Method for improving extraction yield of ganoderma lucidum polysaccharide powder through combination of enzymolysis and ultrasonic waves

Abstract

The invention discloses a method for improving the extraction yield of powder-recovered ganoderma lucidum polysaccharide by combining enzymolysis with ultrasonic waves, which comprises the following steps: performing enzymolysis on powder-recovered ganoderma lucidum by using cellulase which can hydrolyze cellulose in cell walls of the powder-recovered ganoderma lucidum so as to damage the cell walls; hot water and ultrasonic waves are used for extraction, the hole effect of the ultrasonic waves can enhance the movement frequency and speed of substance molecules, the combined effect of enzymolysis and ultrasonic waves is beneficial to dissolution of polysaccharide combined with cellulose in cell walls, and the extraction yield is remarkably improved. Compared with extraction without enzymolysis, the method for extracting the powder-collected ganoderma lucidum polysaccharide through combination of enzymolysis and ultrasonic assistance has the advantage that the extraction yield of the powder-collected ganoderma lucidum polysaccharide can be increased by 82.9%.

Description

(1) Technical Field

[0001] The present invention belongs to the field of biotechnological applications, and particularly relates to a method for improving the extraction rate of Ganoderma lucidum polysaccharide from harvested-powder Ganoderma lucidum by enzymatic hydrolysis combined with ultrasonic waves. (2) Background Art

[0002] The Chinese medicinal material Ganoderma lucidum is the dried fruiting body of the fungus Ganoderma lucidum or Ganoderma sinense of the family Polyporaceae. Ganoderma lucidum contains various bioactive components such as polysaccharides, triterpenoids, polypeptides, nucleosides, sterols, and alkaloids. Among them, polysaccharides and triterpenoids have received the most attention, and their contents are important indicators for measuring the quality of Ganoderma lucidum. For example, the first volume of the Chinese Pharmacopoeia (2020 edition) stipulates that the polysaccharide content of the Chinese medicinal material Ganoderma lucidum shall not be less than 0.90%, and the content of triterpenoids and sterols (calculated as oleanolic acid) shall not be less than 0.50%.

[0003] In recent years, with the continuous exploration of the food and medicinal values of Ganoderma lucidum, consumers' enthusiasm for Ganoderma lucidum products has been increasing continuously. The cultivation technology and deep processing technology of Ganoderma lucidum have developed rapidly, and a series of intensive processing products such as Ganoderma lucidum spore powder, Ganoderma lucidum syrup, Ganoderma lucidum spore oil, and Ganoderma lucidum extract have emerged, and the market scale has been continuously expanding. Among these products, Ganoderma lucidum spore powder has become the most popular product among consumers due to its significant efficacy, and it occupies more than 40% of the Ganoderma lucidum product market. Therefore, the cultivation of Ganoderma lucidum for spore powder accounts for more than half of its total cultivation area.

[0004] Ganoderma lucidum spore powder is the spore naturally ejected from under the Ganoderma lucidum cap after Ganoderma lucidum grows and matures. The Ganoderma lucidum fruiting body with spore production as the main target product is habitually called "harvested-powder Ganoderma lucidum", "bagged Ganoderma lucidum" or "sleeved Ganoderma lucidum" in the industry. The growth period of harvested-powder Ganoderma lucidum is often as long as 9 - 10 months, which is 3 - 5 times that of ordinary Ganoderma lucidum cultivation cycle, and it is uprooted after the fruiting body stops producing spores. Due to the long growth period, high degree of fibrosis, and low content of active ingredients of harvested-powder Ganoderma lucidum, it is not suitable for direct use as medicinal Ganoderma lucidum. For example, when used for extracting polysaccharides or triterpenoids, due to the low extraction rate and poor economic benefits, harvested-powder Ganoderma lucidum has gradually become a low-value by-product in the Ganoderma lucidum spore powder industrial chain. With the continuous development and growth of the spore powder industry, a large number of uprooted harvested-powder Ganoderma lucidum are often disposed of as waste by growers.

[0005] A large number of harvested-powder Ganoderma lucidum have not received sufficient attention and full utilization due to their low economic added value, resulting in potential waste of industrial resources. If a new technology suitable for the deep processing of harvested-powder Ganoderma lucidum can be developed to enhance its economic value, further release the development potential of the Ganoderma lucidum industry, and improve the economic benefits of the industry, it will have important practical significance for increasing the production and income of harvested-powder Ganoderma lucidum growers and for energy conservation and emission reduction in the industry.

[0006] Ganoderma lucidum polysaccharide has attracted much attention due to its wide range of physiological activities. It plays a role in immune regulation, adjuvant anti-tumor, anti-aging, anti-diabetes, protecting cardiovascular and cerebrovascular systems, liver protection and detoxification, etc., and is widely used in the medical, health care, and cosmetics industries. At present, there are many research reports on the extraction of common Ganoderma lucidum polysaccharide. The basic method is the hot water extraction method. On this basis, ultrasonic, microwave, and enzymatic hydrolysis and other auxiliary measures are used for extraction, which can significantly improve the polysaccharide extraction rate. However, the fibrosis degree of harvested Ganoderma lucidum is high, and these methods or the enzymatic hydrolysis conditions used are not applicable to the extraction of its polysaccharide.

[0007] In recent years, enzymatic hydrolysis technology has been widely used in the extraction of edible fungi and plant polysaccharides. This technology uses cellulase, pectinase, or protease, etc., to hydrolyze edible fungi or plant raw materials under appropriate conditions, hydrolyze their cell walls, which is beneficial to the dissolution of intracellular polysaccharides during extraction. The enzymatic hydrolysis-assisted method can not only significantly improve the extraction rate, but also has mild conditions, will not damage the structure of active ingredients in the extract, and can also reduce the molecular weight of polysaccharides, making the extracted polysaccharides have better biological activity.

[0008] In order to develop an extraction method suitable for harvested Ganoderma lucidum polysaccharide, the present invention adopts an enzymatic hydrolysis combined with ultrasonic-assisted extraction method, so that the extraction rate of harvested Ganoderma lucidum polysaccharide is relatively high, thereby ensuring better economic benefits in the industrial production of extracting polysaccharides from harvested Ganoderma lucidum as raw materials. (III) Summary of the Invention

[0009] The object of the present invention is to provide a method for improving the extraction rate of harvested Ganoderma lucidum polysaccharide by enzymatic hydrolysis combined with ultrasonic. The present invention applies the enzymatic hydrolysis technology of cellulase and ultrasonic extraction technology to the extraction of harvested Ganoderma lucidum polysaccharide. After the harvested Ganoderma lucidum is enzymatically hydrolyzed by cellulase, the polysaccharide is then extracted by hot water and ultrasonic, and its polysaccharide extraction rate is significantly improved.

[0010] The technical solution adopted by the present invention is:

[0011] The present invention provides a method for improving the extraction rate of harvested Ganoderma lucidum polysaccharide by enzymatic hydrolysis combined with ultrasonic, and the method comprises the following steps:

[0012] (1) The harvested Ganoderma lucidum is sliced, dried, and pulverized. The obtained harvested Ganoderma lucidum powder is added with deionized water and cellulase, and stirred evenly to form a harvested Ganoderma lucidum enzymatic hydrolysis system;

[0013] (2) The harvested Ganoderma lucidum enzymatic hydrolysis system is enzymatically hydrolyzed in a water bath at a temperature of 35 - 45 °C for 3 - 5 h, then extracted in boiling water for 2 - 3 h, and then in ultrasonic, the water temperature is 30 - 40 °C for extraction for 30 - 45 min and then filtered, and the filtrate is collected to obtain a harvested Ganoderma lucidum water extract;

[0014] (3) The harvested Ganoderma lucidum water extract is concentrated to 1 / 20 - 1 / 30 of the original volume, ethanol is added to precipitate the polysaccharide, the precipitate is washed with absolute ethanol, and after vacuum drying, a harvested Ganoderma lucidum polysaccharide extract is obtained.

[0015] Further, the Ganoderma lucidum for spore collection in step (1) is the fruiting body of artificially cultivated Ganoderma lucidum, which is used to collect Ganoderma lucidum spore powder, pulled out after no more spores are produced, and naturally air-dried; the Ganoderma lucidum powder for spore collection is obtained by cutting the Ganoderma lucidum for spore collection into thin slices with a thickness of 2 - 3 mm, drying to a constant weight at 85°C, and then pulverizing and passing through a 20-mesh sieve to obtain a coarse powder.

[0016] Further, the pH of the deionized water in step (1) is 5.0 - 5.5 (adjusted with 1 mol / L HCl or NaOH); the added volume is 30 - 35 mL / g based on the mass of the Ganoderma lucidum powder for spore collection [i.e., the material-to-water ratio is 1:30 - 1:35 (g:mL)].

[0017] Further, the dosage of cellulase in step (1) is 1750 - 2100 U activity / g based on the mass of the Ganoderma lucidum powder for spore collection.

[0018] Further, the frequency of the ultrasonic wave in step (2) is 40 kHz, and the power is 120 - 160 W.

[0019] Further, the preparation method of the Ganoderma lucidum polysaccharide extract in step (3) is as follows: the water extract of Ganoderma lucidum for spore collection is concentrated under reduced pressure to 1 / 20 - 1 / 30 of the original volume at 60°C and a relative vacuum degree of -0.1 MPa, then anhydrous ethanol with a volume 3 - 4 times that of the concentrated solution is added to make the ethanol volume concentration in the system reach 75% - 80%, left to stand at 4°C for 12 - 18 h, centrifuged at 8000×g for 5 - 10 min, and the precipitate is collected; the precipitate is washed once with anhydrous ethanol at 2 - 4 mL / g based on the mass of the raw material Ganoderma lucidum powder for spore collection, and after centrifugation again, the precipitate is vacuum-dried to a constant weight at 65°C and a relative vacuum degree of -0.1 MPa to obtain the Ganoderma lucidum polysaccharide extract.

[0020] Further, the method for enzymolysis combined with ultrasonic wave assistance to improve the extraction rate of Ganoderma lucidum polysaccharide of the present invention is specifically operated according to the following steps:

[0021] (1) The naturally air-dried Ganoderma lucidum for spore collection is cut into thin slices with a thickness of 2 - 3 mm, dried to a constant weight at 85°C, pulverized, and passed through a 20-mesh sieve to obtain the Ganoderma lucidum powder for spore collection;

[0022] (2) In a beaker, the Ganoderma lucidum powder for spore collection is added with deionized water at 30 - 35 mL / g and pH = 5.0 - 5.5 based on the mass of the Ganoderma lucidum powder for spore collection [i.e., the material-to-water ratio is 1:30 - 1:35 (g:mL)], and cellulase at 1750 - 2100 U activity / g based on the mass of the Ganoderma lucidum powder for spore collection is added, and stirred evenly to form a Ganoderma lucidum enzymolysis system;

[0023] (3) The enzymatic hydrolysis system of Ganoderma lucidum karst. is enzymatically hydrolyzed in a water bath at a temperature of 35-45 °C for 3-5 h, transferred to boiling water for extraction for 2-3 h, and then in an ultrasonic wave (frequency 40 kHz) with a power of 120-160 W and a water temperature of 30-40 °C for extraction for 30-45 min. The enzymatic hydrolysis system is filtered, and the filtrate is collected to obtain the water extract of Ganoderma lucidum karst. The water extract of Ganoderma lucidum karst. is concentrated under reduced pressure to 1 / 20-1 / 30 of the original volume at 60 °C and a relative vacuum of -0.1 MPa, and then anhydrous ethanol 3-4 times the volume of the concentrated solution is added to make the ethanol volume concentration of the system reach 75%-80%. After standing at 4 °C for 12-18 h, it is centrifuged at 8000×g for 5-10 min, and the polysaccharide precipitate is collected; the polysaccharide precipitate is washed once with anhydrous ethanol at 2-4 mL / g based on the mass of the raw material Ganoderma lucidum karst. powder, and after centrifugation again, the precipitate is vacuum dried to constant weight at 65 °C and a relative vacuum of -0.1 MPa to obtain the Ganoderma lucidum karst. polysaccharide extract.

[0024] The present invention provides a Ganoderma lucidum polysaccharide prepared by the above method.

[0025] The present invention also provides an application of the Ganoderma lucidum polysaccharide in the preparation of an antioxidant.

[0026] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0027] In the present invention, Ganoderma lucidum karst. is enzymatically hydrolyzed with cellulase. Cellulase can hydrolyze the cellulose in the cell wall of Ganoderma lucidum karst., causing the cell wall to be damaged; then it is extracted with hot water and ultrasonic waves. The cavitation effect of ultrasonic waves can enhance the movement frequency and speed of substance molecules. The combined action of enzymatic hydrolysis and ultrasonic waves helps the polysaccharides combined with cellulose in the cell wall to dissolve out, and the extraction rate is significantly improved. The method for extracting Ganoderma lucidum karst. polysaccharide by combined enzymatic hydrolysis and ultrasonic wave assistance provided by the present invention can increase the extraction rate of Ganoderma lucidum karst. polysaccharide by 82.9% compared with the extraction method without enzymatic hydrolysis. (IV) Description of the Drawings

[0028] Figure 1 It is the standard curve for the determination of polysaccharides by the phenol-sulfuric acid method (using glucose as the standard).

[0029] Figure 2 It is the DPPH free radical scavenging rate curve of the polysaccharide extracted from Ganoderma lucidum karst. (V) Specific Embodiments

[0030] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0031] The raw materials, instruments and equipment used in the present invention are all common raw materials, instruments and equipment in the art without special instructions; the methods used in the present invention are all conventional methods in the art without special instructions.

[0032] In the embodiments of the present invention, the spore-collecting Ganoderma lucidum is the fruiting body of artificially cultivated Ganoderma lucidum, which is used for collecting Ganoderma lucidum spore powder, pulled out after no more spores are produced, and naturally air-dried; the spore-collecting Ganoderma lucidum powder is the thick powder obtained by cutting the spore-collecting Ganoderma lucidum into thin slices about 3 mm thick, drying at 85 °C to constant weight, and then pulverizing and passing through a 20-mesh sieve.

[0033] In the embodiments of the present invention, the polysaccharide content of the spore-collecting Ganoderma lucidum is determined by the phenol-sulfuric acid method. The specific method is as follows: the spore-collecting Ganoderma lucidum polysaccharide extract is appropriately diluted with deionized water (estimating that the concentration of polysaccharide in the sample is within the range of the standard curve determination); the solid spore-collecting Ganoderma lucidum polysaccharide extract is prepared into a sample solution with a concentration of 0.2 mg / mL with deionized water as the sample to be measured. Pipette 1 mL of the sample solution into a 10-mL stoppered tube, add 1 mL of a 5% phenol aqueous solution by volume, shake well, and then quickly add 5 mL of concentrated sulfuric acid (mass concentration 98%), shake well, heat in boiling water for 15 min, and cool to room temperature. Using the same treatment with 1 mL of deionized water instead of the sample solution as the reference, measure the absorbance (A 490 ) at a wavelength of 490 nm. Measure the A 490 of glucose samples with different concentrations in the same way, and plot the glucose concentration - A 490 standard curve (attached Figure 1 ), obtain the regression equation, and calculate the polysaccharide content in the spore-collecting Ganoderma lucidum sample by the regression equation.

[0034] The extraction rate of the spore-collecting Ganoderma lucidum polysaccharide is calculated according to formula 1:

[0035]

[0036] Example 1: Selection of enzyme types

[0037] In the process of extracting polysaccharides from edible or medicinal fungi, common enzymes include cellulase, pectinase, and protease. In order to determine the enzyme suitable for enzymolysis of spore-collecting Ganoderma lucidum, the polysaccharide extraction rates after enzymolysis of spore-collecting Ganoderma lucidum by different types of enzymes were investigated. The specific method is as follows:

[0038] (1) Put 1 g of spore-collecting Ganoderma lucidum powder into a 150-mL beaker, add 35 mL of deionized water with pH = 5.0 (adjusted with 1 mol / L HCl or NaOH), and add cellulase, pectinase, protease, cellulase + pectinase, or cellulase + protease with an activity of 1400 U / g based on the mass of the spore-collecting Ganoderma lucidum powder, respectively. The addition amount of each enzyme is 700 U / g based on the activity. Stir well to form a spore-collecting Ganoderma lucidum enzymolysis system;

[0039] (2) The Ganoderma lucidum karst enzyme hydrolysis system was hydrolyzed in a water bath at 35°C for 5 h, transferred to boiling water for extraction for 2.5 h, and then extracted in an ultrasonic wave (frequency 40 kHz) with a power of 140 W and a water temperature of 30°C for 45 min. After filtration, the filtrate was collected to obtain the water extract of Ganoderma lucidum karst.

[0040] (3) 1 mL of the water extract of Ganoderma lucidum karst was placed in a centrifuge tube, 4 mL of absolute ethanol (the volume concentration of ethanol in the system was 80%) was added, and after standing at 4°C for 12 h, centrifuged at 8000×g for 5 min. The supernatant was discarded, and the precipitate was dissolved with 10 mL of deionized water. The polysaccharide content in the polysaccharide solution was determined by the phenol-sulfuric acid method.

[0041] According to the above steps, after Ganoderma lucidum karst was hydrolyzed by different types of enzymes, the extraction yields of polysaccharides are shown in Table 1.

[0042] Table 1 Extraction yields of polysaccharides from Ganoderma lucidum karst after hydrolysis by different enzymes

[0043]

[0044] It can be seen from the results in Table 1 that after Ganoderma lucidum karst was hydrolyzed by cellulase, the polysaccharide yield increased significantly, from 0.920% without enzyme to 1.31%, an increase of 42.4%; while after hydrolysis by pectinase and protease, the extraction yield of polysaccharides did not increase significantly; when cellulase was mixed with pectinase and protease at a ratio of 1:1 in terms of activity, the polysaccharide yield was lower than that of using cellulase alone. Therefore, the preferred enzyme for hydrolyzing Ganoderma lucidum karst to extract polysaccharides is cellulase.

[0045] Example 2: Selection of the enzyme addition amount for hydrolyzing Ganoderma lucidum karst

[0046] After determining to hydrolyze Ganoderma lucidum karst with cellulase, the extraction yields of polysaccharides under different enzyme addition amounts were investigated to determine the preferred enzyme addition amount. The specific steps were as follows:

[0047] (1) 1 g of Ganoderma lucidum karst powder was placed in a 150-mL beaker, 35 mL of deionized water with pH = 5.0 (adjusted with 1 mol / L HCl or NaOH) was added, and cellulase with activities of 1050, 1400, 1750, 2100, and 2450 U / g based on the mass of Ganoderma lucidum karst powder was added respectively, and stirred evenly to form the Ganoderma lucidum karst enzyme hydrolysis system.

[0048] (2) The Ganoderma lucidum karst enzyme hydrolysis system was hydrolyzed in a water bath at 35°C for 5 h, transferred to boiling water for extraction for 2.5 h, and then extracted in an ultrasonic wave (frequency 40 kHz) with a power of 140 W and a water temperature of 30°C for 45 min. After filtration, the filtrate was collected to obtain the water extract of Ganoderma lucidum karst.

[0049] (3) Place 1 mL of the aqueous extract of Ganoderma lucidum Karst. spores in a centrifuge tube, add 4 mL of absolute ethanol (the volume concentration of ethanol in the system is 80%), let it stand at 4 °C for 12 h, centrifuge at 8000×g for 5 min, discard the supernatant, add 10 mL of deionized water to dissolve the precipitate, and determine the polysaccharide content in the polysaccharide solution by the phenol-sulfuric acid method.

[0050] According to the above steps, the polysaccharide extraction yields of Ganoderma lucidum Karst. spores enzymatically hydrolyzed under different cellulase dosages are shown in Table 2.

[0051] Table 2 Polysaccharide extraction yields of Ganoderma lucidum Karst. spores under different cellulase dosages

[0052]

[0053] It can be seen from the data in Table 2 that the highest value is reached when the enzyme dosage is 1750 U / g activity; when the enzyme dosage is greater than 2100 U / g activity, the polysaccharide yield decreases. Therefore, the preferred cellulase dosage is 1750 - 2100 U / g activity.

[0054] Example 3: Selection of the pH of the enzymatic hydrolysis system of Ganoderma lucidum Karst. spores

[0055] After determining the preferred cellulase dosage, the polysaccharide extraction yields of Ganoderma lucidum Karst. spores enzymatically hydrolyzed under different system pH conditions were investigated to determine the preferred pH of the enzymatic hydrolysis system. The specific steps are as follows:

[0056] (1) Place 1 g of Ganoderma lucidum Karst. spore powder in a 150-mL beaker, add 35 mL of deionized water with pH values of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0 respectively (adjusted with 1 mol / L HCl or NaOH), add cellulase with an activity of 1750 U / g based on the mass of Ganoderma lucidum Karst. spore powder, stir evenly to form an enzymatic hydrolysis system of Ganoderma lucidum Karst. spores;

[0057] (2) The enzymatic hydrolysis system of Ganoderma lucidum Karst. spores is enzymatically hydrolyzed in a water bath at 35 °C for 5 h, transferred to boiling water for extraction for 2.5 h, and then extracted in an ultrasonic wave (frequency 40 kHz) with a power of 140 W and a water temperature of 30 °C for 45 min, filtered, and the filtrate is collected to obtain the aqueous extract of Ganoderma lucidum Karst. spores;

[0058] (3) Place 1 mL of the aqueous extract of Ganoderma lucidum Karst. spores in a centrifuge tube, add 4 mL of absolute ethanol (the volume concentration of ethanol in the system is 80%), let it stand at 4 °C for 12 h, centrifuge at 8000×g for 5 min, discard the supernatant, add 10 mL of deionized water to dissolve the precipitate, and determine the polysaccharide content in the polysaccharide solution by the phenol-sulfuric acid method.

[0059] According to the above steps, after enzymatically hydrolyzing Ganoderma lucidum Karst. spores under different pH conditions, the polysaccharide extraction yields are shown in Table 3.

[0060] Table 3 Polysaccharide extraction yields of Ganoderma lucidum Karst. under different pH values of the enzymatic hydrolysis system

[0061]

[0062] Note: The relative increase percentage refers to the increase percentage of the polysaccharide extraction yield under different pH conditions relative to the lowest value (the yield at pH = 7.0).

[0063] It can be seen from the data in Table 3 that, comparatively, within the range of system pH = 5.0 - 5.5, after enzymatic hydrolysis of Ganoderma lucidum Karst., the polysaccharide extraction yield is relatively high. Therefore, the preferred pH value of the enzymatic hydrolysis system is 5.0 - 5.5.

[0064] Example 4: Selection of the enzymatic hydrolysis temperature of Ganoderma lucidum Karst.

[0065] After determining the preferred addition amount of cellulase and the system pH in the enzymatic hydrolysis system of Ganoderma lucidum Karst., the polysaccharide extraction yields under different enzymatic hydrolysis temperature conditions were investigated to determine the preferred enzymatic hydrolysis temperature. The specific steps were as follows:

[0066] (1) 1 g of Ganoderma lucidum Karst. powder was placed in a 150-mL beaker, 35 mL of deionized water with pH = 5.0 (adjusted with 1 mol / L HCl or NaOH) was added, and cellulase with an activity of 1750 U / g based on the mass of Ganoderma lucidum Karst. powder was added and stirred evenly to form the enzymatic hydrolysis system of Ganoderma lucidum Karst.;

[0067] (2) The enzymatic hydrolysis system of Ganoderma lucidum Karst. was enzymatically hydrolyzed in a water bath at temperatures of 30, 35, 40, 45, and 50 °C for 5 h, transferred to boiling water for extraction for 2.5 h, and then extracted in an ultrasonic wave (frequency 40 kHz) with a power of 140 W and a water temperature of 30 °C for 45 min, filtered, and the filtrate was collected to obtain the water extract of Ganoderma lucidum Karst.;

[0068] (3) 1 mL of the water extract of Ganoderma lucidum Karst. was placed in a centrifuge tube, 4 mL of absolute ethanol (the volume concentration of ethanol in the system was 80%) was added, and after standing at 4 °C for 12 h, centrifuged at 8000×g for 5 min, the supernatant was discarded, 10 mL of deionized water was added to dissolve the precipitate, and the polysaccharide content in the polysaccharide solution was determined by the phenol-sulfuric acid method.

[0069] According to the above steps, after enzymatic hydrolysis of Ganoderma lucidum Karst. under different temperature conditions, the polysaccharide extraction yields are shown in Table 4.

[0070] Table 4 Polysaccharide extraction yields of Ganoderma lucidum Karst. under different enzymatic hydrolysis temperatures

[0071]

[0072] Note: The relative increase percentage refers to the increase percentage of the polysaccharide extraction yield under different enzymatic hydrolysis temperature conditions relative to the lowest value (the yield at 50 °C).

[0073] As can be seen from the data in Table 4, by comparison, within the range of enzymatic hydrolysis temperature from 35 to 45 °C, the extraction rate of polysaccharides from Ganoderma lucidum Karst. after enzymatic hydrolysis is relatively high. Therefore, the preferred enzymatic hydrolysis temperature is 35 to 45 °C.

[0074] Example 5: Selection of the enzymatic hydrolysis time of Ganoderma lucidum Karst.

[0075] After determining the preferred addition amount of cellulase, the system pH, and the enzymatic hydrolysis temperature in the enzymatic hydrolysis system of Ganoderma lucidum Karst., the extraction rates of polysaccharides under different enzymatic hydrolysis times were investigated to determine the preferred enzymatic hydrolysis time. The specific steps are as follows:

[0076] (1) 1 g of Ganoderma lucidum Karst. powder was placed in a 150-mL beaker, 35 mL of deionized water with pH = 5.0 (adjusted with 1 mol / L HCl or NaOH) was added, and cellulase with an activity of 1750 U / g based on the mass of Ganoderma lucidum Karst. powder was added and stirred evenly to form an enzymatic hydrolysis system of Ganoderma lucidum Karst.

[0077] (2) The enzymatic hydrolysis system of Ganoderma lucidum Karst. was placed in a water bath at 45 °C and enzymatically hydrolyzed for 1, 2, 3, 4, and 5 h respectively, then transferred to boiling water for extraction for 2.5 h, and then extracted in an ultrasonic wave (frequency 40 kHz) with a power of 140 W and a water temperature of 30 °C for 45 min, filtered, and the filtrate was collected to obtain the water extract of Ganoderma lucidum Karst.

[0078] (3) 1 mL of the water extract of Ganoderma lucidum Karst. was placed in a centrifuge tube, 4 mL of absolute ethanol (the volume concentration of ethanol in the system was 80%) was added, and after standing at 4 °C for 12 h, centrifuged at 8000 × g for 5 min, the supernatant was discarded, 10 mL of deionized water was added to dissolve the precipitate, and the polysaccharide content in the polysaccharide solution was determined by the phenol-sulfuric acid method.

[0079] According to the above steps, after Ganoderma lucidum Karst. was enzymatically hydrolyzed for different times, the extraction rates of polysaccharides are shown in Table 5.

[0080] Table 5 Extraction rates of polysaccharides from Ganoderma lucidum Karst. after enzymatic hydrolysis for different times

[0081]

[0082] Note: The relative increase percentage refers to the increase percentage of the polysaccharide extraction rate under different enzymatic hydrolysis times relative to the lowest value (1 h).

[0083] As can be seen from the data in Table 5, within the range of enzymatic hydrolysis time from 3 to 4 h, the extraction rate of polysaccharides from Ganoderma lucidum Karst. after enzymatic hydrolysis is relatively high. Therefore, the preferred enzymatic hydrolysis time is 3 to 4 h.

[0084] Based on the results of Examples 1 - 5, the preferred conditions for enzymatic hydrolysis of Ganoderma lucidum for powder collection are as follows: Add cellulase with an activity of 1750 - 2100 U / g based on the mass of Ganoderma lucidum for powder collection to the Ganoderma lucidum for powder collection, and carry out enzymatic hydrolysis at 35 - 45 °C for 3 - 4 h in a system with a pH of 5.0 - 5.5. Among them, the preferred enzymatic hydrolysis conditions are: Add cellulase with an activity of 1750 U / g based on the mass of Ganoderma lucidum for powder collection to the Ganoderma lucidum for powder collection, and carry out enzymatic hydrolysis at 45 °C for 4 h in a system with a pH of 5.0.

[0085] Example 6: Method for extracting polysaccharides from Ganoderma lucidum for powder collection by enzymatic hydrolysis combined with ultrasonic assistance 1

[0086] A method for extracting polysaccharides from Ganoderma lucidum for powder collection by enzymatic hydrolysis combined with ultrasonic assistance. Under the preferred enzymatic hydrolysis conditions, the operation can be carried out according to the following steps:

[0087] (1) Place 5 g of Ganoderma lucidum powder for powder collection in a 250 - mL beaker, add 175 mL of deionized water with a pH of 5.0, add cellulase with an activity of 1750 U / g based on the mass of Ganoderma lucidum powder for powder collection, and stir evenly to form an enzymatic hydrolysis system of Ganoderma lucidum for powder collection;

[0088] (2) Hydrolyze the enzymatic hydrolysis system of Ganoderma lucidum for powder collection in a water bath at 45 °C for 4 h, transfer it to boiling water for extraction for 2 h, then extract it in an ultrasonic wave (frequency: 40 kHz) with a power of 120 W and a water temperature of 30 °C for 45 min, filter, collect the filtrate, and obtain the water extract of Ganoderma lucidum for powder collection;

[0089] (3) Concentrate all the filtrate in step (2) under reduced pressure to 8.75 mL (1 / 20 of the original filtrate volume) at 60 °C and a relative vacuum degree of - 0.1 MPa, then add 35 mL of absolute ethanol (4 times the volume of the concentrated solution, and the ethanol volume concentration in the system is 80%). After standing at 4 °C for 12 h, centrifuge at 4 °C and 8000 × g for 5 min, discard the supernatant, wash the precipitate with 20 mL of absolute ethanol (4 mL / g based on the mass of the raw Ganoderma lucidum powder for powder collection) once, centrifuge again, and dry the precipitate to a constant weight at 65 °C and a relative vacuum degree of - 0.1 MPa to obtain the polysaccharide extract of Ganoderma lucidum for powder collection.

[0090] According to the above steps, 0.134 g of polysaccharide extract was obtained from 5 g of Ganoderma lucidum for powder collection, and the polysaccharide content was 61.1%, that is, 0.0819 g of Ganoderma lucidum polysaccharide was obtained, and the extraction rate was 1.64%.

[0091] Comparative Example 1: Extracting polysaccharides from Ganoderma lucidum for powder collection by the conventional water extraction and alcohol precipitation method (compared with Example 6)

[0092] (1) Put 5 g of Ganoderma lucidum powder in a 250-mL beaker, add 175 mL of deionized water with pH = 5.0, keep it warm in a water bath at 45 °C for 4 h, transfer it to boiling water for extraction for 2 h, and then extract it in an ultrasonic wave (frequency 40 kHz) with a power of 120 W and a water temperature of 30 °C for 45 min. Filter it and collect the filtrate to obtain the water extract of Ganoderma lucidum powder.

[0093] (2) Concentrate all the filtrate in step (1) under reduced pressure at 60 °C and a relative vacuum of -0.1 MPa to 8.75 mL (1 / 20 of the original filtrate volume), then add 35 mL of absolute ethanol (4 times the volume of the concentrated solution, and the ethanol volume concentration in the system is 80%). After standing at 4 °C for 12 h, centrifuge at 4 °C and 8000×g for 5 min, discard the supernatant, wash the precipitate with 20 mL of absolute ethanol (4 mL / g calculated based on the mass of the raw Ganoderma lucidum powder), centrifuge again, and dry the precipitate at 65 °C and a relative vacuum of -0.1 MPa to constant weight to obtain the Ganoderma lucidum polysaccharide extract.

[0094] According to the above steps, 0.0819 g of polysaccharide extract was obtained from 5 g of Ganoderma lucidum powder, and the polysaccharide content was 56.4%, that is, 0.0462 g of Ganoderma lucidum polysaccharide was obtained, and the extraction rate was 0.924%.

[0095] Comparing the results of Example 6 and Comparative Example 1, it can be seen that: before hot water ultrasonic extraction of Ganoderma lucidum polysaccharide, adding enzymatic hydrolysis treatment with cellulase increased the polysaccharide extraction rate from 0.924% without enzymatic hydrolysis treatment to 1.64%, an increase of 77.5%.

[0096] Example 7. Method 2 for enzymatic hydrolysis combined with ultrasonic-assisted extraction of Ganoderma lucidum polysaccharide

[0097] A method for enzymatic hydrolysis combined with ultrasonic-assisted extraction of Ganoderma lucidum polysaccharide. Under relatively optimal enzymatic hydrolysis conditions, it can be operated according to the following steps:

[0098] (1) Put 10 g of Ganoderma lucidum powder in a 500-mL beaker, add 350 mL of deionized water with pH = 5.0, and add cellulase with an activity of 1750 U / g calculated based on the mass of Ganoderma lucidum powder, stir evenly to form a Ganoderma lucidum enzymatic hydrolysis system;

[0099] (2) Hydrolyze the Ganoderma lucidum enzymatic hydrolysis system in a water bath at 45 °C for 4 h, transfer it to boiling water for extraction for 2.5 h, and then extract it in an ultrasonic wave (frequency 40 kHz) with a power of 140 W and a water temperature of 35 °C for 40 min. Filter it and collect the filtrate to obtain the water extract of Ganoderma lucidum powder.

[0100] (3) All the filtrate from step (2) was concentrated under reduced pressure at 60 °C and a relative vacuum of –0.1 MPa to 14 mL (1 / 25 of the original filtrate volume), then 49 mL of absolute ethanol (3.5 times the volume of the concentrated solution, and the ethanol volume concentration in the system was 77.8%) was added. After standing at 4 °C for 15 h, centrifugation was performed at 4 °C and 8000×g for 8 min. The supernatant was discarded, and the precipitate was washed once with 30 mL of absolute ethanol (3 mL / g based on the mass of the starting Ganoderma lucidum powder). After centrifugation again, the precipitate was dried to a constant weight at 65 °C and a relative vacuum of –0.1 MPa to obtain the Ganoderma lucidum polysaccharide extract.

[0101] According to the above steps, 0.267 g of polysaccharide extract was obtained from 10 g of Ganoderma lucidum powder. The polysaccharide content was 62.4%, that is, 1.67 g of Ganoderma lucidum polysaccharide was obtained, and the extraction yield was 1.67%.

[0102] Comparative Example 2: Extraction of Ganoderma lucidum polysaccharide by the conventional water extraction and alcohol precipitation method (compared with Example 7)

[0103] (1) 10 g of Ganoderma lucidum powder was placed in a 500-mL beaker, 350 mL of deionized water with pH = 5.0 was added, and it was kept warm in a water bath at 45 °C for 4 h. Then it was transferred to boiling water for extraction for 2.5 h, and then extracted in an ultrasonic wave (frequency 40 kHz) with a power of 140 W and a water temperature of 35 °C for 40 min. After filtration, the filtrate was collected to obtain the water extract of Ganoderma lucidum.

[0104] (2) All the filtrate from step (1) was concentrated under reduced pressure at 60 °C and a relative vacuum of –0.1 MPa to 14 mL (1 / 25 of the original filtrate volume), then 49 mL of absolute ethanol (3.5 times the volume of the concentrated solution, and the ethanol volume concentration in the system was 77.8%) was added. After standing at 4 °C for 15 h, centrifugation was performed at 4 °C and 8000×g for 8 min. The supernatant was discarded, and the precipitate was washed once with 30 mL of absolute ethanol (3 mL / g based on the mass of the starting Ganoderma lucidum powder). After centrifugation again, the precipitate was dried to a constant weight at 65 °C and a relative vacuum of –0.1 MPa to obtain the Ganoderma lucidum polysaccharide extract.

[0105] According to the above steps, 0.166 g of polysaccharide extract was obtained from 10 g of Ganoderma lucidum powder. The polysaccharide content was 55.9%, that is, 0.0928 g of Ganoderma lucidum polysaccharide was obtained, and the extraction yield was 0.928%.

[0106] Comparing the results of Example 7 and Comparative Example 2, it can be seen that: before hot water ultrasonic extraction of Ganoderma lucidum polysaccharide, adding enzymatic hydrolysis treatment with cellulase increased the extraction yield of polysaccharide from 0.928% without enzymatic hydrolysis treatment to 1.67%, an increase of 80.0%.

[0107] Example 8: Method for Extracting Ganoderma lucidum Polysaccharide by Enzymolysis Combined with Ultrasonic Assisted Extraction 3

[0108] A method for extracting Ganoderma lucidum polysaccharide by enzymolysis combined with ultrasonic assisted extraction. Under optimal enzymolysis conditions, the operation can be carried out according to the following steps:

[0109] (1) 20 g of Ganoderma lucidum powder is placed in a 1-L beaker, 600 mL of deionized water with pH = 5.0 is added, and cellulase with an activity of 1750 U / g based on the mass of Ganoderma lucidum powder is added, and stirred evenly to form a Ganoderma lucidum enzymolysis system;

[0110] (2) The Ganoderma lucidum enzymolysis system is placed in a water bath at 45 °C and enzymolyzed for 4 h, then transferred to boiling water for extraction for 3 h, and then extracted in an ultrasonic wave (frequency 40 kHz) with a power of 160 W and a water temperature of 40 °C for 30 min, filtered, and the filtrate is collected to obtain a Ganoderma lucidum water extract;

[0111] (3) All the filtrate in step (3) is concentrated under reduced pressure to 20 mL (1 / 30 of the original filtrate volume) at 60 °C and a relative vacuum of -0.1 MPa, then 60 mL of absolute ethanol (3 times the volume of the concentrated solution, and the ethanol volume concentration in the system is 75%) is added, left standing at 4 °C for 18 h, centrifuged at 4 °C and 8000 × g for 10 min, the supernatant is discarded, the precipitate is washed once with 40 mL of absolute ethanol (2 mL / g based on the mass of the raw Ganoderma lucidum powder), centrifuged again, and the precipitate is dried to constant weight at 65 °C and a relative vacuum of -0.1 MPa to obtain a Ganoderma lucidum polysaccharide extract.

[0112] According to the above steps, 0.553 g of polysaccharide extract is obtained from 20 g of Ganoderma lucidum. The polysaccharide content is 61.7%, that is, 0.341 g of Ganoderma lucidum polysaccharide is obtained, and the extraction yield is 1.71%.

[0113] Comparative Example 3: Extracting Ganoderma lucidum polysaccharide by the conventional water extraction and alcohol precipitation method (compared with Example 8)

[0114] (1) 20 g of Ganoderma lucidum powder is placed in a 1000-mL beaker, 600 mL of deionized water with pH = 5.0 is added, kept warm in a water bath at 45 °C for 4 h, transferred to boiling water for extraction for 3 h, and then extracted in an ultrasonic wave (frequency 40 kHz) with a power of 160 W and a water temperature of 40 °C for 30 min, filtered, and the filtrate is collected to obtain a Ganoderma lucidum water extract;

[0115] (2) All the filtrate from step (1) was concentrated under reduced pressure to 20 mL (1 / 30 of the original filtrate volume) at 60 °C and a relative vacuum of –0.1 MPa, and then 60 mL of absolute ethanol (3 times the volume of the concentrated solution, with the ethanol volume concentration in the system being 75%) was added. After standing at 4 °C for 18 h, centrifugation was carried out at 4 °C and 8000×g for 10 min. The supernatant was discarded, and the precipitate was washed once with 40 mL of absolute ethanol (2 mL / g based on the mass of the starting Ganoderma lucidum powder). After centrifugation again, the precipitate was dried to a constant weight at 65 °C and a relative vacuum of –0.1 MPa to obtain the Ganoderma lucidum polysaccharide extract.

[0116] According to the above steps, 0.337 g of polysaccharide extract was obtained from 20 g of Ganoderma lucidum powder. The polysaccharide content was 55.6%, that is, 0.187 g of Ganoderma lucidum polysaccharide was obtained, and the extraction yield was 0.935%.

[0117] Comparing the results of Example 8 and Comparative Example 3, it can be seen that: before hot water ultrasonic extraction of Ganoderma lucidum polysaccharide, adding enzymatic hydrolysis treatment with cellulase increased the extraction yield of polysaccharide from 0.935% without enzymatic hydrolysis treatment to 1.71%, an increase of 82.9%.

[0118] Example 9, Antioxidant Activity of Ganoderma lucidum Polysaccharide Extracted by Enzymatic Hydrolysis Combined with Ultrasonic Assisted Extraction

[0119] Using the DPPH free radical scavenging method, the antioxidant activity of Ganoderma lucidum polysaccharide extracted without enzymatic hydrolysis in Comparative Example 3 and after enzymatic hydrolysis in Example 8 in scavenging DPPH free radicals was measured. The results are shown in Table 6 and Appendix Figure 2 .

[0120] Table 6 DPPH Free Radical Scavenging Rates of Ganoderma lucidum Polysaccharide Extracted without and after Enzymatic Hydrolysis

[0121]

[0122] Calculated from the data in Table 6, the EC 50 of the Ganoderma lucidum polysaccharide extracted after enzymatic hydrolysis in scavenging DPPH free radicals was 0.141 mg / mL, which was significantly lower than that of the Ganoderma lucidum polysaccharide extracted without enzymatic hydrolysis (EC 50 = 0.217 mg / mL), indicating that the antioxidant activity of the Ganoderma lucidum polysaccharide extracted after enzymatic hydrolysis was significantly better than that of the polysaccharide extracted without enzymatic hydrolysis. The EC 50 refers to the median effect concentration, that is, the dose required for the test substance to cause a 50% change in a certain biological effect.

[0123] The method for determining the DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging rate is as follows: Prepare aqueous solutions with polysaccharide concentrations of 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, and 2.0 mg / mL. Add 1 mL of the sample solution and 1 mL of the DPPH absolute ethanol solution (0.2 mmol / L) to a test tube. After thorough mixing, react in the dark for 30 min. Using absolute ethanol as the reference, measure the absorbance at a wavelength of 517 nm, denoted as Ai. According to the above steps, replace the DPPH ethanol solution and the sample solution with absolute ethanol respectively, measure the absorbance at a wavelength of 517 nm, denoted as Aj and A0 respectively, and calculate the DPPH free radical scavenging rate (I) according to the following formula 2.

[0124]

[0125] As mentioned above, it is only a preferred embodiment of the present invention and does not impose any limitations on the present invention. Any simple modifications, changes, amplifications, and equivalent transformations made to the above embodiments according to the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A method for improving the extraction rate of ganoderma lucidum polysaccharides by combining enzymatic hydrolysis with ultrasound, characterized in that: The method comprises the following steps: (1) Slicing, drying and crushing the collected ganoderma lucidum, adding deionized water and cellulase to the obtained collected ganoderma lucidum powder, and stirring evenly to form a collected ganoderma lucidum enzymatic hydrolysis system; (2) The enzymatic hydrolysis system of the powdered Ganoderma lucidum is hydrolyzed in a water bath at a temperature of 35 to 45° C. for 3 to 5 hours, then extracted in boiling water for 2 to 3 hours, and then extracted in an ultrasonic bath at a water temperature of 30 to 40° C. for 30 to 45 minutes, and then filtered to collect the filtrate to obtain the powdered Ganoderma lucidum water extract; (3) The powdered Ganoderma lucidum water extract is concentrated to 1 / 20 to 1 / 30 of the original volume, ethanol is added to precipitate the polysaccharide, the precipitate is washed with anhydrous ethanol, and vacuum dried to obtain the powdered Ganoderma lucidum polysaccharide extract.

2. The method according to claim 1, characterized in that The collected ganoderma lucidum in step (1) is the fruiting body of artificially cultivated Ganoderma lucidum, which is used to collect ganoderma lucidum spore powder. After no more spores are produced, the collected ganoderma lucidum is plucked out and naturally dried. The collected ganoderma lucidum powder is obtained by cutting the collected ganoderma lucidum into slices with a thickness of 2 to 3 mm, drying at 85° C. to constant weight, and then crushing and passing through a 20-mesh sieve to obtain a coarse powder.

3. The method according to claim 1, characterized in that The pH of the deionized water in step (1) is 5.0-5.5, and the added volume is 30-35 mL / g based on the mass of the collected Ganoderma lucidum powder.

4. The method according to claim 1, characterized in that The amount of cellulase added in step (1) is 1750-2100 U activity / g based on the mass of the collected Ganoderma lucidum powder.

5. The method according to claim 1, characterized in that The frequency of the ultrasonic wave in step (2) is 40kHz, and the power is 120-160W.

6. The method according to claim 1, characterized in that The preparation method of the powdered Ganoderma lucidum polysaccharide extract in step (3) is as follows: the powdered Ganoderma lucidum water extract is concentrated under reduced pressure at 60° C. and relative vacuum degree of -0.1 MPa to 1 / 20 to 1 / 30 of the original volume, and then anhydrous ethanol is added in an amount 3 to 4 times the volume of the concentrated solution to make the ethanol volume concentration of the system reach 75% to 80%, and after standing at 4° C. for 12 to 18 hours, the extract is centrifuged at 8000×g for 5 to 10 minutes to collect the precipitate; the precipitate is washed once with anhydrous ethanol at a concentration of 2 to 4 mL / g based on the mass of the raw material powdered Ganoderma lucidum powder, and after centrifugation again, the precipitate is vacuum dried at 65° C. and relative vacuum degree of -0.1 MPa to constant weight to obtain the powdered Ganoderma lucidum polysaccharide extract.

7. A powdered Ganoderma lucidum polysaccharide extract prepared by the method of claim 1.

8. Use of the powdered Ganoderma lucidum polysaccharide extract according to claim 7 in the preparation of antioxidants.

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