Ceramides, methods for their purification, preparation and use

Abstract

This invention discloses a ceramide, its purification method, preparation method, and applications, relating to the field of natural compound separation technology. The purification method for ceramide includes membrane separation of crude ceramide extract, using a membrane material with a molecular weight cutoff of 150–1000 Da. By employing membrane separation to purify the crude ceramide and controlling the membrane material's molecular weight cutoff, ceramide can be enriched on one side of the membrane, while odorous impurities are enriched on the other side. This results in significant removal of odor from the purified ceramide, and the odor does not return after a period of time. This method is simple, has good odor removal effect, and can complete purification without complex organic solvents, leading to a wider range of applications for the obtained ceramide.

Description

Technical Field

[0001] This invention relates to the field of natural compound separation technology, and more specifically, to a ceramide and its purification, preparation, and applications. Background Technology

[0002] Ceramides are present in all eukaryotic cells and play an important regulatory role in cell differentiation, proliferation, apoptosis, aging, and other vital activities. As a major component of the intercellular matrix, ceramides have a strong ability to associate water molecules and maintain skin moisture by forming a network structure in the stratum corneum. Therefore, they play a crucial role in maintaining the moisture balance of the stratum corneum and are widely used in the cosmetics industry.

[0003] Ceramides mainly include ceramides extracted from natural plants and synthetic ceramides. Among them, ceramides extracted from natural plants are one of the main raw materials for ceramide products due to their superior ability to maintain the moisture balance of the stratum corneum and their wide availability. With the increasing demand for skin care, the demand for ceramide products is also increasing, thus there is an urgent need to provide a method for extracting ceramides from natural plants.

[0004] Glucosylceramides are widely found in nature, found in plants such as konjac, rice bran, wheat, corn, apples, and pineapples. However, the content of glucosylceramides in these plants is very low, generally between 0.01% and 0.2%, and the ceramide content in their extracts is also below 5%. Furthermore, due to the rich variety of natural compounds in plants, the extracted ceramides contain other natural components, resulting in a noticeable pungent odor. If naturally extracted ceramides are to be used in food, cosmetics, and other fields, post-processing is necessary to remove the odor-causing components. However, existing odor removal methods suffer from incomplete odor removal, recurrence of the odor after a period of time, and complex processes. Additionally, the use of ceramides in the food industry restricts the types of organic solvents used in the preparation process, further increasing the difficulty of removing odors from naturally extracted ceramides.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a ceramide, its purification method, preparation method, and application.

[0007] This invention is implemented as follows:

[0008] In a first aspect, the present invention provides a method for purifying ceramide, comprising membrane separation of crude ceramide extract, wherein the membrane material used for membrane separation has a molecular weight cutoff of 150 to 1000 Da.

[0009] Secondly, the present invention provides a method for preparing ceramide, comprising extracting plant raw materials with ethanol to obtain crude ceramide extract, and then preparing ceramide according to the purification method of ceramide provided in any of the foregoing embodiments.

[0010] Thirdly, the present invention provides a ceramide, which is prepared by the purification method of ceramide provided in any of the foregoing embodiments or by the preparation method of ceramide provided in any of the foregoing embodiments.

[0011] Fourthly, the present invention provides the use of ceramide as provided in the embodiments in any field of preparing pharmaceuticals, cosmetics or food.

[0012] The present invention has the following beneficial effects:

[0013] This invention provides a ceramide, its purification method, preparation method, and applications. By employing membrane separation to purify crude ceramide, and controlling the molecular weight cutoff of the membrane material, the ceramide can be enriched on one side of the membrane, while odorous impurities are enriched on the other side. This results in significant odor removal from the purified ceramide, and the odor will not return even after a period of time. This method is simple, has good odor removal effect, and can complete purification without complex organic solvents, leading to a wider range of applications for the obtained ceramide. Attached Figure Description

[0014] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 The GCMS chromatograms of ceramide extracts 1-4 provided in Example 1 of this invention;

[0016] Figure 2 This is a distribution diagram of the odor components of the four ceramide-containing substances in Example 1 of the present invention;

[0017] Figure 3 This is a distribution diagram of the odor components of the four ceramide-containing substances in Example 1 of the present invention;

[0018] Figure 4 This is a distribution diagram of the odor component content of the final ceramide extracts obtained in Example 1, Comparative Example 1, and Comparative Example 2 of the present invention.

[0019] Figure 5This is a distribution diagram of the odor component content of the final ceramide extracts obtained in Example 1, Comparative Example 1, and Comparative Example 2 of the present invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0021] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0022] The inventors discovered that crude ceramide extracts obtained from natural raw materials such as plants contain a large amount of neutral lipids, free fatty acids, and cholesterol lipids. These other substances may contain components with odors, such as nonanal and camphor, or some odors may be produced by a combination of multiple substances. Therefore, the purification of plant-derived ceramides is particularly crucial.

[0023] However, the crude ceramide extract obtained from natural raw materials contains different components depending on the selected natural raw materials. Existing purification methods are mainly aimed at removing single types of impurities. Therefore, how to provide a purification method for plant-derived ceramides that can remove more types of impurities, so that more types of natural raw materials can be used to extract ceramide products of better quality, is one of the common goals of this field.

[0024] Currently, ceramides are commonly used in food and cosmetics. Both food and cosmetics have certain requirements regarding the odor of the product, especially in the food industry, where odor can have a crucial impact on the product's edibility. Therefore, in the purification process of plant-derived ceramides, removing the off-odors from the crude extract is one of the key aspects of purification.

[0025] However, the odor components in crude plant-derived ceramide extracts are numerous and complex, and their content varies greatly. Therefore, existing odor removal methods are difficult to completely remove the odor components in crude plant-derived ceramide extracts. They can only reduce the content of the odor components, and the purified ceramides obtained still have an odor, which can easily affect the user experience of food and cosmetics.

[0026] Furthermore, some ceramides may not have a noticeable odor initially after purification, but after a period of storage, the odor may reappear. This could be because some odor components are transformed into other substances during purification, but these other substances are less stable after long-term storage and may also transform into odor components, affecting the application of the ceramides.

[0027] Furthermore, the food and cosmetics industries have even higher requirements for the safety of raw materials, especially the food sector. The food sector requires all raw materials used in food products to be food-grade, which places stricter demands on the production process of ceramides added to food. It necessitates ensuring that all solvents used in the production process are non-toxic, further increasing the difficulty of purifying plant-derived ceramides for food use, particularly in removing odors. Therefore, providing a purification method for plant-derived ceramides applicable to more fields, especially the food sector, is one of the common goals of this field.

[0028] In order to reduce the off-odor components in crude plant-derived ceramide extracts, prevent the purified plant-derived ceramides from having an off-odor or developing an odor after being left for a period of time, and ensure that the purified plant-derived ceramides can be used in food, cosmetics and other fields with higher safety requirements, the inventors proposed the following solution.

[0029] In a first aspect, the present invention provides a method for purifying ceramide, comprising membrane separation of crude ceramide extract, wherein the membrane material used for membrane separation has a molecular weight cutoff of 150 to 1000 Da.

[0030] It should be noted that the "crude ceramide extract" described in this invention refers to a component containing ceramides obtained from natural raw materials. If the selected crude ceramide extract is extracted directly by an organic solvent, such as ethanol, without further separation, pretreatment of the directly extracted crude ceramide extract is included before membrane filtration. If the selected crude ceramide extract can already be enriched in an organic solvent (ethanol), pretreatment is not necessary, and the crude ceramide extract can be directly subjected to membrane filtration.

[0031] Preferably, the pretreatment includes dissolving the crude ceramide extract obtained directly in ethanol and dispersing it in water, and then adding inorganic salts to carry out the reaction; more preferably, the reaction can be carried out by stirring or other existing reaction methods.

[0032] Inorganic salts include any one of sodium chloride, sodium sulfate, and ammonium chloride.

[0033] Crude ceramides can be purified using membrane separation. By controlling the molecular weight cutoff of the membrane material, ceramides can be concentrated on one side of the membrane, while odorous impurities are concentrated on the other side. This results in significant odor removal from the purified ceramide, physical separation of odor components, and no recurrence of odor after a period of time. This method is simple, effectively removes odors, and eliminates the need for organic solvents such as acetone that could compromise the safety of ceramides. The resulting ceramides can be widely used in the food and cosmetic industries.

[0034] In an optional embodiment, the membrane material used for membrane separation has a molecular weight cutoff of 150-1000 Da, more preferably 300-600 Da.

[0035] Preferably, the membrane separation method includes nanofiltration or dialysis, so the corresponding membrane material can be a nanofiltration membrane or a dialysis bag.

[0036] This invention enables the small-batch separation of ceramides by adjusting the membrane separation method and the type of membrane material, and can also be used for large-scale industrial separation of ceramides, making the purification method more versatile.

[0037] When dialysis is used as the membrane separation method, a dialysis bag is selected as the membrane material. Preferably, the dialysate added during the dialysis process is 75-100% ethanol; more preferably, the dialysate added during the dialysis process is 80% ethanol. Using ethanol as the dialysate in the dialysis process can ensure the safety of using the separated ceramide. Preferably, the dialysis process includes dissolving the solid ceramide to be treated in a portion of the dialysate and placing it in a dialysis bag, relying on the concentration difference inside and outside the bag for diffusion, and continuously refreshing the dialysate outside the bag to achieve the purpose of removing small molecule substances.

[0038] By controlling the molecular weight cutoff of the membrane material used in membrane separation, ceramides are retained in the dialysis bag during dialysis, while off-odor components are transferred outside the dialysis bag. After dialysis is completed, the liquid phase in the dialysis bag is collected, concentrated, and dried to obtain purified ceramides. This method can not only increase the ceramide content in the purified product, but also remove off-odors from the purified product and improve the quality of ceramides.

[0039] When nanofiltration is used as the membrane separation method, nanofiltration membranes are selected as the membrane material. Preferably, the nanofiltration solution used in the nanofiltration process is ethanol, with an ethanol concentration of 75% to 100%, more preferably 80%, to ensure the safety of using the separated ceramides.

[0040] The nanofiltration process includes: dissolving the solid ceramide to be treated in a portion of the nanofiltration solution to obtain the ceramide solution to be treated, and then performing nanofiltration on the ceramide solution to be treated. During the nanofiltration process, the remaining nanofiltration solution is continuously replenished to ensure the smooth progress of the nanofiltration process.

[0041] Preferably, the volume ratio of the nanofiltration solution used to dissolve the solid ceramide to be treated to the nanofiltration solution replenished during the nanofiltration process is 1:1 to 10, more preferably 1:3.

[0042] Preferably, the pressure of the nanofiltration process is 0.1 MPa to 3.0 MPa, and the temperature is 15 to 60 °C.

[0043] Preferably, after nanofiltration, the filtrate inside the nanofiltration membrane is concentrated and dried to obtain purified ceramide.

[0044] The present invention can significantly reduce the off-odor components in crude plant-derived ceramide extract by using membrane separation, and the obtained product will not have a back-odor phenomenon after being stored for a period of time. However, in order to further reduce the off-odor components in crude ceramide extract, improve the separation efficiency of membrane separation, and reduce the separation pressure of membrane material in membrane separation, in optional embodiments, the purification method of the present invention further includes acid-base purification and / or column chromatography before membrane separation.

[0045] In some implementations, when the purification method includes membrane separation, acid-base purification, and column chromatography, the order of acid-base purification and column chromatography is not limited. Acid-base purification can be performed first, followed by column chromatography, or column chromatography can be performed first, followed by acid-base purification.

[0046] Preferably, when the purification method includes membrane separation, acid-base purification, and column chromatography, the purification process is performed in the following order: acid-base purification, column chromatography, and membrane separation. By adjusting the purification sequence of the crude ceramide extract to the above manner, not only can more off-odor components be removed, but the processing pressure of both column chromatography and membrane separation steps can also be reduced, thereby improving the separation efficiency of ceramide and impurity components.

[0047] The following sections explain the specific steps involved in acid-base purification and column chromatography:

[0048] Acid-base purification: Before membrane separation, the crude ceramide extract undergoes acid-base purification, which includes acid treatment and alkali treatment. By purifying the crude ceramide extract with acid and alkali, some alkaline impurities (including alkaline odor components) and acidic impurities (including acidic odor components) can be removed, which helps reduce odor components in the crude ceramide extract and improves the membrane separation effect.

[0049] In an optional embodiment, the ceramide raw materials for both acid treatment and alkali treatment are ethanol solutions of ceramide, that is, solutions obtained by dissolving crude ceramide extract in ethanol.

[0050] It should be noted that the order of acid treatment and alkali treatment is not limited. The crude ceramide extract can be acid-treated first to remove alkaline impurities, and then the solid obtained after acid treatment can be alkali-treated to remove acidic impurities. Alternatively, the crude ceramide extract can be alkali-treated first to remove acidic impurities, and then the solid obtained after alkali treatment can be acid-treated to remove alkaline impurities.

[0051] In an optional embodiment, the acid treatment includes adding the crude ceramide extract to an aqueous solution of an organic acid for reaction. The reaction can be carried out by stirring or other existing reaction methods. By reacting the organic acid with alkaline impurities in the crude ceramide extract, alkaline impurities, such as some alkaline odor components, can be removed from the crude ceramide extract.

[0052] Preferably, the organic acid includes, but is not limited to, at least one of malic acid, citric acid and tartaric acid; more preferably, citric acid is used, as it is safer to purify crude ceramide extract and has a better effect on impurity removal.

[0053] Preferably, the organic acid constitutes 1% to 10% of the mass fraction of the crude ceramide extract. For example, it can be any value among 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, or a range between any two values, or other values ​​within that range. Controlling the mass fraction of the organic acid within the above range is beneficial for improving the removal effect of impurities while ensuring the safety of using ceramide.

[0054] Preferably, in order to promote the separation of ceramide from impurities, the aqueous solution of the organic acid also includes an inorganic salt, which is the same as the inorganic salt in the pretreatment, including but not limited to any one of sodium chloride, sodium sulfate or ammonium chloride.

[0055] More preferably, the inorganic salt is sodium chloride, in order to balance the safety of ceramide use and the ability to separate ceramide from impurities.

[0056] Preferably, the mass of sodium chloride is 0.1 to 10 times the mass of the crude ceramide extract.

[0057] Preferably, in order to ensure the separation of ceramides from impurities, the acid treatment time is 10 min to 30 min.

[0058] Preferably, after the acid treatment is completed, the solids are collected to obtain a ceramide extract with alkaline impurities removed.

[0059] In an optional embodiment, the alkaline treatment includes adding the crude ceramide extract to a weakly alkaline solution for reaction, which can be carried out by stirring or other existing reaction methods. By reacting the crude ceramide extract with a weakly alkaline solution, acidic impurities, such as some acidic odor components, can be removed from the crude ceramide extract.

[0060] Preferably, the solute in the weakly alkaline solution includes, but is not limited to, any one of sodium carbonate, sodium hydroxide, sodium bicarbonate, potassium phosphate, and disodium hydrogen phosphate.

[0061] Preferably, the pH of the mixture obtained by dispersing the crude ceramide extract in a weakly alkaline solution is 7-10, in order to remove acidic impurities from the crude ceramide extract.

[0062] Preferably, in order to promote the separation of ceramides and impurities during heating, the weakly alkaline solution also includes inorganic salts.

[0063] Preferably, the inorganic salt includes, but is not limited to, any one of sodium chloride or sodium sulfate.

[0064] Preferably, the mass of the inorganic salt is 0.1 to 10 times the mass of the crude ceramide extract.

[0065] Preferably, in order to ensure the separation of ceramides from impurities, the alkali treatment time is 10 min to 30 min.

[0066] Preferably, after the alkali treatment is completed, the solids are collected to obtain a ceramide extract with acidic impurities removed.

[0067] The odor of the crude ceramide extract was reduced after acid-base purification, which also improved the membrane separation effect.

[0068] Column chromatography purification: This includes separating ceramides and impurities using a chromatography column. The crude ceramide extract obtained in this step is also dissolved in ethanol.

[0069] Preferably, the packing material of the chromatography column is ODS packing material or low-polarity macroporous resin.

[0070] Preferably, the ODS packing is a C18 reverse-phase packing, and the low-polarity macroporous resin includes Mitsubishi SP850 or Mitsubishi SP207 to improve the separation efficiency of ceramides and impurities.

[0071] Preferably, during column chromatography purification, the eluent is an 80% ethanol / water solution and a 100% ethanol solution. During elution, the 80% ethanol / water solution is used first, followed by the 100% ethanol solution.

[0072] The purification method for ceramide provided by this invention does not use solvents such as acetone that are dangerous to the human body throughout the entire process, and the application fields of the product are wider; the purification method is simple and easy to implement, and is suitable for production of various scales; moreover, the purification method has a good purification effect on crude ceramide extract, has a significant ability to remove off-odors, and the ceramide will not have a back-odor phenomenon after being stored for a period of time, resulting in better quality ceramide.

[0073] Secondly, the present invention provides a method for preparing ceramide, comprising extracting plant raw materials with ethanol to obtain crude ceramide extract, and then preparing ceramide according to the purification method of ceramide provided in any of the foregoing embodiments.

[0074] In an optional embodiment, during the preparation of the crude ceramide extract, the concentration of ethanol is 90%–100%, the ethanol extraction time is 15 min–240 min, the ratio of plant material to ethanol is 1:(1–10), and the extraction temperature is 15–75°C.

[0075] Preferably, the plant-based raw materials include, but are not limited to, at least one of rice bran, konjac, wheat, corn, apple, or pineapple.

[0076] Preferably, to facilitate subsequent purification, the preparation of crude ceramide extract further includes filtering, concentrating, dissolving, and re-concentrating the extracted material after ethanol extraction.

[0077] Thirdly, the present invention provides a ceramide, which is prepared by the purification method of ceramide provided in any of the foregoing embodiments or by the preparation method of ceramide provided in any of the foregoing embodiments.

[0078] Fourthly, the present invention provides the use of ceramide as provided in the embodiments in any field of preparing pharmaceuticals, cosmetics or food.

[0079] The following embodiments and comparative examples of the present invention involve the analysis of ceramide content using HPLC: Konjac ceramide standards were prepared into solutions with concentrations of 0.05 mg / ml, 0.1 mg / ml, 0.2 mg / ml, 0.5 mg / ml, and 1.0 mg / ml using a solvent with a chloroform / methanol ratio of 9:1. The test sample was prepared into a solution of approximately 4.0 mg / ml. HPLC-ELSD was used for analysis, with a mobile phase of chloroform:methanol ratio of 90:10, a flow rate of 1.0 ml / min, and detection was performed at 37°C.

[0080] Example 1

[0081] This embodiment provides a method for preparing ceramides, the specific steps of which are as follows:

[0082] S01, Extraction of ceramides

[0083] Take 1 kg of konjac starch and extract it with 2 L of 95% ethanol at 30℃ with mechanical stirring for 2 h. After filtering to remove the residue, concentrate the filtrate to dryness to obtain 11.88 g of viscous brown crude ceramide extract 1-1.

[0084] HPLC-ELSD analysis showed that the ceramide content in crude ceramide extract 1-1 was 7.29%, with a noticeable off-odor.

[0085] Purification of SO2 and ceramides

[0086] S021, Acid-Base Purification

[0087] Crude ceramide extract 1-1 from step S01 was dissolved in 20 mL of anhydrous ethanol. The solution was then dispersed in 200 mL of an aqueous solution of organic acid (containing 600 mg citric acid), and stirred under this dispersion condition. After 20 minutes, 5.94 g of sodium chloride was added, and the mixture was stirred for another 10 minutes. The mixture was then filtered to obtain brown solid I.

[0088] The brown solid I was dissolved in 20 mL of anhydrous ethanol and dispersed in 200 mL of a weakly alkaline solution (pH adjusted to 7–10 using sodium carbonate). The mixture was stirred under these conditions. After 20 minutes, 5.94 g of sodium chloride was added, and the mixture was stirred for another 10 minutes. The mixture was then filtered to obtain ceramide extracts 1–2.

[0089] HPLC-ELSD analysis showed that the ceramide content in the ceramide extracts 1-2 was 11.70%, and the odor was average.

[0090] S022, column chromatography

[0091] Ceramide extracts 1-2 from step S021 were dissolved in 60 mL of anhydrous ethanol. The ethanol solution of ceramide extracts 1-2 was then injected into a C18 packed chromatography column. Elution was first performed using an 80% ethanol / water solution as the eluent, followed by elution using 100% ethanol. The fraction containing ceramides was collected and concentrated under reduced pressure to obtain 1.97 g of ceramide extracts 1-3.

[0092] HPLC-ELSD analysis showed that the ceramide content in the ceramide extracts 1-3 was 26.40%, and there was virtually no off-odor.

[0093] S023, membrane filtration

[0094] Ceramide extracts 1-3 from step S022 were dissolved in 20 mL of 80% ethanol. The ethanol solution of ceramide extracts 1-3 was then placed into a dialysis bag with a molecular weight cutoff of 500 Da and dialyzed in an 80% ethanol / water solution. After dialysis, the liquid in the dialysis bag was collected and concentrated to dryness under reduced pressure to obtain 0.96 g of ceramide extracts 1-4.

[0095] HPLC-ELSD analysis showed that the ceramide content in ceramide extracts 1-4 was 38.70%, and there was no off-odor.

[0096] Example 2

[0097] This embodiment provides a method for preparing ceramides, the specific steps of which are as follows:

[0098] S01, Extraction of ceramides

[0099] Take 1 kg of konjac starch, use 2 L of 95% ethanol, control the temperature at 30℃, and mechanically stir for 2 h. After filtering to remove the residue, concentrate the filtrate to dryness to obtain 11.97 g of viscous brown crude ceramide extract 2-1.

[0100] HPLC-ELSD analysis showed that the ceramide content in crude ceramide extract 2-1 was 7.24%, with a noticeable off-odor.

[0101] Purification of SO2 and ceramides

[0102] S021. Pretreatment: Promotes the separation of ceramide and some impurities in crude ceramide extract 2-1, so that the column chromatography purification process can proceed smoothly.

[0103] Ceramide crude extract 2-1 from step S01 was dissolved in 20 mL of anhydrous ethanol. The solution was then dispersed in 200 mL of water and stirred under this dispersed state. After 20 minutes, 5.99 g of sodium chloride was added, and the mixture was stirred for another 10 minutes. After filtration, 5.28 g of ceramide extract 2-2 was obtained.

[0104] HPLC-ELSD analysis showed that the ceramide content in the ceramide extract 2-2 was 12.23%, and the odor was average.

[0105] S022, column chromatography

[0106] Ceramide extract 2-2 from step S021 was dissolved in 60 mL of anhydrous ethanol. The ethanol solution of ceramide extract 2-2 was then injected into a C18 packed chromatography column. Elution was first performed using an 80% ethanol / water solution as the eluent, followed by elution using 100% ethanol. The fraction containing ceramide was collected and concentrated under reduced pressure to obtain 2.66 g of ceramide extract 2-3.

[0107] HPLC-ELSD analysis showed that the ceramide content in the ceramide extract 2-3 was 23.65%, and it had virtually no odor.

[0108] S023, membrane filtration

[0109] Ceramide extract 2-3 from step S022 was dissolved in 20 mL of anhydrous ethanol. The ethanol solution of ceramide extract 2-3 was then placed into a dialysis bag with a molecular weight cutoff of 500 Da and dialyzed in an 80% ethanol / water solution. After dialysis, the liquid in the dialysis bag was collected and concentrated to dryness under reduced pressure to obtain 0.81 g of ceramide extract 2-4.

[0110] HPLC-ELSD analysis showed that the ceramide content in ceramide extracts 2-4 was 36.32%, and there was no off-odor.

[0111] Example 3

[0112] This embodiment provides a method for preparing ceramides, the specific steps of which are as follows:

[0113] S01, Extraction of ceramides

[0114] Take 1 kg of konjac starch and extract it with 2 L of anhydrous ethanol at 30℃ with mechanical stirring for 4 h. After filtering to remove the residue, concentrate the filtrate to dryness to obtain 11.37 g of viscous brown crude ceramide extract 3-1.

[0115] HPLC-ELSD analysis showed that the ceramide content in crude ceramide extract 3-1 was 7.65%, with a noticeable off-odor.

[0116] Purification of SO2 and ceramides

[0117] S021, Acid-Base Purification

[0118] Crude ceramide extract 3-1 from step S01 was dissolved in 20 mL of anhydrous ethanol. The solution was then dispersed in 200 mL of an aqueous solution of organic acid (containing 570 mg citric acid), and stirred under this dispersion condition. After 20 minutes, 5.69 g of sodium chloride was added, and the mixture was stirred for another 10 minutes. The mixture was then filtered to obtain brown solid I.

[0119] The brown solid I was dissolved in 20 mL of anhydrous ethanol and dispersed in 200 mL of a weakly alkaline solution (pH adjusted to 7–10 using sodium carbonate). The mixture was stirred under these conditions. After 20 minutes, 5.69 g of sodium chloride was added, and the mixture was stirred for another 10 minutes. The mixture was then filtered to obtain ceramide extract 3–2.

[0120] HPLC-ELSD analysis showed that the ceramide content in the ceramide extract 3-2 was 12.55%, and the odor was average.

[0121] S022, membrane filtration

[0122] Ceramide extract 3-2 from step S021 was dissolved in 20 mL of 85% ethanol. The ethanol solution of ceramide extract 3-2 was then placed into a dialysis bag with a molecular weight cutoff of 500 Da and dialyzed in an 80% ethanol / water solution. After dialysis, the liquid in the dialysis bag was collected and concentrated to dryness under reduced pressure to obtain 2.72 g of ceramide extract 3-3.

[0123] HPLC-ELSD analysis showed that the ceramide content in ceramide extract 3-3 was 24.55%, and it had no off-odor.

[0124] Example 4

[0125] This embodiment provides a method for preparing ceramides, the specific steps of which are as follows:

[0126] S01, Extraction of ceramides

[0127] Take 1 kg of konjac starch and extract it with 2 L of 95% ethanol at 30℃ with mechanical stirring for 2 h. After filtering to remove the residue, concentrate the filtrate to dryness to obtain 11.86 g of viscous brown crude ceramide extract 4-1.

[0128] HPLC-ELSD analysis showed that the ceramide content in crude ceramide extract 4-1 was 7.34%, with a noticeable off-odor.

[0129] Purification of SO2 and ceramides

[0130] S021. Pretreatment: Promotes the separation of ceramide and some impurities in the crude ceramide extract 4-1, so that the membrane filtration process can proceed smoothly.

[0131] Ceramide crude extract 4-1 from step S01 was dissolved in 20 mL of anhydrous ethanol. The solution was then dispersed in 200 mL of water and stirred under this dispersed state. After 20 minutes, 5.93 g of sodium chloride was added, and the mixture was stirred for another 10 minutes. After filtration, 5.96 g of ceramide extract 4-2 was obtained.

[0132] HPLC-ELSD analysis showed that the ceramide content in the ceramide extract 4-2 was 12.05%, and it had a noticeable off-odor.

[0133] S022, membrane filtration

[0134] Ceramide extract 4-2 from step S021 was dissolved in 20 mL of 80% ethanol. The ethanol solution of ceramide extract 4-2 was then placed into a dialysis bag with a molecular weight cutoff of 500 Da and dialyzed in an 80% ethanol / water solution. After dialysis, the liquid in the dialysis bag was collected and concentrated to dryness under reduced pressure to obtain 2.70 g of ceramide extract 4-3.

[0135] HPLC-ELSD analysis showed that the ceramide content in ceramide extract 4-3 was 23.48%, and it had no off-odor.

[0136] Example 5

[0137] This embodiment provides a method for preparing ceramides, the specific steps of which are as follows:

[0138] S01, Extraction of ceramides

[0139] Take 40 kg of konjac starch and extract it with 80 L of 95% ethanol at 30 °C with mechanical stirring for 2 h. After filtering to remove the residue, concentrate the filtrate to dryness to obtain 511.34 g of viscous, brown crude ceramide extract 5-1.

[0140] HPLC-ELSD analysis showed that the ceramide content in crude ceramide extract 5-1 was 6.95%, with a noticeable off-odor.

[0141] Purification of SO2 and ceramides

[0142] S021. Pretreatment: Promotes the separation of ceramide and some impurities in the crude ceramide extract 5-1, so that the membrane filtration process can proceed smoothly.

[0143] Ceramide crude extract 5-1 from step S01 was dissolved in 1L of anhydrous ethanol, and the solution was dispersed in 10L of water and stirred under this dispersion condition. After 20 minutes, 255.67g of sodium chloride was added, and the mixture was stirred for another 10 minutes. After filtration, 248.42g of ceramide extract 5-2 was obtained.

[0144] HPLC-ELSD analysis showed that the ceramide content in the ceramide extract 5-2 was 11.28%, and it had a noticeable off-odor.

[0145] S022, membrane filtration

[0146] 100g of ceramide extract 5-2 from step S021 was dissolved in 10L of 80% ethanol, and then the ethanol solution of ceramide extract 5-2 was nanofiltered using a nanofiltration membrane with a molecular weight cutoff of 500 Da.

[0147] The nanofiltration pressure was 2.0 MPa, the temperature was ≤45℃, and the flow rate of the ethanol solution of ceramide extract 5-2 was 54 L / h. When the nanofiltration effluent was approximately 5 L, 30 L of 80% ethanol was gradually added to ensure the smooth progress of the nanofiltration process.

[0148] After nanofiltration, the filtrate inside the nanofiltration membrane was concentrated and dried to obtain 40.5g of ceramide extract 5-3.

[0149] HPLC-ELSD analysis showed that the ceramide content in ceramide extract 5-3 was 20.76%, and it had no off-odor.

[0150] Comparative Example 1

[0151] This comparative example provides a method for preparing ceramides, the specific steps of which are as follows:

[0152] S01, Extraction of ceramides

[0153] Take 2 kg of konjac starch and extract it with 4 L of 95% ethanol at 30°C with mechanical stirring for 2 h. After filtering to remove the residue, concentrate the filtrate to dryness to obtain 24.32 g of crude ceramide extract.

[0154] The crude ceramide extract was dissolved in 40 mL of anhydrous ethanol, and the solution was dispersed in 400 mL of water and stirred under this dispersion condition. After 20 minutes, 12.16 g of sodium chloride was added, and the mixture was stirred for another 10 minutes. After filtration, 12.32 g of ceramide extract 6-1 was obtained.

[0155] HPLC-ELSD analysis showed that the ceramide content in crude ceramide extract 6-1 was 11.54%, with a noticeable off-odor.

[0156] SO2 and ceramide purification: activated carbon treatment

[0157] Dissolve 1.0g of crude ceramide extract 6-1 from step S01 in 20mL of 99% ethanol, then add 0.1g of activated carbon to the solution, stir at room temperature for 2h, filter, collect the filtrate and concentrate to obtain 0.94g of ceramide extract 6-2.

[0158] HPLC-ELSD analysis showed that the ceramide content in the ceramide extract 6-2 was 11.82%, and the odor was average.

[0159] Comparative Example 2

[0160] This comparative example provides a method for purifying ceramides, the specific steps of which are as follows:

[0161] S01. Take 1.0 g of the crude ceramide extract 6-1 from Comparative Example 1, dissolve it in 3 mL of anhydrous ethanol, then disperse it in 15 mL of distilled water, stir for 2 h to obtain a crude ceramide extract dispersion.

[0162] S02. Add 40 mg of chitosan to 2 mL of distilled water and stir to disperse to obtain a chitosan dispersion. Then add 0.04 g of acetic acid to the chitosan dispersion to obtain a solution.

[0163] S03. Add the solution from step S02 to the dispersion of crude ceramide extract from step S01. A yellow solid appears in the solution. Filter and collect the solid. Then dissolve the solid in 3 mL of anhydrous ethanol, filter to remove insoluble matter, collect the filtrate and concentrate to obtain 0.87 g of ceramide extract 7-1.

[0164] HPLC-ELSD analysis showed that the ceramide content in the ceramide extract 7-1 was 12.50%, and the odor was average.

[0165] Comparative Example 3

[0166] This comparative example provides a method for purifying ceramides, the specific steps of which are as follows:

[0167] Dissolve 1.0 g of crude ceramide extract 6-1 from Comparative Example 1 in 2 mL of anhydrous ethanol. Disperse the solution in 20 mL of an aqueous solution of organic acid (containing 50 mg citric acid) and stir under this dispersion condition. After 20 minutes, add 0.5 g of sodium chloride and stir for another 10 minutes. Filter to obtain 0.88 g of ceramide extract 8-1.

[0168] HPLC-ELSD analysis showed that the ceramide content in the ceramide extract 8-1 was 11.78%, and it had virtually no odor.

[0169] Comparative Example 4

[0170] This comparative example provides a method for purifying ceramides, the specific steps of which are as follows:

[0171] Dissolve 1.0 g of crude ceramide extract 6-1 from Comparative Example 1 in 10 mL of 95% ethanol. Then, put the ethanol solution of ceramide extract 6-1 into a dialysis bag with a molecular weight cutoff of 500 Da and dialyze it in 95% ethanol / water solution. After dialysis, collect the liquid in the dialysis bag and concentrate it to dryness under reduced pressure to obtain 0.38 g of ceramide extract 9-1.

[0172] HPLC-ELSD analysis showed that the ceramide content in ceramide extract 9-1 was 20.46%, and it had no off-odor.

[0173] Comparative Example 5

[0174] This comparative example provides a method for purifying ceramides, the specific steps of which are as follows:

[0175] Dissolve 1.0 g of crude ceramide extract 6-1 from Comparative Example 1 in 10 mL of 95% ethanol. Then, put the ethanol solution of ceramide extract 6-1 into a dialysis bag with a molecular weight cutoff of 1000 Da and dialyze it in 95% ethanol / water solution. After dialysis, collect the liquid in the dialysis bag and concentrate it to dryness under reduced pressure to obtain 0.48 g of ceramide extract 10-1.

[0176] HPLC-ELSD analysis showed that the ceramide content in ceramide extract 10-1 was 16.62%, and it had virtually no odor.

[0177] Comparative Example 6

[0178] This comparative example provides a method for purifying ceramides, the specific steps of which are as follows:

[0179] 1.0 g of the crude ceramide extract 6-1 from Comparative Example 1 was dissolved in 2 mL of anhydrous ethanol. The solution was then dispersed in 20 mL of a weakly alkaline solution (pH > 10 adjusted with sodium carbonate) and stirred under this dispersion condition. After 20 minutes, 0.5 g of sodium chloride was added, and the mixture was stirred for another 10 minutes. It was found that no solids could be obtained, and subsequent membrane filtration could not be performed.

[0180] Comparative Example 7

[0181] This comparative example provides a method for purifying ceramides, the specific steps of which are as follows:

[0182] Dissolve 1.0 g of crude ceramide extract 6-1 from Comparative Example 1 in 2 mL of anhydrous ethanol. Disperse the solution in 20 mL of acid solution (adjust pH < 2 with hydrochloric acid) and stir under this dispersion condition. After 20 minutes, add 0.5 g of sodium chloride and stir for another 10 minutes. Filter to obtain 0.68 g of ceramide extract 11-1.

[0183] HPLC-ELSD analysis showed that the ceramide content in the ceramide extract 11-1 was 8.15%, and the odor was average.

[0184] Comparative Example 8

[0185] This comparative example provides a method for purifying ceramides, the specific steps of which are as follows:

[0186] 1.0 g of crude ceramide extract 6-1 from Comparative Example 1 was dissolved in 10 mL of anhydrous ethanol. The ethanol solution of ceramide extract 6-1 was then injected into a macroporous resin (Lanshen Company, model LS-316) packed chromatography column. Elution was first performed with an 80% ethanol / water solution as the eluent, followed by elution with 100% ethanol. The fraction containing ceramide was collected and concentrated under reduced pressure to obtain 0.69 g of ceramide extract 12-1.

[0187] HPLC-ELSD analysis showed that the ceramide content in the ceramide extract 12-1 was 11.72%, and it had virtually no odor.

[0188] Comparative Example 9

[0189] This comparative example provides a method for purifying ceramides, the specific steps of which are as follows:

[0190] Take 100.0g of the crude ceramide extract 5-2 from Example 5, dissolve it in 10L of 80% ethanol, and then use a nanofiltration membrane with a molecular weight cutoff of 1000Da to nanofiltration the ethanol solution of the ceramide extract 5-2 of this comparative example.

[0191] The nanofiltration pressure was 1.5 MPa, the temperature was ≤45℃, and the flow rate of the ethanol solution of ceramide extract 5-2 was 49 L / h. During the nanofiltration process, 30 L of 80% ethanol was added continuously to ensure the smooth progress of the nanofiltration process.

[0192] After nanofiltration, the retentate in the nanofiltration membrane was concentrated and dried to obtain 15.64g of ceramide extract 13-1.

[0193] HPLC-ELSD analysis showed that the ceramide content in ceramide extract 13-1 was 17.13%, and it had virtually no odor.

[0194] Comparative Example 10

[0195] This comparative example provides a method for purifying ceramides, the specific steps of which are as follows:

[0196] Take 50.0g of the crude ceramide extract 5-2 from Example 5, dissolve it in 5L of 100% ethanol, and then use a nanofiltration membrane with a molecular weight cutoff of 150Da to nanofiltration the ethanol solution of the ceramide extract 5-2 of this comparative example.

[0197] The nanofiltration pressure was 2.0 MPa, the temperature was ≤45℃, and the flow rate of the ethanol solution of ceramide extract 5-2 was 52 L / h. During the nanofiltration process, 15 L of 100% ethanol solution was added continuously to ensure the smooth progress of the nanofiltration process.

[0198] After nanofiltration, the retentate in the nanofiltration membrane was concentrated and dried to obtain 31.23g of ceramide extract 14-1.

[0199] HPLC-ELSD analysis showed that the ceramide content in ceramide extract 14-1 was 11.97%, and the odor was average.

[0200] Experimental Example 1: Odor Component Analysis

[0201] Analytical methods: Odor components of the samples were analyzed by headspace solid-phase microextraction and gas chromatography-mass spectrometry (GCMS-MS). The odor components were derived from 150 odor components recorded in the Shimadzu Odor Component Database.

[0202] 1) The crude ceramide extract 1-1 from Example 1 was tested and compared with 150 odorous components in the database. A total of 27 odorous compounds, including aldehydes, ketones, alcohols, esters, and phenols, were detected. After purification using the method in Example 1, some odorous components were significantly removed. Specific test results are shown in Table 1 and... Figures 1-3 As shown.

[0203] Table 1. Odor component content (ng / g) of samples obtained in each step of Example 1.

[0204]

[0205]

[0206]

[0207] From Table 1 and Figures 1-3 It can be seen that in the preparation method of ceramide provided in Example 1 of the present invention, the crude ceramide extract 1-1 with obvious odor obtained by extraction can be gradually purified until the odor components are significantly reduced, and 14 odor components cannot be detected after purification. This shows that the purification method of ceramide provided in the present invention can significantly remove odor components from the crude ceramide extract and has a significant odor removal effect.

[0208] 2) Further, using the same method described above, 27 odor components in ceramide extracts 6-2 of Comparative Example 1 and ceramide extracts 7-1 of Comparative Example 2 were detected and compared with ceramide extracts 1-4 of Example 1, as shown in Table 2. Figure 4 and Figure 5 The results are shown.

[0209] Table 2. Odor component content (ng / g) of samples obtained in Example 1, Comparative Examples 1 and 2.

[0210]

[0211]

[0212]

[0213] From Table 2, Figure 4 and Figure 5 It can be seen that the purification method of the present invention has a significantly greater effect on removing various odor components compared with comparative examples 1 and 2, indicating that the purification method provided by the present invention can obtain ceramide products with less odor compared with existing purification methods.

[0214] Experimental Example 2: The Relationship Between Odor and Time

[0215] The ceramide extracts obtained in Examples 1-5 and Comparative Examples 1-10 (taking Example 1 as an example, here we verify the ceramide extracts 1-4 in Example 1, and the other examples and comparative examples are the same) were used as samples to conduct an odor-time effect experiment, and the results are shown in Table 3.

[0216] Experimental method: Each sample was placed in 15 sealed 10ml glass sample bottles. The sample mass in each glass sample bottle was 0.1g. Ten volunteers smelled the product in the 15 10ml glass sample bottles. The smelling time was 0 days, 1 day, 3 days, 1 week, 2 weeks, 1 month, 2 months and 4 months after the sample preparation.

[0217] Among them, no odor is recorded as Level 1, almost no odor is recorded as Level 2, odor is generally recorded as Level 3, and odor is obvious as Level 4.

[0218] Table 3. Odor characteristics of the purified ceramide extract

[0219]

[0220]

[0221] As shown in Table 3, the ceramides obtained by the purification method provided in the embodiments of the present invention are odorless and do not exhibit any odor return within 4 months of storage. This indicates that the ceramides provided in the embodiments of the present invention have higher purity, more stable quality, and less odor, and can be applied to fields with high requirements for odor, such as food and cosmetics, as well as other commonly used fields of ceramides.

[0222] Comparative Example 1 used activated carbon for purification, while Comparative Example 2 used chitosan. Both methods aim to adsorb odorous impurities from the crude ceramide. However, the activated carbon and chitosan adsorption methods can only adsorb a portion of these impurities, and the odor worsens after 1-2 months of storage. This may be due to interactions between some odorous components, leading to increased odor. This indicates that the ceramides provided by Comparative Examples 1 and 2 have poorer quality stability and stronger odors than the original examples. Their application in food and cosmetics may negatively impact the user experience of the final product.

[0223] Comparative Example 3 used a single acid treatment, which removed some odorous impurities, but the removal was incomplete, and the odor increased over time. Comparative Example 4 changed the concentration of the dialysate outside the dialysis bag during the dialysis process; the obtained ceramide initially had no odor, but the odor intensified after two weeks. Comparative Example 5 increased the molecular weight cutoff of the dialysis bag compared to Comparative Example 4; the obtained ceramide had a stronger odor on day 0 than that of Comparative Example 4, and the odor also intensified over time. Comparative Example 6 adjusted the pH to a strongly alkaline state, causing the ceramide to be unable to separate, making it impossible to verify the odor. Comparative Example 7 adjusted the pH to a strongly acidic state; the separated ceramide had an odor, and the odor became increasingly noticeable over time. Comparative Example 8 used ordinary macroporous resin for column chromatography purification; the separated ceramide had an increased odor compared to the examples, and the odor became increasingly noticeable over time. Comparative Examples 9 and 10 respectively increased or decreased the molecular weight cutoff of the nanofiltration membrane during the nanofiltration process. When the molecular weight cutoff of the nanofiltration membrane increased, the odor of the separated ceramide increased compared to the examples, and the odor gradually became more obvious over time. When the molecular weight cutoff of the nanofiltration membrane decreased, the separated ceramide had an odor, and the odor gradually became more obvious over time.

[0224] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for purifying ceramide, characterized by, This includes membrane separation of crude ceramide extract, wherein the membrane material used for membrane separation has a molecular weight cutoff of 150–1000 Da.

2. The method for purification of ceramide according to claim 1, characterized in that, The membrane material used for membrane separation has a molecular weight cutoff of 300–600 Da; Preferably, the membrane separation method includes dialysis or nanofiltration; Preferably, the dialysate used in the dialysis is 75-100% ethanol; Preferably, the nanofiltration solution used is 75-100% ethanol; Preferably, the nanofiltration pressure is 0.1–3.0 MPa and the temperature is 15–60 °C.

3. The method for purification of ceramide according to claim 1, characterized in that, Before performing the membrane separation, the crude ceramide extract is further subjected to acid-base purification, which includes acid treatment and alkali treatment.

4. The method for purification of ceramides according to claim 3, characterized in that, The acid treatment includes reacting crude ceramide extract with an aqueous solution of organic acid; Preferably, the organic acid includes at least one selected from malic acid, citric acid, and tartaric acid; Preferably, the organic acid accounts for 1% to 10% of the mass fraction of the crude ceramide extract; Preferably, the aqueous solution of the organic acid further includes an inorganic salt, which includes any one of sodium chloride, sodium sulfate, and ammonium chloride; Preferably, the mass of the sodium chloride is 0.1 to 10 times the mass of the crude ceramide extract; Preferably, the acid treatment time is 10 min to 30 min.

5. The method for purification of ceramide according to claim 3, characterized in that, The alkaline treatment includes adding crude ceramide extract to a weakly alkaline solution for reaction; Preferably, the solute in the weakly alkaline solution includes any one of sodium carbonate, sodium hydroxide, sodium bicarbonate, potassium phosphate, and disodium hydrogen phosphate. Preferably, the weakly alkaline solution also includes inorganic salts; Preferably, the inorganic salt includes either sodium chloride or sodium sulfate; Preferably, the mass of the inorganic salt is 0.1 to 10 times the mass of the crude ceramide extract; Preferably, the pH of the mixture obtained by dispersing the crude ceramide extract in the weakly alkaline solution is 7-10; Preferably, the alkali treatment time is 10 min to 30 min.

6. The method for purifying ceramide according to claim 1 or 3, characterized by, This also includes column chromatography purification of crude ceramide extract; Preferably, the parameters of the column chromatography include: the packing material is ODS packing material or low polarity macroporous resin; Preferably, the ODS filler is a C18 reverse phase filler, and the low polarity macroporous resin includes Mitsubishi SP850 or Mitsubishi SP207.

7. A method for producing a ceramide, characterized by, The method includes obtaining a crude ceramide extract by extracting plant raw materials with ethanol, and then preparing ceramide by purifying ceramide according to any one of claims 1 to 6.

8. The method for preparing ceramide according to claim 7, characterized by, In the process of preparing crude ceramide extract, the concentration of ethanol is 90% to 100%, the ethanol extraction time is 15 min to 240 min, the ratio of plant raw material to ethanol is 1:(1 to 10), and the extraction temperature is 15 to 75℃. Preferably, the plant material includes at least one of rice bran, konjac, wheat, corn, apple, or pineapple; Preferably, the preparation of the crude ceramide extract further includes filtering, concentrating, dissolving, and re-concentrating the extracted material after ethanol extraction.

9. A ceramide, characterized in that, It is prepared by the purification method of any one of claims 1 to 6 or by the preparation method of claim 7 or 8.

10. The use of the ceramide as described in claim 9 in any field of preparing pharmaceuticals, cosmetics or food.

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