Application and method of gold nanomaterials in improving yield of ganoderma lucidum active products polysaccharide and triterpene
By adding gold nanomaterials to the liquid fermentation medium of Ganoderma lucidum, the problem of low yield of Ganoderma lucidum polysaccharides and triterpenes was solved, and the yield of polysaccharides was significantly increased and the yield of triterpenes was steadily improved, providing a new method for the synthesis of active products of Ganoderma lucidum.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- HAINAN UNIV
- Filing Date
- 2023-05-19
- Publication Date
- 2026-05-26
Smart Images
Figure CN116814441B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial fermentation technology, specifically providing an application and method of gold nanomaterials in increasing the yield of polysaccharides and triterpenes, active products of Ganoderma lucidum. Background Technology
[0002] In the research of nanomaterials, metallic nanomaterials have become a research hotspot due to their unique physical and chemical properties, such as small size effect, quantum size effect, surface effect, and macroscopic quantum tunneling effect, as well as their relatively low cost, stable properties, and good biocompatibility. The size of metals plays a crucial role in their physical and chemical properties. Bulk metals differ from metallic nanomaterials in their properties. Gold nanomaterials, due to their unique small size, good biocompatibility, and photostability, are particularly noteworthy. Gold nanomaterials mainly include gold nanoparticles, gold nanorods, and gold nanoclusters. The properties of various gold nanomaterials are as follows:
[0003] Gold nanoparticles, as the earliest studied gold nanomaterials, have well-developed synthesis and preparation techniques, allowing for the control of their size. Many researchers have also modified the surface of synthesized gold nanoparticles, further expanding the application range of nanomaterials. Gold nanoparticles possess unique optical, electric, and magnetic field properties.
[0004] Gold nanorods (AuNRs), as rod-shaped gold nanoparticles, are suitable for in vivo biological applications. AuNRs have been synthesized using various methods, including template methods, electrochemical methods, and seed growth methods. Currently, despite significant efforts to improve AuNR synthesis, high-yield, reproducible, and size- and morphology-controlled synthesis methods still require further development.
[0005] Gold nanoclusters are aggregates of several to hundreds of metal atoms, typically ranging in size from 1 to 10 nm. They exhibit fluorescence properties with a significant size-dependent effect. Their unique quantum size effect endows them with characteristics such as ultra-small size, good photostability, low toxicity, large Stokes shift, no flicker, and high catalytic activity.
[0006] The excellent properties of gold nanomaterials make them play an important role in biomedical research such as bioimaging, biosensing, quantitative protein analysis, and ion detection.
[0007] Ganoderma lucidum (G. lingzhi), a basidiomycete of the Ganoderma genus in the fungal kingdom, has fermentation products (such as Ganoderma lucidum polysaccharides, triterpenes, sterols, etc.) also known as secondary metabolites, which have antioxidant, anti-aging, anti-tumor, and lipid-lowering effects, and have extremely high medicinal and health value.
[0008] There are more than 200 kinds of Ganoderma lucidum polysaccharide components. According to the monosaccharide composition, they can be divided into two major categories: homopolysaccharides and heteropolysaccharides. Homopolysaccharides are composed of glucose or galactose. Heteropolysaccharides have a complex spatial structure, which is composed of a variety of monosaccharides linked in different proportions and with different types of glycosidic bonds. Their main chain is composed of monosaccharides mainly composed of glucose, galactose and mannose, and the side chains are composed of sugar units such as fucose, xylose and arabinose.
[0009] Ganoderma lucidum triterpenoids are diverse, with over 200 species isolated from Ganoderma lucidum fruiting bodies, artificially cultured mycelia, and culture media. The vast majority of Ganoderma lucidum triterpenoids are highly oxidized lanosterol derivatives, composed of isoprene units, including ganoderic acid, ganoderol, ganoderic acid, and ganoderic ketone, etc. They are classified according to the number of carbon atoms in their molecules into C... 24 C 27 and C 30 There are three main categories. Generally, triterpenoids containing carboxyl groups are called ganoderic acids. Ganoderic acids are the most important triterpenoids in ganoderic acid, and their representative group is the same as that of ursolic acid.
[0010] As people's demand for these substances increases, traditional methods of obtaining effective medicinal components from Ganoderma lucidum can no longer satisfy them. Instead, they are increasing yields by changing external conditions to create the best culture environment, such as temperature, inoculum size, pH, and environmental conditions. This method starts from the microorganisms themselves and selects a suitable living environment to achieve high yields of fermentation products. Another method is to add exogenous substances to stimulate the microorganisms to ferment and produce more secondary metabolites.
[0011] Currently reported exogenous additives for Ganoderma lucidum fermentation include traditional Chinese medicine substances, methyl jasmonate, metal ions, ethanol, and even rare earth elements. At present, more and more scientists are shifting their research focus to the effects of nanomaterials on microbial fermentation, but the effects of gold nanomaterials on microbial liquid fermentation have not been studied. Summary of the Invention
[0012] To address the low yield of polysaccharides and triterpenoids in Ganoderma lucidum fungi in existing technologies, this invention provides a method that can stably increase the yield of polysaccharides and triterpenoids with a simple production process. This invention utilizes purified gold nanomaterials as an exogenous additive to study its effect on the yield of polysaccharides and triterpenoids in Ganoderma lucidum liquid fermentation products, aiming to explore novel functional additives that promote the fermentation synthesis of active Ganoderma lucidum products and providing insights into the application of nanomaterials in the field of fungal fermentation.
[0013] To solve the above-mentioned technical problems, the present invention provides the following technical solution:
[0014] On the one hand, the present invention provides an application of gold nanomaterials in increasing the yield of polysaccharides and triterpenoids in Ganoderma lucidum fungi.
[0015] Furthermore, the polysaccharide compounds include, but are not limited to, glucose, and also contain one or more of the following: galactose, galactosamine, glucosamine, mannose, arabinose, xylose, fucose, rhamnose, xylose, and fructose.
[0016] Furthermore, the triterpenoid compounds include, but are not limited to, one or more of the following: ganoderic acid, tetracyclic triterpenoids, pentacyclic triterpenoids, ganoderic acid, ganoderic acid, ganoderic oxalic acid, ganoderic-22-enoic acid, ganoderic acid, ganoderic aldehyde, epoxyganoderic acid, ganoderic sterone, ganoderic terpene diol, ganoderic terpene triol, ganoderic terpene ketone diol, ganoderic terpene ketone triol, ganoderic lactone, etc.
[0017] Furthermore, the application involves adding gold nanomaterials to a culture medium containing Ganoderma lucidum fermentation broth, wherein the final concentration of the gold nanomaterials is 5%. 200 mg / L. Preferably, the final concentration of the gold nanomaterial in the fermentation medium is 136 mg / L. The gold nanoparticles have a particle size of 30 nm and are purified by centrifugation to remove residual reducing agent; the gold nanorods have a diameter of 10 nm and a length of 30 nm and are purified by centrifugation to remove residual template and reducing agent.
[0018] On the other hand, this invention also provides a method for increasing the yield of polysaccharides and triterpenes in Ganoderma lucidum using gold nanomaterials: first, Ganoderma lucidum is activated, then the activated strain is used for seed culture preparation, then the seed culture is transferred to a fermentation medium, and gold nanomaterials are added for further fermentation to obtain fermented mycelium, from which the content of polysaccharides and triterpenes in the mycelium can be measured. Specifically, this includes:
[0019] Step 1: Activation of the Ganoderma lucidum strain: Take a piece of mycelium with a diameter of 1 cm and inoculate it onto a PDA plate under sterile conditions. Activate and culture it in a constant temperature incubator at 28℃ for 7 days.
[0020] The method for preparing Ganoderma lucidum mycelium seed liquid is as follows: Under sterile conditions, take 2-3 pieces of Ganoderma lucidum mycelium with a diameter of 1 cm from the edge of the activated PDA plate, chop them up, inoculate them into the seed culture medium, and place them in a shaker at a constant temperature of 28℃ and 160 r / min for about 7 days to obtain the seed liquid.
[0021] Step 2: The method for producing polysaccharides and triterpenes by liquid fermentation of Ganoderma lucidum is as follows: the seed liquid is inoculated into the liquid fermentation medium at an inoculation rate of 10% (V / V). During the stable period (fermentation in the fermentation medium for 168 hours), gold nanomaterials are added and cultured in a constant temperature shaker at 28℃ and 180 r / min for about 7 days.
[0022] Step 3: The method further filters the fermentation broth obtained from fermentation through an 80-mesh sieve, washes it, and dries it at 60°C to constant weight to obtain the fermentation mycelium;
[0023] Step 4: Determine the content of polysaccharides and triterpenes in the fermentation mycelium.
[0024] Furthermore, the initial pH of the fermentation medium is ≥5.5. Preferably, the initial pH of the fermentation medium is 5.5. 7.5.
[0025] Preferably, each liter of the seed culture medium contains: 35g glucose, 5g corn flour, 5g peptone, 2.5g yeast extract, 1g K₂HPO₄, and MgSO₄. 0.5g of 7H2O and 0.05g of VB1.
[0026] Preferably, the potato glucose solid culture medium (PDA) contains: 200.0 g potato extract in 1000 mL, glucose 10.0 g / L, KH2PO4 3.0 g / L, and MgSO4. 7H2O 1.5g / L, VB1 0.05g / L, agar powder 15~20.0g / L.
[0027] In one embodiment, the fermentation medium contains per liter: 40.47 g soluble starch, 7.86 g peptone, 1.5 g K2HPO4, 0.05 g VB1, and the pH is adjusted to approximately 6.75.
[0028] This invention also claims protection for Ganoderma lucidum mycelium rich in polysaccharides and triterpenes prepared using the method described herein.
[0029] The present invention also provides the application of the method in the production of polysaccharides and triterpenes or products containing polysaccharides and triterpenes.
[0030] Compared with the prior art, the present invention has the following beneficial effects:
[0031] This invention provides a novel application of gold nanomaterials with different morphologies in enhancing the production of polysaccharides and triterpenoids through liquid fermentation of Ganoderma lucidum. The interaction between these materials and Ganoderma lucidum cells was investigated. By adding a certain amount of gold nanomaterials to the fermentation medium, the polysaccharide yield increased by 50.37% and the triterpenoid yield increased by approximately 42.78% compared to the result without gold nanomaterials, demonstrating a stable promotion of polysaccharide and triterpenoid synthesis. Furthermore, this invention provides culture conditions for the synthesis of polysaccharides and triterpenoids in Ganoderma lucidum fermentation broth under the induction of gold nanomaterials, providing a theoretical and practical foundation for research on improving the biosynthesis of polysaccharides and triterpenoids in Ganoderma lucidum. Attached Figure Description
[0032] Figure 1 The images show the fluorescence imaging results of the hyphae in the AuNPs group; among them, the bright field image (a) of the hyphae in the AuNPs group, the fluorescence image (b), the hyphae in the control group (c), and the bright field image (d) fluorescence image.
[0033] Figure 2 The images show the fluorescence imaging results of AuNRs hyphae; in the AuNRs group, the bright field image (a) and fluorescence imaging image (b) are shown; the control group hyphae (c) and bright field image (d) are shown.
[0034] Figure 3 The images show the fluorescence imaging results of the interaction between AuNCs and Ganoderma lucidum cells; in the AuNCs group, the bright field image of hyphae (a) and the fluorescence image (b) are shown; the control group, the bright field image of hyphae (c) and the fluorescence image (d) are shown.
[0035] Figure 4 The image shows the scanning electron microscope (SEM) results of Ganoderma lucidum hyphae in the control group without nanomaterials; Ganoderma lucidum hyphae (a) magnified 50,000 times, (b) magnified 2,000 times, (c) elemental analysis results;
[0036] Figure 5 Scanning electron microscopy results of the interaction between AuNPs and hyphae; Ganoderma lucidum hyphae and AuNPs (a) magnified 50,000 times, (b) magnified 2,000 times; (c) elemental analysis results;
[0037] Figure 6 Scanning electron microscopy images of the interaction between AuNRs and hyphae; Ganoderma lucidum hyphae and AuNRs (a) magnified 50,000 times, (b) magnified 2,000 times, (c) elemental analysis results;
[0038] Figure 7 Scanning electron microscopy images of the interaction between AuNCs and hyphae: Ganoderma lucidum hyphae and AuNCs (a) magnified 50,000 times, (b) magnified 2,000 times, (c) elemental analysis results;
[0039] Figure 8 The effects of different types of gold nanomaterials on Ganoderma lucidum fermentation products are shown in the figure; the effects of different types of gold nanomaterials on Ganoderma lucidum fermentation products (a) polysaccharide production and (b) triterpenoid production are shown in the figure.
[0040] Figure 9 The effect of different AuNPs concentrations on the fermentation products of Ganoderma lucidum (a) polysaccharide yield and (b) triterpenoid yield is shown in the figure.
[0041] Figure 10 The effect of different AuNRs concentrations on the fermentation products of Ganoderma lucidum (a) polysaccharide yield and (b) triterpenoid yield is shown in the figure.
[0042] Figure 11The effect of different AuNCs concentrations on the fermentation products of Ganoderma lucidum (a) polysaccharide yield and (b) triterpenoid yield is shown in the figure.
[0043] Figure 12 The graph shows the effect of adding 136 mg / L AuNPs at different time points on the yield of Ganoderma lucidum polysaccharides and the graph shows the effect of adding 6.8 mg / L AuNPs at different time points on the yield of Ganoderma lucidum triterpenes.
[0044] Figure 13 The graph shows the effect of adding 136 mg / L AuNRs at different time points on the yield of Ganoderma lucidum polysaccharides and the graph shows the effect of adding 136 mg / L AuNRs on the yield of triterpenes.
[0045] Figure 14 The graph shows the effect of adding 136 mg / L AuNCs at different times on the yield of Ganoderma lucidum polysaccharides and the graph shows the effect of adding 136 mg / L AuNCs at different times on the yield of triterpenes. Detailed Implementation
[0046] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with specific embodiments and accompanying drawings.
[0047] Unless otherwise specified, all reagents and materials used in the examples and comparative examples are commercially available. Each liter of seed culture medium contains: 35g glucose, 5g corn flour, 5g peptone, 2.5g yeast extract, 1g K₂HPO₄, and MgSO₄. 0.5g of 7H2O and 0.05g of VB1.
[0048] Potato glucose solid medium (PDA) contains: 200.0 g potato extract in 1000 mL, glucose 10.0 g / L, KH2PO4 3.0 g / L, MgSO4 7H2O 1.5g / L, VB1 0.05g / L, agar powder 15g / L.
[0049] Each liter of fermentation medium contains: 40.47g soluble starch, 7.86g peptone, 1.5g K2HPO4, 0.05g VB1, and the pH is adjusted to 6.75.
[0050] This invention provides an application and method for gold nanomaterials in increasing the yield of polysaccharides and triterpenes, active products of Ganoderma lucidum. Specific embodiments are as follows.
[0051] Example 1
[0052] Preparation of gold nanoparticles:
[0053] Add 200 mL of 0.01% HAuCl4 to a 500 mL beaker, place it in a boiling water bath, and seal the mouth of the beaker with sealing film. Take 3.7 mL of 1% sodium citrate solution and quickly add it to the boiling 0.01% HAuCl4. React for 10 min, then remove the beaker and cool to room temperature. Aliquot the cooled solution into 10 mL centrifuge tubes, centrifuge at 6000 rpm for 15 min, remove the supernatant, and redissolve the solution with 200 mL of ultrapure water. Repeat twice to remove residual sodium citrate, yielding 2.9 × 10⁻⁶ ppm. -4 mol / L AuNPs solution.
[0054] Example 2
[0055] Preparation of gold nanorods:
[0056] Add 2.5 mL of CTAB solution (0.2 mol / L) and 1 mL of ultrapure water to 0.12 mL of 0.1% HAuCl4 solution. While stirring continuously, add 0.6 mL of freshly prepared NaBH4 solution. After the solution turns light brown, stir for another 2 min to obtain the seed solution for later use. Take 88.7 mL of CTAB solution (0.1 mol / L) and add it to a 250 mL Erlenmeyer flask. Add 4 mL of AgNO3 solution, 5 mL of 0.1% HAuCl4 solution, and 1.24 mL of ascorbic acid solution in sequence. Stir thoroughly until the solution becomes transparent and colorless. Add 1 mL of the seed solution and incubate at 27.5℃ for 12 h. The solution turns reddish-brown. Centrifuge at 8000 rpm for 10 min, remove the supernatant, and redissolve in 100 mL of ultrapure water. Repeat twice. Remove residual reagents to obtain the 1.47 × 10⁻⁶ CTAB solution. -4 mol / L AuNRs solution.
[0057] Comparative Example 1
[0058] Preparation of gold nanoclusters:
[0059] Add 135.3 mL of ultrapure water to a 250 mL Erlenmeyer flask, followed by 10.2 mL of 1% HAuCl4 and 4.5 mL of 100 mmol / L GSH. After mixing for 5 min, place the flask in a preheated water bath at 70 °C and react for 24 h. During the reaction, add water to the water bath as needed. After the reaction, remove the Erlenmeyer flask and cool it to room temperature. Transfer 150 mL of the obtained AuNCs to an 8000 Da dialysis bag and purify by dialysis. Recover the dialyzed AuNCs to obtain 2 × 10⁻⁶ AuNCs. - 4The mol / L AuNCs solution was stored in a refrigerator at 4°C away from light.
[0060] Example 3
[0061] A method for increasing the yield of polysaccharides and triterpenes, active products of Ganoderma lucidum, specifically includes:
[0062] Step 1: Take one piece of mycelium from the Ganoderma lucidum culture medium and inoculate it into freshly prepared PDA medium. Seal the gaps between the petri dishes with sealing film. Incubate at 28℃ for 7 days. When the plate is covered with white mycelium, remove it from the incubator and store it in a 4℃ refrigerator. To ensure the viability of the mycelium, a new plate is usually inoculated about once a month to prevent the strain from aging.
[0063] The method for preparing Ganoderma lucidum mycelium seed liquid is as follows: Under sterile conditions, take 2-3 pieces of Ganoderma lucidum mycelium with a diameter of 1 cm from the edge of the activated PDA plate, cut the Ganoderma lucidum pieces into pieces with an inoculation knife, inoculate them into the seed culture medium, and place them in a shaker at a constant temperature of 28℃ and 160 r / min for about 7 days to obtain the seed liquid.
[0064] Step 2: The method for producing polysaccharides and triterpenes by liquid fermentation of Ganoderma lucidum is as follows: After the seed liquid is crushed by electric mixer, it is inoculated into liquid fermentation medium at an inoculation rate of 10% (V / V). When the fermentation reaches the stable period after 168 hours in the fermentation medium, the gold nanoparticles prepared in Example 1 are added to make the final concentration in the fermentation medium 136 mg / L. Then, it is put into a 250 mL Erlenmeyer flask and cultured in a constant temperature shaker at 28℃ and 180 r / min for about 7 days to obtain the fermentation broth.
[0065] Step 3: Filter the fermentation broth obtained from fermentation through an 80-mesh sieve, wash it, and dry it at 60℃ to constant weight to obtain fermentation mycelium.
[0066] Example 4
[0067] In this embodiment, gold nanorods prepared in Example 2 were added, and the other conditions were the same as in Example 4.
[0068] Comparative Example 2
[0069] In this embodiment, gold nanoclusters prepared in Comparative Example 1 were added, and the other conditions were the same as in Example 4.
[0070] Example 5: Interaction between gold nanomaterials and Ganoderma lucidum hyphae cells
[0071] The present invention uses the following method to verify whether gold nanomaterials damage mycelia.
[0072] 1. Soak new glass slides in 1 N HCl solution for 3 h, rinse under running water for 30 min, rinse again with ultrapure water, and store in 95% ethanol for later use. Take 1 mL of the bacterial fermentation broth (fermentation broth of gold nanomaterials and Ganoderma lucidum fermented for 7 days in step 2) prepared in Examples 4-5 and Comparative Example 2, respectively, centrifuge at 7000 rpm for 10 min, remove the culture medium, and wash 3 times with PBS. Take 10 µL of the bacterial cell and PBS mixture and drop it onto a glass slide. Place the sample under a fluorescence microscope and observe the hyphae under bright field and DAPI channels, respectively. The results are shown in [Figure number missing]. Figure 1-3 .
[0073] The fermentation broth without added gold nanomaterials was used as a control group.
[0074] Figure 1 The image shows the fluorescence imaging results of AuNPs on the hyphae. The hyphae in the AuNPs group have normal morphology, proving that AuNPs do not cause significant damage to the fungal cells. Figure 2 The image shows the fluorescence imaging results of the hyphae in the AuNRs group. Compared with the control group, the morphology of the hyphae in the AuNRs group was not significantly different from that in the control group. Figure 3 The fluorescence imaging of the interaction between AuNCs and Ganoderma lucidum cells in Comparative Example 2 shows that the hyphae of the AuNCs group are more active and the fluorescence imaging is clearer compared to the control group and the first two gold nanomaterial groups.
[0075] 2. Take 1 mL of the bacterial fermentation broth of the three nanomaterials prepared in Examples 4-5 and Comparative Example 2 (fermentation broth of gold nanomaterials and Ganoderma lucidum fermented for 7 days in step 2), centrifuge at 7000 rpm for 10 min, remove the supernatant, add 2.5% glutaraldehyde fixative (mycelium 1, glutaraldehyde 2) at a volume ratio of 1:2, fix for 20 min, centrifuge at 7000 rpm for 10 min, and wash twice with PBS. Perform gradient elution with 50%, 70%, 90%, and 100% ethanol concentrations, eluting for 5 min each time. Drop 10 µL of the bacterial cell and anhydrous ethanol mixture onto a silicon wafer and allow it to dry naturally at room temperature overnight. Fix the silicon wafer with conductive adhesive, spray with platinum for 30 s, and then observe the state of the gold nanomaterials and mycelium under a scanning electron microscope.
[0076] The fermentation broth without added gold nanomaterials was used as a control group.
[0077] Figures 4, 5, 6, and 7 show the SEM images of the control group, various nanomaterials, and Ganoderma lucidum fermentation broth, respectively. The scanning electron microscopy images and elemental analysis results show that AuNPs and AuNRs adsorb onto the surface of the mycelium. The mycelia in the AuNPs group are indistinguishable from those in the control group, indicating minimal damage to the mycelium. In the AuNCs group, due to their small size, AuNCs can penetrate the intercellular spaces. No AuNCs or their aggregates were observed at 50,000x and 2,000x magnification. The mycelial surface was smooth and indistinguishable from the control group, proving that AuNCs cause virtually no damage to the mycelium. Therefore, the gold nanomaterials in this invention do not harm the Ganoderma lucidum mycelial cells.
[0078] Example 6: Effects of different gold nanomaterials on the yield of polysaccharides and triterpenes
[0079] The content of polysaccharides and triterpenes in the fermentation mycelium was determined, specifically:
[0080] 1) Fermentation mycelium powders using three types of gold nanomaterials were obtained according to Examples 4-6 respectively;
[0081] 2) Take 7 sets of clean test tubes, with 3 replicates per set. Add 0 mL, 0.02 mL, 0.04 mL, 0.06 mL, 0.08 mL, 0.10 mL, and 0.12 mL of the above glucose standard solution to the test tubes, respectively, and add ultrapure water to a final volume of 2 mL. Add 1 mL of 6% phenol solution and 5 mL of concentrated sulfuric acid to each tube, let stand for 10 min, then shake well and allow to cool to room temperature. Measure the absorbance (A) at 490 nm. Plot a standard curve with glucose content (mg) on the x-axis and absorbance on the y-axis.
[0082] 3) Weigh 1g of fermented mycelium powder and use ultrapure water as the extraction solvent at a material-to-liquid ratio of 1:50 (1g fermented mycelium, 50mL ultrapure water). Soak at room temperature for 24h. Extract in hot water at 90℃ for 1h, followed by ultrasonic shaking for 1h. After treatment, centrifuge at 8000rpm for 10min, collect the supernatant, and dilute to a final volume. The thoroughly mixed solution is used as the sample for crude polysaccharide determination. Place 10µl of the sample in a dry test tube, add ultrapure water to 2mL, add 1mL of 6% phenol solution and 5mL of concentrated sulfuric acid to the test tube, let stand for 10min, shake well, and wait for it to cool to room temperature. Measure the absorbance (A) at 490nm. Calculate the content of crude polysaccharides from Ganoderma lucidum using a glucose standard curve.
[0083] 4) Take 8 sets of clean test tubes, with 3 replicates per set. Add 0 mL, 0.02 mL, 0.04 mL, 0.06 mL, 0.08 mL, 0.10 mL, 0.12 mL, and 0.14 mL of ursolic acid standard solution to the test tubes respectively, and evaporate the solvent in boiling water. Add 0.4 mL of 5% vanillin-glacial acetic acid solution and 1 mL of perchloric acid to each test tube, shake well, and place in a 60℃ water bath for 15 min. After the water bath, allow it to cool to room temperature, and add 5 mL of glacial acetic acid to each test tube. Shake well and let stand for 20 min, then measure the absorbance (A) at 548 nm. Plot a standard curve with ursolic acid content (mg) on the x-axis and absorbance on the y-axis.
[0084] 5) Weigh 1 g of fermentation mycelium powder and use anhydrous ethanol as the extraction solvent at a material-to-liquid ratio of 1:50 (1 g fermentation mycelium, 50 mL anhydrous ethanol). Soak at room temperature for 24 h. After soaking, extract in a 75℃ water bath for 1 h, followed by sonication for 1 h. After treatment, centrifuge at 8000 rpm for 10 min, collect the supernatant, and dilute to volume. Mix thoroughly and use as the sample for triterpenoid determination. Place 500 µL of sample in a dry test tube and evaporate the solvent in a boiling water bath until dry. First, add 0.4 mL of 5% vanillin-glacial acetic acid solution to each test tube, then quickly add 1 mL of perchloric acid, shake well, and place in a 60℃ water bath for 15 min. After the water bath, allow it to cool to room temperature, and add 5 mL of glacial acetic acid to each test tube. After shaking and standing for 20 min, the absorbance (A) was measured at 548 nm. The content of intracellular triterpenes in Ganoderma lucidum was calculated by using the ursolic acid standard curve.
[0085] The effects of different gold nanomaterials on the yield of Ganoderma lucidum polysaccharides and triterpenes are shown in Figure 8. The results show that gold nanomaterials all promoted the yield of Ganoderma lucidum polysaccharides. AuNRs promoted the content of Ganoderma lucidum triterpenes, AuNPs had little effect on the content of Ganoderma lucidum triterpenes, while AuNCs had a certain inhibitory effect on the yield of triterpenes.
[0086] Example 7 Effect of gold nanomaterial concentration on polysaccharide and triterpene yield
[0087] The obtained seed culture was homogenized using an electric mixer and then inoculated into liquid fermentation medium at an inoculum rate of 10% (v / v). Three gold nanomaterials with five different Au atom addition concentrations (6.8 mg / L, 34 mg / L, 68 mg / L, 102 mg / L, and 136 mg / L) were added during the stationary phase (168 h of fermentation in the fermentation medium) and cultured. Each group was in triplicate. The dried samples were collected in 2 mL centrifuge tubes according to the experimental procedure for later use in determining the polysaccharide and triterpenoid content.
[0088] Figures 9, 10, and 11 show the effects of different concentrations of gold nanomaterials on the yield of Ganoderma lucidum polysaccharides and triterpenes. As can be seen from the figures, gold nanomaterials affect the content of both Ganoderma lucidum polysaccharides and triterpenes, but the effects are different. They have a promoting effect on Ganoderma lucidum polysaccharides, and a promoting effect as well as an inhibitory effect on Ganoderma lucidum triterpenes.
[0089] Example 8: Effect of the timing of gold nanomaterial addition on the yield of Ganoderma lucidum polysaccharides and triterpenes
[0090] The concentrations of each gold nanomaterial that showed the most significant difference from the control group were selected to investigate the effects of adding gold nanomaterials at different time points on the yield of Ganoderma lucidum polysaccharides and triterpenes. The obtained seed culture was mixed with an electric mixer to break up the mycelial cells, and then inoculated into the fermentation medium at an inoculum rate of 10% (V / V). During fermentation, three types of gold nanomaterials were added at the lag phase (24 h), growth phase (120 h), and plateau phase (168 h) of mycelial growth, and then cultured together for 8 days. The fermentation products were then measured.
[0091] Screening using AuNP concentration gradients revealed the optimal AuNP concentration for polysaccharide production to be 136 mg / L, and the optimal AuNP concentration for triterpenoid production to be 6.8 mg / L. Screening using AuNR concentration gradients also revealed the optimal AuNR concentration for both polysaccharide and triterpenoid production to be 136 mg / L. The optimal AuNC concentration for polysaccharide production was 102 mg / L, and the optimal AuNC concentration for triterpenoid production was 68 mg / L.
[0092] Figures 12, 13, and 14 show the effects of adding different concentrations of gold nanomaterials at different time points on the fermentation products of Ganoderma lucidum. Ultimately, the addition of AuNPs at a concentration of 136 mg / L during the stable period resulted in the highest Ganoderma lucidum polysaccharide yield (333.83 mg / g), a 50.37% increase compared to the control group (165.68 mg / g). The Ganoderma lucidum triterpenoid content was not significantly different from the control group, remaining around 12 mg / g. The addition of AuNRs at a concentration of 136 mg / L during the stable period resulted in the highest Ganoderma lucidum triterpenoid yield (23.33 mg / g), a 42.78% increase compared to the control group (13.35 mg / g). The highest Ganoderma lucidum polysaccharide yield reached 291.04 mg / g, a 43.08% increase compared to the control group (165.68 mg / g). AuNCs had a relatively small effect on the yield of Ganoderma lucidum polysaccharides and triterpenoids.
[0093] In summary, the Ganoderma lucidum mycelial cells of the present invention can produce triterpenoids and polysaccharides through liquid fermentation. By adding a certain amount of gold nanomaterials to its liquid fermentation culture medium, the polysaccharide yield increased by 50.37% and the triterpenoid yield increased by about 42.78% compared with the time when no gold nanomaterials were added, which can stably promote the synthesis of polysaccharides and triterpenoids.
[0094] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for increasing the production of active products polysaccharides and triterpenes of Ganoderma lucidum, characterized in that, Gold nanomaterials are added during the stable growth period of Ganoderma lucidum mycelia. When the gold nanomaterials are gold nanoparticles, the particle size of the gold nanoparticles is 30 nm, and their concentration in the fermentation medium is 136 mg / L to increase the yield of polysaccharides, an active product of Ganoderma lucidum. When the gold nanomaterials are gold nanorods, the diameter of the gold nanorods is 10 nm, the length is 30 nm, and their concentration in the fermentation medium is 136 mg / L to increase the yield of polysaccharides and triterpenes, active products of Ganoderma lucidum.
2. The method of claim 1, wherein, The method for preparing the gold nanoparticles is as follows: 200 mL of 0.01% HAuCl4 is added to a 500 mL beaker, placed in a boiling water bath, and the mouth of the beaker is covered with sealing film; 3.7 mL of 1% sodium citrate solution is taken and quickly added to the boiling 0.01% HAuCl4, reacted for 10 min, removed from the beaker, and cooled to room temperature; the cooled solution is dispensed into 10 mL centrifuge tubes, centrifuged at 6000 rpm for 15 min, the supernatant is removed, and the solution is reconstituted with 200 mL of ultrapure water. This process is repeated twice to remove residual sodium citrate, thus obtaining AuNPs; The preparation method of the gold nanorods is as follows: 2.5 mL of 0.2 mol / L CTAB solution and 1 mL of ultrapure water are added to 0.12 mL of 0.1% HAuCl4 solution. While stirring continuously, 0.6 mL of freshly prepared NaBH4 solution is added. After the solution turns light brown, it is stirred for another 2 min to obtain a seed solution for later use. 88.7 mL of 0.1 mol / L CTAB solution is added to a 250 mL Erlenmeyer flask. 4 mL of AgNO3 solution, 5 mL of 0.1% HAuCl4 solution, and 1.24 mL of ascorbic acid solution are added sequentially. After stirring thoroughly, the solution becomes transparent and colorless. 1 mL of the seed solution is added, and the solution is kept at 27.5℃ for 12 h. The solution turns reddish-brown. After centrifugation at 8000 rpm for 10 min, the supernatant is removed, and the solution is reconstituted with 100 mL of ultrapure water. This process is repeated twice. After removing the residual reagents, AuNRs are successfully prepared.
3. The method of claim 2, wherein, include: Step 1: Take a piece of mycelium with a diameter of 1 cm and inoculate it onto a PDA plate under aseptic conditions. Activate and culture it in a constant temperature incubator at 28℃ for 7 days. Then, under aseptic conditions, take 2-3 pieces of Ganoderma lucidum mycelium with a diameter of 1 cm from the edge of the activated PDA plate, chop them up, inoculate them into the seed culture medium, and place them in a shaker at 28℃ and 160 r / min for 7 days to obtain the activated seed liquid. Step 2: Inoculate the seed liquid activated in Step 1 into the fermentation medium at a volume ratio of 10% and ferment at a temperature of 28℃ and a speed of 180 r / min. Add gold nanomaterials during the stable growth period of Ganoderma lucidum mycelium, and then culture in a constant temperature shaker for 7 days. Step 3: Filter the fermentation broth obtained in Step 2 through an 80-mesh sieve, wash it, and dry it at 60℃ to constant weight to obtain mycelium; Step 4: Determine the content of polysaccharides and triterpenes in the mycelium.
4. The method of claim 3, wherein, In step 2, the initial pH of the fermentation medium is ≥5.
5.
5. The method according to claim 4, characterized in that, The seed culture medium contains per L: glucose 35 g, corn flour 5 g, protein peptone 5 g, yeast extract 2.5 g, K2HPO4 1 g, MgSO4 7H2O 0.5 g, VB1 0.05 g; The fermentation medium contains per liter: 40.47 g soluble starch, 7.86 g peptone, 1.5 g K2HPO4, 0.05 g VB1, and pH adjusted to 6.75.