Co-production method of ganoderan and ergothioneine

Abstract

The invention provides a method for co-producing ganoderan and ergothioneine, which adopts a continuous fermentation method and comprises the following steps: performing sterile coarse filtration on mycelia, flushing with a sterile fresh fermentation medium, returning to the fermentation medium for continuous fermentation, performing polysaccharide extraction on coarse filtrate, and continuously collecting supernate for extracting ergothioneine, and after continuously and circularly fermenting for 3-10 times, mechanically crushing mycelium, and further extracting polysaccharide and ergothioneine. According to the method, through continuous fermentation and combined extraction of ganoderan and ergothioneine, the fermentation efficiency is improved, meanwhile, emission of waste liquid and waste residues is reduced, and the resource utilization value of the waste liquid and mycelia is greatly improved.

Description

Technical Field

[0001] The invention relates to the technical field of biochemical engineering, and in particular to a method for co-producing ganoderma lucidum polysaccharide and ergothioneine. Background Art

[0002] Ganoderma lucidum is known as the "Oriental Fairy Grass" and is rich in polysaccharides, triterpenes, proteins, peptides, vitamins and other substances. Among them, Ganoderma lucidum polysaccharide (GLP) is an important biologically active ingredient of Ganoderma lucidum fungus and is widely developed and utilized because of its extremely high medicinal value.

[0003] Ergothioneine (EGT) is a rare natural amino acid, also known as a natural intracellular antioxidant. It cannot be synthesized by the human body and can only be ingested from food and absorbed and accumulated in the body. Studies have shown that ergothioneine exists in edible and medicinal fungi such as boletus, oyster mushroom, golden top pleurotus, cordyceps, and ganoderma lucidum. It can effectively resist aging, improve sleep difficulties, inhibit and repair cell damage, etc. There is also a large amount of literature proving that the polysaccharides in ganoderma lucidum and ergothioneine play a synergistic role in anti-oxidative damage, cardiovascular protection, anti-inflammatory, and anti-anxiety and depression.

[0004] In the fermentation process, ergothioneine in conventional Ganoderma lucidum fermentation is often treated as waste liquid or waste residue due to its low content and high extraction cost. Therefore, the present invention provides a method for simultaneously extracting Ganoderma lucidum polysaccharide and ergothioneine in a continuous fermentation process, which helps to improve the utilization of effective ingredients in Ganoderma lucidum fermentation. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for co-producing ganoderma lucidum polysaccharide and ergothioneine.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] The invention provides a method for co-producing ganoderma polysaccharide and ergothioneine, which comprises: (1) sequentially performing fermentation treatment and aseptic filtration treatment on a ganoderma strain to obtain a filtrate and mycelium; (2) extracting polysaccharide from the filtrate to obtain ganoderma polysaccharide and supernatant; washing the mycelium with a sterile and fresh fermentation medium and returning the mycelium for further fermentation treatment and aseptic filtration treatment, and continuously collecting the supernatant generated during the treatment process; after continuous cyclic fermentation treatment for 3 to 10 times, mechanically crushing the mycelium and performing enzymatic extraction treatment to obtain ganoderma polysaccharide and ergothioneine.

[0008] Furthermore, the step (2) specifically comprises: adding 4-8 times the volume of 95% anhydrous ethanol to the filtrate, standing at 4-8°C until flocculent stratification occurs, centrifuging at 3000-8000 r / min for 10 minutes, and collecting the supernatant and the centrifugal precipitate respectively; wherein the centrifugal precipitate is the crude extract of Ganoderma lucidum polysaccharide; inoculating the mycelium into the fermentation medium again, and replenishing the fermentation medium with fresh fermentation medium to the initial fermentation volume, and cyclic fermentation treatment for 3-10 times; collecting the mycelium obtained after 3-10 consecutive cyclic fermentations, adding 3-10 times the volume of pure water, and obtaining a solution containing mycelium; mechanically crushing the solution containing mycelium for 30-40 minutes, until until it becomes a viscous solution; add 0.1-0.1% of the composite enzyme solution to the viscous solution, and perform enzymolysis at 45-55° C. for 2.5-3h to obtain an enzymolysis solution; first centrifuge the enzymolysis solution at 3000-4000r / min, extract the obtained centrifuge liquid, and obtain a supernatant and a centrifugal precipitate again; collect and combine the supernatants, and then perform adsorption treatment and elution treatment in sequence to obtain an eluate; decolorize the obtained supernatant to obtain a relatively clear decolorized liquid, filter the decolorized liquid, and let the obtained clear liquid stand overnight; combine and mix the clear liquids, perform agarose gel chromatography filtration, and further obtain a filtrate; perform ultrafiltration on the filtrate to obtain the Ganoderma lucidum polysaccharide and ergothioneine.

[0009] Furthermore, the complex enzyme includes one or more of cellulase, lysozyme and glucanase.

[0010] Furthermore, the adsorption treatment includes adsorbing the supernatant with a macroporous adsorption resin, and the usage ratio of the macroporous adsorption resin is 1-3%; the elution treatment is eluted with pure water; wherein the pore size of the macroporous adsorption resin is 0.3-1.25 mm.

[0011] Further, the ultrafiltration treatment includes: selecting a rolled ultrafiltration membrane for ultrafiltration, a total of two layers of interception filter membranes are provided, the extract passes through a first layer of ultrafiltration membrane, the molecular retention is 6-12kDa, the pressure difference is 0.5-1.5MP, the temperature is 35-40°C, the filter membrane intercepts the liquid as a Ganoderma lucidum polysaccharide solution, and the remaining liquid passes through a second layer of ultrafiltration membrane, the molecular retention is 3k-6kDa, the pressure difference is 0.5-1.5MP, and the filter membrane permeates the liquid as an ergothioneine solution.

[0012] Furthermore, the step (1) specifically includes: culturing the Ganoderma lucidum strain on a solid culture medium to obtain a bacterial block; inoculating the bacterial block into a liquid seed culture medium, and culturing on a shaking table for 4-6 days to obtain a seed solution; inoculating the seed solution into a fermentation medium at an inoculation rate of 5-10%, and fermenting to obtain a fermentation liquid; collecting 30%-50% of the fermentation liquid in the fermentation medium, and aseptically filtering the fermentation liquid to obtain a filtrate and mycelium.

[0013] Furthermore, the conditions of the shaking culture and fermentation culture are both 25-35°C and 100-200r / min.

[0014] Furthermore, the method also includes: preparing Ganoderma lucidum strains and preparing solid culture medium, liquid seed culture medium and fermentation culture medium.

[0015] Furthermore, the steps of preparing the solid culture medium, the liquid seed culture medium and the fermentation culture medium specifically include: using 20% ​​potato juice, 2% glucose, 2% agar powder or agar strips, natural pH, sterilizing at 121°C for 15-20min to obtain a solid culture medium; the solid culture medium is used to culture the ganoderma lucidum strain to obtain a bacterial block; using 20% ​​potato juice, 2% glucose, natural pH, as a liquid seed culture medium, sterilizing at 121°C for 15-20min to obtain a liquid seed culture medium; the liquid seed culture medium is used to culture the bacterial block to obtain a seed liquid; using 2-5% starch, 1-3% glucose, 3-6% yeast powder and 0.01-0.05% potassium dihydrogen phosphate, adjusting the pH to 6.5, sterilizing at 121°C for 15-20min to obtain a fermentation culture medium; the fermentation culture medium is used to ferment the ganoderma lucidum strain to obtain ganoderma lucidum polysaccharide and ergothioneine.

[0016] Furthermore, the step of preparing the Ganoderma lucidum strain specifically includes: obtaining the Ganoderma lucidum strain from a preservation center; wherein the preservation number of the Ganoderma lucidum strain is CCGMC5.1816.

[0017] Compared with the prior art, the technical solution provided by the present invention has at least the following advantages:

[0018] The present invention provides a method for co-producing ganoderma lucidum polysaccharide and thioneine, adopts a continuous fermentation method, mycelium is rinsed with aseptic fresh fermentation medium after aseptic coarse filtration and returned to the fermentation tank for continued fermentation, after the coarse filtrate is subjected to polysaccharide extraction, the supernatant is continuously collected to extract thioneine, after continuous cycle fermentation 3-10 times, the mycelium is mechanically crushed, and polysaccharide and thioneine are further extracted. The method is through continuous fermentation, and ganoderma lucidum polysaccharide and thioneine are jointly extracted, and the fermentation efficiency is improved, while the discharge of waste liquid and waste residue is reduced, and thioneine is jointly extracted, and the resource utilization value of waste liquid and mycelium is greatly improved.

[0019] The present invention utilizes a continuous fermentation method to extract intracellular and extracellular ganoderma lucidum polysaccharides and its byproduct ergothioneine after culturing, thereby improving the utilization value of the byproducts, increasing the yield of the products, achieving the utilization rate of the filter residues, greatly reducing the discharge of wastewater and waste residues, and achieving high-value utilization.

[0020] The invention utilizes a continuous cycle fermentation method to culture ganoderma lucidum strains to generate ganoderma lucidum polysaccharides and ergothioneine, continuously adds sterile fresh culture medium raw materials into the fermentation process, continuously generates products, can realize 3-10 fermentation cycles, realizes joint extraction of the byproduct ergothioneine, improves the ganoderma lucidum fermentation efficiency, and realizes high value and resource utilization of the byproducts through the jointly extracted ergothioneine. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The present invention provides a flow chart of a method for co-producing ganoderma lucidum polysaccharide and ergothioneine. DETAILED DESCRIPTION

[0022] At present, Ganoderma lucidum polysaccharide is produced by batch fermentation method, and its fermentation liquid and mycelium are rich in Ganoderma lucidum polysaccharide, amino acids, triterpenes and other active compounds. Ergothioneine is a rare amino acid, which is present in higher content in Ganoderma lucidum fruiting bodies or fermented mycelium.

[0023] The inventors have found that in the prior art, Ganoderma lucidum fermentation is carried out in batches, the main component of the fermentation liquid is extracellular polysaccharide, and the mycelium also contains a certain amount of intracellular polysaccharide. In conventional Ganoderma lucidum fermentation, ergothioneine is often treated as waste liquid or waste residue due to its low content and high extraction cost.

[0024] Based on this, the present invention provides a method for co-producing ganoderma lucidum polysaccharide and ergothioneine, such as Figure 1 As shown, the method includes: the following are all volume fractions.

[0025] S1. Preparation of bacterial strains and culture medium

[0026] S11. Ganoderma lucidum strain: obtained from a collection center. The strain used in the present invention is numbered CCGMC5.1816.

[0027] S12. Solid culture medium and liquid seed culture medium: Use 20% potato juice, 2% glucose, 2% agar powder or agar strips, natural pH, sterilize at 121°C for 15-20 minutes to obtain a solid culture medium.

[0028] 20% potato juice, 2% glucose and natural pH are used as liquid seed culture medium, which is sterilized at 121° C. for 15-20 minutes to obtain liquid seed culture medium.

[0029] S13, fermentation medium: 2-5% starch, 1-3% glucose, 3-6% yeast powder and 0.01-0.05% potassium dihydrogen phosphate are used, the pH is adjusted to 6.5, and sterilized at 121° C. for 15-20 minutes to obtain a fermentation medium. Among them, potato starch is the best.

[0030] S2. Fermentation and culture of Ganoderma lucidum polysaccharide

[0031] S21. Preparation of liquid seeds of Ganoderma lucidum: inoculate the fungus blocks from the slant or plate of Ganoderma lucidum into liquid seed culture medium, and culture in a shaking incubator at 25-35°C and 100-200 r / min for 4-6 days.

[0032] S22, inoculating the cultured seed liquid into a shake flask containing fermentation medium (hereinafter referred to as fermentation shake flask) at an inoculation rate of 5-10%, and fermenting and culturing at 25-35°C and 100-200 r / min.

[0033] S23, continuous fermentation: 30%-50% of the fermentation liquid in the fermentation shake flask is collected, and the fermentation liquid is filtered through 3-8 layers of sterile gauze, and the filtrate and mycelium are collected respectively. The collected mycelium is re-inoculated into the fermentation shake flask, and fresh fermentation medium is added to the initial fermentation volume. This step can be repeated for 3-10 cycles.

[0034] S3, Ganoderma lucidum polysaccharide and ergothioneine co-extraction step

[0035] S31. Ethanol extraction of Ganoderma lucidum polysaccharide: add 4-8 times volume of 95% anhydrous ethanol to the filtrate collected by continuous fermentation, place in a refrigerator at 4-8°C overnight or for 24 hours until flocculent stratification occurs, centrifuge at 3000-8000r / min for 10 minutes in a high-speed centrifuge, and collect the supernatant and centrifugal precipitate respectively. The precipitate is the crude extract of Ganoderma lucidum polysaccharide.

[0036] S32, collecting the fermented mycelium of Ganoderma lucidum obtained by continuous cycle fermentation for 3-10 times, adding 3-10 times volume of pure water, and mechanically crushing the solution containing mycelium in a high-speed homogenizer for 30-40 minutes until it becomes a viscous solution.

[0037] S33, in order to completely dissolve the mycelium, add 0.1-0.1% of complex enzyme solution to the solution obtained in S32, the complex enzyme includes cellulase, lysozyme and glucanase, and the ratio is not limited. Perform enzymolysis at 45-55°C for 2.5-3h to obtain an enzymolysis solution.

[0038] S34, centrifuge the enzymatic solution with a 3000-4000r / min centrifuge, and extract the crude polysaccharide from the obtained centrifuge using the same operation steps as S31. Collect and combine the supernatant, adsorb it with a macroporous adsorption resin, and the amount of the macroporous adsorption resin is 1-3% (w / v). Elute with pure water and collect the eluate; wherein the pore size of the macroporous adsorption resin is 0.3-1.25mm.

[0039] S35, add 2-4% (w / v) activated carbon to the supernatant obtained in S31 and S34 for decolorization to obtain a relatively clear decolorized liquid. Filter the decolorized liquid and let the obtained clear liquid stand.

[0040] S36, combining and mixing the clarified liquids, and filtering them by agarose gel chromatography to further obtain a filtrate.

[0041] S37, filtrate is ultrafiltered, further separation of ganoderma lucidum polysaccharide and thioneine is filtered and classified.Selection roll ultrafiltration membrane to carry out ultrafiltration, two layers of interception filter membrane are set, extract crosses the first layer of ultrafiltration membrane, and molecular cutoff is 6-12kDa, pressure difference 0.5-1.5MP, temperature is 35-40 DEG C, and the filter membrane interception liquid is ganoderma lucidum polysaccharide solution, and the remaining liquid crosses the second layer of ultrafiltration membrane, and molecular cutoff is 3k-6kDa, pressure difference 0.5-1.5MP, and the filter membrane sees through liquid is thioneine solution.

[0042] That is, the method provided by the present invention finally jointly extracts Ganoderma lucidum polysaccharide and the byproduct ergothioneine from the continuous fermentation process of Ganoderma lucidum polysaccharide.

[0043] The present invention is described in detail below in conjunction with specific implementation modes.

[0044] Example 1

[0045] 1. Preparation of Ganoderma lucidum liquid seeds: dig 1cm thick liquid seeds from the inclined surface or flat plate of Ganoderma lucidum. 2 The bacterial block was inoculated in a liquid seed culture medium. The liquid culture medium in the shake flask was 50 mL (using a total volume of 250 mL shake flask). The culture was carried out in a shaker at 26°C and 100 r / min for 4 days. A total of 4 seed shake flasks were configured.

[0046] 2. Inoculate 4-day-cultured Ganoderma lucidum seed liquid into a fermentation shake flask (fermentation shake flask capacity 500mL, liquid volume 100mL) at a 5% inoculation rate, and ferment and culture for 5 days at 25°C and a shaker speed of 100r / min. A total of 10 flasks were prepared.

[0047] 3. Release and collect 30% of the fermentation liquid in the fermentation shake flask, that is, collect 30mL of fermentation liquid. The specific method is: filter the fermentation liquid with 8 layers of sterile gauze on a clean bench, and collect the filtrate and mycelium respectively. Among them, the collected mycelium is re-inoculated into the fermentation shake flask, and the initial fermentation volume is supplemented with fresh fermentation medium. This step is repeated 3 times, and a total of (30×3+100)×10=1900mL of fermentation liquid is collected. Experiments have shown that the filtrate after filtration is 85%, that is, the filtrate is 1615mL, the wet mycelium accounts for 15%, and the volume is 285mL.

[0048] 4. Combined extraction of Ganoderma lucidum polysaccharide and ergothioneine

[0049] (1) First, the extracellular polysaccharide of the filtrate was extracted, and the crude polysaccharide was extracted by alcohol extraction: 4 times the volume of 95% anhydrous ethanol was added to 1615 mL of the fermentation filtrate, and the total volume was 8075 mL. The mixture was allowed to stand in a refrigerator at 4°C overnight until flocculent stratification appeared, and then centrifuged at 8000 r / min for 10 min in a high-speed centrifuge. The resulting precipitate was crude Ganoderma lucidum polysaccharide. The supernatant was poured out and the total volume of the supernatant was 7720 mL. The volume of the precipitated Ganoderma lucidum polysaccharide was 355 mL, accounting for 4.396%, and ergothioneine was in the supernatant and could be further extracted.

[0050] (2) Separation of mycelial intracellular polysaccharide from ergothioneine: 285 mL of wet thalline (by volume) obtained in step 3 was added with 3 times the volume of pure water to obtain a total volume of 1140 mL. The diluted mycelial solution was mechanically crushed in a high-speed homogenizer for 30 min until it became a viscous solution.

[0051] (3) Cellulase, lysozyme and glucanase are configured into an enzymolysis solution in a mass ratio of 1:1:1, and enzymolysis is performed on 1140 mL of the broken viscous solution. The addition ratio of the enzymolysis solution is 0.1%, i.e., 1.14 mL, in order to further cause the mycelium to be enzymolyzed and broken, and release intracellular polysaccharides and intracellular ergothioneine.

[0052] (4) The enzymatically hydrolyzed mycelium solution was centrifuged at 3000 r / min for 10 min to remove impurities and mycelial fragments, and 1014.6 mL of supernatant was collected. 223.2 mL of Ganoderma lucidum crude polysaccharide was extracted from the 1014.6 mL of supernatant according to the method (1), and 791.4 mL of supernatant was obtained. The supernatant was collected and combined with the supernatant in (1) to obtain 8866.4 mL of supernatant.

[0053] (5) Adsorb with macroporous adsorption resin for 24 hours, the dosage ratio of macroporous adsorption resin is 1% (w / v), then elute with 1x ethanol for 6 hours, and collect 16846.16 mL of eluate.

[0054] (6) 2% (w / v) activated carbon was added to the 16846.16 mL eluate for decolorization for 6 hours to obtain a relatively clear decolorized solution.

[0055] (7) Agarose gel chromatography filtration was performed, and the filtrate loss was 7%, and 15498.47 mL of filtrate was further obtained.

[0056] (8) The filtrate is further ultrafiltered, and a roll-type ultrafiltration membrane is selected for ultrafiltration. Two layers of interception filter membranes are set. The extract passes through the first layer of ultrafiltration membrane, and the molecular cutoff is 6-12 kDa, the pressure difference is 0.5 MP, and the temperature is 35 ° C. The filter membrane intercepts the liquid as Ganoderma lucidum polysaccharide, and the yield is 3.5%, that is, 542.45 mL of Ganoderma lucidum polysaccharide liquid is obtained, and the remaining liquid passes through the second layer of ultrafiltration membrane, and the molecular cutoff is 3-6 kDa, the pressure difference is 0.5 MP, and the filter membrane permeates the liquid as thioneine solution. The collected liquid is 11778.83 mL, and the yield is 76%.

[0057] (9) Collect 355 mL, 223.2 mL, and 542.45 mL of Ganoderma lucidum crude polysaccharide solution

[0058] The total volume was 1120.65 mL. The solution was evaporated using a rotary evaporator, and ethanol was collected to obtain 257.75 g of Ganoderma lucidum polysaccharide.

[0059] (10) 11778.83 mL of the ultrafiltered thioneine solution was first concentrated by rotary evaporation and then subjected to low-temperature crystallization at -4°C to obtain 10.07 g of thioneine.

[0060] In summary, in this embodiment, the Ganoderma lucidum strain was fermented for 3 cycles to obtain 1900 mL of fermentation liquid, and then after the combined extraction of Ganoderma lucidum polysaccharide and thioneine step, 257.75 g of Ganoderma lucidum polysaccharide and 10.07 g of thioneine were obtained. The yields of Ganoderma lucidum polysaccharide and thioneine were 13.56% and 0.53%, respectively.

[0061] Example 2

[0062] 1. Preparation of Ganoderma lucidum liquid seeds: dig 1cm thick liquid seeds from the inclined surface or flat plate of Ganoderma lucidum. 2 The bacterial block was inoculated in a liquid seed culture medium. The liquid culture medium in the shake flask was 50 mL (using a total volume of 250 mL shake flask). The culture was carried out in a shaker at 26°C and 100 r / min for 4 days. A total of 4 seed shake flasks were configured.

[0063] 2. Inoculate 4-day-cultured Ganoderma lucidum seed liquid into a fermentation shake flask (fermentation shake flask capacity 500mL, liquid volume 100mL) at a 5% inoculation rate, and ferment and culture for 5 days at 25°C and a shaker speed of 100r / min. A total of 10 flasks were prepared.

[0064] 3. Release and collect 30% of the fermentation liquid in the fermentation shake flask, that is, collect 30mL of fermentation liquid. The specific method is: filter the fermentation liquid with 8 layers of sterile gauze on a clean bench, and collect the filtrate and mycelium respectively. Among them, the collected mycelium is re-inoculated into the fermentation shake flask, and the initial fermentation volume is supplemented with fresh fermentation medium. This step is repeated 6 times, and a total of (30×6+100)×10=2800mL of fermentation liquid is collected. Experiments have shown that the filtrate after filtration is 82%, that is, the filtrate is 2296mL, the wet mycelium accounts for 18%, and the volume is 504mL.

[0065] 4. Combined extraction of Ganoderma lucidum polysaccharide and ergothioneine

[0066] (1) First, the extracellular polysaccharide of the filtrate was extracted, and the crude polysaccharide was extracted by alcohol extraction: 6 volumes of 95% anhydrous ethanol were added to 2296 mL of the fermentation filtrate, and the total volume was 16072 mL. The mixture was allowed to stand in a refrigerator at 4°C overnight until flocculent stratification occurred, and then centrifuged at 8000 r / min for 10 min in a high-speed centrifuge. The resulting precipitate was crude Ganoderma lucidum polysaccharide. The supernatant was poured out and the total volume of the supernatant was 15,324.65 mL. The volume of the precipitated Ganoderma lucidum polysaccharide was 747.35 mL, accounting for 4.65%, and ergothioneine was in the supernatant and could be further extracted.

[0067] (2) Separation of mycelial intracellular polysaccharide from ergothioneine: 504 mL of wet thalline (by volume) obtained in step 3 was added with 5 times the volume of pure water to obtain a total volume of 3024 mL. The diluted mycelial solution was mechanically crushed in a high-speed homogenizer for 30 min until it became a viscous solution.

[0068] (3) Cellulase, lysozyme and glucanase are configured into an enzymolysis solution in a mass ratio of 1:1:1, and 3024 mL of the broken viscous solution is subjected to enzymolysis, and the addition ratio of the enzymolysis solution is 0.1%, i.e. 3.024 mL, in order to further cause the mycelium to be enzymolyzed and broken, and release intracellular polysaccharides and intracellular ergothioneine.

[0069] (4) The enzymatically hydrolyzed mycelium solution was centrifuged at 3000 r / min for 10 min to remove impurities and mycelial fragments, and 2646 mL of supernatant was collected. 854.11 mL of crude Ganoderma lucidum polysaccharide was extracted from the 2646 mL of supernatant according to the method (1), and 1791.89 mL of supernatant was obtained. The supernatant was collected and combined with the supernatant in (1) to obtain 17116.54 mL of supernatant.

[0070] (5) Adsorb with macroporous adsorption resin for 24 hours, the dosage ratio of macroporous adsorption resin is 1% (w / v), then elute with 1x ethanol for 6 hours, and collect 32589.89 mL of eluate.

[0071] (6) 2% (w / v) activated carbon was added to the 32589.89 mL eluate for decolorization for 6 hours to obtain a relatively clear decolorized solution.

[0072] (7) Agarose gel chromatography filtration was performed, and the filtrate loss was 7.1%, and 30276.01 mL of filtrate was further obtained.

[0073] (8) The filtrate is further ultrafiltered, and a roll-type ultrafiltration membrane is selected for ultrafiltration. Two layers of interception filter membranes are set. The extract passes through the first layer of ultrafiltration membrane, and the molecular retention capacity is 6-12 kDa, the pressure difference is 0.5 MP, and the temperature is 35 ° C. The filter membrane intercepts the liquid as Ganoderma lucidum polysaccharide, and the yield is 3.65%, that is, 1105.07 mL of Ganoderma lucidum polysaccharide liquid is obtained, and the remaining liquid passes through the second layer of ultrafiltration membrane, and the molecular retention capacity is 3-6 kDa, the pressure difference is 0.5 MP, and the filter membrane permeates the liquid as thioneine solution. The collected liquid is 23312.53 mL, and the yield is 77%.

[0074] (9) The crude polysaccharide component solutions of Ganoderma lucidum were collected, and the total amount was 2706.5 mL. The solution was evaporated by a rotary evaporator, and ethanol was collected to obtain 649.56 g of Ganoderma lucidum polysaccharide.

[0075] (10) 23312.53 mL of the ultrafiltered thioneine solution was first concentrated by rotary evaporation and then subjected to low-temperature crystallization at -4°C to obtain 20.1 g of thioneine.

[0076] In summary, in this embodiment, after 6 cycles of fermentation, 2800 mL of fermentation liquid was obtained from the Ganoderma lucidum strain, and after the combined extraction of Ganoderma lucidum polysaccharide and thioneine, 649.56 g of Ganoderma lucidum polysaccharide and 36.37 g of thioneine were obtained respectively. The yields of Ganoderma lucidum polysaccharide and thioneine were 23.2% and 0.72%, respectively.

[0077] Example 3

[0078] 1. Preparation of Ganoderma lucidum liquid seeds: dig 1cm thick liquid seeds from the inclined surface or flat plate of Ganoderma lucidum. 2 The bacterial block was inoculated in a liquid seed culture medium. The liquid culture medium in the shake flask was 50 mL (using a total volume of 250 mL shake flask). The culture was carried out in a shaker at 26°C and 100 r / min for 4 days. A total of 4 seed shake flasks were configured.

[0079] 2. Inoculate 4-day-cultured Ganoderma lucidum seed liquid into a fermentation shake flask (fermentation shake flask capacity 500mL, liquid volume 100mL) at a 5% inoculation rate, and ferment and culture for 5 days at 25°C and a shaker speed of 100r / min. A total of 10 flasks were prepared.

[0080] 3. Release and collect 30% of the fermentation liquid in the fermentation shake flask, that is, collect 30mL of fermentation liquid. The specific method is: filter the fermentation liquid with 8 layers of sterile gauze on a clean bench, and collect the filtrate and mycelium respectively. Among them, the collected mycelium is re-inoculated into the fermentation shake flask, and the initial fermentation volume is supplemented with fresh fermentation medium. This step is repeated 10 times, and a total of (30×10+100)×10=4000mL of fermentation liquid is collected. Experiments have shown that the filtrate after filtration is 81%, that is, the filtrate is 3240mL, the wet mycelium accounts for 19%, and the volume is 760mL.

[0081] 4. Combined extraction of Ganoderma lucidum polysaccharide and ergothioneine

[0082] (1) First, the extracellular polysaccharide of the filtrate was extracted, and the crude polysaccharide was extracted by alcohol extraction: 8 times the volume of 95% anhydrous ethanol was added to 3240 mL of the fermentation filtrate, and the total volume was 29160 mL. The mixture was allowed to stand in a refrigerator at 4°C overnight until flocculent stratification appeared, and then centrifuged at 8000 r / min for 10 min in a high-speed centrifuge. The resulting precipitate was crude Ganoderma lucidum polysaccharide. The supernatant was poured out and the total volume of the supernatant was 27842 mL. The volume of the precipitated Ganoderma lucidum polysaccharide was 1341.94 mL, accounting for 4.602%, and ergothioneine was in the supernatant and could be further extracted.

[0083] (2) Separation of mycelial intracellular polysaccharide from ergothioneine: 760 mL of wet thalline (by volume) obtained in step 3 was added with 8 times the volume of pure water to obtain a total volume of 6840 mL. The diluted mycelial solution was mechanically crushed in a high-speed homogenizer for 30 min until it became a viscous solution.

[0084] (3) Cellulase, lysozyme and glucanase are configured into an enzymolysis solution in a mass ratio of 1:1:1, and enzymolysis is performed on 6840 mL of the broken viscous solution. The addition ratio of the enzymolysis solution is 0.1%, i.e. 6.84 mL, in order to further cause the mycelium to be enzymolyzed and broken, and release intracellular polysaccharides and intracellular ergothioneine.

[0085] (4) The enzymatically hydrolyzed mycelium solution was centrifuged at 3000 r / min for 10 min to remove impurities and mycelial fragments, and 6060.24 mL of supernatant was collected. 2510.03 mL of Ganoderma lucidum crude polysaccharide was extracted from the 6060.24 mL of supernatant according to the method of (1), and 3550.21 mL of supernatant was obtained. The supernatant was collected and combined with the supernatant of (1) to obtain 32710.21 mL of supernatant.

[0086] (5) Adsorb with macroporous adsorption resin for 24 hours, the dosage ratio of macroporous adsorption resin is 1% (w / v), then elute with 1x ethanol for 6 hours, and collect 62672.76 mL of eluate.

[0087] (6) 2% (w / v) activated carbon was added to the 62672.76 mL eluate for decolorization for 6 hours to obtain a relatively clear decolorized solution.

[0088] (7) Agarose gel chromatography filtration was performed, and the filtrate loss was 7.08%, and 58235.53 mL of filtrate was further obtained.

[0089] (8) The filtrate is further ultrafiltered, and a roll-type ultrafiltration membrane is selected for ultrafiltration. Two layers of interception filter membranes are set. The extract passes through the first layer of ultrafiltration membrane, and the molecular cutoff is 6-12 kDa, the pressure difference is 0.5 MP, and the temperature is 35 ° C. The filter membrane intercepts the liquid as Ganoderma lucidum polysaccharide, and the yield is 3.6%. That is, 2096.5 mL of Ganoderma lucidum polysaccharide liquid is obtained, and the remaining liquid passes through the second layer of ultrafiltration membrane, and the molecular cutoff is 3-6 kDa, the pressure difference is 0.5 MP, and the filter membrane permeates the liquid as thioneine solution. The collected liquid is 44608.41 mL, and the yield is 76.6%.

[0090] (9) The crude polysaccharide component solutions of Ganoderma lucidum were collected, including 1341.94 mL, 2510.03 mL, and 2096.5 mL, totaling 5948.47 mL. The solution was evaporated using a rotary evaporator, and ethanol was collected to obtain 1314.5 g of Ganoderma lucidum polysaccharide.

[0091] (10) 44608.41 mL of the ultrafiltered thioneine solution was first concentrated by rotary evaporation and then subjected to low-temperature crystallization at -4°C to obtain 39.25 g of thioneine.

[0092] In summary, in this embodiment, after 10 cycles of fermentation, 4000 mL of fermentation liquid was obtained from the Ganoderma lucidum strain, and after the combined extraction of Ganoderma lucidum polysaccharide and thioneine step, 1314.5 g of Ganoderma lucidum polysaccharide and 39.25 g of thioneine were obtained respectively. The yields of Ganoderma lucidum polysaccharide and thioneine were 32.86% and 0.98%, respectively.

[0093] Those skilled in the art can understand that the above-mentioned embodiments are specific examples for implementing the present application, and in practical applications, various changes can be made to them in form and detail without departing from the spirit and scope of the present application. Any person skilled in the art can make their own changes and modifications without departing from the spirit and scope of the present application, so the scope of protection of the present application shall be based on the scope defined in the claims.

Claims

1. A method for co-producing ganoderma lucidum polysaccharide and ergothioneine, characterized in that: The method includes: (1) subjecting the Ganoderma lucidum strain to fermentation treatment and sterile filtration treatment in sequence to obtain a filtrate and mycelium; (2) extracting polysaccharides from the filtrate to obtain ganoderma polysaccharides and a supernatant; rinsing the mycelium with a sterile, fresh fermentation medium and returning the medium for continued fermentation and sterile filtration, and continuing to collect the supernatant produced during the process; after 3-10 consecutive cycles of fermentation, mechanically crushing the mycelium and performing enzymatic extraction to obtain ganoderma polysaccharides and ergothioneine.

2. The method for co-producing ganoderma lucidum polysaccharide and ergothioneine according to claim 1, characterized in that: The step (2) specifically comprises: Add 4-8 times the volume of 95% anhydrous ethanol to the filtrate, let it stand at 4-8°C until flocculent layers appear, centrifuge at 3000-8000 r / min for 10 minutes, and collect the supernatant and centrifugal precipitate respectively; wherein the centrifugal precipitate is the crude extract of Ganoderma lucidum polysaccharide; The mycelium is inoculated into the fermentation medium again, and the fermentation medium is supplemented to the initial fermentation volume, and the fermentation process is repeated for 3-10 times; the mycelium obtained after the continuous fermentation for 3-10 times is collected, and 3-10 times the volume of pure water is added to obtain a solution containing mycelium; the solution containing mycelium is mechanically crushed for 30-40 minutes until it becomes a viscous solution; Adding 0.1-0.1% of the complex enzyme solution to the viscous solution, and performing enzymolysis at 45-55° C. for 2.5-3 hours to obtain an enzymolysis solution; The enzymatic hydrolysis solution is first centrifuged at 3000-4000 r / min, and the obtained centrifuge is extracted to obtain a supernatant and a centrifugal precipitate again; the supernatant is collected and combined, and then subjected to adsorption treatment and elution treatment in sequence to obtain an eluate; The obtained supernatant is decolorized to obtain a relatively clear decolorized solution, the decolorized solution is filtered, and the obtained clear solution is allowed to stand overnight; The clarified solutions are combined and mixed to perform agarose gel chromatography filtration to further obtain a filtrate; The filtrate is subjected to ultrafiltration treatment to obtain the ganoderma lucidum polysaccharide and ergothioneine.

3. The method for co-producing ganoderma lucidum polysaccharide and ergothioneine according to claim 2, characterized in that: The complex enzyme comprises one or more of cellulase, lysozyme and glucanase.

4. The method for co-producing ganoderma lucidum polysaccharide and ergothioneine according to claim 2, characterized in that: The adsorption treatment includes adsorbing the supernatant with a macroporous adsorption resin, the dosage of which is 1-3%; the elution treatment includes eluting with pure water; wherein the pore size of the macroporous adsorption resin is 0.3-1.25 mm.

5. The method for co-producing ganoderma lucidum polysaccharide and ergothioneine according to claim 2, characterized in that: The ultrafiltration treatment comprises: selecting a rolled ultrafiltration membrane for ultrafiltration, arranging two layers of interception filter membranes, the extract passes through the first layer of ultrafiltration membrane, the molecular interception is 6-12 kDa, the pressure difference is 0.5-1.5 MP, the temperature is 35-40 DEG C, the filter membrane intercepts the liquid which is a ganoderma polysaccharide solution, the remaining liquid passes through the second layer of ultrafiltration membrane, the molecular interception is 3k-6 kDa, the pressure difference is 0.5-1.5 MP, and the filter membrane permeates the liquid which is an ergothioneine solution.

6. The method for co-producing ganoderma lucidum polysaccharide and ergothioneine according to claim 1, characterized in that: The step (1) specifically comprises: The ganoderma lucidum strain is cultured on a solid culture medium to obtain a bacterial block; Inoculating the bacterial block into a liquid seed culture medium, and culturing on a shaking table for 4-6 days to obtain a seed liquid; Inoculating the seed liquid into a fermentation medium at an inoculation rate of 5-10%, fermenting and culturing to obtain a fermentation liquid; 30%-50% of the fermentation liquid in the fermentation medium is collected, and the fermentation liquid is sterile filtered to obtain the filtrate and mycelium.

7. The method for co-producing ganoderma lucidum polysaccharide and ergothioneine according to claim 6, characterized in that: The conditions of the shaking culture and the fermentation culture are both 25-35° C. and 100-200 r / min.

8. The method for co-producing ganoderma lucidum polysaccharide and ergothioneine according to claim 1, characterized in that: The method also includes: preparing ganoderma lucidum strains and preparing solid culture medium, liquid seed culture medium and fermentation culture medium.

9. The method for co-producing ganoderma lucidum polysaccharide and ergothioneine according to claim 8, characterized in that: The steps of preparing the solid culture medium, the liquid seed culture medium and the fermentation culture medium specifically include: 20% potato juice, 2% glucose, 2% agar powder or agar strips, natural pH, sterilized at 121° C. for 15-20 minutes to obtain a solid culture medium; the solid culture medium is used to culture the Ganoderma lucidum strain to obtain a bacterial block; Using 20% ​​potato juice and 2% glucose at a natural pH as a liquid seed culture medium, sterilizing at 121° C. for 15-20 minutes to obtain a liquid seed culture medium; the liquid seed culture medium is used to culture bacterial blocks to obtain seed liquid; The method comprises using 2-5% starch, 1-3% glucose, 3-6% yeast powder and 0.01-0.05% potassium dihydrogen phosphate, adjusting the pH to 6.5, sterilizing at 121° C. for 15-20 minutes, and obtaining a fermentation medium; the fermentation medium is used for fermenting a ganoderma lucidum strain to obtain ganoderma lucidum polysaccharide and ergothioneine.

10. The method for co-producing ganoderma lucidum polysaccharide and ergothioneine according to claim 8, characterized in that: The step of preparing the Ganoderma lucidum strain specifically includes: obtaining the Ganoderma lucidum strain from a preservation center; wherein the preservation number of the Ganoderma lucidum strain is CCGMC5.1816.

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