Method for extracting ceramide and glycosphingolipid from liquor byproduct waste

Abstract

The invention discloses a method for extracting ceramide and glycosphingolipid from the byproduct of liquor, the waste lees, and belongs to the technical field of compound extraction. The invention provides a method for extracting ceramide and glycosphingolipid, which uses the byproduct of liquor, the waste lees, as raw materials, and sequentially undergoes the steps of drying, extraction, alkali adjustment, filtration, acid adjustment, concentration, extraction, recrystallization, etc., to finally obtain free ceramide and glycosphingolipid. The invention extracts ceramide and glycosphingolipid with high economic value from the byproduct of liquor, turns waste into treasure, recycles resources, improves resource utilization, and has significant economic and environmental benefits.

Description

Technical Field

[0001] The invention belongs to the technical field of small molecule compound extraction, and particularly relates to a method for extracting ceramide and glycosphingolipid from waste grains, a byproduct of liquor. Background Art

[0002] Ceramide is a type of amide compound formed by dehydration of long-chain fatty acids and the amino group of sphingosine, namely N-acylceramide. It is a signal transduction substance widely present in eukaryotic cells, mainly existing as a structural component of the cell membrane or as a free molecule in the cell. 40% to 50% of sebum in the stratum corneum is composed of ceramide, which is the main part of the intercellular matrix and plays an important role in maintaining the balance of moisture in the stratum corneum. Ceramide has a strong ability to associate with water molecules. It maintains skin moisture by forming a reticular structure in the stratum corneum, can enhance the skin's anti-aging function, keep the skin elastic, smooth and delicate, and reduce the formation of facial wrinkles; it plays an important messenger role in the biological effects induced by various cytokines, vitamin D3, Fas and CD28 ligands; it has the effects of inhibiting blood pressure rise, activating immunity, hindering lipase activity, and inhibiting cancer cell proliferation. Therefore, in recent years, ceramide has been widely used in medicines, foods and cosmetics as a bioactive ingredient. Ceramide and sugar connected by glycosidic bonds are glycosphingolipids, which also exist in the cell membranes of animals and plants.

[0003] Waste generated during the production and brewing of liquor is called liquor by-products. Waste lees is one of them. Liquor waste has a high moisture content and high acidity. It is very easy to mold and rot when piled up and stored. If it is discarded or incinerated at will, it will cause serious environmental pollution. The reuse of liquor waste has become a growth point for the sustainable development of the circular economy in liquor factories. The main ways to deal with waste lees are: directly use it for animal breeding or biofertilizer; use it as a raw material for biological culture; ferment it into biofeed, etc.

[0004] Although there are reports on methods for preparing ceramides by fermentation with different bacteria (CN113735731A, CN109943602A, CN109206336A, CN106119310A, CN111358732), so far, there are no reports on methods for directly extracting ceramides and glycosphingolipids from the by-product of liquor, the spent grains. Summary of the invention

[0005] The present invention provides a method for extracting ceramide and glycosphingolipid from the waste dregs, a byproduct of liquor, in order to improve the resource utilization rate of the waste dregs and recover high value-added products. The method comprises the following steps:

[0006] A. The by-product of liquor, the lees, is dried and crushed to obtain lees powder;

[0007] B. The waste lees powder is extracted 2 to 6 times by heating and refluxing with an extraction solvent, and the extracts are combined and concentrated to obtain a concentrated extract; wherein the extraction solvent is at least one of 80% to 100% ethanol, a mixture of dichloromethane and methanol in a volume ratio of 5 to 1:1 to 5, and a mixture of chloroform and methanol in a volume ratio of 5 to 1:1 to 5;

[0008] C. Add lime milk to the concentrated extract to adjust the pH of the system to 8-11, let stand for solid-liquid separation, then adjust the pH of the filtrate to 3-6 with acid, concentrate and remove the organic solvent to obtain a concentrated solution;

[0009] D. Add water to the concentrate, extract with a non-polar solvent, extract 1 to 3 times, and collect the aqueous phase; then extract the aqueous phase with a low-polarity solvent, extract 1 to 3 times, and collect the aqueous phase; finally, extract the aqueous phase with a mixture of a low-polarity solvent and methanol in a volume ratio of 1 to 4:1, extract 1 to 3 times, and collect and combine the organic phases; the non-polar solvent is at least one of n-hexane, cyclohexane or petroleum ether; the low-polarity solvent is at least one of ether, dichloromethane or chloroform;

[0010] E. Concentrate the organic phase obtained in step D to remove the organic solvent to obtain an oily substance, add a crystallization solvent, cool in an ice bath, and filter. The obtained solid is dried to obtain a crude product;

[0011] F. Recrystallize the crude product obtained in step E in a recrystallization solvent to obtain free ceramide and glycosphingolipids.

[0012] Wherein, in step A of the above method, the particle size of the waste dregs powder is 20-120 meshes.

[0013] Wherein, in step B of the above method, during each extraction, the ratio of the volume of the extraction solvent to the mass of the waste powder is controlled to be 3-20 mL: 1 g.

[0014] Preferably, in step B of the above method, during each extraction, the ratio of the volume of the extraction solvent to the mass of the dregs powder is controlled to be 4-15 mL:1 g.

[0015] More preferably, in step B of the above method, during each extraction, the ratio of the volume of the extraction solvent to the mass of the dregs powder is controlled to be 5-10 mL:1 g.

[0016] Wherein, in step B of the above method, the time for each extraction is 1 to 6 hours.

[0017] Wherein, in step B of the above method, the extraction solvent is 80% to 95% ethanol.

[0018] Wherein, in step B of the above method, the ratio of the volume of the obtained concentrated extract to the mass of the waste grains powder used is 3-10 mL:1 g.

[0019] Wherein, in step C of the above method, the lime milk is prepared by the following method: 10-15g of quicklime is mixed with 100mL of water and then ground into an emulsion.

[0020] Preferably, in step C of the above method, lime milk is added to adjust the pH of the system to 9-10.

[0021] Wherein, in step C of the above method, the standing time is 4 to 24 hours.

[0022] Wherein, in step C of the above method, the acid is selected from at least one of glacial acetic acid, hydrochloric acid, sulfuric acid and citric acid.

[0023] Preferably, in step C of the above method, the acid is glacial acetic acid or hydrochloric acid.

[0024] Preferably, in step C of the above method, acid is added to adjust the pH to 3-5.

[0025] Wherein, in step D of the above method, the ratio of the volume of water to the volume of the concentrated solution is 1 to 3:1.

[0026] Wherein, in step D of the above method, during the extraction with the non-polar solvent, the ratio of the volume of the non-polar solvent to the volume of the concentrated solution is controlled to be 1 to 3:1 for each extraction.

[0027] Wherein, in step D of the above method, during extraction with a low polarity solvent, the ratio of the volume of the low polarity solvent to the volume of the concentrated solution is controlled to be 1 to 3:1 for each extraction.

[0028] Wherein, in step D of the above method, when the low polarity solvent and methanol mixed solution are extracted, the ratio of the volume of the low polarity solvent and methanol mixed solution to the volume of the concentrated solution is controlled to be 1 to 3:1 for each extraction.

[0029] Wherein, in step E of the above method, the crystallization solvent is at least one of methanol, acetone, acetonitrile or a mixture of chloroform and methanol in a volume ratio of 2 to 10:1.

[0030] Wherein, in step E of the above method, the ratio of the volume of the crystallization solvent to the mass of the oily substance is 0.5-2 mL: 1 g.

[0031] Wherein, in step E of the above method, the ice bath cooling time is 2 to 10 hours.

[0032] Wherein, in step F of the above method, the recrystallization solvent is at least one of methanol, acetone, acetonitrile or a mixture of chloroform and methanol in a volume ratio of 2 to 10:1.

[0033] Wherein, in step F of the above method, the number of recrystallization is 2 to 3 times.

[0034] The present invention uses the waste grains, a byproduct of liquor, as raw materials to prepare high-value-added ceramide and glycosphingolipids, analyzes the molecular ion peak through a 6V ionization energy high-resolution mass spectrometer to obtain a molecular formula, and then analyzes the fragment ion peak through a 20-40V ionization energy high-resolution mass spectrometer to determine the structure of sphingosine, and the long-chain acyl structure is mainly a saturated fatty chain without hydroxyl substitution, a saturated fatty chain with one or two hydroxyl substitutions, and if the compound is a glycoside, its sugar is a hexose. The detected molecular ion peaks and fragment peaks are:

[0035] ESI-MS 894.7022[M+Na] + ,280.2635[M+H] + ,262.2529[M+H] + ;

[0036] ESI-MS 880.6859[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0037] ESI-MS 866.6706[M+Na] + ,280.2635[M+H] + ,262.2529[M+H] + ;

[0038] ESI-MS 850.6749[M+Na] + ,280.2635[M+H] + ,262.2529[M+H] + ;

[0039] ESI-MS 848.6597[M+Na] + ,262.2529[M+H] + ;

[0040] ESI-MS 820.6279[M+Na] + ,262.2532[M+H] + ;

[0041] ESI-MS 794.6121[M+Na] + ,280.2635[M+H] + ,262.2529[M+H] + ;

[0042] ESI-MS 792.5968[M+Na] + ,262.2532[M+H] + ;

[0043] ESI-MS 764.6773[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0044] ESI-MS 762.6985[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0045] ESI-MS 748.6836[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0046] ESI-MS 736.6434[M+Na] + ,282.2798[M+H] + ,264.2632[M+H] + ;

[0047] ESI-MS 734.6639[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0048] ESI-MS 728.6835[M+Na] + ,264.2698[M+H] + ;

[0049] ESI-MS 722.6273[M+Na] + ,282.2798[M+H] + ,264.2698[M+H] + ;

[0050] ESI-MS 720.6486[M+Na] + ,282.2798[M+H] + ,264.2698[M+H] + ;

[0051] ESI-MS 706.6349[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0052] ESI-MS 704.6170[M+Na] + ,280.2635[M+H] + ,262.2529[M+H] + ;

[0053] ESI-MS 702.6714[M+Na] + ,284.2877[M+H] + ;

[0054] ESI-MS 692.6168[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0055] ESI-MS 690.6377[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0056] ESI-MS 678.6009[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0057] ESI-MS 676.5885[M+Na] + ,280.2624[M+H] + ,262.2549[M+H] + ;

[0058] ESI-MS 630.5491[M+Na] + ,262.2532[M+H] + ;

[0059] ESI-MS 602.5154[M+Na] + ,262.2532[M+H] + ;

[0060] ESI-MS 576.4986[M+H] + ,264.2686[M+H]+ ;

[0061] ESI-MS 574.4815[M+Na] + ,262.2559[M+H] + .

[0062] The ceramide compounds are as follows:

[0063]

[0064] At least one of;

[0065] The glycosphingolipids include the following compounds:

[0066]

[0067]

[0068] At least one of .

[0069] Beneficial effects of the present invention:

[0070] The invention uses the by-product waste grains as raw materials, and sequentially undergoes the steps of drying, extraction, alkali adjustment, filtration, acid adjustment, concentration, extraction, recrystallization, etc., to finally obtain free ceramide and glycosphingolipids, turning the waste grains into treasure, improving the comprehensive utilization rate of resources, and having huge economic and environmental benefits; and the invention has a simple preparation process, low cost, and good industrial application prospects. DETAILED DESCRIPTION

[0071] Specifically, the method for extracting ceramide and glycosphingolipid from the liquor byproduct waste comprises the following steps:

[0072] A. The by-product of liquor, the lees, is dried and crushed to obtain lees powder;

[0073] B. The waste lees powder is extracted 2 to 6 times by heating and refluxing with an extraction solvent, and the extracts are combined and concentrated to obtain a concentrated extract; wherein the extraction solvent is at least one of 80% to 100% ethanol, a mixture of dichloromethane and methanol in a volume ratio of 5 to 1:1 to 5, and a mixture of chloroform and methanol in a volume ratio of 5 to 1:1 to 5;

[0074] C. Add lime milk to the concentrated extract to adjust the pH of the system to 8-11, let stand for solid-liquid separation, then adjust the pH of the filtrate to 3-6 with acid, concentrate and remove the organic solvent to obtain a concentrated solution;

[0075] D. Add water to the concentrate, extract with a non-polar solvent, extract 1 to 3 times, and collect the aqueous phase; then extract the aqueous phase with a low-polarity solvent, extract 1 to 3 times, and collect the aqueous phase; finally, extract the aqueous phase with a mixture of a low-polarity solvent and methanol in a volume ratio of 1 to 4:1, extract 1 to 3 times, and collect and combine the organic phases; the non-polar solvent is at least one of n-hexane, cyclohexane or petroleum ether; the low-polarity solvent is at least one of ether, dichloromethane or chloroform;

[0076] E. Concentrate the organic phase obtained in step D to remove the organic solvent to obtain an oily substance, add a crystallization solvent, cool in an ice bath, and filter. The obtained solid is dried to obtain a crude product;

[0077] F. Recrystallize the crude product obtained in step E in a recrystallization solvent to obtain free ceramide and glycosphingolipids.

[0078] In step A of the method of the present invention, the liquor by-product, the lees, is dried and then crushed to a particle size of 20-120 meshes to facilitate extraction.

[0079] In step B of the method of the present invention, at least one of 80% to 100% ethanol, a dichloromethane and methanol mixture with a volume ratio of 5 to 1: 1 to 5, and a chloroform and methanol mixture with a volume ratio of 5 to 1: 1 to 5 is used to extract the lees powder. In order to ensure that ceramide and glycosphingolipids in the lees powder are extracted as much as possible, the present invention controls the number of extractions to 2 to 6 times, and in each extraction, the ratio of the volume of the extraction solvent to the mass of the lees powder is controlled to be 3 to 20 mL: 1 g, preferably 4 to 15 mL: 1 g, and more preferably 5 to 10 mL: 1 g; the time for each extraction is 1 to 6 hours.

[0080] In step B of the method of the present invention, the extraction solvent is preferably 80% to 95% ethanol.

[0081] The extract obtained in step B has very complex ingredients and a large amount of impurities, and most of the impurities need to be removed first. Therefore, the present invention first adds lime milk to adjust the pH value to 8-11, so that most of the acidic impurities are converted into fatty acid calcium precipitation, and after filtering the precipitated impurities, acid is added to adjust the pH of the filtrate to 3-6. No precipitation occurs here, but the alkaline impurities have been converted into salts, so that the alkaline impurities can be removed by subsequent fractional extraction to obtain a concentrated solution containing neutral ceramide and glycosphingolipids. If the opposite is true, it is difficult to achieve the impurity removal effect.

[0082] The concentrated solution obtained in step B still contains a large amount of impurities, so the present invention further purifies the solution through graded extraction. The graded extraction operation is as follows: water is added to the concentrated solution, and then a non-polar solvent is added for extraction, the extraction is performed 1 to 3 times, the organic phases are combined and the non-polar solvent is recovered, and the aqueous phase is retained; then a low-polarity solvent is added to the aqueous phase for extraction, the extraction is performed 1 to 3 times, the organic phases are combined and the low-polarity solvent is recovered, and the aqueous phase is retained; finally, a mixture of a low-polarity solvent and methanol is added to the aqueous phase for extraction, the extraction is performed 1 to 3 times, and the organic phases are combined (the aqueous phase is discarded).

[0083] In the graded extraction, the ratio of the volume of water to the volume of the concentrated solution is 1 to 3:1; in the case of non-polar solvent extraction, the ratio of the volume of the non-polar solvent to the volume of the concentrated solution is controlled to be 1 to 3:1 for each extraction; in the case of low-polar solvent extraction, the ratio of the volume of the low-polar solvent to the volume of the concentrated solution is controlled to be 1 to 3:1 for each extraction; in the case of low-polar solvent and methanol mixed solution extraction, the ratio of the volume of the low-polar solvent and methanol mixed solution to the volume of the concentrated solution is controlled to be 1 to 3:1 for each extraction.

[0084] In step E of the method of the present invention, after the organic phase obtained by fractional extraction is concentrated and the organic solvent is removed, an oily substance is obtained, which is first dissolved in a crystallization solvent, and then cooled in an ice bath for 2 to 10 hours to precipitate a solid, which is dried to obtain a crude product. In step E, the crystallization solvent is at least one of methanol, acetone, acetonitrile, or a mixed solution of chloroform and methanol in a volume ratio of 2 to 10:1, and the ratio of its volume to the mass of the oily substance is 0.5 to 2 mL: 1 g.

[0085] In step F of the method of the present invention, at least one of methanol, acetone, acetonitrile or a mixture of chloroform and methanol in a volume ratio of 2 to 10:1 is used as a recrystallization solvent to recrystallize the crude product; the recrystallization is performed 2 to 3 times, and the solvent obtained from each recrystallization does not need to be exactly the same.

[0086] The scheme of the present invention will be explained below in conjunction with embodiments. It will be understood by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0087] In the embodiment of the present invention, ceramide and glycosphingolipids were detected by the following method (UPLC-QTOF liquid chromatography-mass spectrometry): ES source, positive ion mode, molecular weight scanning range: 50-1500, ionization energy: 6V and 20-40V; chromatographic conditions: mobile phase: methanol / 0.1% formic acid water, flow rate: 0.2mL / min, chromatographic column: ACQUITY UPLCBEH C18, column temperature: 40°C.

[0088] Example 1

[0089] A. Put Wuliangye lees in an oven to dry, then grind them with a grinder to obtain lees powder (40 mesh);

[0090] B. Take 700 g of wine lees powder, add 3.5 L of 95% ethanol each time, stir, heat and reflux for 2 h, extract 3 times in total, filter the extracts 3 times, combine the extracts 3 times and concentrate to about 3 L;

[0091] C. Add lime milk (take 10-15g quicklime (CaO) and place it in a clean small mortar, add 100mL of water and grind it into an emulsion) to adjust the pH value of the extract to about 10, produce a large amount of fatty acid calcium precipitation, let it stand overnight, filter out the fatty acid calcium, adjust the pH of the filtrate to about 4 with 6N hydrochloric acid, concentrate the solution, and recover ethanol until there is no alcohol taste to obtain a concentrated solution;

[0092] D. Add 2 times the volume of water to the concentrate, then extract once with 3 times the volume of petroleum ether and dichloromethane respectively, collect the aqueous phase, concentrate the petroleum ether layer and dichloromethane layer respectively, recover the solvent, add 2 times the volume of dichloromethane and methanol mixture (the volume ratio of dichloromethane to methanol is 3:1) to the aqueous phase, extract once, and collect the organic phase;

[0093] E. Concentrate the organic phase and remove the organic solvent to obtain an oily substance. Add 1 volume of acetone, cool in an ice bath for 4 hours, and then filter. Dry the solid part to obtain 0.25 g of a crude product.

[0094] F. The crude product was recrystallized twice using acetone as the crystallization solvent to obtain 0.16 g of free ceramide and glycosphingolipid products. The identification results are as follows:

[0095] ESI-MS 894.7022[M+Na] + ,280.2635[M+H] + ,262.2529[M+H] + ;

[0096] ESI-MS 880.6859[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0097] ESI-MS 866.6706[M+Na] + ,280.2635[M+H] + ,262.2529[M+H] + ;

[0098] ESI-MS 850.6749[M+Na] +,280.2635[M+H] + ,262.2529[M+H] + ;

[0099] ESI-MS 848.6597[M+Na] + ,262.2529[M+H] + ;

[0100] ESI-MS 820.6279[M+Na] + ,262.2532[M+H] + ;

[0101] ESI-MS 794.6121[M+Na] + ,280.2635[M+H] + ,262.2529[M+H] + ;

[0102] ESI-MS 792.5968[M+Na] + ,262.2532[M+H] + ;

[0103] ESI-MS 764.6773[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0104] ESI-MS 762.6985[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0105] ESI-MS 748.6836[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0106] ESI-MS 736.6434[M+Na] + ,282.2798[M+H] + ,264.2632[M+H] + ;

[0107] ESI-MS 734.6639[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0108] ESI-MS 728.6835[M+Na] + ,264.2698[M+H] + ;

[0109] ESI-MS 722.6273[M+Na] + ,282.2798[M+H] + ,264.2698[M+H] + ;

[0110] ESI-MS 720.6486[M+Na] + ,282.2798[M+H] + ,264.2698[M+H] + ;

[0111] ESI-MS 706.6349[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0112] ESI-MS 704.6170[M+Na] + ,280.2635[M+H] + ,262.2529[M+H] + ;

[0113] ESI-MS 702.6714[M+Na] + ,284.2877[M+H] + ;

[0114] ESI-MS 692.6168[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0115] ESI-MS 690.6377[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0116] ESI-MS 678.6009[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0117] ESI-MS 676.5885[M+Na] + ,280.2624[M+H] + ,262.2549[M+H] + ;

[0118] ESI-MS 630.5491[M+Na] + ,262.2532[M+H] + ;

[0119] ESI-MS 602.5154[M+Na] + ,262.2532[M+H] + ;

[0120] ESI-MS 576.4986[M+Na] + ,264.2686[M+H] + ;

[0121] ESI-MS 574.4815[M+Na] + ,262.2559[M+H] + .

[0122] Example 2

[0123] A. Place Wuliangye lees in the sun to dry, then grind them with a grinder to obtain lees powder (80 mesh);

[0124] B. Take 700 g of wine lees powder, add 4.2 L of 90% ethanol each time, stir, heat and reflux for 4 h, extract 3 times in total, filter the extracts 3 times, combine the extracts 3 times and concentrate to about 4 L;

[0125] C. Add lime milk (take 10-15g quicklime (CaO) and place it in a clean small mortar, add 100mL of water and grind it into an emulsion) to adjust the pH value of the extract to about 9, produce a large amount of fatty acid calcium precipitation, stand overnight and separate the solid and liquid, filter out the fatty acid calcium, adjust the pH of the filtrate to about 3 with 2N hydrochloric acid, concentrate the solution, and recover ethanol until there is no alcohol taste to obtain a concentrated solution;

[0126] D. Add 1 volume of water to the concentrate, then extract with an equal volume of n-hexane for 2 times, collect the aqueous phase, combine and concentrate the n-hexane layer, recover the solvent, extract the aqueous phase with an equal volume of chloroform again, extract 2 times, collect the aqueous phase, combine and concentrate the chloroform layer, recover the solvent, and finally add an equal volume of chloroform and methanol mixture (the volume ratio of chloroform to methanol is 4:1) to the aqueous phase for extraction, extract 2 times, collect and combine the organic phase;

[0127] E. Concentrate the organic phase and remove the organic solvent to obtain an oily substance. Add 0.5 times the volume of methanol, cool in an ice bath for 8 hours, and filter. Dry the solid part to obtain 0.28 g of a crude product.

[0128] F. The crude product was recrystallized twice using methanol as the crystallization solvent to obtain 0.13 g of free ceramide and glycosphingolipid products. The identification results are as follows:

[0129] ESI-MS 894.7022[M+Na] + ,280.2635[M+H] + ,262.2529[M+H] + ;

[0130] ESI-MS 880.6859[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0131] ESI-MS 866.6706[M+Na] + ,280.2635[M+H] + ,262.2529[M+H] + ;

[0132] ESI-MS 850.6749[M+Na] + ,280.2635[M+H] + ,262.2529[M+H] + ;

[0133] ESI-MS 848.6597[M+Na] + ,262.2529[M+H] + ;

[0134] ESI-MS 820.6279[M+Na] + ,262.2532[M+H] + ;

[0135] ESI-MS 794.6121[M+Na] + ,280.2635[M+H] + ,262.2529[M+H] + ;

[0136] ESI-MS 792.5968[M+Na] + ,262.2532[M+H] + ;

[0137] ESI-MS 764.6773[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0138] ESI-MS 762.6985[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0139] ESI-MS 748.6836[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0140] ESI-MS 736.6434[M+Na] + ,282.2798[M+H] + ,264.2632[M+H] + ;

[0141] ESI-MS 734.6639[M+Na] +,282.2791[M+H] + ,264.2686[M+H] + ;

[0142] ESI-MS 728.6835[M+Na] + ,264.2698[M+H] + ;

[0143] ESI-MS 722.6273[M+Na] + ,282.2798[M+H] + ,264.2698[M+H] + ;

[0144] ESI-MS 720.6486[M+Na] + ,282.2798[M+H] + ,264.2698[M+H] + ;

[0145] ESI-MS 706.6349[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0146] ESI-MS 704.6170[M+Na] + ,280.2635[M+H] + ,262.2529[M+H] + ;

[0147] ESI-MS 702.6714[M+Na] + ,284.2877[M+H] + ;

[0148] ESI-MS 692.6168[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0149] ESI-MS 690.6377[M+Na] + ,282.2791[M+H] +,264.2686[M+H] + ;

[0150] ESI-MS 678.6009[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0151] ESI-MS 676.5885[M+Na] + ,280.2624[M+H] + ,262.2549[M+H] + ;

[0152] ESI-MS 630.5491[M+Na] + ,262.2532[M+H] + ;

[0153] ESI-MS 602.5154[M+Na] + ,262.2532[M+H] + ;

[0154] ESI-MS 576.4986[M+Na] + ,264.2686[M+H] + ;

[0155] ESI-MS 574.4815[M+Na] + ,262.2559[M+H] + .

[0156] Example 3

[0157] A. Dry the Wuliangye lees in the sun, and then grind them with a grinder to obtain lees powder (100 mesh);

[0158] B. Take 700 g of wine lees powder, add 7 L of 80% ethanol each time, stir, heat and reflux for 6 h, extract 3 times in total, filter the extracts 3 times, combine the extracts 3 times and concentrate to about 7 L;

[0159] C. Add lime milk (take 10-15g quicklime (CaO) and place it in a clean small mortar, add 100mL of water and grind it into an emulsion) to adjust the pH value of the extract to about 10, produce a large amount of fatty acid calcium precipitation, stand overnight and separate the solid and liquid, filter out the fatty acid calcium, adjust the pH of the filtrate to about 5 with acetic acid, concentrate the solution, and recover ethanol until there is no alcohol taste to obtain a concentrated solution;

[0160] D. Add 3 times the volume of water to the concentrate, then extract with an equal volume of cyclohexane for 3 times, collect the aqueous phase, combine and concentrate the cyclohexane layer, recover the solvent, extract the aqueous phase with an equal volume of chloroform again, extract 3 times, collect the aqueous phase, combine and concentrate the chloroform layer, recover the solvent, and finally add 3 times the volume of chloroform and methanol mixture (the volume ratio of chloroform to methanol is 2:1) to extract the aqueous phase, extract 2 times, collect and combine the organic phase;

[0161] E. Concentrate the organic phase and remove the organic solvent to obtain an oily substance. Add an equal volume of acetonitrile, cool in an ice bath for 6 hours, and then filter. Dry the solid part to obtain 0.21 g of a crude product.

[0162] F. The crude product was recrystallized three times using acetone as the crystallization solvent to obtain 0.15 g of free ceramide and glycosphingolipid products. The identification results are as follows:

[0163] ESI-MS 894.7022[M+Na] + ,280.2635[M+H] + ,262.2529[M+H] + ;

[0164] ESI-MS 880.6859[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0165] ESI-MS 866.6706[M+Na] + ,280.2635[M+H] + ,262.2529[M+H] + ;

[0166] ESI-MS 850.6749[M+Na] + ,280.2635[M+H] + ,262.2529[M+H] + ;

[0167] ESI-MS 848.6597[M+Na] + ,262.2529[M+H] + ;

[0168] ESI-MS 820.6279[M+Na] + ,262.2532[M+H] + ;

[0169] ESI-MS 794.6121[M+Na] + ,280.2635[M+H] + ,262.2529[M+H] + ;

[0170] ESI-MS 792.5968[M+Na] + ,262.2532[M+H] + ;

[0171] ESI-MS 764.6773[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0172] ESI-MS 762.6985[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0173] ESI-MS 748.6836[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0174] ESI-MS 736.6434[M+Na] + ,282.2798[M+H] + ,264.2632[M+H] + ;

[0175] ESI-MS 734.6639[M+Na] +,282.2791[M+H] + ,264.2686[M+H] + ;

[0176] ESI-MS 728.6835[M+Na] + ,264.2698[M+H] + ;

[0177] ESI-MS 722.6273[M+Na] + ,282.2798[M+H] + ,264.2698[M+H] + ;

[0178] ESI-MS 720.6486[M+Na] + ,282.2798[M+H] + ,264.2698[M+H] + ;

[0179] ESI-MS 706.6349[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0180] ESI-MS 704.6170[M+Na] + ,280.2635[M+H] + ,262.2529[M+H] + ;

[0181] ESI-MS 702.6714[M+Na] + ,284.2877[M+H] + ;

[0182] ESI-MS 692.6168[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0183] ESI-MS 690.6377[M+Na] + ,282.2791[M+H] +,264.2686[M+H] + ;

[0184] ESI-MS 678.6009[M+Na] + ,282.2791[M+H] + ,264.2686[M+H] + ;

[0185] ESI-MS 676.5885[M+Na] + ,280.2624[M+H] + ,262.2549[M+H] + ;

[0186] ESI-MS 630.5491[M+Na] + ,262.2532[M+H] + ;

[0187] ESI-MS 602.5154[M+Na] + ,262.2532[M+H] + ;

[0188] ESI-MS 576.4986[M+Na] + ,264.2686[M+H] + ;

[0189] ESI-MS 574.4815[M+Na] + ,262.2559[M+H] + 。

Claims

1. A method for extracting ceramide and glycosphingolipids from the by-product of liquor, waste grains, characterized in that: The following steps are involved: A. The by-product of liquor, the lees, is dried and crushed to obtain lees powder; B. The waste lees powder is extracted 2 to 6 times by heating and refluxing with an extraction solvent, and the extracts are combined and concentrated to obtain a concentrated extract; wherein the extraction solvent is at least one of 80% to 100% ethanol, a mixture of dichloromethane and methanol in a volume ratio of 5 to 1:1 to 5, and a mixture of chloroform and methanol in a volume ratio of 5 to 1:1 to 5; C. Add lime milk to the concentrated extract to adjust the pH of the system to 8-11, let stand for solid-liquid separation, adjust the pH of the filtrate to 3-6 with acid, and concentrate to remove the organic solvent to obtain a concentrated solution; D. Add water to the concentrate, extract with a non-polar solvent, extract 1 to 3 times, and collect the aqueous phase; then extract the aqueous phase with a low-polarity solvent, extract 1 to 3 times, and collect the aqueous phase; finally, extract the aqueous phase with a mixture of a low-polarity solvent and methanol in a volume ratio of 1 to 4:1, extract 1 to 3 times, and collect and combine the organic phases; the non-polar solvent is at least one of n-hexane, cyclohexane or petroleum ether; the low-polarity solvent is at least one of ether, dichloromethane or chloroform; E. Concentrate the organic phase obtained in step D to remove the organic solvent to obtain an oily substance, add a crystallization solvent, cool in an ice bath, and filter. The obtained solid is dried to obtain a crude product; F. Recrystallizing the crude product obtained in step E in a recrystallization solvent to obtain free ceramide and glycosphingolipids; In step E, the crystallization solvent is at least one of methanol, acetone, acetonitrile, or a mixture of chloroform and methanol in a volume ratio of 2 to 10:1; In step F, the recrystallization solvent is at least one of methanol, acetone, acetonitrile or a mixture of chloroform and methanol in a volume ratio of 2 to 10:

1.

2. The method for extracting ceramide and glycosphingolipids from the by-product of liquor, waste grains, according to claim 1, characterized in that: In step A, the particle size of the waste powder is 20-120 meshes.

3. The method for extracting ceramide and glycosphingolipid from the by-product of liquor, waste grains, according to claim 1, characterized in that: In step B, each time during extraction, the ratio of the volume of the extraction solvent to the mass of the dregs powder is controlled to be 3-20 mL: 1 g.

4. The method for extracting ceramide and glycosphingolipid from the by-product of liquor, waste grains, according to claim 3, characterized in that: In step B, during each extraction, the ratio of the volume of the extraction solvent to the mass of the dregs powder is controlled to be 4-15 mL: 1 g.

5. The method for extracting ceramide and glycosphingolipid from the by-product of liquor, waste grains, according to claim 4, characterized in that: In step B, each time during extraction, the ratio of the volume of the extraction solvent to the mass of the dregs powder is controlled to be 5-10 mL: 1 g.

6. The method for extracting ceramide and glycosphingolipid from the by-product of liquor, waste grains, according to claim 1, characterized in that: In step B, the extraction time for each time is 1 to 6 hours.

7. The method for extracting ceramide and glycosphingolipid from the by-product of liquor, waste grains, according to claim 1, characterized in that: In step B, the extraction solvent is 80% to 95% ethanol.

8. The method for extracting ceramide and glycosphingolipid from the by-product of liquor, waste grains, according to claim 1, characterized in that: In step B, the ratio of the volume of the concentrated extract obtained to the mass of the spent grains powder used is 3-10 mL: 1 g.

9. The method for extracting ceramide and glycosphingolipid from the by-product of liquor, waste grains, according to claim 1, characterized in that: In step C, the lime milk is prepared by the following method: 10-15 g of quicklime is mixed with 100 mL of water and then ground into an emulsion.

10. The method for extracting ceramide and glycosphingolipid from the by-product of liquor, waste grains, according to claim 1, characterized in that: In step C, lime milk is added to adjust the pH of the system to 9-10.

11. The method for extracting ceramide and glycosphingolipid from the by-product of liquor, waste grains, according to claim 1, characterized in that: In step C, the standing time is 4 to 24 hours.

12. The method for extracting ceramide and glycosphingolipid from the by-product of liquor, waste grains, according to claim 1, characterized in that: In step C, the acid is selected from at least one of glacial acetic acid, hydrochloric acid, sulfuric acid and citric acid.

13. The method for extracting ceramide and glycosphingolipid from the by-product of liquor, waste grains, according to claim 12, characterized in that: In step C, the acid is glacial acetic acid or hydrochloric acid.

14. The method for extracting ceramide and glycosphingolipid from the by-product of liquor, waste grains, according to claim 1, characterized in that: In step C, acid is added to adjust the pH to 3-5.

15. The method for extracting ceramide and glycosphingolipid from the by-product of liquor, waste grains, according to claim 1, characterized in that: In step D, the ratio of the volume of water to the volume of the concentrated solution is 1 to 3:

1.

16. The method for extracting ceramide and glycosphingolipid from the by-product of liquor, waste grains, according to claim 1, characterized in that: In step D, during the extraction with the non-polar solvent, the ratio of the volume of the non-polar solvent to the volume of the concentrated solution is controlled to be 1 to 3:1 for each extraction.

17. The method for extracting ceramide and glycosphingolipid from the by-product of liquor, waste grains, according to claim 1, characterized in that: In step D, during extraction with a low polarity solvent, the ratio of the volume of the low polarity solvent to the volume of the concentrated solution is controlled to be 1 to 3:1 for each extraction.

18. The method for extracting ceramide and glycosphingolipid from the by-product of liquor, waste grains, according to claim 1, characterized in that: In step D, when the low polarity solvent and methanol mixed solution are extracted, the ratio of the volume of the low polarity solvent and methanol mixed solution to the volume of the concentrated solution is controlled to be 1 to 3:1 for each extraction.

19. The method for extracting ceramide and glycosphingolipid from the by-product of liquor, waste grains, according to claim 1, characterized in that: In step E, the ratio of the volume of the crystallization solvent to the mass of the oil is 0.5-2 mL: 1 g.

20. The method for extracting ceramide and glycosphingolipid from the by-product of liquor, waste grains, according to claim 1, characterized in that: In step E, the ice bath cooling time is 2 to 10 hours.

21. The method for extracting ceramide and glycosphingolipid from the by-product of liquor, waste grains, according to claim 1, characterized in that: In step F, the number of recrystallizations is 2 to 3 times.

22. The method for extracting ceramide and glycosphingolipid from the by-product of liquor, waste grains, according to any one of claims 1 to 21, characterized in that: The ceramide is selected from the following compounds: At least one of; The glycosphingolipids are selected from the following compounds: At least one of .

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