Extraction method of ganoderan

Abstract

The invention provides an extraction method of ganoderan, and particularly belongs to the technical field of plant natural product extraction. The invention provides an extraction method of ganoderma lucidum polysaccharide, which comprises the following steps: mixing ganoderma lucidum powder with an ethanol water solution, refluxing, degreasing, carrying out suction filtration to obtain filter residues, and carrying out vacuum drying to obtain degreased ganoderma lucidum powder; and mixing the degreased ganoderma lucidum powder, cellulase and water, performing enzymolysis, performing hot water extraction, performing filtration to obtain filtrate, performing concentration, and performing freeze drying to obtain the ganoderma lucidum polysaccharide. The extraction method disclosed by the invention is efficient and high in polysaccharide extraction rate.

Description

Technical Field

[0001] The invention belongs to the technical field of plant natural product extraction, and particularly relates to a method for extracting ganoderma lucidum polysaccharide. Background Art

[0002] Ganoderma lucidum is the dried fruiting body of the Polyporaceae fungus Ganoderma lucidum or Ganoderma rubrum, which is known as the "immortal grass". Ancient and modern pharmacological and clinical studies have proved that Ganoderma lucidum has the effect of preventing and curing diseases and prolonging life. Ganoderma lucidum polysaccharide is the main active ingredient of Ganoderma lucidum, which has a wide range of pharmacological activities such as immunomodulation, anti-tumor, anti-oxidation, anti-radiation, anti-aging, and blood circulation and blood stasis. Ganoderma lucidum polysaccharide is also a hot spot in Ganoderma lucidum research.

[0003] The existing extraction processes of Ganoderma lucidum polysaccharides mainly include the following: ① hot water extraction; ② ultrasonic extraction; ③ microwave extraction; ④ enzyme extraction, etc. Ganoderma lucidum polysaccharides exist in the cell wall of the fruiting body, and the fruiting body has a compact structure, so the mass transfer resistance is relatively large during the extraction process, and the relative molecular weight of Ganoderma lucidum polysaccharides is relatively large, so the extraction efficiency of the traditional hot water extraction method alone is low. The enzymatic method is a method that destroys the cell structure by enzymatic hydrolysis to enhance the mass transfer of effective ingredients. It can maximize the extraction of effective ingredients of traditional Chinese medicine and is receiving more and more attention. However, the extraction efficiency of Ganoderma lucidum polysaccharides still needs to be improved. Summary of the invention

[0004] The purpose of the present invention is to provide a method for extracting ganoderma lucidum polysaccharide. The extraction method of the present invention is efficient and has a high polysaccharide extraction rate.

[0005] The present invention provides a method for extracting ganoderma lucidum polysaccharide, comprising the following steps:

[0006] The ganoderma lucidum powder and the ethanol aqueous solution are mixed and refluxed for degreasing, the residue is filtered out by suction, and vacuum dried to obtain the defatted ganoderma lucidum powder;

[0007] The defatted ganoderma lucidum powder, cellulase and water are mixed, enzymolysis is performed, hot water extraction is performed, the filtrate is filtered, concentrated and freeze-dried to obtain ganoderma lucidum polysaccharide.

[0008] Preferably, the preparation method of the ganoderma lucidum powder comprises: washing the ganoderma lucidum, drying at 55-65° C., and crushing to obtain the ganoderma lucidum powder.

[0009] Preferably, the mass ratio of the ganoderma lucidum powder to the ethanol aqueous solution is 1:(20-30); the number of degreasing is 2-3 times; the degreasing time is 1-6 hours; the degreasing temperature each time is 40-90°C; and the volume percentage of ethanol in the ethanol aqueous solution is 50-90%.

[0010] Preferably, the vacuum drying temperature is 55-65°C.

[0011] Preferably, the mass ratio of the defatted Ganoderma lucidum powder to water is 1:(20-40).

[0012] Preferably, the addition amount of the cellulase is 1000-2500 U / g defatted Ganoderma lucidum powder; the temperature of the enzymolysis is 40-55° C.; and the time of the enzymolysis is 30-120 min.

[0013] Preferably, the cellulase comprises industrial cellulase.

[0014] Preferably, the temperature of the hot water extraction is 85° C.; and the time of the hot water extraction is 2 hours.

[0015] Preferably, the filtration comprises sequentially performing gauze filtration and suction filtration.

[0016] Preferably, the concentration further includes centrifugation; the centrifugation condition is 12000r / min, 5min, and the number of centrifugation is 2 to 3 times.

[0017] The present invention provides a method for extracting ganoderma lucidum polysaccharide. The extraction method of the present invention is efficient, green and environmentally friendly. The present invention firstly performs ethanol degreasing treatment on the crushed ganoderma lucidum powder to remove lipid substances in the raw materials. The degreasing process uses ethanol as a solvent, and the extraction process uses water as a solvent. The whole process is free of toxic and harmful reagents, and the green environment is good; then, the ganoderma lucidum polysaccharide is extracted by using cellulase combined with hot water (cellulase is used to first destroy the cell wall structure of ganoderma lucidum, and then hot water is used for extraction to improve the dissolution efficiency of ganoderma lucidum polysaccharide), the extraction efficiency is high, and the structure is complete; the cellulase used is an industrial enzyme, which has low activity and low price (the price is about 150 yuan / kg), and the cost is low, but the yield of the obtained polysaccharide is high; the process is simple and easy to industrialize. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0019] Figure 1 This is a result diagram showing the effect of the amount of cellulase provided by the present invention on the yield of Ganoderma lucidum polysaccharide;

[0020] Figure 2 This is a result diagram showing the effect of the material-liquid ratio on the yield of Ganoderma lucidum polysaccharide provided by the present invention;

[0021] Figure 3 This is a result diagram showing the effect of the enzymatic hydrolysis temperature on the yield of Ganoderma lucidum polysaccharide provided by the present invention;

[0022] Figure 4 This is a graph showing the effect of the enzymatic hydrolysis time on the yield of Ganoderma lucidum polysaccharides provided by the present invention. DETAILED DESCRIPTION

[0023] The present invention provides a method for extracting ganoderma lucidum polysaccharide, comprising the following steps:

[0024] The ganoderma lucidum powder and the ethanol aqueous solution are mixed and refluxed for degreasing, the residue is filtered out by suction, and vacuum dried to obtain the defatted ganoderma lucidum powder;

[0025] The defatted ganoderma lucidum powder, cellulase and water are mixed, enzymolysis is performed, hot water extraction is performed, the filtrate is filtered, concentrated and freeze-dried to obtain ganoderma lucidum polysaccharide.

[0026] In a specific embodiment, the preparation method of the ganoderma lucidum powder comprises: washing the ganoderma lucidum, drying at 55-65°C, and crushing to obtain the ganoderma lucidum powder. In a specific embodiment, the present invention can use deionized water for washing. In a specific embodiment, the drying can be vacuum drying; the temperature of the vacuum drying can be 55-65°C. In a specific embodiment, the vacuum drying is dried to constant weight. The vacuum drying of the present invention can be carried out using a vacuum oven. In a specific embodiment, the crushing can pass through a 0.5 mm sieve. The present invention does not specifically limit the method of the crushing, and the crushing can be carried out using a crusher, such as a FT102 micro soil crusher.

[0027] After obtaining the ganoderma lucidum powder, the present invention mixes the ganoderma lucidum powder with an ethanol aqueous solution and refluxes to perform defatting, extracts the filter residue by suction filtration, and performs vacuum drying to obtain defatted ganoderma lucidum powder. In a specific embodiment, the mass ratio of the ganoderma lucidum powder to the ethanol aqueous solution is 1:(20-30), specifically 1:25; the number of defatting is 2-3 times, specifically 2 times; the time of each defatting is 1-6 hours, specifically 3 hours; the temperature of the defatting is 40-90°C, specifically 80°C; the volume percentage of ethanol in the ethanol aqueous solution is 50-90%, specifically 90%. In a specific embodiment, the temperature of the vacuum drying is 55-65°C, specifically 60°C. In a specific embodiment, the vacuum drying is performed to constant weight.

[0028] After obtaining defatted ganoderma powder, the present invention mixes defatted ganoderma powder, cellulase and water, performs enzymolysis, performs hot water extraction, filters the filtrate, concentrates, and freeze-dries to obtain ganoderma polysaccharide. In a specific embodiment, the mass volume ratio of the defatted ganoderma powder and water is 1: (20-40) g / mL, specifically 1: 20 g / mL, 1: 25 g / mL or 1: 30 g / mL. In a specific embodiment, the amount of cellulase added is 1000-2500 U / g defatted ganoderma powder, specifically 1000 U / g, 1500 U / g, 2000 U / g or 2500 U / g; the temperature of the enzymolysis is 40-55°C, specifically 40°C, 45°C, 50°C or 55°C; the time of the enzymolysis is 30-120 min, specifically 30 min, 60 min, 90 min or 120 min. In a specific embodiment, the cellulase includes industrial cellulase. After the enzymatic hydrolysis and cell wall breaking of the present invention, sufficient extraction is carried out in hot water. In a specific embodiment, the temperature of the hot water extraction is 85°C; the time of the hot water extraction is 2h. In a specific embodiment, the filtration includes sequentially performing gauze filtration and suction filtration. In a specific embodiment, the gauze can be a 200-mesh gauze. After gauze filtration, the filtrate is collected. In a specific embodiment, the suction filtration can be performed using a Buchner funnel to obtain the filtrate. In a specific embodiment, the concentration can be rotary evaporation concentration. In a specific embodiment, the rotary evaporation temperature can be 60-70°C, and the time is based on the concentration to 1 / 5 of the original volume. In a specific embodiment, the concentration can be concentrated to 1 / 5 of the original volume. In a specific embodiment, the concentration further includes centrifugation; the centrifugation conditions are 12000r / min, 5min, and the number of centrifugation is 2-3 times, specifically 2 times. The centrifugation of the present invention is used to remove impurities. Freeze drying is performed after centrifugation. The present invention uses freeze drying, low-temperature rotary evaporation and other methods to retain the pharmacological activity of Ganoderma lucidum polysaccharide to a great extent.

[0029] The invention cleans and removes impurities from the raw material of ganoderma lucidum, crushes and sieves it after vacuum drying, defats it by ethanol reflux, breaks the cell wall by cellulase, fully extracts it with hot water, and filters, filtrates, concentrates, and freezes the extract to obtain ganoderma lucidum polysaccharide. The extraction method of the invention has simple process, no toxic reagents are involved, is green and environmentally friendly, has high polysaccharide yield, and is easy to industrialize.

[0030] In order to further illustrate the present invention, a method for extracting Ganoderma lucidum polysaccharide provided by the present invention is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1

[0032] Effects of different cellulase addition amounts on the yield of Ganoderma lucidum polysaccharides

[0033] Take defatted (90% ethanol aqueous solution as solvent reflux degreasing, solid-liquid ratio of (1:25), temperature of (80°C), time of (3h). Repeat twice, the product is filtered to obtain defatted Ganoderma lucidum powder, and dried in a vacuum oven at 60°C to constant weight) Ganoderma lucidum powder 4.0g is mixed with deionized water, the solid-liquid ratio is fixed at 1:30, the hydrolysis temperature is 50°C, and the hydrolysis time is 90min, the addition amount of cellulase (SUNSON Xiasheng enzyme cellulase (SPE-017 for plant extraction), net content 1kg. Production batch number 22204001, production date 20220328) is changed to 1000U / g (defatted Ganoderma lucidum powder), 1500U / g, 2000U / g, 2500U / g respectively, the subsequent hot water extraction conditions (temperature 85°C, time 2h) remain unchanged, and four parallel experiments are performed. The content of crude Ganoderma lucidum polysaccharide under four different conditions was measured by phenol-sulfuric acid method (using phenol-sulfuric acid method, measuring the absorbance at 486nm by ultraviolet absorption spectrum, and then calculating the polysaccharide content in the extract using the standard curve, polysaccharide yield = polysaccharide content / mass of raw material after defatting). The results of the effect of cellulase amount on Ganoderma lucidum polysaccharide yield are shown in Figure 2. Figure 1 shown.

[0034] Depend on Figure 1 It can be seen that when the amount of cellulase added is between 1000 and 2500 U / g, as the amount of enzyme increases, the enzyme concentration in the whole system rises, the contact opportunity between the enzyme and the substrate increases at the same time, and the number of substrate molecules hydrolyzed by the enzyme increases, so that Ganoderma lucidum polysaccharide can be extracted more efficiently. When the enzyme amount reaches 2500 U / g, although the yield of Ganoderma lucidum polysaccharide increases slightly, considering factors such as cost, it is more reasonable to choose an enzyme amount of 2000 U / g.

[0035] Example 2

[0036] Effects of different solid-liquid ratios on the yield of Ganoderma lucidum polysaccharides

[0037] Take 4.0g of defatted Ganoderma lucidum powder (prepared by the same method as in Example 1), fix the amount of cellulase added to 2000U / g, the hydrolysis temperature to 50°C, the hydrolysis time to 90min, change the solid-liquid ratio to 1:10, 1:20, 1:30, 1:40, and the subsequent hot water extraction conditions to be the same as in Example 1, and conduct four parallel experiments. The content of crude Ganoderma lucidum polysaccharide under four different conditions was measured by phenol-sulfuric acid method. The effect of solid-liquid ratio on the yield of Ganoderma lucidum polysaccharide is shown in the figure. Figure 2 shown.

[0038] Depend on Figure 2 It can be seen that with the increase of the solid-liquid ratio, the yield of crude Ganoderma lucidum polysaccharide increases. When the solid-liquid ratio is 1:20, the yield of crude Ganoderma lucidum polysaccharide is the highest. As the solid-liquid ratio continues to increase, the yield of Ganoderma lucidum polysaccharide is relatively stable. Therefore, it is more reasonable to choose a solid-liquid ratio of 1:20 to extract crude Ganoderma lucidum polysaccharide.

[0039] Example 3

[0040] Effects of different enzymatic hydrolysis temperatures on the yield of Ganoderma lucidum polysaccharides

[0041] Take 4.0g of defatted Ganoderma lucidum powder (prepared by the same method as in Example 1), add 2000U / g of fixed cellulase, set the solid-liquid ratio to 1:30, and set the enzymolysis time to 90min. Change the enzymolysis temperature to 40℃, 45℃, 50℃, and 55℃ respectively. The subsequent hot water extraction conditions are the same as in Example 1. Four parallel experiments are performed. The content of crude Ganoderma lucidum polysaccharide under four different conditions is measured by the phenol-sulfuric acid method. The effect of enzymolysis temperature on the yield of Ganoderma lucidum polysaccharide is shown in the figure. Figure 3 shown.

[0042] Depend on Figure 3 It can be seen that the yield of Ganoderma lucidum polysaccharide increases with the increase of temperature at 40-55℃, and the yield of crude Ganoderma lucidum polysaccharide is the highest at 50℃. At 55℃, the yield of polysaccharide decreases, which may be due to the high temperature and the inhibition of cellulase activity. Therefore, the enzymatic hydrolysis temperature of Ganoderma lucidum polysaccharide is most efficient at 50℃.

[0043] Example 4

[0044] Effects of different enzymatic hydrolysis times on the yield of Ganoderma lucidum polysaccharides

[0045] Take 4.0g of defatted Ganoderma lucidum powder (prepared by the same method as in Example 1), fix the amount of cellulase added to 2000U / g, the solid-liquid ratio to 1:30, the enzymolysis temperature to 50°C, change the enzymolysis time to 30min, 60min, 90min, 120min respectively, and the subsequent hot water extraction conditions are the same as in Example 1, and conduct four parallel experiments. The content of crude Ganoderma lucidum polysaccharide under four different conditions was measured by phenol-sulfuric acid method. The effect of enzymolysis time on the yield of Ganoderma lucidum polysaccharide is shown in the following figure. Figure 4 shown.

[0046] Depend on Figure 4 It can be seen that with the increase of enzymatic hydrolysis time, the yield of Ganoderma lucidum polysaccharide also increases. The yield of Ganoderma lucidum polysaccharide is the highest at 60 minutes, and then decreases slightly. Therefore, choosing an enzymatic hydrolysis time of 60 minutes is more conducive to improving the yield.

[0047] Example 5

[0048] Design of orthogonal experiment to explore the optimal extraction method of Ganoderma lucidum polysaccharide

[0049] The single factor test showed that the solid-liquid ratio of 1:20, the enzyme addition of 2000U / g, the enzymatic hydrolysis temperature of 50°C and the enzymatic hydrolysis time of 60min were beneficial to the extraction of Ganoderma lucidum polysaccharide. Therefore, the present invention designed a three-factor three-level orthogonal test to seek the optimal combination.

[0050] Table 1 Orthogonal experimental design

[0051]

[0052]

[0053] Table 2 Analysis of orthogonal test results

[0054]

[0055] The results of orthogonal analysis showed that the maximum mean yield of each factor level was A1, B1 and C3, that is, the best extraction process optimized by orthogonal experimental design was an enzymatic hydrolysis temperature of 45°C, an extraction time of 30 min, and a liquid-to-solid ratio of 1:25. The order of the extreme differences was: liquid-to-solid ratio C>liquid-to-solid temperature B>liquid-to-solid time A, indicating that the liquid-to-solid ratio had the greatest impact on the polysaccharide yield, and the extraction temperature had the least impact. Therefore, the liquid-to-solid ratio must be strictly controlled during the extraction process.

[0056] Table 3 Analysis of variance table

[0057]

[0058]

[0059] In order to further verify the influence of each factor on the yield of Ganoderma lucidum polysaccharide, the results of the orthogonal test of variance analysis were used. The results of variance analysis Table 3 showed that the influence of each factor on the yield of Ganoderma lucidum polysaccharide was as follows: solid-liquid ratio C>enzyme hydrolysis time B>enzyme hydrolysis temperature A, which was consistent with the results of the range analysis. The significance analysis showed that the solid-liquid ratio C had a significant difference (p<0.05) and was a major factor.

[0060] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for extracting ganoderma lucidum polysaccharide, characterized in that: The following steps are involved: The ganoderma lucidum powder and the ethanol aqueous solution are mixed and refluxed for degreasing, the residue is filtered out by suction, and vacuum dried to obtain the defatted ganoderma lucidum powder; The defatted ganoderma lucidum powder, cellulase and water are mixed, enzymolysis is performed, hot water extraction is performed, the filtrate is filtered, concentrated and freeze-dried to obtain ganoderma lucidum polysaccharide.

2. The extraction method according to claim 1, characterized in that The preparation method of the ganoderma lucidum powder comprises: washing the ganoderma lucidum, drying at 55-65° C., and crushing to obtain the ganoderma lucidum powder.

3. The extraction method according to claim 1, characterized in that The mass ratio of the ganoderma lucidum powder to the ethanol aqueous solution is 1: (20-30); the number of degreasing is 2-3 times; the time of each degreasing is 1-6 hours; the degreasing temperature is 40-90° C.; the volume percentage of ethanol in the ethanol aqueous solution is 50-90%.

4. The extraction method according to claim 1, characterized in that The vacuum drying temperature is 55-65°C.

5. The extraction method according to claim 1, characterized in that The mass ratio of the defatted ganoderma lucidum powder to water is 1:(20-40).

6. The extraction method according to claim 1, characterized in that The added amount of the cellulase is 1000-2500 U / g defatted ganoderma lucidum powder; the temperature of the enzymolysis is 40-55° C.; and the time of the enzymolysis is 30-120 min.

7. The extraction method according to claim 6, characterized in that The cellulase includes industrial cellulase.

8. The extraction method according to claim 1, characterized in that The temperature of the hot water extraction is 85° C.; the time of the hot water extraction is 2 hours.

9. The extraction method according to claim 1, characterized in that The filtration comprises sequentially performing gauze filtration and suction filtration.

10. The extraction method according to claim 1, characterized in that The concentration is followed by centrifugation; the centrifugation is performed at 12000 r / min for 5 min, and the number of centrifugation is 2 to 3 times.

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