product solution obtained in step 2 was loaded onto a Sep-Pak silica column (0.65 g, Waters Corporation, Milford, Massachusetts, USA), which had been pre-equilibrated with a hexane-ethyl ether mixed solvent (volume ratio of 80:20, hereinafter referred to as 'mixed solvent').

[0077] The following elution gradient was then performed: First, fatty acid ethyl esters (FAEE) were eluted and collected with 10 mL of the mixed solvent; second, diacylglycerols were removed by elution with 20 mL of the mixed solvent; finally, the target product 2-MAG was collected by elution with 10 mL of diethyl ether.

[0078] The same method was used to process quality control samples 1 and 2.

[0079] Step 4. Fatty acid composition analysis: The sn-2 monoglycerides purified in step 3 were subjected to methyl esterification according to the method described in GB 5009.168 to prepare the corresponding fatty acid methyl ester derivatives. Subsequently, the fatty acid composition at the sn-2 position was analyzed by gas chromatography normalization method as described in GB 5009.168.

[0080] Simultaneously, the raw oil samples (without enzymatic hydrolysis and thin-layer chromatography separation) of the test sample, quality control sample 1, and quality control sample 2 were subjected to methyl esterification according to the method in GB 5009.168. The total fatty acid composition of the triglycerides was analyzed using gas chromatography normalization.

[0081] Step 5. Data Calculation: Calculate the proportion of the target fatty acid at the sn-2 position using the following formula: Sn% (Y) in quality control sample 1 = (content of Y in sn-2 monoglyceride in quality control sample 1) / (content of Y in total fatty acids of triglycerides in quality control sample 1) × 100%; Sn% (Y) in quality control sample 2 = (content of Y in sn-2 monoglyceride in quality control sample 2) / (content of Y in total fatty acids of triglycerides in quality control sample 2) × 3) × 100%; The content is a mass percentage (wt%). Correction factor = Sn% (Y) in quality control sample 2 / Sn% (Y) in quality control sample 1 Sn-2% (X) = [(X content in sn-2 monoglycerides %) / (X content in total fatty acids of triglycerides % × 3)] × (correction factor) × 100%.

[0082] In the formula, X and Y are both fatty acids at the sn-2 position. X represents the target fatty acid to be tested, which is generally a long-chain polyunsaturated fatty acid, and Y represents the calibration fatty acid, which is a medium-to-long-chain unsaturated fatty acid with a carbon chain length of 16-20 in quality control sample 1.

[0083] Comparative Example 3 The steps and methods of Example 3 were repeated to detect and analyze the test sample, quality control sample 1, and quality control sample 2 respectively. The difference was that no antioxidant measures were taken during the detection and analysis process, and the proportion of the target fatty acid at the sn-2 position was calculated according to the following formula.

[0084] Sn-2% (X) = [ (X content in sn-2 monoglyceride %) / (X content in total fatty acids of triglycerides % × 3) ] × 100%.

[0085] The distribution ratio (occupancy ratio) of each polyunsaturated fatty acid at the sn-2 position of sn-2 monoglyceride in Example 3 and Comparative Example 3 is summarized in Table 3. The fatty acid used for correction is oleic acid or linoleic acid.

[0086] Table 3 Comparative Example 4 A mixture of DHA and ARA oils was used as the test sample, and coconut oil was used as the quality control sample 1. The test sample and quality control sample 1 were mixed at a mass ratio of 1:3 to obtain quality control oil sample 2 (hereinafter referred to as quality control sample 2).

[0087] The remaining operations were performed exactly as described in Example 3. The distribution ratio (occupancy ratio) of each polyunsaturated fatty acid at the sn-2 position of the sn-2 monoglyceride is summarized in Table 4. The fatty acid used for correction was lauric acid or myristic acid.

[0088] Table 4 For existing national standard testing methods, the lack of consideration for oxidation losses during enzymatic hydrolysis, as well as issues such as enzymatic hydrolysis efficiency and separation recovery rate, often leads to the acceptance of distorted results as correct or directly accepting problematic results in actual testing. Through research during actual testing, the applicant discovered that the detection of the sn-2 position ratio in oils with a carbon chain length of 16-20 (more preferably 18) is almost unaffected by enzymatic hydrolysis and oxidation. When only oils with a carbon chain length of 18 are present, the detection level is very stable. However, when oils with a carbon chain length of 18 are mixed with polyunsaturated fatty acids with a carbon chain length exceeding 20, a very large error occurs. Therefore, the detection results are accurate when only oils with a carbon chain length of 18 are tested, but the results are distorted when mixed with polyunsaturated fatty acids with a carbon chain length exceeding 20. Therefore, the detection results of quality control sample 1 as defined in this invention are generally very stable and considered equivalent to the theoretical value. Furthermore, the ratio of two actual test results (quality control sample 1: quality control sample 2) is used to correct the enzymatic hydrolysis reaction.

[0089] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for determining the distribution of fatty acids at the sn-2 position in oils rich in long-chain polyunsaturated fatty acids, characterized in that, Includes any combination of the following steps: (a) Prepare the oil sample to be tested and the quality control oil sample, wherein the quality control oil sample contains a correction fatty acid, which is bound to any acyl position of the triglyceride molecule; (b) The oil sample to be tested is subjected to enzymatic hydrolysis with a specific lipase at position sn-1,3 to obtain the hydrolysis product; (c) The quality control oil sample is subjected to enzymatic hydrolysis with a specific lipase at the sn-1,3 position to obtain the hydrolysis product; (d) Separate the enzymatic hydrolysis products obtained in steps (b) and (c) respectively to obtain the sn-2 position monoglyceride from the oil sample to be tested and the sn-2 position monoglyceride from the quality control oil sample. (e) Determine the content of the target fatty acid in the sn-2 position monoglyceride obtained from the oil sample to be tested in step (d), and the total content of the target fatty acid in the oil sample to be tested; (f) The content of correction fatty acids in the sn-2 position monoglyceride obtained from the quality control oil sample in step (d) and the total content of correction fatty acids in the quality control oil sample are determined respectively. (g) Based on the determination results of steps (e) and (f), the proportion of the target fatty acid in the sn-2 position monoglyceride of the tested oil sample is calculated using the following formula: Sn-2% (X) = [(content of X in sn-2 monoglycerides in the tested oil sample %) / (content of X in total fatty acids of triglycerides in the tested oil sample % × 3)] × (F) × 100%; Wherein, X represents the target fatty acid to be tested; F is a correction factor, which is calculated using the measured data of the quality control oil samples and is used to correct for enzymatic hydrolysis efficiency and analytical system bias.

2. The determination method according to claim 1, characterized in that, The quality control oil samples include quality control oil sample 1 and quality control oil sample 2. The quality control oil sample 1 is selected from medium-chain fatty acid oils, including at least one of sunflower seed oil, soybean oil, corn oil, rapeseed oil, tea seed oil, peanut oil, olive oil, flaxseed oil and avocado oil; the quality control oil sample 2 is prepared by mixing the quality control oil sample 1 and the oil sample to be tested at a mass ratio of 1:10 to 2:

1.

3. The determination method according to claim 2, characterized in that, The correction factor F is calculated using the following formula: For quality control oil sample 1: Sn-2%(Y)1 = (content of Y in sn-2 monoglyceride in quality control oil sample 1) / (content of Y in total fatty acids in triglycerides in quality control oil sample 1) × 3) × 100%; For quality control oil sample 2: Sn%(Y)2 = (content of Y in sn-2 monoglyceride in quality control oil sample 2) / (content of Y in total fatty acids of triglycerides in quality control oil sample 2) × 3) × 100%; Correction factor = Sn%(Y)2 ÷ Sn%(Y)1; Y represents the corrective fatty acid, which is one of the long-chain unsaturated fatty acids with a carbon chain length of 16-20, including at least one of oleic acid, linoleic acid, linolenic acid, and eicosatrienoic acid.

4. The determination method according to claim 1, characterized in that, The reaction temperature for enzymatic hydrolysis described in steps (b) and (c) is ≤40℃ and the reaction time is ≤3h.

5. The determination method according to claim 1, characterized in that, The sn-1,3 specific lipases mentioned in steps (b) and (c) include at least one of pancreatic lipase, pregastric lipase, Candida antarcticis-derived lipase, Mucor milch lipase, Rhizopus oryzae lipase, Aspergillus oryzae lipase, Rhizopus dellemarum EU O93 / Rd lipase, Yersinia lipolytica lipase, Thermophilic filamentous lipase, Penicillium camenbellum lipase, Rhizopus lipase, Penicillium roje lipase, and Candida leucovora lipase.

6. The determination method according to claim 1, characterized in that, The separation described in step (d) includes at least one of thin-layer chromatography or silica gel solid-phase extraction.

7. The determination method according to claim 1, characterized in that, The determination method described in steps (e) and (f) is gas chromatography.

8. The determination method according to claim 1, characterized in that, All processes that may involve contact with air are subjected to antioxidant protection treatment, which includes at least one of inert gas protection treatment, light protection treatment, and the addition of antioxidants.

9. The determination method according to claim 8, characterized in that, The inert gas includes at least one of nitrogen and argon; and / or the antioxidant includes at least one of vitamin E, BHT and TBHQ.

10. The determination method according to claim 1, characterized in that, The test samples and quality control samples can be selectively purified by alumina column chromatography before the start of the test.

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