阅读说明：本技术 一种神经酰胺和葡糖苷（脂）酰鞘氨醇的联产制备方法 (Co-production preparation method of ceramide and glucoside (lipid) acyl sphingosine ) 是由 吴宪君 刘玲 蒋昊月 吴昱隆 于 2021-10-23 设计创作，主要内容包括：本发明提供一种神经酰胺和葡糖苷(脂)酰鞘氨醇的联产制备方法,以魔芋飞粉或米糠渣为原料,依次经提取、调pH值、过滤、调pH值、浓缩、萃取、重结晶等步骤,得到高纯度的游离神经酰胺和葡糖苷(脂)酰鞘氨醇。本发明将魔芋飞粉和米糠渣变废为宝进行资源再利用生产出经济价值高的神经酰胺和葡糖苷(脂)酰鞘氨醇,提高了资源综合利用率,具有巨大的经济效益和环境效益。(The invention provides a coproduction preparation method of ceramide and glucoside (lipid) acylsphingosine, which takes konjac flying powder or rice bran residues as raw materials and sequentially comprises the steps of extracting, adjusting the pH value, filtering, adjusting the pH value, concentrating, extracting, recrystallizing and the like to obtain high-purity free ceramide and glucoside (lipid) acylsphingosine. The invention changes the konjac flying powder and the rice bran residues into valuable materials and recycles the resources to produce ceramide and glucoside (lipid) acyl sphingosine with high economic value, improves the comprehensive utilization rate of resources and has great economic benefit and environmental benefit.)

1. A process for the preparation of ceramide and glucosylceramide, comprising the steps of:

(1) extraction: taking raw material konjac flying powder or rice bran residues, performing reflux extraction for 2-3 times by using 80% -95% ethanol which is 3-10 times of the weight of the konjac flying powder or the rice bran residues, and combining extracting solutions;

(2) adjusting the pH value: adding appropriate amount of lime milk into the extractive solution to adjust its pH to 8-11, generating a large amount of fatty acid calcium precipitate, standing for 2-8 hr;

(3) and (3) filtering: filtering to remove calcium fatty acid precipitate to obtain filtrate;

(4) adjusting the pH value and concentrating: adjusting the pH of the filtrate obtained in the step (3) to 4-6 with acid, concentrating and recovering ethanol until no alcohol smell exists, and supplementing 1-2 times of water into the concentrated solution to obtain a solution;

(5) and (3) fractional extraction: carrying out fractional extraction on the solution obtained in the step (4) by using a mixed organic solvent according to the polarity from low to high, wherein the mixed organic solvent consists of a low-polarity solvent, methanol and glacial acetic acid, and the volume ratio of the low-polarity solvent to the methanol to the glacial acetic acid is 10:1:0.1-10:5: 1;

(6) concentrating to obtain a crude product: concentrating the fractional extractive solutions obtained in step (5) respectively to separate out solid, cooling, filtering to remove liquid part, and drying the solid part to obtain free ceramide and glucoside (lipid) ceramide sphingosine crude products respectively;

(7) recrystallizing the crude product obtained in step (6) in acetone for 2-3 times to obtain pure product of free ceramide and glucoside (lipid) ceramide.

2. The method of claim 1, wherein step (1) is performed with 5-8 times 80% -95% ethanol.

3. The method of claim 2, wherein step (1) is performed with 6 times 80% to 95% ethanol.

4. The method of claim 1, wherein the pH of step (4) is adjusted using an acid selected from at least one of citric acid, glacial acetic acid, hydrochloric acid, and sulfuric acid.

5. The method according to claim 1, wherein the low polarity solvent of step (5) is at least one selected from the group consisting of chloroform, dichloromethane, dichloroethane, n-hexane, cyclohexane, and petroleum ether.

6. The method of any one of claims 1 to 5, wherein the pH is adjusted to 9.8 to 10.3 in step (2) and 4.9 to 5.2 in step (4).

7. The method of claim 5 or 6, wherein the fractional extraction in step (5) is carried out by using a mixed solvent prepared from a low-polarity solvent, methanol and a glacial acetic acid mixed organic solvent according to the volume ratio of 10:1:0.1, 10:1.5:0.1, 10:2:0.1 and 10:2.5:0.1 to extract the solution obtained in step (4) sequentially to obtain extracts F1, F2, F3 and F4; concentrating the extractive solutions F2 and F4 respectively according to step (6) to precipitate solid, cooling, filtering to remove liquid part, drying the solid part to obtain crude products of free ceramide and glucoside (lipid) ceramide respectively, and recrystallizing in acetone for 2-3 times according to step (7) to obtain pure products of free ceramide and glucoside (lipid) ceramide; the low polarity solvent is selected from dichloromethane or trichloromethane.

8. The method of claim 5 or 6, wherein the step (5) of fractional extraction adopts a mixed solvent prepared by mixing a low-polarity solvent, methanol and a glacial acetic acid mixed organic solvent according to the volume ratio of 10:1:0.2, 10:2:0.3, 10:3:0.5, 10:4:0.6 and 10:5:0.8 to extract the solution obtained in the step (4) to obtain extracts F1, F2, F3, F4 and F5 in sequence; concentrating the extractive solutions F2 and F5 respectively according to step (6) to precipitate solid, cooling, filtering to remove liquid part, drying the solid part to obtain crude products of free ceramide and glucoside (lipid) ceramide respectively, and recrystallizing in acetone for 2-3 times according to step (7) to obtain pure products of free ceramide and glucoside (lipid) ceramide; the low polarity solvent is selected from cyclohexane or petroleum ether.

Technical Field

The invention relates to a method for coproducing ceramide and glucoside (ester) acylsphingosine by using konjac flying powder or rice bran residues as raw materials, belonging to the technical field of deep processing of agricultural and sideline products.

Background

Ceramides, also known as sphingolipids, are lipids present in the skin and play an important role in the formation of the stratum corneum of the epidermis. Are common structural units of (neuro) sphingolipids. The fatty acid has a structure of an acid amide bond at the amino group of sphingosine. On which glycosphingolipids are formed if sugars are bound and (neuro) sphingomyelins if phosphorylcholine is bound. Ceramides are intermediate metabolites of sphingolipids, and particularly occupy important positions in biosynthesis. Ceramide exists in cell membranes of animal and plant tissues, and molecules of the ceramide expose monosaccharide or oligosaccharide residues to extracellular spaces to participate in reactions with glycolipid head groups of other cells, antibodies, bacterial toxins and virus envelope proteins, so that the ceramide plays an important role in cell adhesion, regulation of cellular immunity, determination of blood type and serving as a tumor antigen. Ceramide has effects in maintaining skin barrier function, enhancing adhesion between keratinocytes, improving skin dryness, and reducing desquamation of skin. And has a strong ability to associate water molecules, which maintains skin moisture by forming a network structure in the stratum corneum. Can improve the conditions of dry skin, desquamation, roughness, etc.; meanwhile, ceramide can increase the thickness of the horny layer of epidermis, improve the water holding capacity of the skin, reduce wrinkles, enhance the elasticity of the skin and delay the skin aging. Also has activity against other leukemia cells and malignant cells in tissue culture; ceramides can regulate nerve cell growth and variation. Ceramides are sphingolipid breakdown products in the bilayer of biological membranes, and are recognized as second messengers; and in the processes of cell growth, proliferation, differentiation, apoptosis and injury, ceramide plays a wide and important role.

Ceramide and glucosylceramide are present in cell membranes of animal and plant tissues, and have wide sources, but have limited market acceptance because animal-derived ceramide products have risks of being accompanied by pathogenic factors, so that the extraction of ceramide and glucosylceramide from plants is significant.

The konjac flying powder and the rice bran slag are used as raw materials to carry out deep processing on the konjac flying powder and the rice bran slag, and jointly produce high-added-value products of ceramide and glucoside (ester) acyl sphingosine, so that the konjac flying powder and the rice bran slag are changed into valuable, the comprehensive utilization rate of resources is improved, and huge economic benefits and environmental benefits are achieved.

Disclosure of Invention

The invention aims to provide a method for coproducing ceramide and glucoside (lipid) acylsphingosine by using konjac flying powder or rice bran residues as raw materials, which adopts the following technical scheme:

there is provided a process for preparing ceramide and glucosylceramide, which comprises the steps of:

(1) extraction and concentration: taking raw material konjac flying powder or rice bran residues, performing reflux extraction for 2-3 times by using 80% -95% ethanol which is 3-10 times of the weight of the konjac flying powder or the rice bran residues, and combining extracting solutions;

(2) adjusting the pH value: adding appropriate amount of lime milk into the extractive solution to adjust its pH to 8-11, generating a large amount of fatty acid calcium precipitate, standing for 2-8 hr;

(3) and (3) filtering: filtering to remove calcium fatty acid precipitate to obtain filtrate;

(4) adjusting the pH value and concentrating: adjusting the pH of the filtrate obtained in the step (3) to 4-6 with acid, concentrating and recovering ethanol until no alcohol smell exists, and supplementing 1-2 times of water into the concentrated solution to obtain a solution;

(5) and (3) fractional extraction: carrying out fractional extraction on the solution obtained in the step (4) by using a mixed organic solvent according to the polarity from low to high, wherein the mixed organic solvent consists of a low-polarity solvent, methanol and glacial acetic acid, and the volume ratio of the low-polarity solvent to the methanol to the glacial acetic acid is 10:1:0.1-10:5: 1;

(6) concentrating to obtain a crude product: concentrating the fractional extraction liquid obtained in the step (5) respectively to separate out solids, cooling, filtering out a liquid part, and drying the solid part to obtain free ceramide and glucoside (lipid) acyl sphingosine crude products;

(7) recrystallizing the crude product obtained in step (6) in acetone for 2-3 times to obtain pure product of free ceramide and glucoside (lipid) ceramide.

In one embodiment, in step (1), 5-8 times of 80% -95% ethanol is used for extraction.

Further, in the step (1), 6 times of 80-95% ethanol is adopted for extraction.

In some embodiments, the acid used in the step (4) for adjusting the pH is at least one selected from the group consisting of citric acid, glacial acetic acid, hydrochloric acid, and sulfuric acid.

In some embodiments, the low polarity solvent of step (5) is selected from at least one of chloroform, dichloromethane, dichloroethane, n-hexane, cyclohexane, petroleum ether.

In one embodiment, the pH is adjusted to 9.8-10.3 in step (2) and 4.9-5.2 in step (4).

In one embodiment, in the step (5), the solution obtained in the step (4) is extracted by using a mixed organic solvent of dichloromethane or chloroform, methanol and glacial acetic acid according to the volume ratio of 10:1:0.1 to 10:1.5:0.1 to 10:2:0.1 and 10:2.5:0.1 in turn to obtain extract solutions F1, F2, F3 and F4 in turn; concentrating the extractive solutions F2 and F4 respectively according to step (6) to precipitate solid, cooling, filtering to remove liquid part, drying the solid part to obtain crude products of free ceramide and glucoside (lipid) ceramide, and recrystallizing in acetone for 2-3 times according to step (7) to obtain pure products of free ceramide and glucoside (lipid) ceramide.

In one embodiment, in the step (5), the solution obtained in the step (4) is extracted by adopting a mixed organic solvent of cyclohexane or petroleum ether, methanol and glacial acetic acid according to the volume ratio of 10:1:0.2, 10:2:0.3, 10:3:0.5, 10:4:0.6 and 10:5:0.8 to obtain extract solutions F1, F2, F3, F4 and F5 in sequence; concentrating the extractive solutions F2 and F5 respectively according to step (6) to precipitate solid, cooling, filtering to remove liquid part, drying the solid part to obtain crude products of free ceramide and glucoside (lipid) ceramide, and recrystallizing in acetone for 2-3 times according to step (7) to obtain pure products of free ceramide and glucoside (lipid) ceramide.

The ceramide and glucosylceramide of the invention can be analyzed for content (HPLC-ELSD method) as follows:

chromatographic conditions are as follows: the mobile phase was acetonitrile/methanol (v/v) ═ 98:2, flow rate: 1ml/min, column XDB-C18, column temperature: 35 ℃, evaporative photodetector temperature 40 ℃, nitrogen flow rate: 1.5L/min, and the detection sensitivity is 2;

preparation of standard solution: accurately weighing standard ceramide or glucoside (lipid) acyl sphingosine 5mg, placing in 50ml volumetric flask, adding 30ml methanol, ultrasonic dissolving, and fixing volume to scale, and shaking;

preparation of sample solution: precisely weighing 5-50 mg of the sample, placing the sample into a 50ml volumetric flask according to the content range, adding 30ml of methanol, ultrasonically dissolving, fixing the volume to the scale, and shaking up.

The method takes the konjac flying powder or the rice bran residues as raw materials to coproduce and prepare the ceramide and glucoside (ester) acyl sphingosine with high added value, changes the konjac flying powder and the rice bran residues into valuable, improves the comprehensive utilization rate of resources, and has great economic benefit and environmental benefit; the preparation method is simple in preparation process, low in cost and easy to implement, and has good industrial application prospects.

Detailed Description

The foregoing summary of the invention is described in further detail below with reference to specific embodiments. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples. Various substitutions and alterations can be made without departing from the technical idea of the invention as described above, according to the common technical knowledge and conventional means in the field, and the scope of the invention is covered.

Example 1 Co-production of ceramide and glucoside (lipid) acylsphingosine Using konjac flying powder as raw Material

Taking 1000 g of a byproduct konjac flying powder in konjac fine powder processing, stirring and heating the konjac flying powder with 80% ethanol in an amount which is 6 times that of the konjac flying powder for extraction for three times, filtering the konjac flying powder for three times (filter cakes can be used for preparing feeds after being dried), combining extracting solutions obtained in the three times, adding lime milk to adjust the pH value to 10.0 to generate a large amount of fatty acid calcium precipitate, standing the precipitated fatty acid calcium for 3-5 hours, filtering the fatty acid calcium, adjusting the pH value of a filtrate to 5.0 with hydrochloric acid (obtained by diluting concentrated hydrochloric acid with water in a volume ratio of 1: 1), concentrating and recovering ethanol until no alcohol smell exists, supplementing water with the same volume to obtain a solution, carrying out fractional extraction on the solution with a mixed organic solvent of petroleum ether/methanol/glacial acetic acid, sequentially taking the mixed solvent of petroleum ether/methanol/glacial acetic acid in a volume ratio of 10:1:0.2, 10:2:0.3, 10:3:0.5, 10:4:0.6 and 10:5:0.8 as an extracting agent, collecting each extracting solution independently, and obtaining each extracting agent by taking the mixed solvent of petroleum ether/methanol/glacial acetic acid in a volume ratio of 10:2:0.3 as an extracting agent to obtain each extracting agent Concentrating the extract liquid to separate out solid, cooling, filtering to remove liquid, drying the solid to obtain 0.41g (content: 67%) of crude product of ceramide, and recrystallizing the crude product for 3 times by using acetone as a crystallization solvent to obtain 0.2 g of 98% content free ceramide product; using mixed solvent of petroleum ether/methanol/glacial acetic acid with volume ratio of 10:5:0.8 as extractant, concentrating until solid is separated out, cooling, filtering liquid part, drying solid part to obtain crude product of glucoside (lipid) ceramide 1.33g (content of 62%), using acetone as crystallization solvent to recrystallize the crude product for 3 times to obtain 0.6 g (content of 98%) glucoside (lipid) ceramide.

Example 2 Co-production of ceramide and glucoside (lipid) acylsphingosine Using Rice bran residue as raw Material

Taking 1000 g of rice bran residues after rice oil processing, stirring and heating the rice bran residues by using 90% ethanol of which the volume is 8 times that of the rice bran residues, extracting for three times by filtering (filter cakes can be used for preparing feeds after being dried), combining extracting solutions of the three times, adding lime milk to adjust the pH value to 10.0, generating a large amount of fatty acid calcium precipitate, standing for 2-4 hours, filtering to remove the fatty acid calcium, adjusting the pH value of filtrate to 5.0 by using hydrochloric acid (obtained by adding water into concentrated hydrochloric acid according to the volume ratio of 1: 1), concentrating and recovering ethanol until no alcohol smell exists, supplementing water of which the volume is 1.5 times that of the concentrated solution to obtain a solution, carrying out fractional extraction on the solution by using a dichloromethane/methanol/glacial acetic acid mixed organic solvent as an extracting agent, sequentially using dichloromethane/methanol/glacial acetic acid mixed solvents of which the volume ratio is 10:1:0.1, 10:2:0.1 and 10:2.5:0.1 as the extracting agent, separately collecting each extracting solution, using dichloromethane/methanol/glacial acetic acid mixed solvent of which the volume ratio is 10:1 as the extracting agent, concentrating to separate out solid, cooling, filtering to remove liquid, drying the solid to obtain 0.25g (content: 64%) of crude product of ceramide, and recrystallizing the crude product for 3 times by using acetone as a crystallization solvent to obtain 0.12 g of 98% content free ceramide product; using mixed solvent of dichloromethane/methanol/glacial acetic acid with volume ratio of 10:2.5:0.1 as extractant to obtain extract, concentrating to precipitate solid, cooling, filtering to remove liquid part, drying to obtain crude product of glucoside (lipid) ceramide 0.91g (content of 60%), and recrystallizing the crude product 3 times with acetone as crystallization solvent to obtain glucoside (lipid) ceramide 0.47 g (content of 98%).

Example 3 Co-production of ceramide and glucoside (lipid) acylsphingosine Using konjac flying powder as raw Material

Taking 1000 g of a byproduct konjac flying powder in konjac fine powder processing, stirring and heating the konjac flying powder with 95% ethanol in an amount which is 4 times that of the konjac flying powder, extracting for three times, filtering (filter cakes can be used for preparing feeds after being dried), combining extracting solutions in three times, adding lime milk to adjust the pH value to 11.0, generating a large amount of fatty acid calcium precipitate, standing for 6-8 hours, filtering to remove the fatty acid calcium, adjusting the pH value of a filtrate to 6.0 with 2M sulfuric acid, concentrating and recovering ethanol until no alcohol smell exists, supplementing 2 times of water into a concentrated solution to obtain a solution, performing fractional extraction with a cyclohexane/methanol/glacial acetic acid mixed organic solvent, sequentially taking a mixed solvent with a cyclohexane/methanol/glacial acetic acid volume ratio of 10:1:0.1, 10:2:0.3, taking an extracting solution with a cyclohexane/methanol/glacial acetic acid mixed solvent with a volume ratio of 10:1: 0.6, 10:4:0.8, 10:5:1.0 as an extracting agent, separately collecting each extracting solution, taking a cyclohexane/methanol/glacial acetic acid mixed solvent with a cyclohexane/glacial acetic acid volume ratio of 10:2:0.3 as an extracting agent, concentrating to separate out solid, cooling, filtering to remove liquid, drying the solid to obtain 0.36g (content: 70%) of crude product of ceramide, and recrystallizing the crude product for 3 times by using acetone as a crystallization solvent to obtain 0.15 g of a free ceramide product with the content of 99%; the extraction liquid obtained by using the mixed solvent of cyclohexane/methanol/glacial acetic acid with the volume ratio of 10:5:1.0 as the extracting agent is concentrated to precipitate solid, the liquid part is filtered after cooling, the solid part is dried to obtain 1.17g (with the content of 66%) of crude product of glucoside (lipid) sphingosine, and the crude product is recrystallized for 3 times by using acetone as the crystallization solvent to obtain 0.49 g (with the content of 98%) of glucoside (lipid) sphingosine.

Example 4 Co-production of ceramide and glucoside (lipid) acylsphingosine Using konjac flying powder as raw Material

Taking 1000 g of a byproduct konjac flying powder in konjac fine powder processing, stirring and heating the konjac flying powder with 90% ethanol of which the volume is 8 times that of the konjac flying powder, extracting the konjac flying powder for three times by filtering (filter cakes can be used for preparing feeds after being dried), combining extracting solutions of the three times, adding lime milk to adjust the pH value to 10.0, generating a large amount of fatty acid calcium precipitate, standing for 3-5 hours, filtering to remove the fatty acid calcium, adjusting the pH value of a filtrate to 5.0 by using hydrochloric acid (obtained by adding water into concentrated hydrochloric acid according to the volume ratio of 1: 1), concentrating and recovering ethanol until no alcohol smell exists, supplementing water of which the volume is 1.5 times that of the concentrated solution to obtain a solution, performing fractional extraction on the solution by using a trichloromethane/methanol/glacial acetic acid mixed organic solvent as an extracting agent, sequentially using the trichloromethane/methanol/glacial acetic acid mixed solvent of which the volume ratio is 10:1:0.1, 10:1.5:0.1, 10:2:0.1 as the extracting agent, collecting each extracting solution independently, and obtaining each extracting solution by using the trichloromethane/methanol/glacial acetic acid mixed solvent of which the trichloromethane/glacial acetic acid is 10:1 Taking the solution, concentrating to separate out a solid, cooling, filtering out a liquid part, drying the solid part to obtain 0.45g (with the content of 68%) of a ceramide crude product, and carrying out 3 times of recrystallization on the crude product by taking acetone as a crystallization solvent to obtain 0.24 g of a free ceramide product with the content of 98%; using chloroform/methanol/glacial acetic acid mixed solvent as extractant, concentrating until solid is separated out, cooling, filtering to remove liquid part, drying to obtain crude product 1.40g (content: 63%), recrystallizing the crude product with acetone for 3 times to obtain 0.67 g (content: 98%) of glucoside (lipid) ceramide.

Example 5 Co-production of ceramide and glucoside (lipid) acylsphingosine Using Rice bran residue as raw Material

Taking 1000 g of rice bran residues after rice oil processing, stirring and heating the rice bran residues by using 85% ethanol in an amount which is 10 times that of the rice bran residues, extracting for three times, filtering (filter cakes can be used for preparing feeds after being dried), combining extracting solutions in three times, adding lime milk to adjust the pH value to 8.0, generating a large amount of fatty acid calcium precipitate, standing for 5-7 hours, filtering to remove the fatty acid calcium, adjusting the pH value of a filtrate to 4.0 by using 0.2M lemon, concentrating and recovering ethanol until no alcohol smell exists, supplementing water with the same volume to the concentrated solution to obtain a solution, carrying out fractional extraction on the solution by using a mixed organic solvent of n-hexane/methanol/glacial acetic acid as an extracting agent, sequentially using a mixed solvent of n-hexane/methanol/glacial acetic acid in a volume ratio of 10:1:0.1, 10:2:0.2, 10:3:0.5, 10:4:0.7 and 10:5:0.8 as an extracting agent, separately collecting each extracting solution, using a mixed solvent of n-hexane/methanol/glacial acetic acid in a volume ratio of 10:3:0.5 as an extracting agent to obtain an extracting agent, concentrating to separate out solid, cooling, filtering to remove liquid, drying solid to obtain crude product of ceramide 0.29g (content 68%), recrystallizing the crude product for 3 times with acetone as crystallization solvent to obtain 0.16 g of free ceramide product with content of 99%; using mixed solvent of n-hexane/methanol/glacial acetic acid with volume ratio of 10:5:0.8 as extractant to obtain extract, concentrating to precipitate solid, cooling, filtering to remove liquid part, drying to obtain crude product of glucoside (lipid) sphingosine (0.96 g, content of 63%), and recrystallizing the crude product for 3 times with acetone as crystallization solvent to obtain 0.50 g of glucoside (lipid) sphingosine (content of 99%).