CN108272822B - Extraction method and application of milk polar lipid - Google Patents

Abstract

The invention discloses an extraction method and application of milk polar lipid, wherein the extraction method comprises the following steps: removing casein from the casein powder by isoelectric precipitation; centrifuging, collecting supernatant, adding chloroform-methanol solution, stirring, centrifuging, collecting lower organic phase, repeatedly extracting lower organic phase, collecting, and rotary evaporating to remove chloroform-methanol to obtain compound lipid; adding acetone into the compound lipid, filtering, and collecting precipitate to obtain milk polar lipid; the application of extracting the milk polar lipid from the buttermilk powder as the raw material in preparing the milk polar liposome is to mix the extracted milk polar lipid with cholesterol, hydrate the mixture after rotary evaporation and then carry out ultrasonic treatment to obtain the milk polar liposome. The method for extracting the milk polar lipid from the buttermilk powder has the advantages of novelty in raw material selection and extraction processes, simple flow, easy realization of industrialization and suitability for popularization and use.

Description

Extraction method and application of milk polar lipid

Technical Field

The invention belongs to the technical field of dairy processing, and particularly relates to an extraction method and application of milk polar lipid.

Background

The milk fat globule membrane is a membrane coated outside the fat globule in milk, and is composed of neutral lipid, polar lipid, membrane protein, etc., wherein the content of the milk polar lipid accounts for about 1% of the total milk fat content, and mainly comprises phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and sphingomyelin. Compared with common egg yolk lecithin or soybean lecithin in the market, the milk polar lipid has more functional characteristics such as the effects of reducing cardiovascular diseases, reducing gastrointestinal tract infection, reducing cholesterol absorption, regulating immunity, promoting infant brain development and the like due to the sphingomyelin contained, and has certain industrial application value because the special amphiphilic molecular structure also has the effects of foamability, emulsibility, texture improvement and the like.

Milk polar lipids reported abroad can be isolated from buttermilk (butter milk) and butterwhey (butter serum). Buttermilk and cream whey are by-products from different stages of the cream making process, containing fragments of milk fat globule membranes, but are generally considered low value products and are often used as animal feed after industrial drying.

Currently, the difficulty in extracting milk polar lipids is how to separate the polar lipids from other components such as proteins, lactose, minerals and triglycerides. The reported extraction methods of polar lipids in milk can be roughly divided into physical extraction and chemical extraction, wherein the physical extraction method is separation extraction by using supercritical extraction technology or membrane technology; the chemical extraction method comprises extracting with organic solvent such as chloroform, methanol, diethyl ether, and ethanol.

Gassi J Y and the like obtain milk polar lipids from cream whey through degreasing, heat treatment, acid precipitation of casein, and removal of minerals, lactose, lactic acid and the like through ultrafiltration and dialysis, but the process is complicated in separation method and high in cost, and the purity of the separated milk polar lipids is not high because proteins (casein, whey protein and fat globule membrane protein), triglyceride, some salts and the like cannot be completely separated.

Extracting cream whey with ten times volume of ethanol by Watanabe S and the like overnight, dissolving the ethanol extract in chloroform-methanol-water mixed solution, placing the mixture in a separating funnel for overnight shaking, collecting the lower layer organic phase, performing rotary evaporation to remove chloroform-methanol, and removing non-polar lipid by silica gel column chromatography to obtain polar lipid.

Disclosure of Invention

Aiming at the defects of the existing problems, the invention aims to provide an extraction method of milk polar lipid and application thereof, and the extraction method has the characteristics of high extraction efficiency, high speed, simple process flow and easy popularization and application.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a method for extracting polar lipid from milk comprises the following steps:

(1) removing casein: dissolving buttermilk powder with distilled water, stirring, adjusting pH of the solution to 4.5-4.9, centrifuging, and collecting supernatant for use;

(2) chloroform-methanol extraction of complex lipids: adding chloroform-methanol solution into the supernatant extracted in the step (1), stirring and centrifuging; after layering, sucking a lower organic phase, adding chloroform-methanol solution into an upper aqueous phase, extracting the organic phase again according to the steps, repeating the steps for two times or more, collecting the organic phase, performing rotary evaporation to remove chloroform-methanol to obtain a viscous liquid, and blowing nitrogen to a solid state to obtain the composite lipid;

(3) acetone precipitation of milk polar lipids: adding acetone into the composite lipid obtained in the step (2) and stirring; collecting the precipitate after filtration to obtain the milk polar lipid.

In the preferable technical scheme, in the step (1), the volume ratio of the mass of the buttermilk powder to the distilled water is 1: 7-10.

As a preferred technical scheme of the application, in the step (1), the centrifugation time is 15-30min, and the rotation speed is 4000-.

In a preferred embodiment of the present invention, in the step (2), the volume ratio of chloroform to methanol is 2: 1.

As a preferable technical scheme of the application, the volume ratio of the chloroform-methanol mixed solution to the supernatant in the step (2) is 1: 4-6.

As a preferred technical scheme of the application, in the step (2), the stirring speed is 400-600 r/min; the centrifugation time is 15-30min, and the rotating speed is 6000-.

The application of the milk polar lipid in preparing the milk polar liposome is prepared by mixing the milk polar lipid extracted from the buttermilk powder and cholesterol according to the mol ratio of 1:0.5-3 and performing a film dispersion method.

As a preferred technical scheme of the application, the milk polar lipid and the cholesterol are mixed according to the molar ratio of 1:2 to prepare the milk polar liposome.

As a preferred technical scheme of the application, the milk polar lipid is dissolved in chloroform, cholesterol is added, the mixture is added into a reactor after being mixed, the organic solvent is removed by decompression and rotary evaporation, pure water is added for hydration, and the liposome suspension after hydration is subjected to ultrasonic treatment to prepare the uniform milk polar liposome.

As the preferable technical scheme of the application, the hydration temperature is 37 ℃ and the time is 1 h; ultrasonic time 5s, ultrasonic gap 7s, 80 times, and ultrasonic power 400W.

The cheese milk powder is subjected to isoelectric point precipitation to remove cheese protein, and then is extracted by chloroform-methanol solution to obtain milk mixed lipid, chloroform-methanol is adopted for multiple extraction in experiments to improve the extraction rate, and stirring is adopted during extraction to improve the extraction efficiency; because the neutral lipid is soluble in acetone and the polar lipid is insoluble in acetone, the polar lipid is separated by adding acetone during subsequent treatment to obtain the milk polar lipid.

The obtained milk polar lipid is mixed with cholesterol in proportion, and the milk polar liposome can be obtained by a film dispersion method. The liposome has unique affinity and targeting property, and can reduce drug toxicity and improve drug stability. Due to the existence of a large amount of sphingomyelin in the milk polar lipid, the milk fat plastid is more stable and has stronger oxidation resistance than the soybean liposome in a certain condition range.

Advantageous effects

Compared with the prior art, the invention has the following beneficial effects:

(1) the milk polar lipid is extracted by taking the buttermilk powder as a raw material, and the buttermilk powder has low content of triglyceride and is easy to extract the milk polar lipid;

(2) the processes for extracting the compound lipid by acid-precipitating casein, extracting the compound lipid by chloroform-methanol and precipitating the polar lipid by acetone are simpler, the cost is low and the effect is good;

(3) the research on the milk polar lipid in China is very little, and no commercialized milk polar lipid exists in the domestic market, so that the patent provides a rapid and efficient extraction method of the milk polar lipid, and the method has simple process flow and is easy to popularize and apply;

(4) according to the invention, the buttermilk powder is adopted to extract the milk polar lipid, so that the source of liposome preparation raw materials is widened, and the stability and the oxidation resistance of the liposome are improved;

drawings

FIG. 1 is a standard graph of phospholipid content determination;

FIG. 2 is a thin layer chromatogram of the milk polar lipids extracted in example 1;

FIG. 3 is a transmission electron micrograph of the milk polar liposomes prepared in example 1;

FIG. 4 is a particle size distribution diagram of the milk polar liposome prepared in example 1;

fig. 5 is a graph of the potential distribution of the milk polar liposomes prepared in example 1.

Detailed Description

The present invention will be described in further detail with reference to examples. The reagents or instruments used are not indicated by manufacturers, and are regarded as conventional products which can be purchased in the market.

Example 1

(1) And (3) precipitating casein: 30g of buttermilk powder is taken and dissolved in 300mL of distilled water, and the solution is evenly stirred and is bathed in water at 40 ℃ for 10 minutes. Adjusting the pH value of the solution to 4.5-4.9 by lactic acid, centrifuging at 8000r/min for 30min, and taking the supernatant in a beaker;

(2) organic solvent extraction: adding 60mL of chloroform-methanol mixed solution (2: 1v/v) into a beaker of the supernatant, stirring for 5 minutes by an ultrasonic stirrer, placing the mixture into a centrifuge tube, centrifuging for 30 minutes at 8000r/min, sucking the lowest organic phase by a rubber head dropper, discarding the protein precipitate in the middle layer, pouring the upper aqueous phase into the beaker, continuously adding 60mL of chloroform-methanol mixed solution to extract lipid, stirring, centrifuging, collecting the lower organic phase, and repeating the steps for three times. Mixing the three organic phases, performing rotary evaporation in water bath at 35 ℃ under the vacuum degree of-0.9 MPa, pouring out the mixture into a beaker when the mixture is evaporated to be viscous liquid, and blowing nitrogen until chloroform is completely volatilized to obtain mixed polar lipid;

(3) acetone precipitation of polar lipids: to the mixed lipid, 10mL of acetone was added and the mixture was thoroughly stirred. Scraping the adherent precipitate, filtering all precipitates, and collecting the precipitate on the filter paper to obtain the polar lipid. 1mg of polar lipid sample is weighed and dissolved in 1mL of chloroform to prepare a sample solution which can be used for subsequent detection.

(4) Determination of polar lipid content

Preparing a soybean lecithin standard solution: precisely weighing 5mg of soybean phospholipid, adding chloroform into a 25mL volumetric flask, dissolving, adding to scale, shaking to obtain 0.2mg/mL standard solution of soybean phospholipid, and storing at 4 deg.C.

Preparing a color developing agent: 2.705g of ferric trichloride and 3.04g of ammonium thiocyanate are accurately weighed into a 100mL volumetric flask, double distilled water is added to dissolve the materials and add the materials to the scale, the materials are shaken up to prepare a developer solution, and the developer solution is stored at room temperature.

And (3) drawing a soybean phospholipid standard curve: accurately weighing 0.2mg/mL of soybean phospholipid standard solution 0.2, 0.4, 0.8, 1.2, 1.6 and 2mL in a centrifuge tube with a plug, adding chloroform to 4mL, adding ammonium thiocyanate solution 4mL, mixing uniformly by vortex for 1min, centrifuging at 2000r/min for 10min, and taking down the lower layer. And (3) measuring a light absorption value A at 485nm of a spectrophotometer by using chloroform as a blank, and performing linear regression on the concentration C (mg/mL) by using A to obtain a standard curve equation. FIG. 1 is a standard curve for phospholipid content determination.

Determination of polar lipid content: accurately weighing 10 mu L of sample solution diluted by two hundred times, paralleling three parts, measuring in a centrifuge tube with a plug according to a soybean phospholipid standard curve method, and finally calculating the polar lipid content in the sample according to a standard curve equation, wherein the concentration of the phospholipid in the chloroform methanol extraction organic phase can be measured to be 37.7 mg/mL.

(5) Thin layer chromatography

Solution preparation: the extracted milk polar lipid sample, Phosphatidylcholine (PC) (standard, available from sigma), Phosphatidylethanolamine (PE) (standard, available from sigma), Phosphatidylinositol (PI) (standard, available from sigma), Phosphatidylserine (PS) (standard, available from sigma), and Sphingomyelin (SM) (standard, available from sigma) were dissolved in chloroform to prepare a 1mg/mL solution.

Sample application: drawing a horizontal line about 1cm at the lower end of the silica gel plate, uniformly drawing scales on the horizontal line, keeping the distance between the two sides, absorbing a certain amount of samples and standard substances by a capillary tube, spotting the samples on the scales, and drying by a blower.

Preparing a developing agent: preparing developing agent from chloroform-methanol-water-ammonia water mixture (3:2:0.3:0.1, v/v/v/v), and pouring into a chromatography tank.

And (4) placing the silica gel plate with the sample in a chromatographic cylinder, blowing the silica gel plate dry after 40 minutes, and placing the silica gel plate in an iodine cylinder for color development.

FIG. 2 is a thin layer chromatography of milk polar lipids, from which it can be seen that the milk polar lipid components include Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Phosphatidylinositol (PI), Phosphatidylserine (PS), and Sphingomyelin (SM).

(6) Preparing liposome: sample solution 5mL, the amount of cholesterol was measured according to the molar ratio of polar lipid to cholesterol 1:2, mixed and added to round bottom flask, the organic solvent was removed by rotary evaporation under reduced pressure at 55 deg.C, and the liposome was attached to the wall of the flask as a thin film.

(7) Observation of milk polar liposome Properties

Observing the shape of the milk polar liposome by adopting a transmission electron microscope: and ultrasonically homogenizing the hydrated liposome suspension (ultrasonic time is 5s, ultrasonic gap is 7s, 80 times), centrifuging under a high-speed freezing centrifuge, (10000r/min, 30min), discarding the supernatant, sucking 2mL of pure water to blow off adherent liposome to form suspension, refrigerating, and observing under an electron microscope. Dripping two drops of prepared liposome on a special copper net by a negative dyeing method through a transmission electron microscope, naturally drying, carrying out negative dyeing by 2.5 percent phosphotungstic acid, naturally volatilizing to concentrate and deposit particles on the copper net, observing by the transmission electron microscope and taking pictures. FIG. 3 is a transmission electron microscope image of liposomes at 100nm, the liposomes have a spherical structure, a rounded shape and uniform dispersion;

the particle size and the potential are measured by a nanometer particle size ZETA potentiometer: a small amount of suspension of creamy plastid is absorbed, diluted and measured for particle size and potential by a nanometer laser particle size potential analyzer, and figure 4 is a particle size distribution diagram of liposome, the average particle size is about 324nm, and the particle size distribution is relatively uniform. FIG. 5 is a graph showing the potential distribution of liposomes having a Zeta potential of-48.3 mV, which are anionic liposomes.

Example 2

The difference from example 1 is that in step (2), the rotary evaporation product was washed with 75mL of chloroform-methanol (2: 1, v/v), placed in a separatory funnel, and left overnight. The polar lipid content in the chloroform-methanol extract organic phase was measured to be 31.9 mg/mL.

Example 3

The difference from example 1 is that step (2) the rotary evaporation was washed with 50mL of chloroform-methanol (2: 1, v/v), placed in a separatory funnel, and left overnight. The polar lipid content in the chloroform-methanol extract organic phase was determined to be 22.9 mg/mL.

Example 4

The difference from example 1 is that, in step (6), 5mL of the sample solution was sampled, and the molar ratio of polar lipid to cholesterol was 1: 1.5 the corresponding cholesterol amount was weighed, mixed and added to a round bottom flask and the organic solvent removed by rotary evaporation under reduced pressure at 55 ℃. The particle size of the creamy body is 391.4nm and the potential is-42.6 mv.

Example 5

The difference from example 1 is that, in step (6), 5mL of the sample solution was sampled, and the molar ratio of polar lipid to cholesterol was 1: 3 weighing corresponding cholesterol amount, mixing, adding into round bottom flask, and rotary evaporating at 55 deg.C under reduced pressure to remove organic solvent. The particle size of the creamy bodies was found to be 434.3nm with a potential of-44.1 mv.

The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept and the scope of the appended claims is intended to be protected.

Claims (1)

1. A method for extracting polar lipids from milk, comprising the steps of:

(1) removing casein: dissolving buttermilk powder with distilled water, stirring uniformly, adjusting the pH value of the solution to 4.5-4.9, carrying out centrifugal separation, and taking supernatant for later use, wherein the volume ratio of the buttermilk powder to the distilled water is 1:7-10, the centrifugal time is 15-30min, and the rotating speed is 4000-;

(2) chloroform-methanol extraction of complex lipids: adding a chloroform-methanol solution into the supernatant extracted in the step (1), stirring and centrifuging, wherein the volume ratio of chloroform to methanol is 2:1, the volume ratio of the chloroform-methanol mixed solution to the supernatant is 1:4-6, the stirring speed is 400-8000 r/min, the centrifuging time is 15-30min, and the centrifuging speed is 6000-8000 r/min; after layering, sucking a lower organic phase, adding chloroform-methanol solution into an upper aqueous phase, extracting the organic phase again according to the steps, repeating the steps for two times or more, collecting the organic phase, performing rotary evaporation to remove chloroform-methanol to obtain a viscous liquid, and blowing nitrogen to a solid state to obtain the composite lipid;

(3) acetone precipitation of milk polar lipids: adding acetone into the composite lipid obtained in the step (2) and stirring; collecting the precipitate after filtration to obtain the milk polar lipid.

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