Extraction and detection of phospholipids in mammalian milk and milk products

Abstract

The present application relates to the field of dairy products, in particular to a method for extracting and detecting phospholipids in mammalian milk and dairy products. The extraction method comprises: taking the dry matter of mammalian milk or dairy products, mixing with dispersing agent, antioxidant and extraction reagent, and then extracting at 50-100 DEG C, 8-10 Mpa. The detection method comprises: using the aforementioned extraction method to separate the phospholipid extract from the sample to be detected, and then detecting it. The extraction method of the present application can efficiently extract the phospholipid material in mammalian milk, and has good reproducibility, is simple, fast, and can significantly reduce the amount of extraction solvent, which significantly reduces the extraction cost of a single sample. The detection method of the present application has good precision and high recovery rate, can effectively separate several phospholipids in mammalian milk, and realize accurate qualitative and quantitative analysis of phospholipid materials including PC, PE, PI, PS and SM.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of dairy products, in particular to a method for extracting and detecting phospholipids in mammalian milk and dairy products. BACKGROUND

[0002] Polar lipids are the main components in milk fat globule membranes, which are mainly composed of phosphatidylcholines (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and sphingomyelin (SM). Phospholipids are important substances for forming biological membranes and are one of the basic substances of life, which are closely related to cell recognition, species specificity and tissue immune function, etc. In addition, phospholipids are important nutritional components and flavor precursor substances of food. In addition, studies have shown that phospholipids have the functions of improving memory, regulating blood lipids, improving immunity and anti-aging, etc. Therefore, it is of great significance to explore phospholipids in mammalian milk or dairy products.

[0003] In order to analyze the phospholipid components in mammalian milk or dairy products, it is necessary to select an appropriate phospholipid extraction method to prevent the loss of some components. The existing milk fat extraction method is petroleum ether-ethyl ether solvent extraction method. For example, "Application of Acid Hydrolysis-Soxhlet System in Determination of Total Fat in Dairy Products" (Hu Peiqin, Zhang Chunhe and Ye Min, Trace Elements and Health Research, 2008, No. 2) discloses a method for determining total fat in liquid dairy products by using FOSS acid hydrolysis-soxhlet total fat analysis system. The method uses acid hydrolysis and then extracts fat with a mixed solvent of petroleum ether and ethanol. The method provides satisfactory results for neutral lipid extraction, but does not mention the extraction of phospholipid components in mammalian milk or dairy products. In addition, the commonly used phospholipid extraction method is chloroform-methanol solvent extraction method. The defect of this method is that when it is applied to mammalian milk or dairy products, the extraction effect is not good, and there is still room for improvement in the extraction rate of phospholipids. At the same time, when using this method, the sample processing time is long, the organic solvent is volatile, the experimental personnel are exposed to harmful substances, and the extraction efficiency is not high. SUMMARY

[0004] In order to solve the above technical problems, the present application provides a method for extracting and detecting phospholipids in mammalian milk and dairy products.

[0005] Specifically, the present application first provides a method for extracting phospholipids in mammalian milk and dairy products, which comprises:

[0006] The dry substance of the mammal milk or milk product is mixed with a dispersing agent, an antioxidant and an extraction reagent, and then extracted at 50-100 DEG C and 8-10 MPa.

[0007] The extraction reagent is selected from one or more of chloroform, n-hexane, ethyl acetate, methanol and ethanol.

[0008] The present application finds that the phospholipid can be efficiently extracted from the mammal milk or milk product without destroying the phospholipid component by the above method.

[0009] Preferably, the extraction reagent contains at least one or more of chloroform, dichloromethane and n-hexane, which can ensure a more optimal extraction effect on the phospholipid substances including PC, PE, PI, PS and SM.

[0010] More preferably, the extraction reagent contains one or both of chloroform and dichloromethane, and the volume content of which in the extraction reagent is 60-75%, which is more optimal for the extraction of each phospholipid substance.

[0011] Preferably, the mass ratio of the extraction reagent to the dry substance is 3-10:1.

[0012] Preferably, the dispersing agent is selected from one or both of diatomite and sea sand, which is more optimal for the extraction.

[0013] More preferably, the mass ratio of the dispersing agent to the dry substance is 1.5-3:1.

[0014] Preferably, the antioxidant is butylated hydroxytoluene and / or butylated hydroxyanisole, which is more beneficial to the protection of the phospholipid component and does not affect the extraction and subsequent detection.

[0015] More preferably, the antioxidant is added in an amount of 0.01-0.05% based on the mass of the extraction solution.

[0016] Preferably, the extraction is carried out at 60-100 DEG C and 8-10 MPa.

[0017] Preferably, the mass ratio of the diatomite to the dry substance is 2:1.

[0018] Preferably, the extraction time is 5-15 min.

[0019] Preferably, the mammal milk or milk product is extracted for more than twice.

[0020] Preferably, the extraction method further comprises:

[0021] After the extraction is completed, the solvent in the obtained liquid is evaporated under vacuum to obtain a phospholipid extract.

[0022] The method for obtaining the dry matter from the mammal milk or milk product can be freeze-drying, and other methods can be used by those skilled in the art without damaging the phospholipid component, which are not particularly limited herein.

[0023] In order to avoid the damage to the phospholipid component, the phospholipid should be stored under light-proof condition at-18℃ or below after being extracted.

[0024] Those skilled in the art can combine the above-mentioned preferred schemes according to the conventional understanding to obtain a better embodiment of the extraction method of the present application.

[0025] As a preferred embodiment, the extraction method of the phospholipid in the mammal milk and milk product is as follows:

[0026] The mammal milk or milk product is freeze-dried to obtain the dry matter thereof, and the dry matter and dispersing agent are weighed at a mass ratio of 1:1.5-3, mixed, and then loaded into an extraction cell. The extraction reagent is added at a solid-liquid ratio of 1:3-10, and then the antioxidant is added at 0.01-0.05% of the mass of the solution. The sample extraction cell is covered with a metal cover and tightened, and then placed into the instrument. The temperature is set to 50-100℃, the pressure is set to 8-10Mpa, the static circulation is set to 1-4 times (more preferably 2 times), and the extraction time is set to 5-15min. Then the combined extract solution is evaporated in a vacuum rotary evaporator, the lipid extract is weighed, and stored in an amber vial at-18℃ until subsequent application (such as chromatographic analysis).

[0027] The present application further provides a detection method of the phospholipid in the mammal milk and milk product, which comprises: using the above-mentioned extraction method to separate the phospholipid extract from the sample to be detected, and then detecting the same.

[0028] As preferred, the detection is performed by HPLC (High Performance Liquid Chromatography), wherein,

[0029] The chromatographic column is a normal phase silica gel chromatographic column;

[0030] Two-element gradient elution is used, the mobile phase A is a mixture of methanol, water, glacial acetic acid and triethylamine at a volume ratio of 75-90:10-25:0.2-0.8:0.05-0.1, and the mobile phase B is a mixture of n-hexane, isopropyl alcohol and the mobile phase A at a volume ratio of 15-25:40-55:25-35;

[0031] The flow rate is 1mL / min;

[0032] The column temperature is 30-40℃;

[0033] The gradient elution degree is shown in Table 1 as follows:

[0034] Table 1

[0035]

[0036] In a preferred embodiment, the mobile phase A is a mixture of methanol, water, glacial acetic acid and triethylamine with a volume ratio of 85:15:0.2-0.8:0.05-0.1; and the mobile phase B is a mixture of n-hexane, isopropyl alcohol and the mobile phase A with a volume ratio of 20:48:32.

[0037] As preferred, in the HPLC, the detector is an evaporative light scattering detector (ELSD) with air as the atomizing gas, a gas pressure of 300-350 kPa and a drift tube temperature of 30-45℃.

[0038] By the above method, each phospholipid substance (including PC, PE, PI, PS and SM) can be well separated in the chromatogram, so that a person skilled in the art can determine the type of phospholipid according to the peak time of each peak.

[0039] Meanwhile, since the above method has good precision and repeatability, the relative content of each phospholipid component can also be calculated according to the standard curve equation of each phospholipid.

[0040] In order to better quantitatively detect each phospholipid component, the detection method preferably further comprises:

[0041] Preparation of a phospholipid standard solution, detection and analysis by the HPLC, drawing a standard curve with the concentration of the standard as the abscissa and the average value of the peak area as the ordinate, and calculating the standard curve equation;

[0042] Substituting the peak area corresponding to the phospholipid in the sample to be tested into the standard curve equation, the concentration of the phospholipid in the mammalian milk is calculated.

[0043] A person skilled in the art can combine the above preferred schemes according to the conventional understanding to obtain a better embodiment of the detection method of the present application.

[0044] In the present application, good results can be obtained when the extraction or detection object is mammalian milk or dairy products. In some embodiments, the mammalian milk or dairy products can be cow milk, goat milk or dairy products thereof.

[0045] In the present application, the "liquid-to-solid ratio" refers to the mass ratio of the dry matter of the mammalian milk or dairy product to the extraction reagent.

[0046] Based on the above technical solution, the present application has the following beneficial effects:

[0047] (1) The extraction method in the present application can efficiently extract phospholipid substances in mammal milk, has good reproducibility, is simple, fast, and can obviously reduce the amount of extraction solvent, which makes the extraction cost of a single sample also significantly reduced.

[0048] (2) The extraction method in the present application adopts completely closed treatment, effectively reduces the volatilization of organic solvent and the time of laboratory personnel contacting organic vapor, is more friendly to the environment, and is more conducive to ensuring the health and safety of the operating personnel.

[0049] (3) The detection method in the present application has good precision and high recovery rate, can effectively separate several phospholipids in mammal milk, and realizes accurate qualitative and quantitative analysis of phospholipid substances including PC, PE, PI, PS and SM. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 It is an HPLC-ELSD detection spectrum of phospholipids in cow milk detected in Example 1.

[0051] Figure 2 It is a phospholipid standard curve in Example 13. DETAILED DESCRIPTION

[0052] The following examples are used to illustrate the present application, but not to limit the scope of the present application.

[0053] In order to facilitate comparison effect, the cow milk detected in the following examples is the same batch of cow milk, and the goat milk is the same batch of goat milk.

[0054] If the specific technology or condition is not specified in the examples, it is carried out according to the technology or condition described in the literature in the art, or according to the product instruction. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be purchased through a regular channel.

[0055] Example 1

[0056] This example first provides an extraction method of phospholipids in cow milk, and the steps are as follows:

[0057] The cow milk is freeze-dried, 2g of freeze-dried milk powder is weighed, mixed with 4g of diatomite, and loaded into an extraction cell. Chloroform-methanol solution (volume ratio of 2:1) is added according to a solid-liquid ratio of 1:5, 0.02% antioxidant is added to the mass of the solution, a metal cover is covered and tightened, and the sample extraction cell is placed in the instrument. The temperature is set to 70℃, the pressure is set to 8Mpa, static circulation is set to 2 times, and the extraction time is set to 10min. After the extraction is completed, solid-liquid separation is carried out, and then the combined solvent extract is evaporated in a vacuum rotary evaporator. The phospholipid extract is weighed and stored in an amber vial, and stored at -18℃ until chromatographic analysis.

[0058] The present embodiment further provides a detection method in bovine milk. The obtained phospholipid extract is detected by high performance liquid chromatography. The high performance liquid chromatography conditions are as follows:

[0059] The detector is an evaporative light scattering detector (ELSD) with air as the atomizing gas, a gas pressure of 300 kPa, and a drift tube temperature of 40 °C. The chromatographic column is a normal phase silica gel chromatographic column. Two-component gradient elution is adopted. The mobile phase A is a mixture of methanol, water, glacial acetic acid and triethylamine with a volume ratio of 85:15:0.2:0.05. The mobile phase B is a mixture of n-hexane, isopropyl alcohol and the mobile phase A with a volume ratio of 20:48:32. The flow rate is 1 mL / min, the column temperature is 35 °C, and the gradient elution program is shown in Table 1. Finally, the animal milk phospholipid is detected. The types of phospholipids are determined according to the peak time of each peak, and the relative content of each phospholipid component is calculated according to the standard curve equation of each phospholipid.

[0060] Figure 1 The HPLC-ELSD detection spectrum of the obtained bovine milk phospholipid is shown in Table 2.

[0061] Table 2

[0062]

[0063] Example 2

[0064] The present embodiment first provides a method for extracting phospholipids in bovine milk. The steps are as follows:

[0065] The bovine milk is freeze-dried, 2 g of freeze-dried milk powder is weighed, mixed with 4 g of sea sand, and loaded into an extraction cell. Ethyl acetate-methanol solution (volume ratio of 2:1) is added according to a solid-liquid ratio of 1:7, 0.01% antioxidant is added to the mass of the solution, a metal cover is covered and tightened, and the sample extraction cell is placed in the instrument. The temperature is set to 65 °C, the pressure is set to 9 Mpa, the static cycle is set to 1, and the extraction time is set to 5 min. After the extraction is completed, solid-liquid separation is performed, and then the combined solvent extract is evaporated in a vacuum rotary evaporator. The phospholipid extract is weighed and stored in an amber vial at -18 °C until chromatographic analysis.

[0066] The present embodiment further provides a detection method in bovine milk. The obtained phospholipid extract is detected by high performance liquid chromatography. The high performance liquid chromatography conditions are as follows:

[0067] The detector is evaporative light scattering detector (ELSD), air as atomizing gas, gas pressure 320 kPa, drift tube temperature 40℃. The column is normal phase silica gel column; two-element gradient elution is used, mobile phase A is a mixture of methanol, water, glacial acetic acid and triethylamine with a volume ratio of 85:15:0.5:0.05; mobile phase B is a mixture of n-hexane, isopropanol and mobile phase A with a volume ratio of 20:48:32. The flow rate is 1 mL / min, column temperature 30℃, and the gradient elution program is shown in Table 1. Finally, animal milk phospholipids are detected. The types of phospholipids are determined according to the peak time of each peak, and the relative content of each phospholipid component is calculated according to the standard curve equation of each phospholipid.

[0068] The results of quantitative analysis of phospholipids in milk are as follows:

[0069] Table 3

[0070]

[0071] Example 3

[0072] This example first provides a method for extracting phospholipids in cow's milk, the steps are as follows:

[0073] The cow's milk is freeze-dried, 2g of freeze-dried milk powder is weighed, mixed with 4g of diatomite, and loaded into an extraction cell. The ratio of solid to liquid is 1:6, n-hexane-methanol solution (volume ratio 2:1) is added, 0.03% antioxidant is added to the mass of the solution, a metal cover is placed on and tightened, and the sample extraction cell is placed in the instrument. The temperature is set to 80℃, the pressure is 10Mpa, static circulation is 3 times, and the extraction time is 8min. After the extraction is completed, solid-liquid separation is performed, and then the combined solvent extract is evaporated in a vacuum rotary evaporator. The phospholipid extract is weighed and stored in an amber vial at -18℃ until chromatographic analysis.

[0074] This example further provides a method for detecting cow's milk, the obtained phospholipid extract is detected by high performance liquid chromatography, and the high performance liquid chromatography conditions are as follows:

[0075] The detector is evaporative light scattering detector (ELSD), air as atomizing gas, gas pressure 350 kPa, drift tube temperature 35℃. The column is normal phase silica gel column; two-element gradient elution is used, mobile phase A is a mixture of methanol, water, glacial acetic acid and triethylamine with a volume ratio of 85:15:0.8:0.1; mobile phase B is a mixture of n-hexane, isopropanol and mobile phase A with a volume ratio of 20:48:32. The flow rate is 1 mL / min, column temperature 35℃, and the gradient elution program is shown in Table 1. Finally, animal milk phospholipids are detected. The types of phospholipids are determined according to the peak time of each peak, and the relative content of each phospholipid component is calculated according to the standard curve equation of each phospholipid.

[0076] The results of the quantitative analysis of phospholipids in the milk are shown in Table 4 below:

[0077] Table 4

[0078]

[0079] Example 4

[0080] This example first provides a method for extracting phospholipids from cow's milk, the steps of which are as follows:

[0081] The cow's milk is freeze-dried, 2 g of the freeze-dried milk powder is weighed out, mixed with 4 g of diatomite, and loaded into an extraction cell. A dichloromethane-methanol solution (volume ratio 2:1) is added at a solid-liquid ratio of 1:10, 0.03% of an antioxidant is added by mass of the solution, a metal lid is placed on the cell and tightened, and the sample extraction cell is placed in the instrument. The temperature is set to 70°C, the pressure is set to 8 MPa, static circulation is performed 4 times, and the extraction time is 12 min. After the extraction is completed, solid-liquid separation is performed, and then the combined solvent extract is evaporated in a vacuum rotary evaporator. The phospholipid extract is weighed and stored in an amber vial at -18°C until chromatographic analysis.

[0082] This example further provides a method for detecting phospholipids in cow's milk. The obtained phospholipid extract is detected by high performance liquid chromatography, and the conditions for high performance liquid chromatography are as follows:

[0083] An evaporative light scattering detector (ELSD) is used as the detector, air is used as the atomizing gas, the gas pressure is 330 kPa, and the drift tube temperature is 40°C. A normal phase silica gel chromatographic column is used, two-component gradient elution is used, mobile phase A is a mixture of methanol, water, glacial acetic acid, and triethylamine at a volume ratio of 85:15:0.2:0.05, and mobile phase B is a mixture of n-hexane, isopropyl alcohol, and mobile phase A at a volume ratio of 20:48:32. The flow rate is 1 mL / min, the column temperature is 40°C, and the gradient elution program is shown in Table 1. Finally, the animal milk phospholipids are detected. The types of phospholipids are determined according to the peak time of each peak, and the relative content of each phospholipid component is calculated according to the standard curve equation of each phospholipid.

[0084] The results of the quantitative analysis of phospholipids in the milk are shown in Table 5 below:

[0085] Table 5

[0086]

[0087] Example 5

[0088] This example first provides a method for extracting phospholipids from cow's milk, the steps of which are as follows:

[0089] The milk was freeze-dried, 2 g of freeze-dried milk powder was weighed, mixed with 4 g of sea sand, and loaded into an extraction cell. Hexane was added at a solid-liquid ratio of 1:5, 0.05% antioxidant was added to the solution, a metal cover was placed on the extraction cell and tightened, and the sample extraction cell was placed in the instrument. The temperature was set to 80°C, the pressure was 9 MPa, static circulation was 2 times, and the extraction time was 15 min. After the extraction was completed, solid-liquid separation was performed, and then the combined solvent extract was evaporated in a vacuum rotary evaporator. The phospholipid extract was weighed and stored in an amber vial at -18°C until chromatographic analysis.

[0090] The present embodiment further provides a detection method in milk. The obtained phospholipid extract is detected by high performance liquid chromatography. The high performance liquid chromatography conditions are as follows:

[0091] An evaporative light scattering detector (ELSD) is used as the detector, air is used as the atomizing gas, the gas pressure is 300 kPa, and the drift tube temperature is 40°C. The chromatographic column is a normal phase silica gel chromatographic column. Two-component gradient elution is used. The mobile phase A is a mixture of methanol, water, glacial acetic acid and triethylamine at a volume ratio of 85:15:0.2:0.05. The mobile phase B is a mixture of n-hexane, isopropanol and mobile phase A at a volume ratio of 20:48:32. The flow rate is 1 mL / min, the column temperature is 30°C, and the gradient elution program is shown in Table 1. Finally, the animal milk phospholipid is detected. The types of phospholipids are determined according to the peak time of each peak, and the relative content of each phospholipid component is calculated according to the standard curve equation of each phospholipid.

[0092] The results of the quantitative analysis of phospholipids in milk are shown in Table 6 below:

[0093] Table 6

[0094]

[0095] Example 6

[0096] The present embodiment first provides a method for extracting phospholipids in milk. The steps are as follows:

[0097] The milk was freeze-dried, 2 g of freeze-dried milk powder was weighed, mixed with 4 g of sea sand, and loaded into an extraction cell. Hexane was added at a solid-liquid ratio of 1:5, 0.05% antioxidant was added to the solution, a metal cover was placed on the extraction cell and tightened, and the sample extraction cell was placed in the instrument. The temperature was set to 80°C, the pressure was 9 MPa, static circulation was 2 times, and the extraction time was 15 min. After the extraction was completed, solid-liquid separation was performed, and then the combined solvent extract was evaporated in a vacuum rotary evaporator. The phospholipid extract was weighed and stored in an amber vial at -18°C until chromatographic analysis.

[0098] The embodiment further provides a detection method in cow milk. The obtained phospholipid extract is detected by high performance liquid chromatography. The high performance liquid chromatography conditions are as follows:

[0099] The detector is an evaporative light scattering detector (ELSD), air is used as atomizing gas, the gas pressure is 330 kPa, and the drift tube temperature is 40℃. The chromatographic column is a normal phase silica gel chromatographic column; two-element gradient elution is adopted, the mobile phase A is a mixture of methanol, water, glacial acetic acid and triethylamine with a volume ratio of 85:15:0.5:0.05; the mobile phase B is a mixture of n-hexane, isopropyl alcohol and the mobile phase A with a volume ratio of 20:48:32. The flow rate is 1 mL / min, the column temperature is 35℃, and the gradient elution program is shown in Table 1. Finally, the animal milk phospholipid is detected. The types of phospholipids are determined according to the peak time of each peak, and the relative contents of each phospholipid component are calculated according to the standard curve equation of each phospholipid.

[0100] The quantitative analysis results of the phospholipids in milk are shown in Table 7.

[0101] Table 7

[0102]

[0103] Example 7

[0104] The embodiment first provides a method for extracting phospholipids in goat milk, and the steps are as follows:

[0105] The goat milk is freeze-dried, 2g of the freeze-dried milk powder is weighed, mixed with 4g of sea sand, and then loaded into an extraction cell. Dichloromethane-n-hexane-methanol solution (volume ratio of 1:1:1) is added in a solid-liquid ratio of 1:9, 0.03% antioxidant is added in an amount of the solution, a metal cover is covered and tightened, and the sample extraction cell is placed in the instrument. The temperature is set to 100℃, the pressure is set to 8Mpa, the static cycle is set to 3 times, and the extraction time is set to 12min. After the extraction is completed, solid-liquid separation is performed, and then the combined solvent extract is evaporated in a vacuum rotary evaporator. The phospholipid extract is weighed and stored in an amber bottle at -18℃ until chromatographic analysis.

[0106] The embodiment further provides a detection method in cow milk. The obtained phospholipid extract is detected by high performance liquid chromatography. The high performance liquid chromatography conditions are as follows:

[0107] The detector is evaporative light scattering detector (ELSD), air as atomizing gas, gas pressure 320 kPa, drift tube temperature 40℃. The chromatographic column is normal phase silica gel chromatographic column; two-element gradient elution is used, mobile phase A is a mixture of methanol, water, glacial acetic acid and triethylamine with a volume ratio of 85:15:0.5:0.05; mobile phase B is a mixture of n-hexane, isopropanol and mobile phase A with a volume ratio of 20:48:32. The flow rate is 1 mL / min, the column temperature is 40℃, and the gradient elution program is shown in Table 1. Finally, animal milk phospholipids are detected. The types of phospholipids are determined according to the peak time of each peak, and the relative content of each phospholipid component is calculated according to the standard curve equation of each phospholipid.

[0108] The results of quantitative analysis of phospholipids in milk are as follows in Table 8:

[0109] Table 8

[0110]

[0111] Example 8

[0112] This example first provides a method for extracting phospholipids in cow's milk, the steps are as follows:

[0113] The cow's milk is freeze-dried, 2g of freeze-dried milk powder is weighed, mixed with 4g of diatomite, and loaded into an extraction cell. Chloroform-n-hexane-methanol solution (volume ratio 1:1:1) is added according to a solid-liquid ratio of 1:7, 0.01% antioxidant is added to the mass of the solution, a metal cover is put on and tightened, and the sample extraction cell is placed in the instrument. The temperature is set to 80℃, the pressure is 10Mpa, static circulation is 2 times, and the extraction time is 15min. After the extraction is completed, solid-liquid separation is performed, and then the combined solvent extract is evaporated in a vacuum rotary evaporator. The phospholipid extract is weighed and stored in an amber vial at -18℃ until chromatographic analysis.

[0114] This example further provides a method for detecting cow's milk, and the obtained phospholipid extract is detected by high performance liquid chromatography. The high performance liquid chromatography conditions are as follows:

[0115] The detector is evaporative light scattering detector (ELSD), air as atomizing gas, gas pressure 330 kPa, drift tube temperature 40℃. The chromatographic column is normal phase silica gel chromatographic column; two-element gradient elution is used, mobile phase A is a mixture of methanol, water, glacial acetic acid and triethylamine with a volume ratio of 85:15:0.5:0.05; mobile phase B is a mixture of n-hexane, isopropanol and mobile phase A with a volume ratio of 20:48:32. The flow rate is 1 mL / min, the column temperature is 35℃, and the gradient elution program is shown in Table 1. Finally, animal milk phospholipids are detected. The types of phospholipids are determined according to the peak time of each peak, and the relative content of each phospholipid component is calculated according to the standard curve equation of each phospholipid.

[0116] The results of the quantitative analysis of phospholipids in the milk are shown in Table 9 below:

[0117] Table 9

[0118]

[0119] Example 9

[0120] This example first provides a method for extracting phospholipids in cow milk, the steps of which are as follows:

[0121] The cow milk is freeze-dried, 2 g of the freeze-dried milk powder is weighed out, mixed with 4 g of sea sand, and loaded into an extraction cell. A chloroform-ethyl acetate-methanol solution (volume ratio of 1:1:1) is added at a solid-liquid ratio of 1:10, 0.05% antioxidant is added by mass of the solution, a metal cover is placed on and tightened, and the sample extraction cell is placed in the instrument. The temperature is set to 60°C, the pressure is set to 10 MPa, static circulation is set to 4 times, and the extraction time is set to 6 min. After the extraction is completed, solid-liquid separation is performed, and then the combined solvent extract is evaporated in a vacuum rotary evaporator. The phospholipid extract is weighed and stored in an amber vial at -18°C until chromatographic analysis.

[0122] This example further provides a method for detecting phospholipids in cow milk. The obtained phospholipid extract is detected by high performance liquid chromatography, and the high performance liquid chromatography conditions are as follows:

[0123] An evaporative light scattering detector (ELSD) is used as the detector, air is used as the atomizing gas, the gas pressure is 350 kPa, and the drift tube temperature is 40°C. A normal phase silica gel chromatographic column is used, two-component gradient elution is used, mobile phase A is a mixture of methanol, water, glacial acetic acid, and triethylamine at a volume ratio of 85:15:0.8:0.1, and mobile phase B is a mixture of n-hexane, isopropyl alcohol, and mobile phase A at a volume ratio of 20:48:32. The flow rate is 1 mL / min, the column temperature is 35°C, and the gradient elution program is shown in Table 1. Finally, the animal milk phospholipids are detected. The types of phospholipids are determined according to the peak time of each peak, and the relative content of each phospholipid component is calculated according to the standard curve equation of each phospholipid.

[0124] The results of the quantitative analysis of phospholipids in the milk are shown in Table 10 below:

[0125] Table 10

[0126]

[0127] Example 10

[0128] This example first provides a method for extracting phospholipids in goat milk, the steps of which are as follows:

[0129] Sheep milk was freeze-dried, 2 g of freeze-dried milk powder was mixed with 4 g of diatomite respectively, and then was loaded into an extraction cell. Chloroform was added according to a solid-liquid ratio of 1:6, and 0.04% antioxidant was added according to the mass of the solution. A metal cover was put on and tightened, and the sample extraction cell was placed in the instrument. The temperature was set to 70°C, the pressure was set to 9 MPa, static circulation was set to 2 times, and the extraction time was set to 13 min. After the extraction was completed, solid-liquid separation was performed, and then the combined solvent extract was evaporated in a vacuum rotary evaporator. The phospholipid extract was weighed and stored in an amber vial at -18°C until chromatographic analysis.

[0130] The example further provides a detection method in bovine milk. The obtained phospholipid extract is detected by high performance liquid chromatography. The high performance liquid chromatography conditions are as follows:

[0131] An evaporative light scattering detector (ELSD) is used as the detector, air is used as the atomizing gas, the gas pressure is 310 kPa, and the drift tube temperature is 40°C. A normal phase silica gel chromatographic column is used. Two-element gradient elution is used. The mobile phase A is a mixture of methanol, water, glacial acetic acid and triethylamine in a volume ratio of 85:15:0.2:0.05. The mobile phase B is a mixture of n-hexane, isopropyl alcohol and mobile phase A in a volume ratio of 20:48:32. The flow rate is 1 mL / min, the column temperature is 35°C, and the gradient elution program is shown in Table 1. Finally, animal milk phospholipids are detected. The types of phospholipids are determined according to the peak time of each peak, and the relative content of each phospholipid component is calculated according to the standard curve equation of each phospholipid.

[0132] The results of the quantitative analysis of phospholipids in milk are shown in Table 11 below:

[0133] Table 11

[0134]

[0135] Example 11

[0136] The example first provides a method for extracting phospholipids in bovine milk. The steps are as follows:

[0137] Bovine milk was freeze-dried, 2 g of freeze-dried milk powder was mixed with 4 g of diatomite respectively, and then was loaded into an extraction cell. Chloroform was added according to a solid-liquid ratio of 1:6, and 0.04% antioxidant was added according to the mass of the solution. A metal cover was put on and tightened, and the sample extraction cell was placed in the instrument. The temperature was set to 70°C, the pressure was set to 9 MPa, static circulation was set to 2 times, and the extraction time was set to 13 min. After the extraction was completed, solid-liquid separation was performed, and then the combined solvent extract was evaporated in a vacuum rotary evaporator. The phospholipid extract was weighed and stored in an amber vial at -18°C until chromatographic analysis.

[0138] The embodiment further provides a detection method in cow milk. The obtained phospholipid extract is detected by high performance liquid chromatography. The high performance liquid chromatography conditions are as follows:

[0139] The detector is an evaporative light scattering detector (ELSD), air is used as atomizing gas, the gas pressure is 330 kPa, and the drift tube temperature is 40℃. The chromatographic column is a normal phase silica gel chromatographic column; two-element gradient elution is adopted, the mobile phase A is a mixture of methanol, water, glacial acetic acid and triethylamine with a volume ratio of 85:15:0.8:0.1; the mobile phase B is a mixture of n-hexane, isopropyl alcohol and the mobile phase A with a volume ratio of 20:48:32. The flow rate is 1 mL / min, the column temperature is 30℃, and the gradient elution program is shown in Table 1. Finally, the animal milk phospholipid is detected. The types of phospholipids are determined according to the peak time of each peak, and the relative contents of each phospholipid component are calculated according to the standard curve equation of each phospholipid.

[0140] The quantitative analysis results of the phospholipids in milk are shown in Table 12.

[0141] Table 12

[0142]

[0143] Example 12

[0144] The embodiment first provides a method for extracting phospholipids in cow milk, and the steps are as follows:

[0145] The cow milk is freeze-dried, 2g of the freeze-dried milk powder is weighed, mixed with 4g of sea sand, and then loaded into an extraction cell. 0.02% antioxidant is added to the methanol solution according to a solid-liquid ratio of 1:8, a metal cover is covered and tightened, and the sample extraction cell is placed in the instrument. The temperature is set to 70℃, the pressure is set to 10Mpa, the static circulation is set to 2 times, and the extraction time is set to 10min. After the extraction is completed, solid-liquid separation is performed, and then the combined solvent extract is evaporated in a vacuum rotary evaporator. The phospholipid extract is weighed and stored in an amber bottle at -18℃ until chromatographic analysis.

[0146] The embodiment further provides a detection method in cow milk. The obtained phospholipid extract is detected by high performance liquid chromatography. The high performance liquid chromatography conditions are as follows:

[0147] The detector is evaporative light scattering detector (ELSD), air as atomizing gas, gas pressure 330 kPa, drift tube temperature 40℃. The chromatographic column is normal phase silica gel chromatographic column; two-element gradient elution is adopted, mobile phase A is a mixture of methanol, water, glacial acetic acid and triethylamine with a volume ratio of 85:15:0.2:0.05; mobile phase B is a mixture of n-hexane, isopropanol and mobile phase A with a volume ratio of 20:48:32. The flow rate is 1 mL / min, the column temperature is 35℃, and the gradient elution program is shown in Table 1. Finally, the animal milk phospholipids are detected. The types of phospholipids are determined according to the peak time of each peak, and the relative content of each phospholipid component is calculated according to the standard curve equation of each phospholipid.

[0148] The results of quantitative analysis of phospholipids in milk are as follows in Table 13:

[0149] Table 13

[0150]

[0151] Example 13

[0152] Accurately weigh 0.01 g of PE, PI, PS, PC and SM standard powder respectively, dissolve in chloroform-methanol solution (2:1, v / v) to prepare 1000 μg / mL standard solution, add chloroform-methanol solution (2:1, v / v) to dilute to 500 μg / mL, 250 μg / mL, 125 μg / mL and 62.5 μg / mL respectively, and sample different standards twice. The high performance liquid chromatography conditions are the same as in Example 1.

[0153] Draw the standard curve with the concentration of the standard as the abscissa and the average of the peak areas of the two injections as the ordinate, and calculate the standard curve equation. The linear equation and correlation coefficient of phospholipids are shown in Table 14 below, Figure 2 as the phospholipid standard curve.

[0154] Table 14

[0155] Phospholipid species Linear equation [R 2 ]] Linear range (mg / mL) Phosphatidylcholine y = 168264x - 14891 0.9922 0.0625-1 Phosphatidylethanolamine y = 289719x - 26940 0.9929 0.0625-1 Sphingomyelin y = 308053x - 19654 0.9944 0.0625-1 Phosphatidylinositol y = 378530x - 4750.9 0.9987 0.0625-1 Phosphatidylserine y = 244636x - 12800 0.9971 0.0625-1

[0156] Substitute the peak area corresponding to each phospholipid in the sample to be tested into the standard curve equation, and the concentration of each phospholipid in the sample to be tested can be calculated.

[0157] Comparative Example 1

[0158] The detection sample in this comparative example is the same as in Example 1, and the specific detection method is as follows:

[0159] The milk was freeze-dried, 2 g of the freeze-dried milk powder was placed in a 50 mL centrifuge tube, and then 15 mL of chloroform-methanol solution (volume ratio of 2:1) was added to the centrifuge tube. The mixture was mechanically stirred for 30 minutes, and centrifuged at 4°C, and as much as possible of the upper organic solvent fraction was carefully removed with a pipette. And 3 mL of 0.9% sodium chloride solution was added to the collected organic solvent fraction and mechanically mixed for 1 minute, and then the test tube was stored at 4°C overnight. The liquid was collected by a separatory funnel, and finally the extract was concentrated by a nitrogen blowing instrument, weighed, and stored at -18°C until chromatographic analysis.

[0160] The high performance liquid chromatography conditions were as follows:

[0161] The HPLC analysis of phospholipids in milk used an evaporative light scattering detector (ELSD) with air as the atomizing gas, a gas pressure of 330 kPa, and a drift tube temperature of 40°C. The chromatographic column was a normal phase silica gel chromatographic column; two-component gradient elution was used, mobile phase A was a mixture of methanol, water, glacial acetic acid and triethylamine in a volume ratio of 85:15:0.5:0.05; and mobile phase B was a mixture of n-hexane, isopropyl alcohol and mobile phase A in a volume ratio of 20:48:32. The flow rate was 1 mL / min, the column temperature was 35°C, and the gradient elution program is shown in Table 1. Finally, the animal milk phospholipids were detected. The types of phospholipids were determined according to the peak time of each peak, and the relative content of each phospholipid component was calculated according to the standard curve equation of each phospholipid.

[0162] The results of the quantitative analysis of phospholipids in milk are as follows in Table 15:

[0163] Table 15

[0164]

[0165] Comparative Example 2

[0166] The detection sample in the present comparative example was the same as in Example 1, and the specific detection method was as follows:

[0167] The milk was freeze-dried, 2 g of the freeze-dried milk powder was placed in a 50 mL centrifuge tube, and then 15 mL of chloroform-methanol solution (volume ratio of 2:1) was added to the centrifuge tube. The mixture was mechanically stirred for 30 minutes, and centrifuged at 4°C, and as much as possible of the upper organic solvent fraction was carefully removed with a pipette. And 3 mL of 0.9% sodium chloride solution was added to the collected organic solvent fraction and mechanically mixed for 1 minute, and then the test tube was stored at 4°C overnight. The liquid was collected by a separatory funnel, and finally the extract was concentrated by a nitrogen blowing instrument, weighed, and stored at -18°C until chromatographic analysis.

[0168] The high performance liquid chromatography conditions were as follows:

[0169] The HPLC analysis of phospholipids in milk was performed with an evaporative light scattering detector (ELSD) using air as the nebulizing gas at a pressure of 350 kPa and a drift tube temperature of 40°C. The column used was a normal-phase silica gel column, and two-component gradient elution was used. The mobile phase A was a mixture of methanol, water, glacial acetic acid and triethylamine at a volume ratio of 85:15:0.8:0.1, and the mobile phase B was a mixture of n-hexane, isopropyl alcohol and mobile phase A at a volume ratio of 20:48:32. The flow rate was 1 mL / min, the column temperature was 40°C, and the gradient elution program is shown in Table 1. Finally, the animal milk phospholipids were detected. The types of phospholipids were determined according to the peak time of each peak, and the relative content of each phospholipid component was calculated according to the standard curve equation of each phospholipid.

[0170] The results of the quantitative analysis of phospholipids in milk are shown in Table 16 below.

[0171] Table 16

[0172]

[0173] Experimental Example 1: Recovery rate experiment

[0174] Three samples of bovine milk were taken, and the bovine milk was pretreated and detected by HPLC according to the method of Example 1. The peak area of each phospholipid component was substituted into the standard curve equation obtained in Example 13 to calculate the original mass concentration. Then, a known amount of each phospholipid component standard solution was added to each of the three bovine milk samples to obtain a mixed solution. The mixed solution was detected by HPLC according to the method of Example 1, and the peak area of each corresponding peak was substituted into the standard curve equation obtained in Example 13 to calculate the mass concentration after spiking. The spiking recovery rate was calculated as follows: spiking recovery rate = (mass concentration after spiking - original mass concentration) / spiking mass concentration. The results are shown in Table 17, and the recovery rate was 92.5% to 104.3%, indicating that the sample lost less during the detection process.

[0175] Table 17

[0176]

[0177] The spiking recovery rates of Examples 2-12 and Comparative Examples 1-2 were determined and calculated according to the above method, and the spiking recovery rates of Examples 2-12 and Comparative Examples 1-2 were all in the range of 90.3% to 107.6%, all meeting the detection requirements.

[0178] Experimental Example 2: Precision experiment

[0179] The mixture solution containing PE, PI, PS, PC and SM 5 standard samples was injected 6 times repeatedly under the high performance liquid chromatography condition of Example 1, the peak area of each time was recorded, and the average value and relative standard deviation of the peak area of each phospholipid component were calculated, and the results were shown in Table 18. It can be seen that the peak area of each phospholipid standard sample has good repeatability, and the relative standard deviation is less than 3%.

[0180] Table 18

[0181]

[0182] The precision of Examples 2-12 and Comparative Examples 1-2 was determined and the relative standard deviation was calculated according to the above method. The peak area of each phospholipid standard sample of Examples 2-12 and Comparative Examples 1-2 has good repeatability, and the relative standard deviation is less than 3%.

[0183] Although the present application has been described in detail with general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application claimed.

Claims

1. A method for extracting phospholipids from mammalian milk and dairy products, characterized in that, include: Dry matter of mammalian milk or dairy products is mixed with a dispersant, an antioxidant, and an extraction reagent, and then extracted at 50-100℃ and 8-10 MPa. After the extraction is completed, solid-liquid separation is performed, and the solvent in the resulting liquid is evaporated under vacuum to obtain a phospholipid extract. in, The mass ratio of the extraction reagent to the dry matter is 3-10:1; The extraction reagent contains a first solvent, methanol, and a second solvent; the second solvent contains at least one or two of chloroform and dichloromethane, and the volume content of the second solvent in the extraction reagent is 60%-75%. The dispersant is selected from diatomaceous earth; the mass ratio of the dispersant to the dry matter is 2:

1. The antioxidant is butylated hydroxytoluene and / or butylated hydroxyanisole; the amount of antioxidant added is 0.01-0.05% based on the mass of the extraction solution.

2. The extraction method according to claim 1, characterized in that, The extraction time is 5-15 minutes, and mammalian milk or dairy products are extracted more than twice.

3. The extraction method according to any one of claims 1-2, characterized in that, Extraction is carried out at 60-100℃ and 8-10 MPa.

4. A method for detecting phospholipids in the dry matter of mammalian milk and the dry matter of dairy products, characterized in that, include: After separating the phospholipid extract from the sample to be tested using the extraction method according to any one of claims 1-3, the extract is then tested.

5. The detection method according to claim 4, characterized in that, The detection was performed by HPLC, wherein... The chromatographic column is a normal-phase silica gel column; A binary gradient elution was used. Mobile phase A was a mixture of methanol, water, glacial acetic acid and triethylamine in a volume ratio of 75-90:10-25:0.2-0.8:0.05-0.1; mobile phase B was a mixture of n-hexane, isopropanol and mobile phase A in a volume ratio of 15-25:40-55:25-35. The flow rate is 1 mL / min; The column temperature is 30-40℃; The gradient elution levels are as follows: 。 6. The detection method according to claim 5, characterized in that, In the HPLC, the detector is an evaporative light scattering detector, with air as the nebulizer gas, a gas pressure of 300-350 kPa, and a drift tube temperature of 30-45℃.

7. The detection method according to claim 6, characterized in that, The detection method further includes: Prepare phospholipid standard solutions and perform detection and analysis by HPLC. Plot a standard curve with the concentration of the standard on the x-axis and the average peak area on the y-axis, and calculate the equation of the standard curve. Substitute the peak area corresponding to the phospholipid in the sample to be tested into the standard curve equation to calculate the concentration of phospholipid in mammalian milk.

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