WO2015103974A1 - Method for extracting and purifying l-ergothioneine - Google Patents

Abstract

A method for extracting and purifying L-ergothioneine comprises the following steps: (1) soaking and extracting a mushroom mycelium fermentation liquid by using hot water, so as to obtain a mushroom and L-ergothioneine water solution; (2) performing ultrafiltration of the mushroom and L-ergothioneine water solution by using an ultrafiltration membrane with a molecular weight cut-off of 1kDa to 30kDa, and collecting an ultrafiltration liquid; and (3) performing chromatographic purification of the ultrafiltration permeation liquid, the chromatographic purification conditions being: a chromatographic filler being an HILIC filler, a mobile phase forming acetonitrile-water with a volume ratio of 80: 20 to 88: 12, and a quantity of feed of L-ergothioneine being 0.01g to 0.323 per 100g.

Description

Method for extracting and purifying ergot sulfur Technical field

The invention belongs to the technical field of biochemical products, food and medicine, and relates to a method for separating and purifying ergot sulfur, in particular to a method for extracting and purifying ergothione from mycelium.

Background technique

L-Ergothioneine (EGT) is a rare natural chiral amino acid. It is an important physiologically active substance in the body. It has the functions of scavenging free radicals, detoxifying, maintaining DNA biosynthesis, normal cell growth, and cellular immunity. Anti-radiation, whitening and anti-aging functions have been identified as a unique and versatile cell physiological protective agent, especially in antioxidants and energy regulation. Therefore, ergot sulfur is used in food, cosmetics and Biomedicine and other industries have broad application prospects.

Marvin S. Hausman MD, CEO of the Global Nutrition Society, pointed out that since ergot can inhibit the damage of various diseases to human cells and tissues, it can be regarded as an important vitamin in the human body and can be used as a dietary food. Ingredients (Grigat S, Harlfinger S, Pal S, et al. Probing the substrate specificity of the ergothioneine transporter with methimazole, hercynine, and organic cations. Biochem Pharmacol, 2007, 74: 309-316.).

Experiments have shown that humans and animals do not produce any toxic side effects after taking ergothione, which is generally recognized as safe (GRAS). Based on the special biological functions and pharmacological activities of ergot sulfur, OXIS International has introduced ErgoFlex, a dietary supplement made of ergothione, which relieves chronic joint pain, promotes cartilage health, reduces inflammation, improves physical fitness and supplements. The role of energy, etc. (Benson KF, Ager DM, Landes B, et al. Improvement of joint range of motion (ROM) and reduction of chronic pain after consumption of an ergothioneine-containing nutritional supplement. Prev Med, 2012, 54: S83- S89.).

Compared with the chemical synthesis method and the natural biological extraction method, the biosynthesis of ergobacteria in the deep fermentation of sputum bacteria has the advantages of high yield, low cost, easy realization of large-scale production and product safety, and is a technical development direction for synthesizing ergot sulfur. Most of the ergot sulfur biosynthesis of sputum biosynthesis is accumulated in the mycelium. Therefore, the preparation of ergothione by deep fermentation of sputum bacteria must be carried out. A technique for extracting and purifying ergothione from mycelium.

Mitsubishi extracts the dried powder of the genus Umbelliferae with acetone or ethanol solution, and further purifies ergothione by column chromatography, thin layer chromatography, ion exchange chromatography and paper electrophoresis (Mitsubishi, Chem Ind. JP58057365-A) Harada E was extracted with ethyl acetate to extract the fruiting body of the tea tree mushroom, and further separated and extracted by cation exchange resin and silica gel chromatography. After lyophilization, it was purified by dextran gel filtration and reversed phase high pressure liquid chromatography to obtain high purity. Erva Nagoya, Iwade Kingaku Kenkyusho Kk, Daicel Chem. Method for producing ergothioneine [P]. JP: JP2009161498A); Nukina M et al. extract ergothione from artificially cultivated mushroom fruit bodies, first drying and pulverizing the mushrooms It is extracted by hot water, filtered and dissolved in ethanol. It is separated and extracted by cation exchange resin and silica gel thin layer chromatography. High-purity liquid chromatography and freeze-drying are then used to prepare high-purity ergothioneine in Nukina M.Preparation of ergothioneine useful in Pharmaceuticals, by fractionating mushroom extract containing ergothioneine, with solvent,separati Ng the ergothioneine, and purifying the fraction liquid containing the ergothioneine [P]. JP: JP2008110988-A); there is no domestic research on the purification of ergothione, only the report of Zhou Nianbo and other extracts of ergot sulphur from the stalk of Agaricus bisporus, first The mushroom stem was pulverized, extracted with hot NaOH solution, and the solid matter was removed by centrifugation. The supernatant was concentrated under reduced pressure, and then separated by alumina chromatography. The recovery rate of ergot sulfur was 91.2%, and the purity was 25.6%. (Zhou Nianbo, Li Yiqun, Yin Qinhong, Alumina column chromatography for extracting ergot sulfur from the stalk of Agaricus bisporus[J]. Anhui Agricultural Sciences, 2010, 38(27): 14842~14843.).

The above purification methods have many steps and complicated procedures; most of the extraction solvents are organic solvents or chemical reagents, and additional organic solvents or other reagents must be removed for further purification; atmospheric pressure chromatography such as ion exchange chromatography, alumina chromatography, etc. Although the method can realize large-scale preparation, the separation efficiency and product purity are low, and the operation is complicated, especially for the separation of samples with complicated components; high-purity ergot sulfur can be purified by high performance liquid chromatography (HPLC method). Because of, but high cost, small amount of preparation, only get milligram-level products, limited to laboratory research applications.

Medium pressure preparative chromatography is a new separation and purification system for purification and purification of natural products and biological products. It has constant pressure in separation process, medium pressure preparation, fast flow rate, large processing capacity, high column efficiency, high product purity, and is suitable for industrial production. . There is no report in the prior art for the purification of ergothione by medium pressure preparative chromatography.

Summary of the invention

The object of the present invention is to overcome the deficiencies of the prior art and provide a method for rapidly extracting and purifying ergothione from a mycelium, thereby providing ergot sulfur product of desired purity for food, health care, medicine and other industries. The method is simple and easy to implement, and is especially suitable for large-scale production of ergot sulfur.

The object of the present invention is achieved by the following technical solutions.

1. Hot water extraction: After the deep fermentation of the mycelium of the mycelium, the mycelium fermentation liquid is separated by solid-liquid separation, and the mycelium is collected, and the wet mycelium weight: water (g: mL) is added 1:0.5-1. 40: water, made into an aqueous suspension of mycelium. The mycelial aqueous suspension is heated to 70 to 100 ° C, and extracted with stirring at 0 to 600 rpm for 1 to 60 minutes, and ergot sulfur is immersed from the cells of the mycelium into an aqueous solution outside the cells. Separation of solid and liquid, respectively, collecting mycelium and ergot sulfur aqueous solution, the mycelium can be repeatedly leached according to the above method, for multiple leaching, combined with leaching ergot sulfur aqueous solution.

The solid-liquid separation method may be centrifugation, filtration or natural sedimentation.

2. Ultrafiltration: The ergot sulfur aqueous solution obtained in the step 1 is filtered by an ultrafiltration membrane to remove macromolecular impurities such as proteins and polysaccharides. The ultrafiltration permeate is collected and dried or concentrated according to actual needs to obtain a dry crude or concentrate. The ergot can be dried as a solid crude or concentrated to a level above 0.1 g/L.

The ultrafiltration membrane used for ultrafiltration may be a membrane of a ceramic membrane or a polymer material. The molecular weight cut-off of the ultrafiltration membrane is selected from 1 kDa to 30 kDa, preferably from 4 kDa to 6 kDa.

Microfiltration can be performed before ultrafiltration. The microfiltration membrane may be a membrane of a ceramic membrane, a metal membrane or a polymer material. The microfiltration membrane has a pore diameter of 0.01 to 5 μm, preferably 0.1 to 0.5 μm.

The drying method may be, but not limited to, spray drying, vacuum drying or freeze drying, and drying to obtain a crude product of ergothione.

The concentration method can be, but is not limited to, evaporation or vacuum concentration.

3. Chromatographic purification: the crude product or concentrate obtained in step 2 is dissolved in 0.5 to 5 volumes of acetonitrile, sonicated for 1 to 30 minutes, filtered through a microfiltration membrane having a pore size of 0.1 to 0.5 μm, and concentrated to a preparative chromatography system. The chromatographic purification is carried out after the injection request, for example, the concentration of ergothione is from 6 g/L to 48 g/L.

The purified chromatography column can be, but not limited to, a flash chromatography column and a dynamic axial compression column. Etc., the chromatographic packing is a HILIC (Hydrophilic Interaction Chromatography) packing. Column specifications are based on actual throughput.

The mobile phase consists of acetonitrile-water, which is isocratically eluted, and the mobile phase volume ratio is selected from 80:20 to 88:12, preferably from 88:12 to 85:15. The chromatographic detection wavelength is 280 nm, the collection wavelength is 254 nm; the ergot sulfur injection amount is 0.01 g to 0.323 g per 100 g of the filler, preferably 0.113 g to 0.225 g, and the flow rate is determined according to the specifications of the column.

According to the ultraviolet detection spectrum, the corresponding products of each chromatographic peak are collected, and the components containing ergothione are concentrated and dried by high pressure liquid chromatography (HPLC), and the purity of the ergot sulfur of the obtained sample is 96.6~ 99.0%.

The concentration method can be, but is not limited to, evaporation or vacuum concentration.

The drying method may be, but not limited to, spray drying, vacuum drying or freeze drying, and after drying, a high purity ergothione product is obtained.

Detection method of ergothione:

High-pressure liquid chromatography (HPLC), the column was HILIC analytical column (250 mm × 4.6 mm, 5 μm), the mobile phase was acetonitrile-water, and the volume ratio was 85:15. The flow was adjusted with 20 mmol/L acetic acid-ammonium acetate buffer. The pH of the phase was 6.0, the detection wavelength was 254 nm, the column temperature was 40 ° C, the flow rate was 1 mL/min, and the injection amount was 10 μL.

The advantages of the invention are:

(1) The steps are small, the time is short, the operation is simple, the yield is high, the purity of the product is high, and various purity ergothione products can be prepared according to different application requirements.

(2) The leaching process of ergot in the mycelium is only using water as the extraction solvent, instead of the low-boiling organic extraction solution, no inorganic chemicals and petrochemical reagents are added during the leaching process; The lowest cost avoids the dangers of using organic solvents in large quantities during operation; without adding impurities, subsequent processes do not have to consider removing artificially added impurities.

(3) The separation of ergot sulfur and macromolecular impurities was achieved by ultrafiltration technology, and the product with ergosulfur purity of 25.2%~34.7% was obtained, which is suitable for applications with low purity requirements. At the same time, the harm of macromolecular impurities to the chromatographic packing used later is avoided, the ultrafiltration process has no phase change, the operation is simple, the energy consumption is low, the cost is low, no chemical test is added, and no pollution is caused.

(4) Further purification using medium-pressure chromatography, compared with atmospheric pressure column chromatography, the ergot sulfur prepared by the method has high purity, easy control of process and product quality, and is suitable for complex components. Separation and purification of ergothione in the product. Compared with the high-pressure liquid chromatography technology, the invention can prepare high-purity ergot sulfur on a large scale, has high output, small equipment investment and low operating cost, and is suitable for industrial production.

DRAWINGS

Figure 1 is a medium pressure chromatographic separation spectrum in Example 2;

2 is a HPLC detection spectrum of ergothione after chromatographic purification in Example 2;

Figure 3 is a medium pressure chromatographic separation map in Example 3;

4 is a HPLC detection spectrum of ergothione after chromatographic purification in Example 3;

Figure 5 is a medium pressure chromatographic separation map in Example 4;

Figure 6 is a chromatogram of ergothione HPLC after chromatographic purification in Example 4.

detailed description

The following examples of the invention are merely illustrative of specific embodiments of the invention, which are not to be construed as limiting the invention. Rather, the invention is not intended to be limited to the details of the invention, and the modifications, modifications, substitutions, combinations, and simplifications.

Experimental instruments, materials and reagents

Medium pressure preparative chromatography system (Dr Flash S type, Lisui Technology (Suzhou) Co., Ltd.); high pressure liquid chromatography (Agela LC-10F type), Flash column (HILIC, 20 ~ 45μm, 80g) and HILIC analytical column (250mm×4.6mm, 5μm, VH510525BXN0068) are all produced by Tianjin Bona Aijieer Technology Co., Ltd.; hollow fiber ultrafiltration membrane (Tianjin Aisheng Membrane Filtration Technology Co., Ltd.); 0.22μm pore size microporous membrane (Tianjin Bo Nai Aier Technology Co., Ltd.; Rotary Evaporator (Hei-VAPPrecsion ML/G6, Heidolph); Freeze Dryer (FreeZone, Labconco); Water Bath (TW20, Julabo); Digital Heating Magnetic Stirrer (MIX Control 20, WIGGENS); high-speed desktop refrigerated centrifuge (TGL-16M, Xiangyi Centrifuge Instrument Co., Ltd.); ultrasonic machine (SB-5200D, Ningbo Xinzhi Biotechnology Co., Ltd.).

The main reagent: ergothione reference substance (purity ≥ 98%, Biomol International Inc.); acetonitrile, acetic acid, ammonium acetate and other reagents are commercially available chromatographically pure. Chemical reagents KH ₂ PO ₄ , MgSO ₄ ·7H ₂ O, etc., were purchased from Sinopharm Chemical Reagent Co., Ltd. Corn flour was purchased from Meihekou Xingda Rice Industry Co., Ltd., soybean meal was purchased from Beijing Zhongmian Ziguang Biotechnology Co., Ltd., α-amylase was purchased from Beijing Suo Laibao Technology Co., Ltd., and glycerin was purchased from Tianjin Sailboat Chemicals. Reagent Technology Co., Ltd., casein 胨 was purchased from Yanshi City Baijia Industry and Trade Co., Ltd.

Example 1: Preparation of Pleurotus ostreatus CGMCC No.6232 Mycelium Fermentation Broth

Incline medium: PDA medium (Becton, Dickinson and Company).

Liquid seed culture medium: corn flour 30g / L, soybean meal 15g / L, α-amylase 54U / L, KH ₂ PO ₄ 3g / L, MgSO ₄ · 7H ₂ O 1.5g / L, the rest is water, at 121 Sterilize at °C for 20 min, and the volume of liquid in a 500 mL flask is 150 mL.

Liquid fermentation medium: glycerin 50g / L, casein 胨 35g / L, KH ₂ PO ₄ 3g / L, MgSO ₄ · 7H ₂ O 1.5g / L, the rest is water, sterilized at 121 ° C for 20min, 500mL triangle bottle The amount of liquid is 150 mL.

The lawn of the slant culture medium strain CGMCC No. 6232 was picked up and seeded, and cultured for 4 days at 25 ° C shaker at 150 rpm to obtain a seed liquid. The seed liquid was introduced into the fermentation medium at a volume ratio of 5% by inoculum, and cultured at 25 ° C for 15 days at 150 rpm to obtain a mycelial fermentation broth.

Example 2: Extraction and purification of ergothione

The mycelium fermentation broth of Pleurotus ostreatus CGMCC No.6232 was centrifuged to collect 50 g of mycelium, and the mycelium was prepared by adding 1:40 water according to wet hyphae weight: water (g: mL). Aqueous suspension. The mycelial aqueous suspension was placed in a water bath at 100 ° C without immersion for 5 min, and the ergot sulfur was immersed in the aqueous solution. After filtration, the mycelium and the filtrate were separately collected to obtain 1700 mL of an aqueous solution of ergothione.

The above aqueous solution of ergot sulfur was ultrafiltered by a hollow fiber ultrafiltration membrane having a pore diameter of 4 kDa to obtain a permeate of 1000 mL, and the purity of ergothione was determined by a high pressure liquid phase to be 31.6%. The permeate was rotary evaporated at 50 ° C to a concentrate having a ergot concentration of 1.72 g/L or spray-dried to obtain a crude product of ergothione.

0.5 volume of acetonitrile was added to the above 300 mL concentrated solution, sonicated for 20 min, filtered through a 0.22 μm pore size microfiltration membrane, and the filtrate was rotary evaporated to 15 mL at 55 ° C to obtain a rotary evaporated concentrate.

The rotary distillation concentrate was further purified by medium pressure preparative chromatography system. The two flash columns were used in series, and the mobile phase was acetonitrile-water with a volume ratio of 88:12. The detection wavelength and collection wavelength were 280 nm and 254 nm, respectively. The flow rate was 40 mL/min, and the injection amount of 100 g of the filler ergothione was 0.323 g.

The corresponding components of each chromatographic peak were collected. The component of the high pressure liquid phase detection of 27.9 to 28.3 min was ergothione, and the purity was 98.93% of ergothione, and the recovery rate was 37.5%.

Example 3: Extraction and purification of ergothione

After the deep fermentation of the mycelium of Pleurotusostreatus CGMCC No.6232, the mycelium fermentation broth was centrifuged to collect 600 g of mycelium, and the wet hyphae weight: water (g: mL) was added to 1:0.5 water. , made into an aqueous suspension of mycelium. The mycelial aqueous suspension was placed in a 70 ° C water bath, and the mixture was immersed for 60 min at 600 rpm, and the ergot sulfur was immersed in the aqueous solution. After filtration, the mycelium and the filtrate were separately collected, and the mycelium was repeatedly extracted 5 times according to the above method, and the filtrate after the leaching was combined to obtain 1100 mL of an aqueous solution of ergothione.

The above aqueous solution of ergot sulfur was ultrafiltered by a hollow fiber ultrafiltration membrane having a pore diameter of 6 kDa to obtain a permeate of 600 mL, and the purity of ergothione was determined by a high pressure liquid phase to be 25.2%. The permeate was rotary evaporated at 50 ° C to a concentrate having a ergot concentration of 0.82 g/L or spray-dried to obtain a crude product of ergothione.

Five volumes of acetonitrile were added to the above 20 mL of the concentrate, sonicated for 20 min, filtered through a 0.22 μm pore size microfiltration membrane, and the filtrate was rotary evaporated at 40 ° C to 15 mL of a rotary concentrated liquid.

The rotary distillation concentrate was further purified by a medium pressure preparative chromatography system. The two flash columns were used in series, and the mobile phase was acetonitrile-water at a volume ratio of 80:20. The detection wavelength and the collection wavelength were 280 nm, respectively. At 254 nm, the flow rate was 60 mL/min, and the injection amount of 100 g of the filler ergothione was 0.01 g.

The corresponding components of each chromatographic peak were collected. The composition of the high pressure liquid phase detection of 15.7 to 16.2 min was ergothione, and the purity was 96.62% of ergothione, and the recovery rate was 28.9%.

Example 4: Extraction and purification of ergothione

After the deep fermentation of the mycelium of Pleurotusostreatus CGMCC No.6232, the mycelium fermentation broth was filtered to collect 500 g of mycelium, according to the wet hyphae weight: water (g: mL) 1:1 water was added to prepare an aqueous suspension of mycelium. The mycelial aqueous suspension was placed in a 90 ° C water bath, and immersed for 10 min at 200 rpm with stirring, and ergot sulfur was immersed in the aqueous solution. After filtration, the mycelium and the filtrate were separately collected, and the mycelium was repeatedly extracted three times according to the above method, and the aqueous solution after the leaching was combined to obtain 1200 mL of an aqueous solution of ergothione.

The above aqueous solution of ergot sulfur was ultrafiltered by a hollow fiber ultrafiltration membrane having a pore diameter of 4 kDa to obtain 650 mL of permeate, and the purity of ergothione was determined by high pressure liquid phase to be 34.7%. The permeate was rotary evaporated at 50 ° C to a concentrate having a ergot concentration of 1.8 g/L or spray dried to obtain a crude product of ergothione.

Two volumes of acetonitrile were added to the above 100 mL of the concentrate, sonicated for 15 min, filtered through a 0.22 μm pore size microfiltration membrane, and the filtrate was rotary evaporated to 15 mL of a rotary concentrated liquid at 40 °C.

The rotary distillation concentrate was further purified by a medium pressure preparative chromatography system. Two flash columns were used in series, and the isocratic elution method was used. The mobile phase was acetonitrile-water, the volume ratio was 85:15, and the detection wavelength and collection wavelength were 280 nm, respectively. At 254 nm, the flow rate was 50 mL/min, and the injection amount of 100 g of the filler ergothione was 0.113 g.

The corresponding components of each chromatographic peak were collected. The component of the high pressure liquid phase detection of 22.8 to 23.3 min was ergothione, and the purity was 98.99% of ergothione, and the recovery rate was 47.6%.

Industrial applicability

The invention provides a method for rapidly extracting and purifying ergothione from a mycelium with few steps, short time, simple operation, high yield, large-scale preparation of high-purity ergot sulfur, suitable for industrial application.

Although the present invention has been described in detail herein, the present invention is not limited thereto, and those skilled in the art can make modifications in accordance with the principles of the present invention. Therefore, various modifications in accordance with the principles of the present invention should be understood as falling within The scope of protection of the present invention.

Claims (6)

1. A method for extracting and purifying ergothione, comprising the steps of:

   (1) a step of extracting the mycelium fermentation broth by hot water, including

   The mycelial fermentation broth is separated by solid-liquid separation, and the mycelium is collected, and water of 1:0.5 to 1:40 is added according to the weight of wet hyphae: water (g: mL) to prepare an aqueous suspension of mycelium.

   The mycelial aqueous suspension is heated to 70-100 ° C, and stirred for 1 to 60 minutes at 0 to 600 rpm, and

   After solid-liquid separation, the mycelium and ergothione aqueous solution are separately collected;

   (2) a step of ultrafiltration of the ergothione aqueous solution with an ultrafiltration membrane having a molecular weight cut off of 1 kDa to 30 kDa, collecting the ultrafiltration permeate, drying or concentrating to obtain a dried crude or concentrate, and

   (3) a step of chromatographically purifying the ultrafiltration permeate, including

   Add 0.5 to 5 volumes of acetonitrile to the dried crude or concentrate, sonicate for 1 to 30 minutes, filter and concentrate to achieve the injection requirements for the preparative chromatography system, and

   The chromatographic purification is carried out under the following conditions: the chromatographic filler is a HILIC filler, the mobile phase composition is acetonitrile-water in a volume ratio of 80:20 to 88:12, and the ergot sulfur is injected in an amount of 0.01 g to 0.323 g per 100 g of the filler. .
2. The method of claim 1 wherein the bacillus is Pleurotus ostreatus CGMCC No. 6232.
3. The method according to claim 1, wherein after said step (1) is finished, further comprising repeating said step (1) on said collected mycelium, performing multiple leaching, and each time The ergot sulfur solution obtained by the leaching is combined and used in the step (2).
4. The method according to any one of claims 1 to 3, wherein the ultrafiltration membrane used in the step (2) has a molecular weight cut off of 4 kDa to 6 kDa.
5. The method according to any one of claims 1 to 3, wherein the mobile phase in the step (3) is composed of acetonitrile-water having a volume ratio of 88:12 to 85:15.
6. The method according to any one of claims 1 to 3, wherein the ergot sulfur injection amount in the step (3) is 0.113 g to 0.225 g per 100 g of the filler.

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