阅读说明：本技术 一种分离纯化芝麻素酚三糖苷的方法 (Method for separating and purifying sesaminol triglycoside ) 是由 高锦鸿 芦鑫 黄纪念 孙强 贾聪 王瑞丹 宋国辉 张丽霞 于 2019-09-23 设计创作，主要内容包括：本发明属于油脂深加工技术领域,具体涉及一种分离纯化芝麻素酚三糖苷的方法,具体为：以粉碎过的芝麻或芝麻饼粕为原料,脱脂后,采用高压均质技术提取法,提取液经过超滤膜分离系统进行分离浓缩,浓缩液经聚酰胺柱层析分离纯化,即可获得98%以上的芝麻素酚三糖苷。相较于传统的芝麻素酚三糖苷提取纯化方法,本发明利用高压均质技术及膜分离结合柱层析分离纯化技术,具有提取率高、处理量大、操作简便、易回收,成本低等优点。本发明方法为芝麻素酚三糖苷的工业化制备提供了新的方法,这对于芝麻的提质增效开发开辟了新的途径,对于芝麻素酚三糖苷的研究及应用奠定了坚实的理论基础,具有积极的学术及经济价值。(The invention belongs to the technical field of deep processing of grease, and particularly relates to a method for separating and purifying sesaminol triglycoside, which specifically comprises the following steps: the method comprises the following steps of taking crushed sesame or sesame cake meal as a raw material, degreasing, extracting by adopting a high-pressure homogenization technology, separating and concentrating an extracting solution by using an ultrafiltration membrane separation system, and separating and purifying a concentrated solution by using polyamide column chromatography to obtain the sesaminol triglycoside with the concentration of more than 98%. Compared with the traditional sesaminol triglycoside extraction and purification method, the method disclosed by the invention has the advantages of high extraction rate, large treatment capacity, simplicity and convenience in operation, easiness in recovery, low cost and the like by utilizing a high-pressure homogenization technology and a membrane separation and column chromatography combined separation and purification technology. The method provides a new method for the industrial preparation of sesaminol triglycoside, opens up a new way for the quality-improving and efficiency-increasing development of sesame, lays a solid theoretical foundation for the research and application of sesaminol triglycoside, and has positive academic and economic values.)

1. A method for separating and purifying sesaminol triglycoside is characterized by comprising the following steps:

1) pretreatment of raw materials:

the method comprises the following steps of (1) deoiling crushed sesame or sesame cake meal serving as a raw material by adopting subcritical extraction or Soxhlet extraction, and then removing residual subcritical fluid or organic solvent by reduced pressure distillation to obtain deoiled sesame cake meal for later use;

2) extraction of sesaminol triglycoside:

crushing the deoiled sesame cake meal obtained in the step 1) to 80-120 meshes according to a feed-liquid ratio of 1 g: adding 60-80% ethanol solution into 20-60ml to obtain suspension, homogenizing under 20-60 MPa for 10-40min, centrifuging, collecting supernatant, and concentrating under reduced pressure to obtain concentrated solution;

3) separating and concentrating sesaminol triglycoside:

diluting the concentrated solution obtained in the step 2) by 2-5 times with ultrapure water, performing ultrafiltration separation by adopting an ultrafiltration membrane, and collecting the trapped concentrated solution in which sesaminol trisaccharide glycoside is positioned, namely a crude extract solution for later use;

4) further separation and purification of sesaminol triglycoside:

and (3) further separating and purifying the crude extract solution obtained in the step 3) by adopting a polyamide chromatographic column, eluting by using ultrapure water, detecting by using an HPLC method, collecting the eluent rich in sesaminol triglycoside, merging, concentrating under reduced pressure, and carrying out vacuum freeze drying to obtain the sesaminol triglycoside with the purity of more than 98%.

2. The method for separating and purifying sesaminol triglycoside as claimed in claim 1, wherein the subcritical extraction conditions in step 1) are as follows: extracting at 30-60 deg.C for 3-5 times, each time for 20-40 min, and adding 2-5L liquefied butane or propane as solvent into 1kg semen Sesami or semen Sesami cake; the Soxhlet extraction conditions were: ether or petroleum ether is selected as a solvent, and heating reflux is carried out for 6-8 h.

3. The method for separating and purifying sesaminol triglycoside as claimed in claim 1, wherein the step 2) is followed by centrifugation at 3000-5000 rpm for 10-30 min after the completion of the high pressure homogenization.

4. The method for separating and purifying sesaminol triglycoside as claimed in claim 3, wherein the precipitate obtained by centrifugation in step 2) is separated into two fractions in a ratio of 1 g: adding 60-80% ethanol solution into 20-60ml to obtain suspension, homogenizing under high pressure, repeating the above steps for 1-3 times, mixing the supernatants, and concentrating under reduced pressure to obtain concentrated solution.

5. The method for separating and purifying sesaminol triglycoside as claimed in claim 1, wherein the ultrafiltration membrane used in step 3) is flat membrane or roll-type membrane, the ultrafiltration membrane is UE or UF series ultrafiltration membrane, the molecular weight cutoff is 1000-100000, and the operation pressure is 0.5-3.0 MPa.

Technical Field

The invention belongs to the technical field of deep processing of oil and byproducts and separation and purification of bioactive components, and particularly relates to a method for separating and purifying sesaminol triglycoside.

Background

Sesame is one of the main traditional high-quality oil plants in the world, has a long planting history, the sesame yield of China is at the top of the world, and the sesame is called sesame kingdom. At present, sesame production is the main processing of sesame oil in China, and 75% of sesame is used for oil pressing. According to measurement and calculation, more than 50 million tons of sesame cake meal are generated in China every year. The sesame cake meal is mainly used as feed and fertilizer in China due to the limitation of processing level, and the sesame cake meal resource is not fully and reasonably utilized. How to realize the resource utilization of the sesame cake meal becomes an industrial problem to be solved urgently.

Sesame and cake thereof contain a certain amount of sesaminol glucoside besides abundant protein resources. The sesaminol glucoside is a water-soluble sesaminol, mainly exists in the form of trisaccharide glucoside, and has a molecular formula of C₃₈H₄₈O₂₂The sesame is the water-soluble lignan with the highest content in sesame, the average content is about 0.68 mg/g, and the sesame has certain oxidation resistance and can inhibit DNA oxidative damage, diminish inflammation and the like. For example, sesaminol triglycoside is converted to mammalian lignans, enterodiols and enterolactones by intestinal microbial action, which can prevent hormonal diseases; sesaminol trisaccharide can protect neuronal cells from damage by its antioxidant properties and is effective in curing oxidative-induced neuronal degenerative diseases such as Alzheimer's disease.

In order to obtain sesaminol triglycoside, the traditional preparation method of the sesaminol triglycoside comprises the steps of degreasing normal hexane, carrying out magnetic stirring or ultrasonic-assisted extraction by using an ethanol water solution, concentrating, freeze-drying, separating and purifying by polyamide column chromatography, and then separating and purifying by a HW-40s sephadex column. Although the method has the advantage of high purity of sesaminol trisaccharide, the method has the defects of small treatment amount, long time consumption, low yield, complicated operation, no contribution to industrial scale-up production and the like. Therefore, there is a need for improvement and innovation of the above process.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a method for separating and purifying sesaminol triglycoside with industrial conversion potential, which has the advantages of high production efficiency, large treatment capacity, simple operation, high product purity, small pollution, easy recovery of raw materials and the like.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for separating and purifying sesaminol triglycoside comprises the following steps:

1) pretreatment of raw materials:

the method comprises the following steps of (1) deoiling crushed sesame or sesame cake meal (preferably low-temperature cold-pressed sesame cake meal) serving as a raw material by subcritical extraction or soxhlet extraction, and then removing residual subcritical fluid or organic solvent by reduced pressure distillation to obtain deoiled sesame cake meal for later use;

2) extraction of sesaminol triglycoside:

crushing the deoiled sesame cake meal obtained in the step 1) to 80-120 meshes according to a feed-liquid ratio of 1 g: adding 60-80% ethanol solution into 20-60ml to obtain suspension, placing in a high pressure homogenizer, homogenizing under 20-60 MPa for 10-40min, centrifuging, collecting supernatant, and concentrating under reduced pressure to obtain concentrated solution;

3) separating and concentrating sesaminol triglycoside:

diluting the concentrated solution obtained in the step 2) by 2-5 times with ultrapure water, performing ultrafiltration separation by adopting an ultrafiltration membrane, collecting the trapped concentrated solution in which sesaminol trisaccharide glycoside is positioned, namely a crude extract solution, and storing in a refrigerator at the temperature of-4 ℃ for later use;

4) further separation and purification of sesaminol triglycoside:

and (3) further separating and purifying the crude extract solution obtained in the step 3) by adopting a polyamide chromatographic column, eluting by using ultrapure water, detecting by using an HPLC method, collecting by using a fractional collector, collecting the eluent rich in sesaminol triglycoside, merging, and carrying out reduced pressure rotary concentration and vacuum freeze drying to obtain the sesaminol triglycoside with the purity of more than 98%.

Specifically, the subcritical extraction conditions in the step 1) are as follows: extracting at 30-60 deg.C for 3-5 times, each time for 20-40 min, and adding 2-5L liquefied butane or propane as solvent into 1kg semen Sesami or semen Sesami cake; the Soxhlet extraction conditions were: diethyl ether or petroleum ether is selected as solvent, and heated and refluxed for 6-8 h (generally at 30-70 ℃). The residual oil content in the sesame cake meal subjected to deoiling in the step 1) is not more than 1%.

Further, the reduced pressure distillation conditions in the step 1) are specifically as follows: distilling at 40-70 deg.C under-0.08-0.12 MPa for 20-100 min. In addition, the cold-pressed sesame cake meal is preferably crushed to 20-80 meshes. When subcritical extraction is adopted, the pulverized sesame cake meal needs to be bagged for extraction, and nylon bags, cotton bags, cloth bags and the like can be used as the bags, and the mesh number of the bags is more than 200 meshes; if Soxhlet extraction is adopted, the crushed sesame cake pulp is packaged by using filter paper.

Specifically, after the high-pressure homogenization in the step 2) is finished, the centrifugation is preferably carried out at 3000-.

Specifically, in the step 2), the sediment obtained by centrifugation is mixed according to the feed-liquid ratio of 1 g: adding 60-80% ethanol solution into 20-60ml to obtain suspension, homogenizing under high pressure in a high pressure homogenizer, repeating the above steps for 1-3 times, mixing the supernatants, and concentrating under reduced pressure (to remove ethanol in solution) to obtain concentrated solution.

Further, the ultrafiltration membrane used in the step 3) is a flat membrane or a roll-type membrane, the ultrafiltration membrane adopts a UE or UF series ultrafiltration membrane, the molecular weight cutoff is 1000-100000, and the operating pressure is 0.5-3.0 MPa. Further preferably, after the concentrated solution obtained in step 2) is diluted by ultrapure water by 2-5 times, ultrafiltration separation is carried out by adopting an ultrafiltration membrane with the molecular weight cut-off of 1000-fold and 100,000, firstly, an ultrafiltration membrane with the larger molecular weight cut-off which is permeable by sesaminol triglycoside is selected for separation and concentration, the concentrated solution is concentrated until the cut-off is 5-20% of the mass of the original ultrafiltration solution, the permeate is collected, then, an ultrafiltration membrane with the smaller molecular weight cut-off which is impermeable by sesaminol triglycoside is selected for further separation and concentration, the concentrated cut-off is collected until the cut-off is 5-20% of the mass of the original ultrafiltration solution, and the concentrated cut-off is collected and stored in a refrigerator at.

The invention firstly applies the high-pressure homogenizing extraction and membrane separation technology to the extraction, separation and concentration of sesaminol triglycoside, wherein the homogenizing technology is a refining and emulsifying technology which is quite widely applied at present and is widely applied to dairy products, beverages, cosmetics, food, chemical industry and the like, the high-pressure homogenizing technology is the best homogenizing technology applied at present, the average particle size of materials can reach below 1 mu m after the materials are homogenized under high pressure, and the technology can crush the cell tissues of the materials to the maximum extent under the combined action of a cavity effect, a ram effect and a shearing effect, has better effect, so that the application of the technology to the extraction of the sesaminol triglycoside is beneficial to the improvement of the extraction rate of the sesaminol triglycoside and the improvement of the resource utilization rate.

The membrane separation technology belongs to the high and new technology category, and has the characteristics of no phase state change, no chemical change, good selectivity, simple molecular-level filtration and filtration process, easy control and amplification, separation, concentration, purification, refining and the like at normal temperature, so that the membrane separation technology is widely applied to a plurality of fields of food, medicine, biology, chemistry and the like at present, and generates great economic benefit and social benefit. Ultrafiltration is an important branch of membrane separation, the pore diameter of the membrane is 1-20nm, the applied pressure difference is 0.1-1.0MPa, and the ultrafiltration is commonly used for removing macromolecules in solution, separating large molecules from small molecules and grading large molecules.

The invention applies high-pressure homogeneous extraction technology and ultrafiltration separation combined column chromatography technology to replace the traditional magnetic stirring or ultrasonic auxiliary extraction technology and single column chromatography purification, reduces the production cost while improving the yield, the treatment capacity and the production efficiency of the sample, is easy to realize automation, and provides theoretical guidance and realization possibility for industrial preparation of sesaminol triglucoside. Compared with the traditional preparation of sesaminol trisaccharide glycoside by twice column chromatography, the method has the advantages and beneficial effects as follows:

1) the high-pressure homogenizing extraction technology adopted in the invention controls the high-pressure homogenizing pressure and the high-pressure homogenizing extraction time, so that the sesaminol triglucoside in sesame (sesame cake meal) is extracted more completely while the extraction time is saved, and compared with ultrasonic-assisted extraction, the extraction yield of the sesaminol triglucoside in sesame (sesame cake meal) raw materials with the same quality is improved by 8.23%;

2) according to the invention, the sesaminol triglycoside is separated by membrane separation, so that the treatment capacity is large, the efficiency and the energy are high, the cleaning and the recovery are easy, the secondary utilization is realized, and the cost is low; the trapped fluid can be diluted and ultrafiltered for multiple times, the loss of a target object in the raw material is reduced, and compared with the separation and purification of crude sesaminol triglucoside extract directly weighed without membrane separation by polyamide column chromatography, the yield of sesaminol triglucoside is improved by 23.65 percent and the purity can reach more than 98 percent by separation and purification by isometric polyamide column chromatography;

3) the sesaminol triglycoside prepared by the invention has high purity and good quality, and can be applied to subsequent research, food, medicine and other industries;

compared with the traditional sesaminol triglycoside extraction and purification method, the method disclosed by the invention has the advantages of high extraction rate, large treatment capacity, simplicity and convenience in operation, easiness in recovery, low cost and the like by using a high-pressure homogenization technology and a membrane separation and column chromatography combined separation and purification technology. The method provides a new method for the industrial preparation of sesaminol triglycoside, opens up a new way for the quality-improving and efficiency-increasing development of sesame, lays a solid theoretical foundation for the research and application of sesaminol triglycoside, and has positive academic and economic values.

Drawings

FIG. 1 is a detection map of a high performance liquid chromatograph of a supernatant obtained by extracting sesaminol trisaccharide solution from sesame meal through high-pressure homogenization in step 2) in example 1 and centrifuging; compared with the detection spectrum of a high performance liquid chromatograph of the ultrasonic-assisted extraction solution in the comparative example 1, the extraction yield of sesaminol triglycoside in the sesame cake raw material with the same quality is improved by 8.23%;

FIG. 2 is a high performance liquid chromatography detection chart of supernatant after centrifugation of a solution obtained by ultrasonic-assisted extraction of sesaminol trisaccharide glycoside from sesame meal in comparative example 1;

FIG. 3 is a high performance liquid detection spectrum obtained from the UE003 trapped fluid in step 3) of example 1, and comparing with FIG. 1, it can be seen that after the solution is separated and purified by an ultrafiltration membrane, the concentration of sesaminol triglycoside is increased from 125 μ g/mL to 1083 μ g/mL, and part of impurities in the solution are removed or reduced, the liquid phase purity of sesaminol triglycoside is increased from 39.67% to 58.53%, and the separation and concentration of sesaminol triglycoside can be realized;

FIG. 4 shows that the crude sesaminol triglycoside extract obtained in step 4) of example 1 after membrane separation in step 3 is used as a raw material, and after separation and purification by a polyamide chromatographic column, a high performance liquid detection spectrum of the part with the highest sesaminol triglycoside content and purity is collected by a fraction collector, compared with the sesaminol triglycoside solution obtained in step 3) of comparative example 2 without membrane separation and directly subjected to separation and purification by a polyamide chromatographic column in step 3), the content of sesaminol triglycoside is increased by 23.65%, and the liquid purity of sesaminol triglycoside is increased from 95.25% to 98.13%;

FIG. 5 is a super-resolution mass spectrometer chromatogram of sesaminol triglycoside obtained in step 4) of example 1 after detection by a super-resolution mass spectrometer;

FIG. 6 is a High Performance Liquid Chromatography (HPLC) chromatogram showing that the sesaminol triglycoside concentrate obtained in step 2) of comparative example 2 is directly subjected to step 3) of polyamide column separation and purification without membrane separation, and the fraction having the highest sesaminol triglycoside content and purity is collected by a fraction collector, wherein the HPLC purity is 95.25%.

Detailed Description

The technical solution of the present invention is further described in detail with reference to the following examples, but the scope of the present invention is not limited thereto.

In the present invention, ethanol refers to a volume concentration unless otherwise specified.