Preparation method of wheat germ extract rich in spermidine

Abstract

The present invention relates to a preparation method of a wheat germ extract rich in spermidine. The method is characterized in that the method realizes the preparation of a wheat germ extract rich in spermidine from wheat germ by using macroporous adsorption resin combined with silica gel column chromatography technology. The raw material of the present invention is wheat germ for extraction, which is the raw material with the highest spermidine content in plants, providing the best choice for the preparation of a wheat germ extract rich in spermidine. And ethanol is used as the extraction solvent, which is simple, commonly used, easy to obtain and safe. Compared with other supercritical carbon dioxide, trichloroethanol, etc., the extraction cost is greatly reduced. The processing process of this method is simple, the preparation time is short, the extraction rate is high, and the thermal degradation of heat-sensitive components can be effectively avoided. The content of spermidine in the obtained wheat germ extract is 220 times that of wheat germ, which can effectively reduce the cost and increase the content of spermidine in the extract.

Description

Technical Field

[0001] The present invention relates to the technical field of food processing, and particularly to a method for preparing a wheat germ extract rich in spermidine. Background Art

[0002] Wheat germ, also known as malt powder and germ, is golden yellow granular, the root of wheat life, and the part with the highest nutritional value in wheat. Wheat germ is extracted from high-quality wheat by a wheat germ cutter, and is rich in various proteins, vitamins, minerals, amino acids and spermidine.

[0003] Spermidine, also known as spermidine trihydrochloride, H2N(CH2)3NH(CH2)4NH2, is widely present in animal, plant and microbial cells, and is biosynthesized from putrescine (butanediamine) and S-adenosylmethionine. Naturally extracted spermidine has anti-aging, anti-cancer, cardiovascular protection, improvement of Alzheimer's disease, improvement of metabolic diseases, improvement of muscle regeneration ability, improvement of memory ability, reduction of neurodegeneration, improvement of blood pressure, reduction of the incidence and mortality of cardiovascular diseases, and has a wide range of anti-inflammatory and antioxidant effects. Research shows that the highest plant source of its content is wheat germ, which is about 2-10 times that of other plants. However, at present, most of the extraction of spermidine comes from other animals and plants, so extracting and separating spermidine from wheat is a better choice.

[0004] In 2017, the European Commission Regulation (EU) 2017 / 2470 included wheat germ extract rich in spermidine in the list of novel foods, and announced that the content of spermidine in this extract is 0.8-2.4 mg / g and the daily intake of spermidine is not more than 6 mg. In March 2020, the European Union improved the specifications of wheat germ extract rich in spermidine as a novel food, and stipulated its definition, aflatoxin limit and microbial standards in the annex. The main methods and techniques for preparing spermidine in the domestic and foreign markets include solvent extraction method, chemical synthesis method, enzymatic method, microbial fermentation method and genetic engineering technology, which have disadvantages such as high energy consumption, many side reactions, large pollution, cumbersome operation and high cost. Summary of the Invention

[0005] Aiming at the above problems existing in the prior art, the purpose of the present invention is to provide a method for preparing a natto extract rich in spermidine. The purpose of the present invention is to provide a method for preparing a wheat germ extract rich in spermidine. This method uses the method of alcohol extraction and column loading, which is relatively convenient and commonly used, simple and easy to operate, can effectively reduce costs, and increase the content of spermidine in the extract.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] Step 1: Weigh an appropriate amount of wheat germ powder, add ethanol for hot reflux extraction. Collect and combine the extraction solution, filter it, and concentrate the filtrate to an extract state.

[0008] Step 2: Add ethanol to the extract, heat and stir until dissolved and clarified, cool overnight, and perform suction filtration to obtain a filtrate for standby.

[0009] Step 3: Perform column chromatography on the above filtrate, elute with ethanol, and collect the eluate of spermidine-like components under gas-phase monitoring.

[0010] Step 4: Concentrate the eluate to dryness, heat and stir with ethanol until dissolved and clarified, cool overnight, and perform suction filtration to obtain a filtrate for standby.

[0011] Step 5: Perform silica gel column chromatography on the above filtrate, elute with ethyl acetate - n-hexane, and obtain an eluate of high-purity spermidine-like components under liquid-phase monitoring.

[0012] Step 6: Concentrate the above eluate of high-purity spermidine-like components, dry under reduced pressure, and determine the content by gas phase.

[0013] As a further illustration of the present invention, the ethanol added in Step 1 is an ethanol solution with a concentration of 60% - 70% and a volume 8 - 10 times that of the raw material, and the hot reflux extraction is carried out 2 - 3 times, 2 - 3 hours each time.

[0014] As a further illustration of the present invention, the ethanol added in Step 2 is an ethanol solution with a concentration of 50% - 60% and a volume 10 - 15 times that of the raw material, and the heating temperature is 80°C.

[0015] As a further illustration of the present invention, the resin used for column chromatography in Step 3 is D101 macroporous adsorption resin, the ethanol used for elution is an ethanol solution with a concentration of 45% - 55%, and the elution flow rate is 3 - 5 BV / h.

[0016] As a further illustration of the present invention, the ethanol used in Step 4 is an ethanol solution with a concentration of 60% - 70% and a volume 8 - 10 times that of the raw material.

[0017] As a further illustration of the present invention, the feed ratio of ethyl acetate - n-hexane in the eluate in Step 5 is 1:(4 - 6).

[0018] The beneficial effects of the present invention include:

[0019] 1. The raw material of the present invention is extracted from wheat germ, which is the raw material with the highest spermidine content in plants, providing the best choice for preparing wheat germ extract rich in spermidine;

[0020] 2. The present invention uses ethanol as the extraction solvent, which is a simple, commonly used, easily obtainable and safe extraction solvent. Compared with other extraction solvents such as supercritical carbon dioxide and trichloroethanol, the extraction cost is greatly reduced, and there are few related studies on extraction with ethanol at present;

[0021] 3. The present invention realizes for the first time the preparation of wheat germ extract rich in spermidine by using macroporous adsorption resin combined with silica gel column chromatography, effectively improving the content of spermidine. The content of spermidine in the obtained wheat germ extract is 220 times that of wheat germ;

[0022] 4. The method of the present invention has a simple process, is safe and reliable, has low production cost and considerable economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the gas phase detection spectrum of spermidine in wheat germ extract. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention will be further described in detail below in conjunction with the specific embodiments. The present invention can be better understood according to the following embodiments. However, those skilled in the art should easily understand that the specific process conditions and their results described in the embodiments are only used to illustrate the present invention, and should not and will not limit the scope of the present invention covered by the claims.

[0025] Example 1

[0026] Weigh 5 kg of wheat germ raw material, add 40 L of 60% ethanol for heat reflux extraction twice, 2 hours each time. Collect and combine the extraction solutions, filter, concentrate the filtrate to an extract paste, and obtain 847 g of extract paste. Add 0.847 L of 50% ethanol to the extract paste, heat and stir at 80 °C until dissolved and clarified, cool overnight, filter by suction, and collect the filtrate. Perform column chromatography on the filtrate with D101 macroporous adsorption resin and elute with 45% ethanol at an elution rate of 3 BV / h. Collect the eluate of spermidine components under gas phase monitoring. Concentrate the eluate to dryness to obtain 262 g of solid, add 2.096 L of 60% ethanol, heat and stir until dissolved and clarified, cool overnight, and perform suction filtration to obtain the filtrate. Perform column chromatography on the filtrate with 200-mesh silica gel and elute with ethyl acetate - n-hexane (1:4) solution. Obtain the eluate of high-purity spermidine components under gas phase monitoring. Concentrate the eluate and dry it under reduced pressure to obtain 13.2 g of wheat germ extract. The gas phase detection content is 4.39%, and the recovery rate is 47.69%;

[0027] Example 2

[0028] Weigh 5 kg of wheat germ raw material, add 45 L of ethanol with a concentration of 65% for heat reflux extraction 3 times, 2 hours each time. Collect and combine the extraction solutions, filter, concentrate the filtrate to an extract state to obtain 906 g of extract. Add 1.09 L of ethanol with a concentration of 50% to the extract, heat and stir at 80 °C until dissolved and clarified, cool overnight, filter by suction, and collect the filtrate. Subject the filtrate to column chromatography using D101 macroporous adsorption resin and elute with ethanol with a concentration of 50%, with an elution rate of 5 BV / h, and collect the eluate of the spermidine-like components under gas phase monitoring. Concentrate the eluate to dryness to obtain 254 g of solid, add 2.286 L of ethanol with a concentration of 60% for heating and stirring until dissolved and clarified, cool overnight, and perform suction filtration to obtain the filtrate. Subject the filtrate to column chromatography using 200-mesh silica gel and elute with an ethyl acetate-n-hexane (1:6) solution to obtain a high-purity eluate of the spermidine-like components under gas phase monitoring. Concentrate the eluate and dry it under reduced pressure to obtain 11.9 g of wheat germ extract, with a gas phase detection content of 4.72% and a recovery rate of 46.23%;

[0029] Example 3

[0030] Weigh 5 kg of wheat germ raw material, add 50 L of ethanol with a concentration of 70% for heat reflux extraction 3 times, 3 hours each time. Collect and combine the extraction solutions, filter, concentrate the filtrate to an extract state to obtain 930 g of extract. Add 1.4 L of ethanol with a concentration of 60% to the extract, heat and stir at 80 °C until dissolved and clarified, cool overnight, filter by suction, and collect the filtrate. Subject the filtrate to column chromatography using D101 macroporous adsorption resin and elute with ethanol with a concentration of 55%, with an elution rate of 4 BV / h, and collect the eluate of the spermidine-like components under gas phase monitoring. Concentrate the eluate to dryness to obtain 247 g of solid, add 2.470 L of ethanol with a concentration of 60% for heating and stirring until dissolved and clarified, cool overnight, and perform suction filtration to obtain the filtrate. Subject the filtrate to column chromatography using 200-mesh silica gel and elute with an ethyl acetate-n-hexane (1:5) solution to obtain a high-purity eluate of the spermidine-like components under gas phase monitoring. Concentrate the eluate and dry it under reduced pressure to obtain 10.9 g of wheat germ extract, with a gas phase detection content of 5.36% and a recovery rate of 48.09%.

[0031] Without departing from the spirit or essential characteristics of the present invention, the present invention can be implemented in other specific forms. The described embodiments are considered to be illustrative in all respects and not restrictive. Therefore, the scope of the present invention is indicated by the appended claims rather than the preceding description. All changes falling within the equivalent meaning and scope of the claims shall be included within its scope.

Claims

1. A preparation method of wheat germ extract rich in spermidine, characterized in that, The method includes the step of preparing a wheat germ extract rich in spermidine from wheat germ raw materials by using an alcohol extraction and column loading technique; The method includes the following steps: (1) Take wheat germ raw materials, add ethanol for thermal reflux extraction multiple times, collect and combine the extraction solution, filter, and concentrate the filtrate to a paste-like state to obtain a paste; (2) Add ethanol to the paste, heat and stir to dissolve until clear, cool overnight, filter by suction, and collect the filtrate of the paste; (3) Subject the filtrate of the paste to column chromatography using a macroporous adsorption resin, and elute with ethanol. Under gas phase monitoring, collect the eluate of the spermidine component; (4) Concentrate the eluate of the spermidine component to dryness to obtain a solid, add ethanol, heat and stir to dissolve until clear, cool overnight, and perform suction filtration to obtain a solid filtrate; (5) Subject the solid filtrate to column chromatography using silica gel, and elute with an ethyl acetate - n - hexane solution. Under gas phase monitoring, obtain a high - purity eluate of the spermidine component; (6) Concentrate the high - purity eluate of the spermidine component, and dry it under reduced pressure to obtain a wheat germ extract.

2. The method according to claim 1, wherein The wheat germ raw materials in step (1) are 5 kg, and the ethanol concentration in steps (1) and (2) is 50 - 70%; the temperature of heating and stirring in step (2) is 80 °C.

3. The method according to claim 1, wherein The thermal reflux extraction of ethanol in step (1) is carried out 2 - 3 times, 2 - 3 hours each time.

4. The method according to claim 3, characterized in that, The thermal reflux extraction of ethanol in step (1) is carried out 3 times, 3 hours each time.

5. The method according to claim 1, wherein The macroporous adsorption resin in step (3) is D101 macroporous adsorption resin, and the ethanol concentration in steps (3) and (4) is 45 - 70%; the elution speed in step (3) is 4 BV / h.

6. The method according to claim 5, characterized in that The ethanol concentration in step (3) is 45% - 55%, and the ethanol concentration in step (4) is 60% - 70%.

7. The method according to claim 1, wherein The silica gel in step (5) is 200 - mesh silica gel, and the ratio of ethyl acetate to n - hexane in the ethyl acetate - n - hexane solution is 1:4 to 1:

6.

8. The method according to claim 7, wherein The ratio of ethyl acetate to n - hexane in the ethyl acetate - n - hexane solution is 1:

5.

9. The method according to claim 1, wherein Specifically, step (1) is to take 5 kg of wheat germ raw materials, add 50 L of 70% - concentration ethanol for thermal reflux extraction 3 times, 3 hours each time; collect and combine the extraction solution, filter, and concentrate the filtrate to a paste - like state to obtain 930 g of paste; Specifically, step (2) is to add 1.4 L of 60% - concentration ethanol to the paste, heat and stir at 80 °C to dissolve until clear, cool overnight, filter by suction, and collect the filtrate; Specifically, step (3) is to subject the filtrate to column chromatography using D101 macroporous adsorption resin, and elute with 55% - concentration ethanol at an elution speed of 4 BV / h. Under gas phase monitoring, collect the eluate of the spermidine component; Specifically, step (4) is to concentrate the eluate to dryness to obtain 247 g of solid, add 2.470 L of 60% - concentration ethanol, heat and stir to dissolve until clear, cool overnight, and perform suction filtration to obtain the filtrate; The specific operation of step (5) is to subject the filtrate to column chromatography on 200-mesh silica gel and elute it with an ethyl acetate - n-hexane (1:5) solution to obtain an eluate of high-purity spermidine components under gas-phase monitoring; The specific operation of step (6) is to concentrate the eluate and dry it under reduced pressure to obtain 10.9 g of wheat germ extract.

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