Background[1][2]
Phospholipids are important components of cell membranes and have important physiological functions. High-purity phospholipid fractionated products such as lecithin and cephalin can be widely used in the nutritional, pharmaceutical and cosmetic industries. However, because the current high-purity lecithin and cephalin are mostly prepared using high-performance liquid chromatography (HPLC) elution mode, the product processing capacity is small and the solvent consumption is large. Therefore, the production cost is high and the price is expensive, thus limiting its application. In order to reduce costs, many scholars have conducted research. For example, Kim et al. tried to simplify the mobile phase and used pure methanol as the mobile phase to separate egg yolk phospholipids. They achieved certain results, but they could not fundamentally solve the problem of leaching. disadvantages inherent in washing chromatography.
Physical and chemical properties[1]
Brain, also known as phosphatidylethanolamine. It is widely distributed in animals, especially concentrated in the brain and spinal cord. Its content is relatively high in soybeans. It can be used clinically as a hemostatic agent and a reagent for liver function tests. A phospholipid composed of glycerol, fatty acids, phosphoric acid, and ethanolamine. Fresh products are colorless solids that easily turn reddish-brown in the air. Hygroscopic. Insoluble in PVC resin water and acetone, slightly soluble in ethanol, soluble in chloroform and ether. It can be used as an antioxidant and also used in medicine. It can be extracted from fresh brains after slaughtering livestock or by-products of soybean oil extraction.
Phosphatidyl Ethanolamine (PE) and phosphatidyl choline (PC) are two important phospholipids, commonly known as cephalin and lecithin, which are widely used in food, medicine, cosmetics, etc. industry. High-purity cephalin is a good natural surfactant with unique biological activity and physiological functions. It is non-toxic, non-polluting, non-irritating and easy to biodegrade. It has attracted great attention from the scientific and industrial circles at home and abroad. Its demand is increasing year by year. Increase, but currently my country still relies on imports of cephalin products with a purity greater than 90%. Therefore, developing a low-cost, high-purity cephalin preparation method suitable for my country’s national conditions has important economic and social value.
The main methods for refining cephalin include extraction, column chromatography, precipitation, membrane separation, etc. Column chromatography has attracted much attention due to its advantages such as simple operation, good separation effect, no need for expensive equipment, and large processing capacity. In the research on column chromatography, currently the main focus is on cheap silica gel and alumina.
Apply[3]
Cephalin is a good natural surfactant with unique biological activity and physiological functions. It is non-toxic, non-polluting, non-irritating, and easy to biodegrade. It has attracted great attention from the scientific and industrial circles at home and abroad. At the same time, cephalin As a new type of rubber vulcanization accelerator and/or antioxidant.
Preparation[2]
Method 1:
Fresh egg yolks were first degreased with acetone three times and vacuum filtered. The filter cake was extracted twice with 95% alcohol. The filtrate was combined and vacuum concentrated to obtain crude phospholipid. After HPLC analysis, the crude phospholipid contained PC75.0% (wt%) and PE16.0% (wt%). Take silica gel with a water content of 10% and wet-pack it with a mixed solvent of dichloromethane containing 10% methanol. Rinse 50ml. Add 60 ml of crude phospholipid solution with a concentration of 100 mg/ml (the solvent is a methylene chloride solution containing 10% methanol), and then rinse with 200 ml of methanol. The flow rate is controlled at 0.4 ml/min. After adding the sample, start collecting the effluent (per portion About 5ml collected). The effluent was analyzed by TLC and then combined with the cephalin fractions. After vacuum concentration and freeze-drying, 0.55g of cephalin was obtained. The content of cephalin in the product was 91% by HPLC analysis. Rinse 150ml of the column with a methanol solution containing 2% (weight percent) ammonia (prepared from ammonia water containing 25% by weight), and then rinse 100ml with a dichloromethane mixed solvent containing 10% methanol. After that, the sample can be re-injected and rinsed in the same way. The flow rate is controlled at 0.4ml/min.
Production method of bactericidal preservative soy cephalin:
1) Ethanol extraction, place the powdered phospholipid in a tank, add ethanol at a ratio of 17L of ethanol with a purity of 96% for 1kg of powdered phospholipid, stir and reflux for 50-70 minutes, extract the extract, extract three times, and combine the three extracts ;
2) Refrigeration, place the above extraction liquid in a cold storage at a temperature of -5–7℃, and refrigerate for no less than 12 hours; 3. Concentrate, dry, temperature 80-90℃, vacuum degree 0.1-0.3MPa, time 1-3h, the finished product is obtained.
Supercritical carbon dioxide removal of fat to produce hydrolyzed brain protein powder and cephalin:
Remove the meninges and blood vessels from fresh or frozen animal brains, squeeze them through a 6-mesh sieve, lay them flat on a baking tray in a vacuum drying oven, and dry them to a moisture content of 3% under a vacuum of -0.08 Mpa and a temperature of 45°C. As follows, the dried product is obtained and stored at -5°C for later use; the dried animal brain powder is loaded into the extraction kettle of the HA221-40-100 supercritical CO2 extraction equipment in batches, with the pressure of the extraction kettle being 32.5 MPa and the temperature being 41 ℃; the pressure of the separation kettle I is 18 Mpa, the temperature is 45°C; the pressure of the separation kettle II is 6Mpa, the temperature is 48°C; the entrainer is 95% ethanol, and the dosage is 20% of the brain dried product; the extraction time is 2 hours; the carbon dioxide flow rate is 60 liters/ Kg/hour; take out the residue in the extraction kettle and use it for enzymatic hydrolysis to produce hydrolyzed brain protein powder; take out the paste in the extraction kettle I and use it to extract cephalin. The paste in the extraction kettle II can be removed;
Weigh the residue in the extraction kettle100 kg of material, add purified water 3 times, add animal protein hydrolysis special enzyme, the dosage is 0.5%, start the mixer and mix evenly, use 20% sodium hydroxide to adjust the pH to 7.5, the temperature is 65℃±1℃, stir for 3 hours for enzymatic hydrolysis ; Add 0.16% flavor enzyme and continue hydrolysis for 3 hours under the above pH value and temperature conditions; raise the temperature to 90°C and inactivate the enzyme for 20 minutes; cool down to 45°C, add 4% activated carbon, stir for 25 minutes, and then let stand for 30 minutes for adsorption ;Plate and frame press filtration to remove solid impurities, the pressure is 0.12Mpa, cycle repeatedly until the filtrate is clear, and then finely filtered through a 0.45µm microporous membrane to obtain the fine filtrate;
Pretreat the macroporous resin HPD-200 according to the instructions. Take 70 kg of the treated HPD-200 macroporous resin and put it into the glass column; take the fine filtrate and pass it through the column at a flow rate of 2BV/h. When 1BV flows out, collect it and pass it through the column. liquid; after adding the fine filtrate, elute the fine filtrate with 4BV purified water, and combine the column liquid and the eluent. The pretreated anion resin 201×4FC and cation resin 001×4 are desalted; the desalted liquid is collected for concentration, Dry; wash the macroporous resin HPD-200 with purified water containing 0.3% hydrochloric acid, and then wash it with purified water until the Ph value of the effluent is 5.0~6.5; then wash the macroporous resin with purified water containing 0.4% sodium hydroxide. Then wash with pure water until the Ph value is 7.0~7.5; until no biuret reaction is detected under alkaline conditions with 1% copper sulfate; the macroporous resin can be reused; anion resin 201×4FC, cation resin 001×4 is regenerated with 2.1~5% sodium hydroxide aqueous solution and 3.0~4.5% hydrochloric acid respectively;
Vacuum membrane concentration desalination solution, vacuum membrane concentration conditions are: the whole process is controlled below 65℃, the vacuum degree is -0.09Mpa; the solid content of the solution after concentration is 22~25%; the concentrated solution is spray dried, spray drying conditions: nozzle The inlet temperature is 170°C; the outlet temperature is 50-70°C, and 5 kg of cerebroprotein hydrolyzate powder is obtained; take out the paste containing cephalin and cholesterol in the separation kettle I, and dry it in a vacuum with a vacuum degree of -0.08 Mpa. The temperature is 62°C and dried for 4 hours to obtain a thick paste;
Use 4 times the volume of acetone to extract the cholesterol and oils in the thick paste three times. Recover the acetone from the extraction solution and the mother liquor respectively. Discard the extraction solution to recover the cholesterol and oils obtained after acetone; put 100 layers into the glass column. Silica gel is used for analysis; another 6 parts of silica gel are mixed with an equal amount of mother liquor after recovering acetone, and the mixture is added to the upper part of the silica gel column, and then 6 times of 95% hot ethanol is used to elute the silica gel column at a temperature of 75°C and a flow rate of 3 parts/minute. , collect the cephalin ethanol solution, and recover the ethanol in a vacuum, with a vacuum degree of -0.06 Mpa and a temperature of 60°C until the distillate has no ethanol smell; the paste is cephalin, with a content of ≥90%, and is stored in a nitrogen-filled seal.
Main reference materials
[1] Li Wei, Shao Youyuan, & Huang Guangdou. (2001). Separation of lecithin and cephalin by column chromatography. Journal of Hubei University of Technology, 16(2), 63-65.
[2] Zhang Weinong, He Haibo, Feng Yuqi, & Da Shilu. (2004). Study on the preparation of high-purity soybean lecithin and cephalin by column chromatography. China Oils and Fats, 29(6), 54-58.
[3] Yang Xiaoping, Dai Yousheng, & Chen Hua. (1994). Effects of fish oil on the fatty acid composition of cephalin in mice. Acta Nutrition (2), 164-168.
