A method for detecting the content of sn-2 dha in dha formulated oil

Abstract

The application provides a method for detecting the content of Sn-2 DHA in DHA formulated oil, comprising: pretreating a sample to be detected; hydrolyzing and separating the pretreated sample to obtain DHA oil; and detecting the content of Sn-2 DHA in the DHA oil; wherein the hydrolyzing comprises: hydrolyzing the pretreated sample by using pancrelipase; the pancrelipase is used in an amount of at least 2000 mg per 1 g of the pretreated sample, and the hydrolysis is performed for at least 10 minutes. The application provides a method for detecting the content of Sn-2 DHA, which can effectively improve the accuracy of detecting the content of Sn-2 DHA in DHA oil, especially the accuracy of detecting the content of Sn-2 DHA in DHA formulated oil, by improving the hydrolysis degree of the hydrolysis process, which is of great significance for quality control of DHA oil products and fatty acid analysis.

Description

Technical Field

[0001] This invention relates to the field of food testing technology, and in particular to a method for detecting the Sn-2 site DHA content in DHA-blended oil. Background Technology

[0002] Docosahexaenoic acid (DHA), commonly known as "brain gold," is a vital omega-3 polyunsaturated fatty acid that plays a crucial role in retinal and brain function, particularly in infant vision and brain development. Numerous studies have confirmed that DHA promotes cognitive abilities and improves vision. Furthermore, research indicates that DHA can improve mental health, reduce the risk of cardiovascular disease, aid in cancer treatment, lower prehepatic lipid levels in the circulatory system, and reduce the risk of diet-induced type 2 diabetes.

[0003] Depending on the source of the lipids used, DHA can be classified based on its distribution at different sites on the triglyceride (TAG) molecule (sn-1, 2, or 3). After DHA is ingested, pancreatic lipases at the sn-1 and 3 positions hydrolyze TAG, generating Sn-2 monoglycerides (MAG) and free fatty acids (FFA). Sn-2 MAG is well absorbed in the small intestinal mucosa and is subsequently used to resynthesize TAG or phospholipids (PL, an important component of brain cell membranes). In contrast, FFA derived from the sn-1 and sn-3 positions is not specifically absorbed. Therefore, TAGs distributed at the Sn-2 position are more readily absorbed and utilized by the body than randomly distributed TAGs. Furthermore, studies have shown that Sn-2 DHA lipids play a positive role in brain function development and in alleviating brain diseases such as anxiety, stress, cognitive decline, schizophrenia, and stroke. However, current guidelines on DHA diets or supplements are primarily limited to recommendations on total DHA intake, with little information on the distribution of DHA at different positions.

[0004] From a pharmacological and nutritional perspective, the positional distribution of DHA in TAG and PL molecules affects brain development and functional maintenance. Therefore, elucidating the distribution of DHA in common oils and the characteristics of diets rich in Sn-2 DHA lipids, and accordingly establishing a method to accurately detect the DHA content at the Sn-2 site in DHA-rich oils, is particularly important. Although there are currently methods for detecting Sn-2 site DHA using nuclear magnetic resonance (NMR), these methods involve expensive equipment, have significant application limitations, and require highly skilled personnel to interpret the spectra. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a method for detecting the Sn-2 site DHA content in DHA blended oils. By employing a specific hydrolysis method, the accuracy of detecting the Sn-2 site DHA content in DHA oils is improved, particularly in blended DHA oils.

[0006] This invention provides a method for detecting the Sn-2 site DHA content in DHA-blended oil, comprising:

[0007] Pre-treatment of the sample to be tested;

[0008] The pretreated sample was hydrolyzed and separated to obtain DHA oil;

[0009] The content of Sn-2 DHA in the DHA oil was determined;

[0010] The hydrolysis includes:

[0011] For the pretreated sample, pancreatic lipase was used for hydrolysis; the amount of pancreatic lipase was at least 2000 mg / (1g of pretreated sample), and the hydrolysis time was at least 10 minutes.

[0012] Currently, the standard GB / T 24894-2010, "Determination of Fatty Acid Components at the 2-position of Triglycerides in Animal and Vegetable Oils," is basically used in China to determine the Sn-2 position DHA in DHA oils. However, due to the demands of different applications, DHA oils are often blended, for example, by adding sunflower seed oil or coconut oil. In an experiment, the inventor accidentally discovered that the Sn-2 position DHA content test results of blended DHA oils using GB / T 24894 were significantly lower than those of unblended oils. For example, the Sn-2 position DHA content in unblended DHA oil (58% DHA content) was 35%, while in blended DHA oil (42% DHA content) it was only 18%. This indicates that existing methods for detecting the Sn-2 position fatty acid composition are not suitable for DHA oils, especially for blended DHA oils.

[0013] In one embodiment of the present invention, the hydrolysis includes:

[0014] The pretreated sample was mixed with sodium cholate and calcium chloride and then processed at 35–45°C.

[0015] Then mix hydrochloric acid and ether, stir well and centrifuge.

[0016] In one embodiment of the present invention, the amount of sodium cholate used is 1-50 mg / (1g of pretreated sample); and / or, the amount of calcium chloride used is 200-1000 mg / (1g of pretreated sample).

[0017] In one embodiment of the present invention, the separation includes:

[0018] The DHA oil was separated from the hydrolysis product using a thin-plate microplate method.

[0019] In one embodiment of the present invention, detecting the content of Sn-2 DHA in the DHA oil includes:

[0020] The separated products were mixed with sodium methoxide and treated at 55–65°C for 20–40 minutes; then mixed with boron trifluoride and treated at 55–65°C for 20–40 minutes; after mixing with n-hexane solution and brine, the layers were separated, and the organic layer was subjected to chromatographic analysis.

[0021] In one embodiment of the present invention, the preprocessing includes:

[0022] The sample to be tested was purified by alumina chromatography.

[0023] In one embodiment of the present invention, the DHA blended oil includes crude DHA and vegetable oil.

[0024] The present invention has the following beneficial effects:

[0025] This invention provides a method for detecting the Sn-2 site DHA content. By increasing the dosage of lipase and the hydrolysis time, the accuracy of detecting the Sn-2 site DHA content in DHA oils is effectively improved, especially for DHA-blended oils containing other ingredients such as sunflower oil and coconut oil. The method for detecting the Sn-2 site DHA content in DHA oils provided by this invention has high accuracy, similar to that of nuclear magnetic resonance (NMR) methods, while reducing detection costs. This provides an important basis for quality control of DHA oil products. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0027] In the following examples, 50% virgin DHA is DHA refined oil obtained directly through fermentation, extraction, and refining processes without any blending, and the DHA content is 50.1%, hereinafter referred to as 50% virgin DHA.

[0028] Example 1

[0029] This embodiment provides a method for detecting the Sn-2 site DHA content in DHA-containing oils. The specific procedure is as follows:

[0030] 1. Sample preparation and purification

[0031] According to the method of GB / T 24894-2010, the acid value of the sample is tested. If the acid value is less than 3%, the sample can be directly purified by alumina chromatography column. If the acid value is greater than 3%, sodium hydroxide is used to neutralize it first, and then alumina chromatography column is used to purify the sample.

[0032] Prepare a suspension by mixing 15g of activated alumina with 50mL of n-hexane or petroleum ether. Pour the suspension into the chromatography column while shaking to ensure uniform sedimentation of the alumina. When the solvent level drops to 1mm-2mm above the adsorbent, carefully pour 5g of oil solution dissolved in 25mL of n-hexane or petroleum ether into the chromatography column. Collect the wash solution flowing out of the column in a 100mL round-bottom flask.

[0033] Most of the solvent is removed by vacuum distillation, then a nitrogen stream is introduced, and the oil is dried at 30℃~40℃ until the solvent is completely removed.

[0034] 2. Hydrolyzed sample

[0035] For the sample obtained in step 1 (in this invention, the acid value of the sample oils does not require neutralization with sodium hydroxide, so direct alumina chromatography purification is sufficient), weigh approximately 0.1 g of the column-passed sample into a test tube. Add 20 mg (40 mg / 100 mg / 200 mg) of pancreatic lipase, 2 ml of buffer solution, 0.5 ml of sodium cholate solution, and 0.2 ml of calcium chloride solution. Shake well at 40°C for 1 min (2 min / 5 min / 10 min / 15 min / 20 min / 30 min). Transfer the test tube to a mixer and shake well for 2 min. Add 1 ml of hydrochloric acid and 1 ml of diethyl ether to the test tube. Vigorously shake the test tube for 10 s. Transfer the test tube to a centrifuge and centrifuge at 4000 rpm for 10 min. Transfer the diethyl ether phase to a vial.

[0036] 3. Separation of DHA lipids

[0037] The hydrolyzed sample obtained in step 2 was separated by thin-layer chromatography, following the separation procedure for 2-monoglycerides in GB / T24894-2010.

[0038] The process is as follows:

[0039] (1) Separation of DHA lipids

[0040] Spot the hydrolyzed sample onto the thin-layer plate 15 mm from the bottom edge using a glass capillary tube. Place the thin-layer plate in a developing tank pre-filled with developing solvent and saturated. Cover and perform chromatography until the solvent reaches 10 mm from the top edge of the thin-layer plate. Dry the thin-layer plate in air and spray the developing solvent onto it using a spray gun. Mark the DHA lipid band under UV light and scrape it off with a micro-scraper to obtain silica gel containing DHA lipids. Prepare fatty acid methyl esters by combining this with 0.1 g of the original sample (pre-column sample) according to GB / T 17376 method, and then determine the fatty acid methyl esters according to GB / T 17377 gas chromatography method.

[0041] The calculation formula is as follows:

[0042]

[0043] in:

[0044] W – The percentage of Sn-2 DHA in total DHA (by mass).

[0045] W2 – DHA content at the Sn-2 position.

[0046] Wt – Total DHA content of the sample.

[0047] Experimental Example 1

[0048] This experimental example uses the existing GB / T 24894-2010 method and the method described in Example 1 to detect the Sn-2 site DHA content in DHA oil, as detailed below:

[0049] 1. Test results according to GB / T 24894-2010 method

[0050] GB / T 24894-2010 was used to test 50% DHA crude oil and 50% DHA crude oil + 10% high-sunflower oil. The 50% DHA crude oil is the refined DHA oil obtained directly through fermentation, extraction, and refining processes of this invention, without any blending. Its DHA content is 50.1%, hence it is simply referred to as 50% DHA crude oil, and can also be referred to as Jiabiyou Sample 1 in the following text. The following results were obtained:

[0051] Table 1 Test results according to GB / T 24894-2010

[0052] 50% DHA crude oil 50% DHA crude oil + 10% high-sunflower oil Sn-2-bit DHA 35.5 18.6

[0053] The results show that the accuracy of the Sn-2 position DHA detection significantly decreased after adding high-sunflower oil (the DHA content in high-sunflower oil is almost negligible).

[0054] 2. Test results using NOVZYM435

[0055] After replacing pancreatic lipase in GB / T 24894-2010 with NOVZYM435, the test results are as follows:

[0056] Table 2 Detection results of NOVZYM435

[0057] 50% DHA crude oil 50% DHA crude oil + 10% high-sunflower oil Sn-2-bit DHA 20.3 13.8

[0058] 3. Determination of Sn-2 DHA content in DHA lipids under different pancreatic lipase hydrolysis conditions

[0059] The samples tested are as follows:

[0060] Sample 1 is a blended oil: 50% raw DHA oil + 10% palm oil; Sample 2 is a blended oil: 50% raw DHA oil + 10% high-sunflower oil; Sample 3 is an unblended oil: 50% raw DHA oil.

[0061] The test results are as follows:

[0062] Table 3. Detection results of Sn-2 DHA content in DHA oils under different hydrolysis conditions (partial representative)

[0063]

[0064]

[0065] As shown in the table above, the meanings of the enzymatic hydrolysis conditions are as follows:

[0066] Sn-2-20mg-1min indicates that the amount of pancreatic lipase used in the hydrolysis step is 20mg, and the hydrolysis time is 1min.

[0067] The results showed that with increasing hydrolysis time and enzyme content, the DHA content at the Sn-2 site in blended oils exhibited a significant increasing trend, reaching a near-equilibrium level at 200 mg / 10 min. The change in unblended oils was relatively smaller, but also showed a certain upward trend.

[0068] In addition, NOVZYM435 was used to replace pancreatic lipase under the same hydrolysis conditions, but the overall test results were significantly lower than those of pancreatic lipase.

[0069] 4. Based on the results of step 3 above, the present invention further uses two conditions, Sn-2-40mg 2min and Sn-2-200mg 10min, to conduct experiments on commercially available oils. The experimental samples are commercially available sample 1 and its blend with high sunflower oil or palm oil; and Jiabiyou sample 2 and its blend with high sunflower oil, palm oil or coconut oil.

[0070] Among them, commercially available sample 1 is a DHA oil with a content of 40% purchased from the market, while sample 2 from Jiabiyou is a 40% DHA oil produced by the present invention. It has a different strain than the 50% DHA crude oil, but the extraction and refining methods are the same.

[0071] The specific results are as follows:

[0072] Table 4. Comparison of Sn-2 DHA content at different hydrolysis times in different blended oils.

[0073]

[0074]

[0075] 5. This invention further detected the DHA content at the Sn-2 site in commercially available samples 2-5, and obtained the following results:

[0076] Table 5 Comparison of Sn-2 DHA content in commercially available oils under different hydrolysis conditions

[0077] Method Name Commercially available sample 2 3 commercially available samples Commercially available sample 4 5 commercially available samples Sn-2-40mg 2min 28.88％ 25.35％ 24.68％ 33.87％ Sn-2-200mg 10min 32.77％ 36.81％ 32.97％ 44.52％

[0078] This invention further employs nuclear magnetic resonance to detect DHA at the Sn-2 site in commercially available samples, with the following results:

[0079] Table 6. Magnetic Resonance Verification Results of Commercially Available Samples

[0080]

[0081] As can be seen from the results above, the method provided by this invention can effectively improve the accuracy of detecting the Sn-2 site DHA content in DHA oils, achieving an accuracy close to that of nuclear magnetic resonance.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting the Sn-2 site DHA content in DHA-blended oil, characterized in that, include: Pre-treatment of the sample to be tested; The pretreated sample was hydrolyzed and separated to obtain DHA oil; The content of Sn-2 DHA in the DHA oil was determined; The hydrolysis includes: For the pretreated sample, pancreatic lipase was used for hydrolysis; for every 1g of pretreated sample, the amount of pancreatic lipase used was at least 2000mg, and the hydrolysis time was at least 10 minutes; the DHA blended oil included crude DHA and vegetable oil. The separation includes: The DHA oil was separated from the hydrolysis product using a thin-plate microplate separation method. The detection of the Sn-2 site DHA content in the DHA oil includes: The separated products were mixed with sodium methoxide and treated at 55-65°C for 20-40 minutes; then mixed with boron trifluoride and treated at 55-65°C for 20-40 minutes; after mixing with n-hexane solution and brine, the layers were separated, and the organic layer was analyzed by chromatography.

2. The method according to claim 1, characterized in that, The hydrolysis includes: The pretreated sample was mixed with sodium cholate and calcium chloride and then processed at 35-45°C. Then mix hydrochloric acid and ether, stir well and centrifuge.

3. The method according to claim 2, characterized in that, For every 1g of pretreated sample, the amount of sodium cholate used is 1~50mg; and / or, for every 1g of pretreated sample, the amount of calcium chloride used is 200~1000mg.

4. The method according to claim 1, characterized in that, The preprocessing includes: The sample to be tested was purified by alumina chromatography.

5. The method according to claim 1, characterized in that, The DHA crude oil is a refined DHA oil obtained through fermentation, extraction, and refining processes.

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