Method for preparing product rich in ergothioneine from ganoderma lucidum spore residues

Abstract

The invention discloses a method for preparing a product rich in ergothioneine from ganoderma lucidum spore residues, which comprises the following steps: carrying out ultrasonic-assisted water extraction on the ganoderma lucidum spore residues and water for 2-3 times according to a solid-liquid ratio of 1: 8-1: 20kg / L, merging filtrate, concentrating under reduced pressure, extracting with ethanol, standing overnight, taking supernate, recovering ethanol, and drying. The method has the advantages of simple process steps, low production cost, high ergothioneine extraction efficiency and high ergothioneine content, and can more comprehensively utilize the whole lucid ganoderma. According to the method, the ergothioneine product is effectively extracted from the ganoderma lucidum spore residue which is a ganoderma lucidum spore powder production waste, and a favorable data support is provided for development and utilization of total ganoderma lucidum in the aspects of health foods and cosmetics in future.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine extraction, and particularly relates to a method for preparing a product rich in ergothioneine from ganoderma lucidum spore residue. Background Art

[0002] Ergothioneine is a rare compound derived from histidine, which is stable and not prone to self-oxidation. As people continue to explore ergothioneine, its physiological functions such as anti-oxidation, protection of nerve cells, anti-aging, and beauty and skin care are constantly analyzed, and the demand for ergothioneine is also growing rapidly. It has been widely used in food, cosmetics, medicine and other fields. Edible fungi (including wild and cultivated) are the largest organisms that can naturally synthesize ergothioneine.

[0003] At present, there is a serious lack of research on ergothioneine in different types of edible fungi. The ergothioneine content of many common edible fungi is still unknown, and many scientific issues related to it also need more systematic research. my country has a huge edible fungi cultivation industry, but how to extract it at low cost is particularly critical and is also one of the technical bottlenecks in the development of related industries. However, there are few existing research reports.

[0004] The invention uses the defatted particles discarded after the broken wall ganoderma lucidum spore powder is subjected to CO2 supercritical extraction, that is, the ganoderma lucidum spore residue as a raw material to extract ergothioneine, thereby turning waste into treasure. There is no such report at present, and the invention lays a foundation for better development and utilization of whole ganoderma lucidum (including ganoderma lucidum and ganoderma lucidum spore powder) in the future. Summary of the invention

[0005] The object of the present invention is to overcome the above-mentioned shortcomings and provide a method for preparing a product rich in ergothioneine from ganoderma lucidum spore residue, which is simple, easy to operate and suitable for large-scale industrial production.

[0006] The object of the present invention is achieved in the following ways:

[0007] A method for preparing a product rich in ergothioneine from ganoderma lucidum spore residue. The method comprises the following steps: mixing the ganoderma lucidum spore residue with water at a solid-liquid ratio of 1:8 to 1:20 kg / L, extracting the mixture 2 to 3 times with ultrasonic-assisted water, filtering, combining the filtrate, concentrating under reduced pressure, extracting with ethanol, standing the mixture overnight, taking the supernatant, recovering the ethanol and then drying the mixture.

[0008] Furthermore, the Ganoderma lucidum spore residue is defatted granules obtained by granulating, drying and CO2 supercritical extraction of broken Ganoderma lucidum spore powder.

[0009] Furthermore, the CO2 supercritical extraction conditions are extraction temperature 35°C, extraction pressure 30MPa, extraction time 3 hours, CO2 flow rate 0.5m 3 / h.

[0010] Furthermore, the ultrasonic-assisted water extraction is performed twice, with the first water extraction material-liquid ratio being 1:10-20 kg / L by weight to volume, and the second water extraction material-liquid ratio being 1:8-15 kg / L by weight to volume.

[0011] Furthermore, the power of the ultrasonic assisted extraction is 50W-500W, the extraction temperature is 20-100°C, and the extraction time is 30min-60min. Preferably, the power of the ultrasonic assisted extraction is 400W-500W, the extraction temperature is 55-65°C, and the extraction time is 30min-45min.

[0012] Furthermore, the reduced pressure concentration is performed to a specific gravity of 1.05 to 1.4 g / cm 3 .

[0013] Furthermore, the added volume concentration is 95% ethanol extraction.

[0014] Furthermore, during the ethanol extraction, the volume ratio of ethanol to liquid is 1:2 to 1:6, and most preferably 1:4.

[0015] Furthermore, the drying is spray drying or reduced pressure drying.

[0016] Most preferably, the method for preparing a product rich in ergothioneine, its specific preparation process is as follows:

[0017] (1) Ganoderma lucidum spore residue is defatted particles obtained by granulation, drying and CO2 supercritical extraction of broken Ganoderma lucidum spore powder. The conditions for CO2 supercritical extraction deoiling are: extraction temperature 35°C, extraction pressure 30MPa, extraction time 3 hours, CO2 flow rate 0.5m 3 / h.

[0018] (2) Ganoderma lucidum spore residue was subjected to ultrasonic-assisted extraction twice with water at different solid-liquid ratios. The solid-liquid ratio of the first water extraction was 1:10-1:20 kg / L, and the solid-liquid ratio of the second water extraction was 1:8-1:15 kg / L. The power was 50 W-500 W, the temperature was 20-100 °C, and the extraction time was 30 min-60 min.

[0019] (3) Reduce pressure and concentrate to a specific gravity of 1.05 to 1.4 g / cm 3 , add 95% ethanol (volume ratio of 1:2 to 1:6) for extraction, let it stand overnight, take the supernatant and recover the ethanol.

[0020] (4) drying (spray drying or reduced pressure drying), the content of ergothioneine is 15% to 20%, and the yield is 1% to 3%.

[0021] Compared with the prior art, the invention has the following beneficial effects: the invention has simple process steps, low production cost, high ergothioneine extraction efficiency, high content, and can more comprehensively utilize whole ganoderma lucidum (including ganoderma lucidum and ganoderma lucidum spore powder). The invention effectively extracts ergothioneine from ganoderma lucidum spore residue, which is a waste material in the production of ganoderma lucidum spore powder, and provides favorable data support for the development and utilization of whole ganoderma lucidum in health food and cosmetics in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the embodiment of the present invention 1 ergothioneine standard substance chromatogram;

[0023] Figure 2 It is the chromatogram containing thioneine product of the embodiment of the present invention 1;

[0024] Figure 3 It is the chromatogram of thioneine product contained in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described in detail below in conjunction with several preferred embodiments. This embodiment is implemented on the premise of the technical solution of the invention, and provides a detailed implementation method and specific operation process.

[0026] The experimental methods described in the following examples are conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. described in the following examples can be obtained from commercial sources unless otherwise specified. The broken spore powder of Ganoderma lucidum was purchased from Anhui Huangshan Yunle Ganoderma Lucidum Co., Ltd., and the broken spore powder with a breaking rate of ≥95% was obtained by breaking the spore powder.

[0027] Example 1

[0028] (1) Ganoderma lucidum spore residue is defatted particles obtained by granulation, drying and CO2 supercritical extraction of broken Ganoderma lucidum spore powder. The conditions for CO2 supercritical extraction deoiling are: extraction temperature 35°C, extraction pressure 30MPa, extraction time 3 hours, CO2 flow rate 0.5m 3 / h.

[0029] (2) Ganoderma lucidum spore residue and water were subjected to ultrasonic-assisted extraction twice at different solid-liquid ratios. The solid-liquid ratio of the first water extraction was 1:20 kg / L, and the solid-liquid ratio of the second water extraction was 1:10 kg / L. The ultrasonic power was 400 W, the temperature was 60 °C, and the extraction time was 45 min. After each extraction, the extraction was performed and the two filtrates were combined.

[0030] (3) The filtrate was concentrated under reduced pressure to a specific gravity of 1.3 g / cm 3 , add 95% ethanol by volume for extraction (volume ratio of material to liquid is 1:4), let it stand overnight, take the supernatant and recover the ethanol.

[0031] (4) drying under reduced pressure obtains product, and the thioneine content of this product after testing is 19.5%, and the yield is 1%.

[0032] Example 2

[0033] (1) Ganoderma lucidum spore residue is defatted particles obtained by granulation, drying and CO2 supercritical extraction of broken Ganoderma lucidum spore powder. The conditions for CO2 supercritical extraction deoiling are: extraction temperature 35°C, extraction pressure 30MPa, extraction time 3 hours, CO2 flow rate 0.5m 3 / h.

[0034] (2) Ganoderma lucidum spore residue and water were subjected to ultrasonic-assisted extraction twice at different solid-liquid ratios. The solid-liquid ratio of the first water extraction was 1:20 kg / L, and the solid-liquid ratio of the second water extraction was 1:8 kg / L. The power was 450 W, the temperature was 65 °C, and the extraction time was 40 min. After each extraction, the extraction was performed and the two filtrates were combined.

[0035] (3) The filtrate was concentrated under reduced pressure to a specific gravity of 1.05 g / cm 3 , add 95% ethanol by volume for extraction (volume ratio of material to liquid is 1:2), let it stand overnight, take the supernatant and recover the ethanol.

[0036] (4) drying under reduced pressure obtains product, and the thioneine content of this product after testing is 17.8%, and the yield is 1.1%.

[0037] Example 3

[0038] (1) Ganoderma lucidum spore residue is defatted particles obtained by granulation, drying and CO2 supercritical extraction of broken Ganoderma lucidum spore powder. The conditions for CO2 supercritical extraction deoiling are: extraction temperature 35°C, extraction pressure 30MPa, extraction time 3 hours, CO2 flow rate 0.5m 3 / h.

[0039] (2) Ganoderma lucidum spore residue and water were subjected to ultrasonic-assisted extraction twice at different solid-liquid ratios. The solid-liquid ratio of the first water extraction was 1:10 kg / L, and the solid-liquid ratio of the second water extraction was 1:15 kg / L. The power was 500 W, the temperature was 55°C, and the extraction time was 45 min. Filtering was performed after each extraction, and the two filtrates were combined.

[0040] (3) The filtrate is concentrated under reduced pressure to a specific gravity of 1.4 g / cm 3 , add 95% ethanol by volume for extraction (volume ratio of material to liquid is 1:6), let it stand overnight, take the supernatant and recover the ethanol.

[0041] (4) drying under reduced pressure obtains product, and the thioneine content of this product after testing is 17.1%, and the yield is 1.15%.

[0042] Comparative Example 1

[0043] (1) Ganoderma lucidum spore residue is defatted particles obtained by granulation, drying and CO2 supercritical extraction of broken Ganoderma lucidum spore powder. The conditions for CO2 supercritical extraction deoiling are: extraction temperature 35°C, extraction pressure 30MPa, extraction time 3 hours, CO2 flow rate 0.5m 3 / h.

[0044] (2) Ganoderma lucidum spore residue and water were subjected to ultrasonic-assisted extraction twice at different solid-liquid ratios. The solid-liquid ratio of the first water extraction was 1:10 kg / L, and the solid-liquid ratio of the second water extraction was 1:5 kg / L. The power was 400 W, the temperature was 60°C, and the extraction time was 45 min. After each extraction, the extraction was performed and the two filtrates were combined.

[0045] (3) The filtrate was concentrated under reduced pressure to a specific gravity of 1.1 g / cm 3 , add 95% ethanol for extraction (volume ratio of solid to liquid is 1:4), let stand overnight, take the supernatant and recover the ethanol.

[0046] (4) drying under reduced pressure obtains product, and the thioneine content of this product after testing is 8.8%, and the yield is 2%.

[0047] Comparative Example 2

[0048] (1) Ganoderma lucidum spore residue is defatted particles obtained by granulation, drying and CO2 supercritical extraction of broken Ganoderma lucidum spore powder. The conditions for CO2 supercritical extraction deoiling are: extraction temperature 35°C, extraction pressure 30MPa, extraction time 3 hours, CO2 flow rate 0.5m 3 / h.

[0049] (2) Ganoderma lucidum spore residue and water were subjected to ultrasonic-assisted extraction twice at different solid-liquid ratios. The solid-liquid ratio of the first water extraction was 1:20 kg / L, and the solid-liquid ratio of the second water extraction was 1:10 kg / L. The ultrasonic power was 400 W, the temperature was 60 °C, and the extraction time was 45 min. After each extraction, the extraction was performed and the two filtrates were combined.

[0050] (3) The filtrate was concentrated under reduced pressure to a specific gravity of 1.3 g / cm 3 , add 75% ethanol by volume for extraction (volume ratio of material to liquid is 1:4), let it stand overnight, take the supernatant and recover the ethanol.

[0051] (4) drying under reduced pressure obtains product, and the thioneine content of this product after testing is 9.4%, and the yield is 1.5%.

[0052] Comparative Example 3

[0053] (1) Ganoderma lucidum spore residue is defatted particles obtained by granulation, drying and CO2 supercritical extraction of broken Ganoderma lucidum spore powder. The conditions for CO2 supercritical extraction deoiling are: extraction temperature 35°C, extraction pressure 30MPa, extraction time 3 hours, CO2 flow rate 0.5m 3 / h.

[0054] (2) Ganoderma lucidum spore residue was subjected to ultrasonic-assisted extraction with water at different solid-liquid ratios. The solid-liquid ratio of water extraction was 1:20 kg / L, the ultrasonic power was 400 W, the temperature was 60° C., the extraction time was 45 min, and then filtered.

[0055] (3) The filtrate was concentrated under reduced pressure to a specific gravity of 1.3 g / cm 3 , add 95% ethanol by volume for extraction (volume ratio of material to liquid is 1:4), let it stand overnight, take the supernatant and recover the ethanol.

[0056] (4) drying under reduced pressure obtains product, and the thioneine content of this product after testing is 13.8%, and the yield is 1.35%.

[0057] The present invention carries out the content determination of effective ingredient thioneine to the product rich in thioneine prepared by embodiment and comparative example method, and the determination method is as follows:

[0058] 1. Preparation of standard working solution

[0059] Weigh thioneine standard substance 10mg, dissolve with water and be settled to 10mL, obtain thioneine standard stock solution (1000mg / L).Accurately pipette a certain amount of thioneine stock solution, obtain a series of standard working solutions (mass concentration is respectively 1.0mg / L, 2.0mg / L, 5.0mg / L, 10.0mg / L, 20.0mg / L, 50.0mg / L) with 0.1% formic acid methanol solution step by step moisture absorption.

[0060] 2. Sample preparation

[0061] Weigh 1g of sample (accurate to 0.01g) and place it in a 50mL centrifuge tube. Add 40.00mL of 70% methanol aqueous solution accurately. After ultrasonic extraction with an ultrasonic cleaner for 30min, centrifuge at 4000r / min for 5min, take 2mL of the extract and blow it to near dryness, re-dissolve it with 100mL of 0.1% formic acid methanol solution, filter it through a 0.22μm filter membrane, and take 1mL of the filtrate for determination by ultra-performance liquid chromatography.

[0062] 3. Chromatographic reference conditions

[0063] Chromatographic column: Agilent Poroshell 120SB-C18 (2.1 mm × 100 mm, 2.7 μm);

[0064] Mobile phase: 0.1% formic acid acetonitrile solution-0.1% formic acid aqueous solution (88+12, V / V);

[0065] Flow rate: 0.4 mL / min;

[0066] Column temperature: 40°C;

[0067] Wavelength: 262nm;

[0068] Injection volume: 3 μL.

[0069] 4. Drawing of standard curve

[0070] Use thioneine series standard working solution, measure with reference to above-mentioned chromatographic condition, take the mass concentration of standard working solution as abscissa, and peak area is the standard curve of thioneine drawn as ordinate.

[0071] 5. Quantitative determination

[0072] The qualitative analysis was performed according to the retention time, and the relative deviation between the sample and the standard retention time at similar concentrations was no more than 2%, and the external standard method was used for quantitative analysis. The response value of ergothioneine in the test solution should be within the range of the standard curve, and if it exceeds the range of 1.0 mg / L to 50.0 mg / L of the standard working curve, it should be diluted and then sampled and analyzed.

[0073] 6. Calculation of results

[0074] The ergothioneine content in the sample is measured in mass fraction w, and numerical value is expressed in percentage (%), and is calculated as follows:

[0075]

[0076] Where:

[0077] c——The value of ergothioneine concentration in the sample solution obtained from the standard curve, in milligrams per liter (mg / L);

[0078] V——The value of the volume of the extraction solution, in milliliters (mL);

[0079] m——the numerical value of the sample mass, in grams (g);

[0080] f——dilution multiple.

[0081] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A method for preparing a product rich in ergothioneine from Ganoderma lucidum spore residue, characterized in that The method comprises the following steps: extracting ganoderma lucidum spore residue and water 2-3 times with ultrasonic-assisted water according to a material-liquid ratio of 1:8-1:20 kg / L, filtering, combining the filtrate, concentrating under reduced pressure, extracting with ethanol, standing overnight, taking the supernatant, recovering the ethanol and drying.

2. The method according to claim 1, characterized in that The ganoderma lucidum spore residue is defatted particles obtained by granulating, drying and CO2 supercritical extraction of broken ganoderma lucidum spore powder.

3. The method according to claim 2, characterized in that The CO2 supercritical extraction conditions are as follows: extraction temperature 35°C, extraction pressure 30MPa, extraction time 3 hours, CO2 flow rate 0.5m 3 / h.

4. The method according to claim 1, characterized in that The ultrasonic-assisted water extraction is performed twice, the first water extraction material-liquid ratio is 1:10-20 kg / L by weight to volume, and the second water extraction material-liquid ratio is 1:8-15 kg / L by weight to volume.

5. The method according to claim 1, characterized in that The power used in the ultrasonic-assisted water extraction is 50W-500W, the extraction temperature is 20-100°C, and the extraction time is 30min-60min.

6. The method according to claim 5, characterized in that The power used in the ultrasonic-assisted water extraction is 400W-500W, the extraction temperature is 55-65°C, and the extraction time is 30min-45min.

7. The method according to claim 1, characterized in that The reduced pressure concentration to a specific gravity of 1.05-1.4 g / cm 3 .

8. The method according to claim 1, characterized in that The added volume concentration is 95% ethanol extraction.

9. The method according to claim 1, characterized in that The ethanol extraction is performed in a volume ratio of ethanol to liquid of 1:2 to 1:

6.

10. The method according to claim 1, characterized in that The drying is spray drying or reduced pressure drying.

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