Method for extracting and detecting lipid in liquid milk raw milk

Abstract

The invention relates to a method for extracting and detecting lipid in liquid raw milk, and provides a method for extracting and detecting lipid in liquid raw milk by verifying and preferably selecting a Bligh-Dyer method for extracting lipid in liquid raw milk and optimizing chromatography and mass spectrometry conditions, so as to improve the separation and identification capability of complex lipid molecules. And the detection sensitivity and stability are obviously improved.

Description

Technical Field

[0001] The invention belongs to the technical field of food safety, and in particular relates to a method for extracting and detecting lipids in liquid milk. Background Art

[0002] As one of the important sources of nutrition in human daily diet, the nutritional value and health benefits of milk have been widely recognized. With the increasing demand of consumers for healthy diet, the study of the nutritional components of milk, especially the lipid components, has gradually become the focus of academic and industrial attention. Not only has traditional cow's milk gradually gained attention, but in recent years, special dairy products such as goat's milk, camel's milk, horse's milk, and donkey's milk have also been favored by the market due to their unique nutritional value and health potential. These special milks have significant differences in lipid composition, which not only has an important impact on the sensory properties and nutritional value of dairy products, but also exhibits unique functional properties in dairy processing, storage and application. Therefore, in-depth research on the lipid composition and characteristics of different special milks is of great significance for the functional development and customized production of milk.

[0003] Lipids are one of the important components of milk, including triglycerides (TG), phosphatidylcholine (PC), sphingomyelin (SM), monoglycerides (MG), diglycerides (DG) and other types. These lipids play an indispensable role in the structural stability of cell membranes, signal transduction, energy storage and other biological functions. Recent studies have shown that the differences in lipid composition of different types of milk may have a profound impact on the emulsification properties, nutritional value and processing properties of the product.

[0004] Many methods have been developed so far for the extraction of milk lipids. The three most commonly used methods are Folch, Bligh-Dyer and methyl tert-butyl ether (MTBE). In addition, in basic research, researchers often use a faster, more economical and more environmentally friendly alternative method - ether / petroleum ether method. However, for different types of lipids, the extraction efficiency of the four methods varies, and there may even be significant differences. In addition, the traditional lipid chromatography-mass spectrometry method shows certain limitations in lipid detection, mainly including wide peaks, many overlapping peaks, unstable baselines and other problems, resulting in less than ideal resolution and detection sensitivity.

[0005] Therefore, in order to detect and analyze lipids in milk more efficiently and accurately, lipid extraction methods and chromatography-mass spectrometry detection conditions with better overall effects still need to be developed. Summary of the invention

[0006] In view of the defects of the prior art, the present invention has verified and optimized the Bligh-Dyer method for extracting lipids in liquid milk and raw milk, and optimized the chromatographic and mass spectrometric conditions, thereby providing a method for extracting and detecting lipids in liquid milk and raw milk, so as to improve the separation and identification capabilities of complex lipid molecules and significantly improve the detection sensitivity and stability.

[0007] In one aspect, the present invention provides a method for extracting and detecting lipids in liquid milk and raw milk, the method comprising the following steps:

[0008] S1: Extraction: extracting lipids from the liquid milk sample, wherein the extraction method is selected from the Folch method, the Bligh-Dyer method, the MTBE method or the ethanol / petroleum ether method to obtain an extract;

[0009] S2: Pretreatment: The extract was redissolved in dichloromethane-methanol to obtain the sample to be tested;

[0010] S3: Sample loading: Load the sample to be tested onto the chromatographic column;

[0011] S4: Elution and detection: The sample was eluted with a mobile phase (containing 70% mobile phase A and 30% mobile phase B), and lipids were detected by coupled chromatography-mass spectrometry; the content of mobile phase B in the mobile phase was continuously increased until the content of mobile phase B in the mobile phase was 100%, and the elution lasted for 38 minutes; wherein, mobile phase A was 60% acetonitrile, 40% water and 10 mM ammonium formate, and mobile phase B was 10% acetonitrile, 90% isopropanol and 10 mM ammonium formate.

[0012] In one or more embodiments, in step S1, the extraction method is the Bligh-Dyer method.

[0013] In one or more embodiments, in step S2, the volume ratio of dichloromethane to methanol in the dichloromethane-methanol is 1:1.

[0014] In one or more embodiments, in step S3, the injection volume of the sample to be tested is 2 μL.

[0015] In one or more embodiments, during the elution and detection process of step S4, the elution is: maintaining 70% mobile phase A and 30% mobile phase B at 0-1 min; reducing mobile phase A from 70% to 20% and increasing mobile phase B from 30% to 80% at 1-3 min; further adjusting mobile phase A to 10% and mobile phase B to 90% at 3-26 min; maintaining 0% mobile phase A and 100% mobile phase B at 26-36 min; maintaining isocratic elution of 0% mobile phase A and 100% mobile phase B at 36-38 min; and returning to 70% mobile phase A and 30% mobile phase B for elution at 38-40 min.

[0016] The present invention selects four classic lipid extraction methods - Folch method, Bligh-Dyer method, MTBE method and ether / petroleum ether method, and compares the differences in their extraction efficiency of liquid milk lipids. Among them, the Bligh-Dyer method performs best in terms of balanced extraction effect and ease of operation, and is used as the preferred extraction method. In addition, the present invention also optimizes the detection method of raw milk lipids, by optimizing the chromatographic and mass spectrometry conditions to improve the separation and identification capabilities of complex lipid molecules, and significantly improve the detection sensitivity and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a diagram of the lipid composition of raw milk samples with different characteristics.

[0018] Figure 2 This is a graph showing the extraction results of four extraction methods for raw milk samples with different characteristics.

[0019] Figure 3 : is the test result diagram of mixed milk before and after the optimization of chromatography-mass spectrometry conditions. Figure 3 A is a graph showing the detection results of the mixed milk before the optimization of the chromatography-mass spectrometry conditions; Figure 3 B is the detection result diagram of the mixed milk after the chromatography-mass spectrometry conditions were optimized.

[0020] English abbreviations in the attached figures: AcCa (Acetyl-CoA Carboxylase), AEA (Arachidonoyl Ethanolamide), Cer (Ceramide), CH (Cholesterol), ChE (Cholesterol Ester), DAP (Diacylglycerol Phosphate), DG (Diglyceride), DGDG (Digalactosyldiacylglycerol), FA (Fatty Acid), Hex1Cer (Monohexosylceramide), Hex2Cer (Dihexosylceramide), LBPA (Lysobisphosphatidic acid), Acid, lysophosphatidic acid), LPC (Lysophosphatidylcholine), LPE (Lysophosphatidylethanolamine), LPG (Lysophosphatidylglycerol), LSM (Lysosphingomyelin), MG (Monoacylglycerol), MGDG (Monogalactosyldiacylglycerol), OAHFA (O-Acyl Hydroxy Fatty Acids), PA (Phosphatidic Acid), PC (Phosphatidylcholine), PE (Phosphatidylethanolamine), Pet (Phosphatidylethanol), PFAA (Polyunsaturated Fatty Acids, polyunsaturated fatty acids), PG (Phosphatidylglycerol, phosphatidylglycerol), PI (Phosphatidylinositol, phosphatidylinositol), PIP (PhosphatidylinositolPhosphate, phosphatidylinositol phosphate), PS (Phosphatidylserine, phosphatidylserine), SM (Sphingomyelin, sphingomyelin), SPH (Sphingosine,Sphingosine), SQDG (Sulfoquinovosyldiacylglycerol), TG (Triacylglycerol), WE (Wax Ester), Relative Abundance. DETAILED DESCRIPTION

[0021] Example 1: Preferred method for extracting lipids from liquid milk

[0022] In this example, the lipid components in each liquid milk sample were detected by using a UHPLC-HRMS (ultra-high performance liquid chromatography-high resolution mass spectrometry) system (purchased from ThermoScientific) to obtain Figure 1 The results are shown in Figure 2. The raw milk samples are camel milk 1 (camel 1), camel milk 2 (camel 2), horse milk, donkey milk, goat milk, buffalo milk, Jersey milk, Holstein milk, yak milk 1 (yak 1) and yak milk 2 (yak 2).

[0023] The lipidomics analysis was performed on the mixed milk lipids extracted by the four different lipid extraction methods, where the mixed milk was a mixture of all the liquid milk and raw milk samples in equal proportions. The data was uploaded to lipid search 5.0 for lipidome search and comparison, and all the lipid species and peak areas obtained were statistically analyzed. The results are shown in the figure. Figure 2 shown.

[0024] from Figure 2 From the data results, it can be seen that the four lipid extraction methods (Folch method, Bligh-Dyer method, MTBE method and ethanol / petroleum ether method) have significant differences in the extraction efficiency of different lipid groups, reflecting their respective adaptive lipid categories and applicability.

[0025] Specifically, the Folch method showed a relatively balanced extraction effect in multiple lipid categories, especially in lipids such as Cer (sphingolipids), DG (diacylglycerol / diglycerol) and WE (wax esters). Although the Folch method is relatively excellent in overall extraction efficiency, it is slightly insufficient in some polar lipid types (such as LPE (lysophosphatidylethanolamine) and PI (phosphatidylinositol)), which may be related to the method's extraction priority for non-polar components, resulting in a low recovery rate of polar lipids. The Bligh-Dyer method showed a high efficiency in the extraction of different types of lipids, especially in the extraction of key lipids such as DG and PC (phosphatidylcholine), which is better than other methods, and its ability to balance water-soluble and fat-soluble components is good, which is suitable for large-scale sample processing. The Bligh-Dyer method has good performance in multiple lipid categories. Although the extraction efficiency of some lipids such as TG and WE is slightly inferior to that of the Folch method, its overall level is still high. Therefore, the Bligh-Dyer method not only performs well in extraction efficiency, but also has advantages in ease of operation. Figure 2 The performance in the MTBE method shows its advantages in the extraction of lipid categories such as TG and PC, and is particularly suitable for studies with specific requirements for TG and PC. However, the MTBE method is insufficient in the extraction of polar lipids such as Cer and PE (phosphatidylethanolamine), which may be due to the fact that MTBE is more compatible with non-polar components, thus limiting the recovery rate of these polar lipids. The ethanol / petroleum ether method shows high efficiency in the extraction of WE and has certain advantages in the extraction of non-polar lipids; however, the extraction efficiency of most polar lipids (such as Cer and FA (fatty acids)) is low.

[0026] In summary, the Bligh-Dyer method has obvious advantages in the balance of lipid extraction from liquid milk and ease of operation, and is particularly suitable for maintaining high efficiency when extracting key lipids such as DG and PC.

[0027] Example 2: Optimization of the detection method of lipids in liquid milk and raw milk

[0028] This embodiment optimizes the chromatographic and mass spectrometric conditions of the UHPLC-HRMS system (purchased from Thermo Scientific). The experiment used an Accucore TMC18 chromatographic column (150×2.1mm, 2.6μm), the column temperature was set to 45°C, and the flow rate was 250μL / min. The mass spectrometry analysis used an electrospray ionization source, which scanned simultaneously in positive and negative ion modes. The ion source temperature in the positive ion mode was set to 300°C, the spray voltage was 3.0kV (positive ion mode) and 2.5kV (negative ion mode), and the ion transfer tube temperature was 350°C. The scanning range of the primary MS is 200-1800Da. Data processing uses LipidSearch software for peak identification, extraction, and secondary identification of lipids. The test sample is a mixed milk, that is, the mixed milk described in the embodiment.

[0029] Before optimization ( Figure 3 A) experimental conditions, lipid samples were extracted by Bligh-Dyer method and then redissolved in isopropanol, and the injection volume was 5 μL. Mobile phase A was composed of 70% acetonitrile, 30% water and 10mM ammonium acetate, and mobile phase B was isopropanol and 10mM ammonium acetate solution. The gradient elution program was: 0-2 minutes to maintain 97% mobile phase A and 3% mobile phase B; 2-25 minutes linear gradient transition from 97% mobile phase A to 0% mobile phase A, 3% mobile phase B to 100% mobile phase B; 25-30 minutes to maintain 0% mobile phase A and 100% mobile phase B; 30-40 minutes to return to 97% mobile phase A and 3% mobile phase B.

[0030] Among them, the detection conditions before optimization can be found in the reference (Noriyuki Tomiyasu et al., Analytical and Bioanalytical Chemistry (2024) 416: 5445–5456). The conditions before optimization showed certain limitations in lipid detection, mainly including wide peaks, many overlapping peaks, unstable baselines, etc., resulting in unsatisfactory resolution and detection sensitivity, especially in the negative ion mode, where the baseline fluctuated greatly.

[0031] After optimization ( Figure 3B), lipid samples were extracted by Bligh-Dyer method and then dissolved in dichloromethane-methanol (1:1, v / v), and the injection volume was reduced to 2 μL. Mobile phase A was changed to 60% acetonitrile, 40% water and 10 mM ammonium formate, and mobile phase B was 10% acetonitrile, 90% isopropanol and 10 mM ammonium formate. The gradient elution program was refined: 70% mobile phase A and 30% mobile phase B were maintained from 0 to 1 minute; mobile phase A was reduced from 70% to 20% and mobile phase B was increased from 30% to 80% from 1 to 3 minutes; mobile phase A was further adjusted to 10% and mobile phase B to 90% from 3 to 26 minutes; 0% mobile phase A and 100% mobile phase B were maintained from 26 to 36 minutes; isocratic elution of 0% mobile phase A and 100% mobile phase B was maintained from 36 to 38 minutes; and 70% mobile phase A and 30% mobile phase B were restored from 38 to 40 minutes.

[0032] Through these optimizations, the lipid separation and detection effects were significantly improved. First, more time buffer was provided for detection, peak tailing and overlap of the latter segment were avoided, and the peak distribution was improved; second, the signal intensity of the chromatographic peak was significantly improved, the peak height was increased, and the noise level was significantly reduced, so that the signal-to-noise ratio was significantly improved. Especially in the negative ion mode, the baseline became more stable and the fluctuation was reduced. This baseline stability is particularly important in lipid detection, because a stable baseline can more effectively identify low-abundance compounds and improve the detection sensitivity of trace lipid molecules; secondly, after changing the reconstitution solvent from a single isopropanol to a dichloromethane-methanol mixed solvent (1:1, v / v), the solubility of lipid molecules was improved, the injection effect was more uniform, and it helped to improve the stability of the detection. In addition, the optimized mobile phase system uses ammonium formate instead of ammonium acetate, which improves the ionization efficiency and enhances the responsiveness and ionization efficiency of the mass spectrometry signal. Six parallel samples of mixed milk were tested before and after optimization. As shown in Table 1, the difference in the number of lipid ions that the LipidSearch software can identify before and after optimization shows that the number of ions that can be identified after optimization is obviously greater, with an average increase of 17.9%.

[0033] Table 1: Number of lipid ions that can be identified by LipidSearch software before and after detection method optimization

[0034]

[0035] In summary, the present invention selected four classic lipid extraction methods - Folch method, Bligh-Dyer method, MTBE method and ether / petroleum ether method, and compared the differences in their lipid extraction efficiency in liquid milk raw milk. Among them, the Bligh-Dyer method performed best in terms of balanced extraction effect and ease of operation, and was used as the preferred extraction method. In addition, the present invention also optimized the detection method of liquid milk raw milk lipids, by optimizing the chromatographic and mass spectrometry conditions to improve the separation and identification capabilities of complex lipid molecules, and significantly improve the detection sensitivity and stability.

Claims

1. A method for extracting and detecting lipids in liquid milk, characterized in that: The method comprises the following steps: S1: Extraction: extracting lipids from the liquid milk sample, wherein the extraction method is selected from the Folch method, the Bligh-Dyer method, the MTBE method or the ethanol / petroleum ether method to obtain an extract; S2: Pretreatment: The extract was redissolved in dichloromethane-methanol to obtain the sample to be tested; S3: Sample loading: Load the sample to be tested onto the chromatographic column; S4: Elution and detection: The sample was eluted with a mobile phase (containing 70% mobile phase A and 30% mobile phase B), and the content of mobile phase B in the mobile phase was continuously increased until the content of mobile phase B in the mobile phase was 100%; the elution process lasted for 40 minutes, and the lipids were continuously detected by chromatography-mass spectrometry during the elution; The mobile phase A was 60% acetonitrile, 40% water and 10 mM ammonium formate, and the mobile phase B was 10% acetonitrile, 90% isopropanol and 10 mM ammonium formate.

2. The method according to claim 1, characterized in that In step S1, the extraction method is the Bligh-Dyer method.

3. The method according to claim 1, characterized in that In step S2, in the dichloromethane-methanol, the volume ratio of dichloromethane to methanol is 1:

1.

4. The method according to claim 1, characterized in that In step S3, the loading volume of the sample to be tested is 2 μL.

5. The method according to claim 1, characterized in that In step S4, during the elution and detection process of step S4, the elution process is: maintaining 70% mobile phase A and 30% mobile phase B at 0-1min; reducing mobile phase A from 70% to 20% and increasing mobile phase B from 30% to 80% at 1-3min; further adjusting mobile phase A to 10% and mobile phase B to 90% at 3-26min; maintaining 0% mobile phase A and 100% mobile phase B at 26-36min; maintaining isocratic elution of 0% mobile phase A and 100% mobile phase B at 36-38min; and returning to 70% mobile phase A and 30% mobile phase B for elution at 38-40min.

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