CN112250706A

CN112250706A - Method for extracting and purifying phospholipid from clams

Info

Publication number: CN112250706A
Application number: CN202011040174.2A
Authority: CN
Inventors: 沈清; 宋恭帅; 王萍亚; 赵巧灵
Original assignee: Zhoushan Institute For Food And Drug Control; Zhejiang Gongshang University
Current assignee: Zhoushan Institute For Food And Drug Control; Zhejiang Gongshang University
Priority date: 2020-09-28
Filing date: 2020-09-28
Publication date: 2021-01-22
Anticipated expiration: 2040-09-28
Also published as: CN112250706B

Abstract

The invention discloses a method for extracting and purifying phospholipid from clams, which comprises the following steps: collecting clam tissues, homogenizing by using a high-speed dispersion machine, and then drying under vacuum freezing to obtain clam powder; ② chloroform methanol ultrasonic treatment is used to extract crude lipid from clam powder; thirdly, washing the crude lipid with cold acetone to obtain a crude lipid extract; and fourthly, using the compound of the silicon dioxide fiber nanoparticles and the graphene oxide nanoparticles as an adsorbent, and performing adsorption extraction on the phospholipid in the crude lipid extract by using a solid phase extraction method. The invention can effectively and reliably extract highly purified phospholipid from clams so as to carry out lipidomics analysis and phenotype analysis.

Description

Method for extracting and purifying phospholipid from clams

Technical Field

The invention relates to a method for extracting and purifying phospholipid from clams, and belongs to the technical field of food manufacturing and detection.

Background

Clams belong to shellfish, are one of the most abundant marine mollusks, and are widely cultivated and harvested worldwide. Asian clams (Corbicula fluminea) are mainly distributed in east Asian countries, especially China, Korea and Japan. The Food and Agriculture Organization (FAO) of the united nations reports that the yield of fishing and aquaculture of clams worldwide (2018) exceeds 660 ten thousand tons, which indicates that clams are the highest yielding species of shellfish. Clams are attractive to customers in coastal areas due to their unique nutritional ingredients, and research on their nutritional ingredients, including minerals, lipids, proteins, etc., has drawn a great deal of attention from the large consumption of clams.

Recent research reports indicate that clams are rich in polyunsaturated phospholipids, which play a key role in maintaining the endogenous system and cell membranes, participating in signaling and acting as binding sites for proteins. Phospholipids are the major components of cell membranes and are composed mainly of Phosphatidic Acid (PA), Phosphatidylinositol (PI), Phosphatidylethanolamine (PE), Phosphatidylcholine (PC), Sphingomyelin (SM) and small amounts of neutral fats. The chemical structure of phospholipids consists of a glycerol backbone and a hydrophilic polar head group and two hydrophobic acyl chains. It is known that long-chain polyunsaturated fatty acyl chains (LC-PUFA), such as eicosapentaenoic acid (EPA, C20: 5) and docosahexaenoic acid (DHA, C22: 6) acyl chains, can relieve neurodegenerative diseases and improve brain function. Phospholipids of the LCPUFA structure are reported to have higher bioavailability, partitioning efficiency and functionality in reducing cardiovascular risk, etc. than ethyl esterified LC-PUFAs and glycerides of the LC-PUFA structure. However, the complexity and diversity of chemical structures present challenges to phospholipid molecular analysis.

In the fields of food industry, life sciences and medicine, scientists have prepared phospholipids by extracting purified phospholipids from substrates and have performed lipidomic and phenotypic analyses. Generally, extraction of crude total lipids using organic solvents (including methanol, dichloromethane, chloroform, etc.) presents the challenge of purifying low abundance phospholipid molecular species MS in impurities and non-polar triglycerides.

Disclosure of Invention

The invention aims to provide a method for extracting and purifying phospholipid from clams. The method can effectively and reliably extract highly purified phospholipids from clams so as to carry out lipidomics analysis and phenotype analysis.

The technical scheme of the invention is as follows: a method for extracting and purifying phospholipid from clams is characterized by comprising the following steps:

collecting clam tissues, homogenizing by using a high-speed dispersion machine, and then drying under vacuum freezing to obtain clam powder;

② chloroform methanol ultrasonic treatment is used to extract crude lipid from clam powder;

thirdly, washing the crude lipid with cold acetone to obtain a crude lipid extract;

and fourthly, using the compound of the silicon dioxide fiber nanoparticles and the graphene oxide nanoparticles as an adsorbent, and performing adsorption extraction on the phospholipid in the crude lipid extract by using a solid phase extraction method.

In the above method for extracting and purifying phospholipids from clams, in the step (iv), the preparation method of the composite of silica fiber nanoparticles and graphene oxide nanoparticles is as follows: dispersing the synthesized graphene oxide nano particles in water and carrying out ultrasonic treatment to obtain a graphene oxide solution, and uniformly dispersing for 1 h; then, adding the silica fiber nanoparticles into the dispersed graphene oxide solution according to the mass ratio of the silica fiber nanoparticles to the graphene oxide nanoparticles of 1: 1, and stirring the mixed solution for 2 hours at 120 ℃ by intense magnetic force; finally, the residue was washed three times or more by using a 50% v/v ethanol aqueous solution to remove unabsorbed silica fiber nanoparticles, to obtain a composite of silica fiber nanoparticles and graphene oxide nanoparticles.

In the method for extracting and purifying phospholipid from clams, the preparation method of the silicon dioxide fiber nanoparticles comprises the following steps: under ultrasonic treatment, after dissolving a mixture of 0.60g of urea and 1.10g of cetylpyridinium bromide (CPB) in 30mL of water for 5min, 30mL of cyclohexane and 0.94mL of isopropanol are added; then, 3mL of tetraethyl orthosilicate (TEOS) was added dropwise to the mixture using a syringe, and the solution was heated to 70 ℃ with stirring; after centrifugation at 8000g for 10min at 4 ℃, the resulting residue was washed three times with acetone, deionized water and absolute ethanol in order and dried under vacuum for 12h to obtain silica fiber nanoparticles by calcination at 500 ℃ for 4 h.

In the foregoing method for extracting and purifying phospholipids from clams, the preparation method of the graphene oxide nanoparticles comprises the following steps: mixing 360mL of sulfuric acid solution (98%) per 40mL of phosphoric acid solution (85%) to obtain a mixed acid solution, adding 18.0g of potassium permanganate and 3.0g of graphite flake into the mixed acid solution to perform reaction, after the reaction is cooled to room temperature, pouring the reaction mixture on ice, and adding 5mL of 30% H by mass concentration₂O₂The aqueous solution was quenched and then centrifuged, and the resulting mixture after centrifugation was neutralized with 4 wt% aqueous HCl (8,000 g at 4 ℃ for 30min), and the residual salts in the resulting solid material were removed by dialysis in ultrapure deionized water and dried under vacuum at room temperature overnight to give graphene oxide nanoparticles.

In the method for extracting and purifying phospholipid from clams, the concrete method of the step II comprises the steps of accurately weighing clam powder obtained in the step I, mixing the clam powder with chloroform-methanol mixed liquor with the volume ratio of 2:1, extracting crude lipid under ultrasonic treatment, and adding deionized water into the mixture to separate hydrophilic impurities; then, the mixture was centrifuged at 8000g for 10min, the bottom organic phase was recovered while the solid residue was extracted twice more with chloroform, and finally, the collected organic phases were combined and evaporated to dryness under a nitrogen stream using 12mL of chloroform-methanol mixture, 3.2mL of deionized water, and 10mL of chloroform per 0.3g of clam powder.

In the method for extracting and purifying phospholipid from clam, in the step (i), the rotating speed of the high-speed disperser is 1500 rpm.

In the method for extracting and purifying the phospholipid from the clams, the specific method of the step (IV) is as follows: packing the adsorbent in a sleeve, arranging two polypropylene gaskets at the bottom and the top of the sleeve to well fix the adsorbent to prepare an SPE column; and then mixing the mixture by using hexane and a volume ratio of 2:1, loading the crude lipid extract on an SPE column under vacuum, then successively activating with a chloroform/isopropanol mixture of 2:1 chloroform/isopropanol mixed solution and 0.1% formic acid ether solution by mass percentage wash SPE column, in order to remove free fatty acid and neutral lipid; finally, the reaction solution is passed through a column at a flow rate of 1.5 mL. min^–1The SPE cartridge was washed with a mixture of chloroform methanol hydrochloric acid (2: 1: 0.01, v/v/v) to obtain a fraction of the objective compound, and the eluate was dried under a nitrogen stream.

Compared with the prior art, the phospholipid in the crude lipid extract is adsorbed and extracted by using a solid phase extraction method after the lipid in the clams is coarsely extracted by using a compound of silicon dioxide fiber nano particles and graphene oxide nano particles as an adsorbent, the method utilizes the characteristic that the surface of the graphene oxide contains a plurality of polar parts and the porous characteristic of the silicon dioxide fiber nano particles, uses the graphene oxide nano particles as a supporting material, compounds the silicon dioxide fiber nano particles on the graphene oxide nano particles, and selectively absorbs the phospholipid by using the adsorbent which is not subjected to solid phase extraction and is prepared from the composite material, so that the phospholipid in the clams can be more fully and reliably purified and extracted. Tests prove that the detection method can quickly and accurately detect the contents of harmful substances and flavor substances in the smoked instant sturgeon fillets, so that the quality of the products can be controlled more conveniently. The phospholipid extracted by the method can identify and quantify 35 PMS in clams at most, and obtains 53.62% of PC molecular species with EPAADHA structure.

Drawings

FIG. 1 is a schematic diagram comparing the structural characterization of GO, KCC-1 and G/KCC-1;

FIG. 2 is an XRD pattern of GO, KCC-1 and G/KCC-1;

FIG. 3 is the UVvis DRS spectra of GO, KCC-1 and G/KCC-1;

FIG. 4 is a graphical representation of the effect of pH on phospholipid recovery;

FIG. 5 is a schematic of the effect of elution solution volume on phospholipid recovery;

FIG. 6 is a graphical representation of the effect of flow rate on phospholipid recovery;

FIG. 7 is a mass spectrum of crude lipid extract (A);

FIG. 8 is a mass spectrum of acetone-washed lipid (B);

FIG. 9 is a mass spectrum of the target phospholipid (C).

Detailed Description

The invention is further illustrated by the following figures and examples, which are not to be construed as limiting the invention.

Examples are given.

1.1 materials and reagents

Graphite, 3-Aminopropyltriethoxysilane (APTES) and tetraethyl orthosilicate (TEOS) were purchased from SigmaAldrich (St. Louis, Mo., USA). Acetonitrile and methanol were purchased from merck, darmstadt, germany, in chromatographic grade. By providing hydrogen peroxide (H)₂O₂) Phosphoric acid (H)₃PO₄) Hydrochloric acid (HCl), acetone, isopropanol, potassium permanganate (KMnO)₄) And urea national chemical agents ltd (shanghai, china). Phosphatidylcholine (PC) 14: 0/14: 0, Phosphatidylethanolamine (PE) 15: 0/15: 0 and Phosphatidylinositol (PI) 16: 0/16: a representative chemical standard of 0 is available from Avanti Polar Lipids, Inc. Using MilliQ seriesUltrapure water (18.2M Ω. cm) was produced systematically (Bedford, Mass., USA).

1.2 Synthesis of composite of silica fiber nanoparticles and graphene oxide nanoparticles (hereinafter referred to as G/KCC-1)

Graphene oxide nanoparticles (hereinafter referred to as GO). And (3) adding 9: 1(360 mL: 40mL) by volume concentrated H was added₂SO₄/H₃PO₄,KMnO₄The mixture of (18.0g) and graphite flakes (3.0g) may develop a slight exotherm during the reaction. After cooling to room temperature (about 25 deg.C), the mixture was poured onto ice and 5mL of 30% H₂O₂And (4) quenching. Then, after centrifugation, the mixture was neutralized with 4 wt% aqueous HCl (8,000 g at 4 ℃ C. for 30 min). The residual salts in the resulting solid material were removed by dialysis in ultrapure deionized water. The synthesized GO was dried under vacuum at room temperature overnight.

Synthesis of silica fiber nanoparticles (hereinafter abbreviated as KCC-1). First, a mixture of urea (0.60g) and CPB (1.10g) was dissolved in water (30mL) for 5min under sonication, followed by the addition of cyclohexane (30mL) and isopropanol (0.94 mL). TEOS (3mL) was then added dropwise to the mixture using a syringe and the solution was heated to 70 ℃ with stirring. After centrifugation at 8000g for 10min at 4 ℃, the residue was washed three times with acetone, deionized water and absolute ethanol in that order and dried under vacuum for 12 h. KCC-1 nanoparticles were obtained by calcination at 500 ℃ for 4 h.

The G/KCC-1 nanocomposite is synthesized by a direct steam-induced internal hydrolysis method. In brief, the synthesized GO nanoparticles were dispersed in water and sonicated to obtain a GO solution that was uniformly dispersed for 1 h. KCC-1 nanoparticles were then added to the GO solution at a G/KCC-1 mass ratio of 1: 1. The mixed solution was stirred vigorously magnetically at 120 ℃ for 2 h. Finally, the unabsorbed KCC-1 was removed by washing the residue three times with 50% v/v ethanol aqueous solution to obtain a G/KCC-1 nanocomposite.

1.3 characterization

Atomic Force Microscope (AFM) images were performed by tapping on a Digital Multimode V scanning probe microscope (Veeco Metrology, USA) to measure the thickness of G/KCC-1 nanocomposites. Transmission electron microscopy (TEM, JEOL Jem-2100F, Thermo Scientific, Mass.) and scanning electron microscopy (SEM, FEI survey F50, Thermo Scientific, Mass.) were used to characterize the G/KCC-1 nanohybrids. The phase composition of the G/KCC-1 nano hybrid was evaluated using X-ray diffraction (XRD) with CuK α X-ray radiation (λ 1.5406nm) (Smartlab9, Rigaku, japan). For the analysis of the elemental composition, an ultraviolet-visible diffuse reflectance spectroscopy (UV vis-DRS) spectrometer (Lambda 950, Perkin Elmer, USA) equipped with a 150mm integrating sphere attachment was used.

1.4 Experimental example of extracting purified phospholipid from clam

Clams (corbicula fluminea) were purchased from Wumei supermarket (hang state, china). Tissues were collected, homogenized using an Ultra Turrax (T25, IKA-Werka, germany) at 1500rpm, and then dried to a powder under vacuum freeze-drying (Beta 2-8LD plus, Christ, germany). 0.3g of dried clam powder was accurately weighed out again and mixed with chloroform methanol (2: 1, v/v, 12 mL). After extraction of crude lipids under sonication, deionized water (3.2mL) was added to the mixture to separate hydrophilic impurities. The mixture was then centrifuged at 8000g for 10 min. The bottom organic phase was recovered while the solid residue was extracted two more times with chloroform (10 mL). Finally, the collected organic phases were combined and evaporated to dryness under a stream of nitrogen. The crude lipids were washed with cold acetone (-20 ℃) before solid phase extraction with G/KCC-1.

The preparation method of the G/KCC-1SPE mini-column is as follows. The synthesized G/KCC1 nanocomposite (120mg, 0.8cm height) was packaged in a sleeve (1 mL). Two polypropylene gaskets were placed at the bottom and top of the cartridge to hold the adsorbent well, thus making a G/KCC-1SPE cartridge. G/KCC-1SPE cartridge by hexane and chloroform/isopropanol (2: 1, V/V) activated. The crude lipid extract (60mg) was loaded onto an SPE cartridge under vacuum. The column was then washed sequentially with chloroform/isopropanol (2: 1, v/v) and 0.1% formic acid in ether to remove free fatty acids and neutral lipids. Finally, the reaction solution is passed through a column at a flow rate of 1.5 mL. min^–1The column was washed with a mixture of chloroform-methanol-hydrochloric acid (2: 1: 0.01, v/v/v) to obtain a fraction of the objective compound. The eluent is placed under nitrogenAnd (4) drying under flowing.

1.5HILIC-MS analysis

The chromatographic separation of phospholipids was carried out using a Cosmosil HILIC chromatography column (250X 4.6mm, 3 μm). Phospholipid analysis was performed using an Agilent 1100 series HPLC instrument (Agilent technologies, Palo alto, Calif.) with a mass spectrometer (4000Q-Trap, Applied biosystems Sciex, Foster, USA with an electrospray ionization (ESI) interface). In gradient mode, the target analytes retained on the column are washed with mobile phases of A (20mM ammonium acetate and 20mM acetic acid in water) and B (20mM acetic acid in ACN). The sample injection amount was set to 2. mu.L, and the flow rate was set to 0.6 mL. min^–1And a column temperature of 30 ℃. Prior to each injection, the column was washed, readjusted to the initial state, and re-equilibrated for 10 minutes to achieve good reproducibility. Phospholipids were ionized by an ESI ion source in negative ion mode (500 ℃ C., -4500V). Nitrogen was controlled at a curtain pressure of 25psi, ion source gas 1(GS1)35psi and ion source gas 2(GS2)35 psi. The Declustering Potential (DP), the Entrance Potential (EP) and the ion spray voltage (IS) were set to-60V and-10V, -4500V, respectively. Analysts 1.6.3 (Applied Biosystems, california, usa) are Applied for system control, spectrum collection and data analysis.

1.6 data analysis and statistics

HILIC-MS data was analyzed by the LIPID MAPS toolkit. One-way anova was used to determine statistical differences in significance levels (p < 0.05). The correlation was checked by Pearson correlation analysis (2 tails) and visualized by R version 3.6.2. The variability present in the data sets obtained was performed by MetabioAnalyst 3.0 (McGill university of Montreal, Canada).

2 results and discussion

We designated the products obtained at each stage of the treatment process (including crude lipid extract, acetone washed lipids and target phospholipids) as a, B and C, respectively.

The structure and morphology of GO, KCC-1 and G/KCC-1 nanocomposites were characterized by SEM, AFM and TEM. In fig. 1 a shows a SEM image of GO, which shows a large layered structure with a smooth surface. B is an AFM image showing the thickness and surface of the GO plate. After strong oxidation of graphite, single-layer and multi-layer GO sheets with smooth surfaces and a thickness of about 0.854nm are obtained. C shows an SEM image of KCC-1 having a wrinkled structure of mesoporous KCC-1. This structure can increase the coating ability and significantly enlarge the surface area. TEM image (D) of KCC-1 shows flower-like spherical morphology of colloidal silica spheres with center-radial nanowrinkles, formed by growth of central dendritic fibers. After KCC-1 is compounded with GO, the surface of GO is covered by KCC-1 particles (E). AFM image (F) showed an increase in the thickness of G/KCC-1 (4.571 nm). The characterization results of scanning electron microscopy, atomic force microscopy and transmission electron microscopy were consistent, which could confirm the successful preparation of GO sheets coated with KCC-1 particles.

FIG. 2 depicts XRD patterns of GO, KCC-1 and G/KCC-1 nanocomposites. After oxidation, the XRD pattern of the prepared GO shows a strong diffraction peak at 10.12 ° 2 θ, which corresponds to an interlayer distance of 0.88 nm. The resulting interlayer spacing can be attributed to the presence of carboxyl, hydroxyl, and epoxy functional groups on the GO surface, indicating that graphite has been successfully oxidized to GO. The diffraction peak of the 24,25G/KCC-1 nano hybrid is 23.5 degrees, which is attributed to the diffraction peak of KCC-1 particles. The optical properties of GO, KCC-1 and G/KCC-1 nano-hybrids were studied by UVvis DRS spectroscopy (FIG. 3). The absorption peak of GO is in the range of 235 to 310 nm. The transition of the aromatic CC bond can be reflected in the absorption peak at 235 nm. Absorption peaks of the G/KCC-1 nano hybrid were observed at both 230.1nm and 271.4nm due to the formation of silane molecules and KCC-1 particles in the graphite structure and on the GO surface by the plasma.

2.2 optimization of the adsorbent Material (SPE matrix)

The G/KCC-1 nanocomposite is an excellent solid material for phospholipid SPE because it has a large surface area of carbon and silica elements. In order to completely recover PMS remaining on G/KCC-1, conditions most influential on the pH of the sample, the volume and flow rate of the elution liquid, and the like were optimized.

The effect of pH on PMS recovery was first investigated. Under very low acid and base conditions, the adsorbent will degrade and cause the KCC-1 moieties to cleave from the GO surface. Thus, the pH was adjusted to 3 to 7, and the results are shown in FIG. 4As shown. The recovery of the representative standards gradually increased as the pH increased from 3 to 6. The recovery of phospholipids was highest (75.4% -88.6%) at pH 6, and dropped dramatically as pH was further increased. Thereafter, the volume of the elution solution in the range of 1-5mL was optimized to completely elute all PMS remaining on the adsorbent. The recovery of the three phospholipid standards was gradually increased with increasing elution solution volume (figure 5). It can be observed that PI is more sensitive to the volume of the elution solution than PC and PE. When the volume was increased to 5mL, the recovery of PI increased greatly to a maximum of 69.7%. The trend of PC and PE changes relatively gradually, which means that PC and PE are relatively easy to desorb from G/KCC-1 nanocomposites compared to PI. This phenomenon also indicates that PC and PE have stronger binding ability to KCC-1 than PI. Finally, 0.5 to 2.5mL min was also tested^–1To investigate its effect on PMS recovery (fig. 6). The PMS exhibits a curve in which the recovery rate increases and then decreases as the flow rate increases. At 1.5 mL/min^–1The highest recovery of PC, PE and PI was in the range of 60.2% to 80.1% at the flow rate of (2). Finally, the pH of the sample was used 6, the eluent was 5mL and the flow rate was 1.5mL min^–1The parameter (c) of (c).

2.3 characterization of lipids

TLC has been used for characterization of biologically active substances and to monitor the progress of the reaction by semi-quantitative or quantitative analysis. It was used in G/KCC-1SPE eluates for rapid analysis of phospholipid content. The profile of phospholipids in the clam samples and standards was analyzed. The results show that the crude lipid of clams consists of PE, PC, PI and a large number of impurities. After washing with cold acetone, some impurities were removed, indicating that cold acetone can effectively remove non-polar lipids. For the G/KCC-1SPE, the eluted fractions appeared clean as observed in TLC plates (as most of the impurities in the head and tail were eliminated.) the composition of the remaining phospholipid species was consistent with the crude lipid sample.

To fully characterize the PMS, we used HILIC-MS technique. In this study, phospholipids with different polarity heads resulted in a significant change in their retention times on HILIC columns, and thus baseline separation of the three phospholipids PC, PE and PI was achieved within a 20min run time. In the positive ion mode, phospholipids cannot be ionized simultaneously at a single time, and the signal is easily suppressed by impurities due to protonation competition, so that the ionization of phospholipids can be handled in the negative ion mode. When formic acid is added to the mobile phase, PE and PI are typically protonated as [ M-H ] -, whereas PC is readily ionized as [ M + HCOO ] -. FIG. 7 shows the-MS spectra of representative crude HILIC lipids, in which the key peaks can be grouped into three groups according to the m/z range, namely m/z 450-550, m/z600-650 and m/z 750-900. Peaks in the ranges of m/z 450-550 and m/z 750-900, respectively, can be identified as lysophospholipids and phospholipids, while peaks in the range of m/z600 to 650 are co-extracted impurities from clam samples. The crude lipids were washed with cold acetone to remove non-polar molecules and interferences and the corresponding product spectra were recorded. Compared to the spectrum of crude lipid, the phospholipid signal in FIG. 8 increased from 2.0e6 cps to 2.3e6 cps, more PMS was observed, and the impurity peaks (e.g., m/z 600.4, m/z 610.4, and m/z 627.3) increased, and decreased significantly. The spectrum of the G/KCC-1SPE eluate is recorded in FIG. 9. The phospholipid signal observed in the m/z 700-900 range was further enhanced to 2.6e6 cps. In addition, background noise is hardly seen, and most of impurities are effectively removed. The HILIC-MS results for these three samples were almost identical to those of TLC analysis. After noise filtering and centroid processing, a total of 35 ions could be effectively identified (table 1). Since the head groups of these ions have been determined by phospholipid standards, the LIPIDMAPS software can unambiguously predict the sum of carbon atoms and double bonds.

TABLE 1 identification and content of phospholipids in clam samples treated by three methods

2.4 quantification of Phospholipids

Each PMS was quantified on the basis of an external calibration model. PC 14 is based on three principles: 0/14: 0, PE 15: 0/15: 0 and PI 16: 0/16: the principle behind 0, the three phospholipid standards, used to quantify all identified PMSs, was that the ionization efficiency of the compounds in MS was largely dependent on the dipole moment, the dipole moment of the phospholipids was mainly concentrated on the head group, and the dipole moment of the fatty acyl chains was negligible. The model was established by plotting a calibration curve using the peak area and the weight coefficient of phospholipid standard concentration at five levels as 11 ×. As shown in table 2, PC 14: 0/14: linear equation of 0 is y ═ 4.76E +06x-57100, R²Is 0.9981, wherein the variables for x and y are concentrations (mg. g)^–1) And peak area. Thus, the absolute content of each PMS was calculated as shown in table 1. After the G/KCC-1SPE, PC 16 was found: 0/20: 5(m/z 824.6) is most important, followed by PC 16: 0/18: 1(m/z 804.6) and PC 16: 0/22: 6(m/z 850.6). Interestingly, the PC molecular species of the epaahha structure account for a significant proportion of 53.62%. It has been reported that this species of PC molecules with EPA/DHA structure has a function in regulating the intestinal immune response, regulating the intestinal flora composition and relieving chronic stress [28 ]]. In the m/z 700-800 range, ions at m/z 716.4 and m/z 790.5 are the two most important PE molecular species, identified as [ PE 16: 0/18: 1-H]And [ PE 18: 0/22: 6-H]-. A total of 7 PI molecular species were identified, with PI 18: 0/18: 1(m/z 863.6) and PI 16: 1/18: 0(m/z 835.6) is the two PI molecular species with the highest content, respectively 1.57 and 1.06 μ g. mg^–1. The results show that the G/KCC-1SPE can enrich PMS in a pure way.

TABLE 2 SPE-HILIC-MS method verification in terms of linearity, sensitivity, accuracy and recovery

2.5 analytical Properties

The performance of the G/KCC-1SPE HILIC-MS lipidomics method has been validated through correlation coefficient (R2), linear range, limit of detection (LOD), limit of quantitation (LOQ), daytime and internal validation of day precision and recovery. As shown in Table 2, the regression equations were linear in the concentration range of 0.39-200. mu.g.mL-1, and the correlation coefficient (R2) was in the range of 0.9965 to 0.9981, indicating a good linear relationship between the regression equations. Peak area (y) and target phospholipid concentration (x). LOD and LOQ are considered to be the lowest concentrations of target analyte that can be unambiguously identified and quantified, and are calculated using signal-to-noise ratios (s/n) of 3 and 10, respectively. The measured LOD and LOQ are respectively 0.19-0.51. mu.g.mL^–1And 0.48-1.47. mu.g.mL^–1Within the range of (1). The accuracy of the method is expressed as an intra-day accuracy and an inter-day accuracy. The day precision was evaluated by calculating the Relative Standard Deviation (RSD) of the six technical replicates, while the day precision was determined by examining the variability for three consecutive days. RSD for the intra-day precision is between 4.64% and 7.16%, and RSD for the inter-day precision is in the range of 5.78% -7.30%. By mixing at 50, 100 and 150. mu.g.mL^–1And adding a standard blank matrix into the phospholipid standard substance to perform a recovery rate experiment. This value is calculated based on the proportion of standards added, which is greater than 88.19%. The result shows that the G/KCC-1SPE HILIC-MS method is reliable, accurate and effective in selective extraction from clams and lipidomics phenotype analysis.

4 conclusion

In the experimental example, G/KCC-1 nano-composite is successfully prepared and used as a self-made absorbent of a micro chromatographic column for carrying out high-selectivity SPE on the phospholipid of clams. PMS in the crude lipid was initially extracted by washing with cold acetone. The PMS was then further purified and recovered by G/KCC-1SPE in one run at optimal conditions of sample pH, elution volume and flow rate. 35 PMS in the clams are identified and quantified together, and 53.62% of PC molecular species with EPAADHA structures are obtained. The present invention provides an efficient and reliable method for selectively purifying phospholipids in clams.

Claims

1. A method for extracting and purifying phospholipid from clams is characterized by comprising the following steps:

2. The method for extracting and purifying phospholipid from clams as claimed in claim 1, wherein in the step (iv), the preparation method of the compound of silica fiber nanoparticles and graphene oxide nanoparticles is as follows: dispersing the synthesized graphene oxide nano particles in water and carrying out ultrasonic treatment to obtain a graphene oxide solution, and uniformly dispersing for 1 h; then, adding the silica fiber nanoparticles into the dispersed graphene oxide solution according to the mass ratio of the silica fiber nanoparticles to the graphene oxide nanoparticles of 1: 1, and stirring the mixed solution for 2 hours at 120 ℃ by intense magnetic force; finally, the residue was washed three times or more by using a 50% v/v ethanol aqueous solution to remove unabsorbed silica fiber nanoparticles, to obtain a composite of silica fiber nanoparticles and graphene oxide nanoparticles.

3. A method for extracting and purifying phospholipid from clams as claimed in claim 2, wherein the method for preparing the silica fiber nano-particles is as follows: under ultrasonic treatment, dissolving a mixture of 0.60g of urea and 1.10g of bromohexadecylpyridine in 30mL of water for 5min, and then adding 30mL of cyclohexane and 0.94mL of isopropanol; then, 3mL of ethyl orthosilicate was added dropwise to the mixture using a syringe, and the solution was heated to 70 ℃ with stirring; after centrifugation at 8000g for 10min at 4 ℃, the resulting residue was washed three times with acetone, deionized water and absolute ethanol in order and dried under vacuum for 12h to obtain silica fiber nanoparticles by calcination at 500 ℃ for 4 h.

4. The method for extracting and purifying phospholipid from clams as claimed in claim 1, wherein the preparation method of the graphene oxide nano-particles is as follows: every 40mL of 85 mass percent phosphoric acid solution is mixed with 360mL of 98 mass percent sulfuric acid solution to obtain a mixed acid solution, 18.0g of potassium permanganate and 3.0g of graphite flake are added into the mixed acid solution for reaction, after the reaction is cooled to room temperature, the mixture obtained by the reaction is poured on ice, and 5mL of H with the mass concentration of 30 percent is used₂O₂Quenching the aqueous solution, then carrying out centrifugal treatment, neutralizing the mixture obtained after the centrifugal treatment by using a 4 wt% HCl aqueous solution, removing residual salt in the obtained solid material by dialysis in ultrapure deionized water, and carrying out vacuum drying at room temperature overnight to obtain the graphene oxide nano-particles.

5. A method for extracting and purifying phospholipid from clam according to claim 1, wherein the specific method of the step II comprises the steps of accurately weighing clam powder obtained in the step I, mixing the clam powder with chloroform-methanol mixed solution with a volume ratio of 2:1, extracting crude lipid under ultrasonic treatment, and adding deionized water into the mixture to separate hydrophilic impurities; then, the mixture was centrifuged at 8000g for 10min, the bottom organic phase was recovered while the solid residue was extracted twice more with chloroform, and finally, the collected organic phases were combined and evaporated to dryness under a nitrogen stream using 12mL of chloroform-methanol mixture, 3.2mL of deionized water, and 10mL of chloroform per 0.3g of clam powder.

6. The method for extracting and purifying phospholipids from clams as claimed in claim 1, which is characterized in that: in the first step, the rotating speed of the high-speed dispersion machine is 1500 rpm.

7. The method for extracting and purifying phospholipid from clams as claimed in claim 1, wherein the specific method of the step (iv) is as follows:packing the adsorbent in a sleeve, arranging two polypropylene gaskets at the bottom and the top of the sleeve to well fix the adsorbent to prepare an SPE column; and then mixing the mixture by using hexane and a volume ratio of 2:1, loading the crude lipid extract on an SPE column under vacuum, then successively activating with a chloroform/isopropanol mixture of 2:1 chloroform/isopropanol mixed solution and 0.1% formic acid ether solution by mass percentage wash SPE column, in order to remove free fatty acid and neutral lipid; finally, the reaction solution is passed through a column at a flow rate of 1.5 mL. min^–1Is 2: 1: the SPE cartridge was washed with a 0.01 chloroform/methanol/hydrochloric acid mixture to obtain a fraction of the target compound, and the eluate was dried under a stream of nitrogen.