Method for extracting ergothioneine from sparassis crispa

Abstract

The invention discloses a method for extracting ergothioneine from sparassis crispa, which comprises the following steps: freeze-drying and crushing fresh sparassis crispa to obtain sparassis crispa powder; performing water extraction on the sparassis crispa powder at 30-50 DEG C, and after water extraction is finished, performing solid-liquid separation and concentration to obtain a sparassis crispa concentrated solution; loading a sample of the sparassis crispa concentrated solution, passing through a macroporous adsorption resin chromatographic column, eluting with water, and recording the loading effluent and the water eluent as macroporous adsorption resin eluent; activating ion exchange resin, washing with water until the pH is nearly neutral, loading the macroporous adsorption resin eluent, passing through an ion exchange resin chromatographic column, eluting with water after loading, eluting with ammonia water, and marking the ammonia water eluent as the ion exchange resin eluent; and concentrating the ion exchange resin eluent, crystallizing with absolute ethyl alcohol at 4 DEG C, and carrying out suction filtration after the crystallization is ended, thereby obtaining the ergothioneine. According to the method, the ergothioneine is extracted from the sparassis crispa and purified, and the method is suitable for industrial production of natural ergothioneine.

Description

Technical Field

[0001] The invention relates to a method for extracting ergothioneine, in particular to a method for extracting ergothioneine from hydrangea. Background Art

[0002] L-Ergothioneine (EGT), chemically known as 2-mercaptohistidine trimethylolate, is the only known natural 2-thioimidazole amino acid. It was discovered in 1909 by Professor Charles Tanret while studying ergot fungi that destroy rye grains. Studies have shown that ergothioneine has antioxidant, anti-inflammatory, neuroprotective, reproductive health, cardiovascular, and eye disease prevention effects. Due to its antioxidant and stability, it is widely used in cosmetics, medical, and food industries.

[0003] Ergothioneine exists in the cells and tissues of most mammals and plants. Its content in most organisms is very low, but it is abundant in some mushroom varieties, red beans, black beans, red meat, etc. Although ergothioneine is widely present in nature, it can only be synthesized by edible fungi, cyanobacteria, mycobacteria and non-yeast fungi, and humans and other mammals can only ingest it through diet.

[0004] At present, the method for producing thioneine has chemical synthesis, biological extraction and biological fermentation.Wherein, biological extraction is mainly purified from edible fungi rich in thioneine content or corresponding fermentation liquid, and studies have shown that the content of thioneine in common edible fungi such as Pleurotus eryngii (P.eryngii), Flammulina velutipes and its scraps is high, and has natural safety, and is a good resource for preparing thioneine.Thioneine is highly soluble in water, and is relatively stable in alkaline solution, so extraction solvent generally selects deionized water, low concentration methanol, acetonitrile or dilute alkaline solution.Reflux extraction is the method for extracting thioneine classics, and also has enzymatic extraction, ultrasonic microwave combined method etc. to extract thioneine in addition.

[0005] However, the ergothioneine content extracted in edible fungi (such as mushroom hyphae fermentation liquid) is still very low, and many impurities cannot be removed, making it difficult to balance product purity and yield, resulting in lower ergothioneine product purity, which is difficult to meet food, medicine and cosmetics production requirements. Summary of the invention

[0006] The invention aims to provide a method for extracting ergothioneine from hydrangea, which solves the problem that it is difficult to balance the purity and yield of the product when extracting ergothioneine from edible fungi in the prior art. The ergothioneine is extracted from hydrangea with a purity of more than 98% and a yield of more than 70%.

[0007] In order to achieve the above object, the present invention provides a method for extracting ergothioneine from hydrangea, which comprises: freeze-drying fresh hydrangea, crushing to 50-80 meshes, and obtaining hydrangea powder; extracting the hydrangea powder at 30-50°C, after the water extraction is completed, separating the solid and the liquid, and concentrating the extract by a low-temperature concentration method not higher than 30°C to obtain a hydrangea concentrated solution; loading the hydrangea concentrated solution, passing it through a macroporous adsorption resin chromatographic column, eluting it with water, combining the loading effluent and the water The eluent is recorded as a macroporous adsorption resin eluent; wherein the macroporous adsorption resin used is a styrene-type macroporous adsorption resin; the ion exchange resin is activated, then eluted with water until the pH value is nearly neutral, the macroporous adsorption resin eluent is loaded, and passed through an ion exchange resin chromatographic column, and then eluted with water after loading, and then eluted with ammonia water, and the ammonia water eluents are combined and recorded as an ion exchange resin eluent; wherein the ion exchange resin used is a polystyrene cation; the ion exchange resin eluent is vacuum concentrated to Brix 55-70, anhydrous ethanol is added, and crystallization is carried out at 4°C. After the crystallization is completed, suction filtration is performed to obtain a solid of ergothioneine.

[0008] The present invention mainly removes slightly smaller polar impurities (such as polyphenol compounds) extracted from hydrangea by macroporous adsorption resin, then removes large polar impurities (such as polysaccharides and polypeptides) by ion exchange resin, and finally further purifies and removes salts brought by ion exchange resin by crystallization.

[0009] Preferably, the macroporous adsorption resin is selected from HP-20, XAD1600N, XR918 or XR3SPA. More preferably, the macroporous adsorption resin is selected from XR918 and XAD1600N.

[0010] Preferably, the ion exchange resin is selected from 001*7, D001H, 112, XR157 or XR84S. More preferably, the ion exchange resin is selected from XR157.

[0011] Preferably, during the water extraction, the mass of water is 8 to 12 times the mass of the hydrangea powder. If the amount of water is too little, the fungus cannot be immersed and the extraction rate will be low; but if the amount of water is too much, the cost will increase.

[0012] Preferably, the low-temperature concentration method adopts membrane reverse osmosis concentration or flash evaporation concentration; the membrane reverse osmosis concentration or flash evaporation concentration extract is concentrated to 1 / 10 of the original volume.

[0013] Preferably, during macroporous adsorption resin column chromatography, the volume ratio of the hydrangea concentrate to the macroporous adsorption resin is 2:1; during water elution, 2 to 3 times the column volume is used for elution.

[0014] Preferably, during ion exchange resin column chromatography, the volume ratio of the macroporous adsorption resin eluent to the ion exchange resin is (3-4):1; during water elution, 2-3 column volumes are used for elution; and 4-5 column volumes are used for elution with ammonia water.

[0015] Preferably, the concentration of the ammonia water is 2%.

[0016] Preferably, during the crystallization, the amount of anhydrous ethanol used is 1 times the amount of the concentrated solution after the ion exchange resin eluate is concentrated; and the crystallization time is 12 to 24 hours.

[0017] Preferably, during the crystallization, the crystallization process is repeated at least once; during the first crystallization, the ion exchange resin eluate is vacuum concentrated to Brix 55-70, anhydrous ethanol is added, and crystallization is carried out at 4°C. After the crystallization is completed, the mother liquor is filtered and re-concentrated to Brix 65-75; during the second crystallization, anhydrous ethanol is added, and crystallization is carried out at 4°C. After the crystallization is completed, the crystals obtained by crystallization are filtered and mixed.

[0018] The method for extracting ergothioneine from hydrangea of ​​the present invention solves the problem that it is difficult to balance product purity and yield in the prior art of extracting ergothioneine from edible fungi, and has the following advantages:

[0019] The present invention finds in research that hydrangea contains abundant natural ergothioneine, so the present invention extracts ergothioneine from hydrangea and purifies ergothioneine, which is suitable for industrial production of natural ergothioneine.

[0020] The ergothioneine extracted by the method of the invention has a purity of more than 98% and a yield of more than 50%. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The ergothioneine extracted from hydrangea of ​​the present invention 1 H NMR spectrum.

[0022] Figure 2 The ergothioneine extracted from hydrangea of ​​the present invention 13 C NMR spectrum. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Experimental Example 1 Detection of ergothioneine in Hydrangea

[0025] The fresh mushrooms of Hydrangea purchased from different channels in the market were studied. 200 g were taken, cut into pieces, added with 600 mL of water, and extracted in a 70° C. water bath for 2 h. The ergothioneine content in the water extract of Hydrangea was detected by HPLC. The results are shown in Table 1 below.

[0026] Table 1 is the test results of thioneine content in the water extract of hydrangea

[0027]

[0028] Experimental Example 2 Effects of Different Drying Methods on Ergothioneine Content in Hydrangea

[0029] Take 500g fresh hydrangea respectively, detect by HPLC, thioneine content is 0.046%, adopt different drying methods to process (referring to Table 2) respectively, detect the change of thioneine amount after drying, and the result is shown in Table 2 below.

[0030] Table 2 Effects of different drying methods on ergothioneine content in Hydrangea

[0031]

[0032] Sparassis latifolia is a rare and precious emerging edible and medicinal fungus, Sparassis family, Sparassis genus. The present invention has been found in research that Sparassis latifolia contains abundant natural ergothioneine, and different drying methods are found to affect the content of ergothioneine, and the loss of ergothioneine by freeze drying is the least, so the present invention adopts Sparassis latifolia to extract ergothioneine, and purifies ergothioneine, which is suitable for industrial production of natural ergothioneine.

[0033] A method for extracting ergothioneine from hydrangea provided by the present invention is as follows:

[0034] A method for extracting ergothioneine from hydrangea, the method comprising:

[0035] (1) Hydrangea pretreatment

[0036] The study found that during the conventional drying process (sun drying or oven drying) of fresh hydrangea, a large amount of ergothioneine will be lost due to the influence of biological enzymes in the hydrangea, up to 60-70%. Therefore, the hydrangea should be pretreated to minimize the loss.

[0037] The invention freeze-dries the hydrangea, so that thioneine can be retained most fully, and the loss of thioneine is within 5%. The freeze-dried hydrangea is crushed to 50-80 meshes, and thioneine has very good water solubility. The hydrangea crushed to 50-80 meshes is easy for water to penetrate, which is beneficial to the subsequent extraction of thioneine.

[0038] (2) Hydrangea Extract Concentration

[0039] The crushed hydrangea is extracted with 8 to 12 times the amount (mass multiple) of water at a temperature of 30 to 50° C. Low-temperature extraction at 30 to 50° C. can minimize impurities, such as the extraction of polysaccharides, proteins, etc., without affecting the extraction of ergothioneine.

[0040] The extract is separated into solid and liquid to remove the hydrangea fungus. The solid-liquid separation can be carried out by plate filter pressing, which can squeeze out the residue as much as possible and reduce the loss.

[0041] The hydrangea extract is concentrated by low temperature concentration methods such as membrane reverse osmosis concentration or flash concentration. During the entire concentration process, the temperature is not higher than 30°C. The purpose is to prevent the generation of new impurities and to prevent the loss of ergothioneine and pigment changes caused by high temperature during the concentration process. The extract is concentrated by reverse osmosis or low temperature flash concentration to 1 / 10 of the original volume for the next step of separation and purification.

[0042] After this step, the concentrated solution extracted contains large polar impurities (such as polysaccharides, oligosaccharides, polypeptides, organic acids) and small polar impurities such as polyphenols. In this step, after testing and calculation, the extraction rate of thioneine is 85~90% (thioneine amount in thioneine amount / Seedlea cocci in the extract).

[0043] (3) Macroporous adsorption resin

[0044] The obtained hydrangea concentrated solution was directly loaded onto the sample, and the volume ratio of the concentrated solution to the macroporous adsorption resin was 2:1 (i.e., 2L of concentrated solution was loaded onto 1L of macroporous adsorption resin by column volume). After loading, 2 to 3 times of column volume of deionized water were used for elution, and the loading effluent and the water eluate were combined and recorded as the macroporous adsorption resin eluate.

[0045] The macroporous adsorption resin used above is a styrene-type macroporous adsorption resin, such as HP-20 (purchased from Mitsubishi Chemical Corporation), XAD1600N (purchased from Rohm and Haas), XR918 (purchased from Shanghai Suner Chemical) or XR3SPA (purchased from Shanghai Suner Chemical) and the like.

[0046] (4) Ion exchange resin

[0047] The ion exchange resin is first activated according to the standard steps (acid-water-base-water-acid-water), and finally eluted with deionized water until the pH is close to neutral. The above macroporous adsorption resin eluate is directly loaded, and the volume ratio of the eluate to the ion exchange resin is (3-4):1 (i.e. 3-4L of concentrated solution on 1L column volume of ion exchange resin). After loading, elute with deionized water for 2-3 column volumes, and then elute with 2% ammonia water for 4-5 column volumes, and combine the ammonia water eluate.

[0048] The ion exchange resin used above is polystyrene cation, such as 001*7, D001H and 112 (all purchased from Suqing), XR157 and XR84S (all purchased from Suner), etc.

[0049] (5) Crystallization

[0050] The ammonia eluate obtained by ion exchange resin is vacuum concentrated to Brix 55-70, 1 times the amount of anhydrous ethanol is added, and the mixture is placed at 4°C for crystallization for 12-24 hours. Filter by suction, the mother liquor is re-concentrated to Brix 65-75, 1 times the amount of anhydrous ethanol is added, and the mixture is placed at 4°C for crystallization for 12-24 hours. Filter by suction. The crystals obtained by crystallization twice are mixed to obtain ergothioneine with a purity of more than 98% and a recovery rate of more than 50%.

[0051] A method for extracting ergothioneine from hydrangea provided by the present invention is described in detail by Example 1 below.

[0052] Example 1

[0053] A method for extracting ergothioneine from hydrangea, the method being specific as follows:

[0054] 100 kilograms (100000g) of fresh Spadina viridis, the content of ergothioneine detected by HPLC is about 50g, after freeze-drying, 11.5 kilograms (i.e. 11500g) of dry Spadina viridis are obtained, which are pulverized, passed through a 60 mesh sieve, and Spadina viridis powder is obtained.

[0055] Add 100 kg of water to the hydrangea powder for low-temperature cold extraction at 40-50°C for 4-5 hours. Stirring is maintained during the process. After the extraction is completed, filter to remove the bacteria. Repeat the low-temperature cold extraction process twice: After the first extraction, filter the bacteria with a plate and frame. Continue to use 100 kg of water for warm and cold extraction. The extraction time is 4-5 hours. Stirring is maintained during the process. After the second extraction, filter the bacteria with a plate and frame. Combine the two extracts.

[0056] The extract is filtered through a ceramic membrane to remove residual fine bacteria, and then concentrated by reverse osmosis or low-temperature flash evaporation to concentrate the extract to about 20L.

[0057] Prepare 10 L of XR918 macroporous adsorption resin and perform pretreatment: rinse with 95% ethanol and then rinse with 30 L of water. The concentrate is directly applied to the macroporous resin and then eluted with 30 L of water at a flow rate of 20 L / h. The applied effluent and the water eluate are combined and recorded as the macroporous adsorption resin eluate.

[0058] Prepare 10 L of XR157 ion exchange resin, pre-treat it according to the process (acid-water-base-water-acid-water), and finally rinse it with water to pH = 6-7. Apply the above macroporous adsorption resin eluate directly to XR157 ion exchange resin. After loading, first elute 30 L with water, then elute 50 L with 2% ammonia water, combine the ammonia eluates, and carry out subsequent vacuum concentration.

[0059] The ammonia eluate obtained by ion exchange resin is vacuum concentrated to Brix 60 (55-70), and 1 times the amount of anhydrous ethanol is added, and the mixture is placed at 4°C for crystallization for 12-24 hours. Filter with suction, re-concentrate the mother liquor to Brix 70 (65-75), add 1 times the amount of anhydrous ethanol, and place the mixture at 4°C for crystallization for 12-24 hours. Filter with suction, mix the crystals crystallized twice, and obtain 30 g of ergothioneine, with a recovery rate of 60% and a purity of more than 98%. Figure 1 and 2 Shown in, for the structural identification of the ergothioneine extracted by the present invention, from 1 H NMR and 13 The C NMR result shows that the obtained compound is ergothioneine, and its chemical structure is shown in Formula I. The optical rotation of the extracted ergothioneine is measured, and the optical rotation is ≥ (+) 122°.

[0060]

[0061] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A method for extracting ergothioneine from hydrangea, characterized in that, The method includes: Fresh hydrangeas are freeze-dried and crushed to 50-80 meshes to obtain hydrangea powder; Extracting the hydrangea powder with water at 30-50° C., separating the solid from the liquid after the water extraction is completed, and concentrating the extract with a low temperature concentration method of no higher than 30° C. to obtain a hydrangea concentrated solution; The hydrangea concentrated solution is loaded onto a sample, passed through a macroporous adsorption resin chromatographic column, eluted with water, and the loaded effluent and the water eluate are combined to be recorded as a macroporous adsorption resin eluate; wherein the macroporous adsorption resin used is a styrene-type macroporous adsorption resin; The ion exchange resin is activated and then eluted with water until the pH is close to neutral, the macroporous adsorption resin eluate is loaded, and the eluate is passed through an ion exchange resin chromatographic column, eluted with water after loading, and then eluted with ammonia water, and the ammonia water eluate is combined and recorded as the ion exchange resin eluate; wherein the ion exchange resin used is polystyrene cation; The ion exchange resin eluate is vacuum concentrated to Brix 55-70, anhydrous ethanol is added, and crystallization is performed at 4° C. After the crystallization is completed, suction filtration is performed to obtain a solid which is ergothioneine.

2. The method according to claim 1, characterized in that The macroporous adsorption resin is selected from HP-20, XAD1600N, XR918 or XR3SPA.

3. The method according to claim 1, characterized in that The ion exchange resin is selected from 001*7, D001H, 112, XR157 or XR84S.

4. The method according to claim 1, characterized in that: During the water extraction, the mass of water is 8 to 12 times the mass of the hydrangea powder.

5. The method according to claim 1, characterized in that The low-temperature concentration method adopts membrane reverse osmosis concentration or flash evaporation concentration; the membrane reverse osmosis concentration or flash evaporation concentration extract is concentrated to 1 / 10 of the original volume.

6. The method according to claim 1, characterized in that During macroporous adsorption resin column chromatography, the volume ratio of the hydrangea concentrated solution to the macroporous adsorption resin is 2:1; during water elution, 2 to 3 times the column volume is used for elution.

7. The method according to claim 1, characterized in that When the ion exchange resin column is used for chromatography, the volume ratio of the macroporous adsorption resin eluent to the ion exchange resin is (3-4):1; when eluting with water, 2-3 column volumes are used for elution; and 4-5 column volumes are used for elution with ammonia water.

8. The method according to claim 1, characterized in that: The concentration of the ammonia water is 2%.

9. The method according to claim 1, characterized in that: During the crystallization, the amount of anhydrous ethanol used is 1 times the amount of the concentrated solution after the ion exchange resin eluate is concentrated; the crystallization time is 12 to 24 hours.

10. The method according to claim 1, characterized in that During the crystallization, the crystallization process is repeated at least once; during the first crystallization, the ion exchange resin eluate is vacuum concentrated to Brix 55-70, anhydrous ethanol is added, and crystallization is performed at 4°C. After the crystallization is completed, the mother liquor is filtered and re-concentrated to Brix 65-75; during the second crystallization, anhydrous ethanol is added, and crystallization is performed at 4°C. After the crystallization is completed, the crystals obtained by the crystallization are filtered and mixed.

Images