Culture medium for high yield of ganoderan through solid fermentation of golden coptis dregs

Abstract

A culture medium for high yield of ganoderan through solid fermentation of Jinlian medicine residues is prepared from, by weight, 10%-20% of the Jinlian medicine residues, 0.5%-2% of wheat bran, 1%-4% of soybean meal, 0.05%-0.2% of urea, 0.05%-0.3% of ammonium sulfate and the balance water, and the total amount is 100%. The preparation method comprises the following steps: mixing all the components together, uniformly stirring, sterilizing for 25 minutes at the temperature of 121 DEG C under the pressure of 0.15 Mpa, and cooling to room temperature. According to the method, the Jinlian medicine residues are used as main raw materials, and the formula of the medicine residue culture medium is optimized, so that the medicine residue culture medium is more suitable for growth of ganoderma lucidum mycelia and high yield of ganoderma lucidum polysaccharides, the problem of waste of the Jinlian medicine residues can be green and efficiently solved, high-added-value ganoderma lucidum polysaccharide products can be produced, economic benefits are increased, waste utilization is achieved, and environmental pollution is reduced; and the method has obvious social and economic benefits.

Description

Technical Field

[0001] The invention relates to the technical field of fungal culture medium, in particular to a culture medium for high-yield ganoderma lucidum polysaccharide by solid fermentation of ginseng root residues. Background Art

[0002] Traditional Chinese medicine has a history of thousands of years. It is the crystallization of the wisdom of the Chinese nation's long-term struggle against diseases from ancient times to the present, and its role in the modern medical field is becoming increasingly important. With the acceleration of the modernization process of traditional Chinese medicine and its promotion in the global medical and health field, the application scale and industrialization degree of Chinese herbal medicine have been significantly improved, and the contribution rate of the Chinese medicine industry has reached one-third of the total national pharmaceutical industry. However, the waste residues of Chinese herbal medicine processing are increasing, and how to deal with and reuse them has become an increasingly serious problem. According to statistics, the solid residue waste generated in the process of Chinese herbal medicine industrialization in my country is as high as 50 million tons each year. This type of waste is rich in cellulose, lignin and other effective ingredients, and has extremely high biomass resource potential, but it is still treated by traditional methods such as incineration, landfill or open-air stacking, which not only causes waste of resources and occupies production space, but may also cause social, economic and environmental problems such as soil pollution and greenhouse gas emissions. Therefore, exploring efficient resource utilization methods for Chinese herbal medicine residues has become an important topic for achieving green and sustainable development of the Chinese medicine industry.

[0003] At present, scholars have proposed different solutions to the problem of recycling Chinese medicine residues. For example, some residues can be used as organic fertilizers to improve the soil environment, increase crop yields, and reduce the use of chemical fertilizers; using residues as animal feed additives can reduce the use of grain feed, alleviate the contradiction between supply and demand between grain and feed, and reduce the use of antibiotics; residues contain a large amount of carbon and nitrogen elements and inorganic salts, and can also be used as a cultivation matrix for edible and medicinal fungi; in addition, some scholars use the loose and porous physical properties of residues to prepare adsorbents for the adsorption and treatment of pollutants (such as heavy metals, fluorides or toluene). These solutions have low investment costs and will have certain benefits in the future; in addition, there are some high-cost and high-return residue solutions, such as using pyrolysis or gasification to convert residues into bio-oil or combustible gas, or using complex processes to make residues into bio-based materials. There are many types of Chinese medicine residues, and reasonable utilization strategies need to be formulated according to the characteristics of different residues. Honeysuckle (Lonicera japonica) and Forsythia suspensa (Forsythia suspensa) are often used as a classic medicine pair (referred to as "Jinlian"). Because of their effects of dispersing wind-heat, clearing away heat and detoxifying, they are widely used in the treatment of colds, inflammation and other diseases, and the market demand is huge. At present, there is little research on the reuse of mixed Jinlian residues. The large amount of residues produced after industrial extraction still faces the dilemma of traditional extensive treatment due to the lack of effective utilization technology. Therefore, how to transform Jinlian residues into high-value-added products has become a technical bottleneck that needs to be broken through.

[0004] Ganoderma lucidum, a rare fungus with both medicinal and edible values, has extremely high application values in many fields such as immunomodulation, anti-tumor, and antioxidant. Its active components such as polysaccharides and triterpenoids are particularly important. Polysaccharides are considered the most core active components in Ganoderma lucidum, with the most research and the widest application. In recent years, the technology of cultivating Ganoderma lucidum using agricultural or traditional Chinese medicine wastes (such as wheat bran, soybean meal, mulberry branches, astragalus, and residues of Panax notoginseng) has gradually matured, which not only reduces production costs but also realizes the recycling of wastes. However, the research on the solid-state fermentation of residues of Forsythia suspensa (Thunb.) Vahl and Lonicera japonica Thunb. with Ganoderma lucidum to produce high-yield Ganoderma lucidum polysaccharides is still blank. The residual active components and abundant lignocellulose in the residues of Forsythia suspensa (Thunb.) Vahl and Lonicera japonica Thunb. can provide a nutrient source for the growth of Ganoderma lucidum mycelium. Therefore, through the solid-state fermentation of the residues of Forsythia suspensa (Thunb.) Vahl and Lonicera japonica Thunb. with Ganoderma lucidum, not only the problem of treating the residues of Forsythia suspensa (Thunb.) Vahl and Lonicera japonica Thunb. can be solved, but also high-yield Ganoderma lucidum polysaccharides can be produced, generating additional economic benefits. Existing data show that although Ganoderma lucidum can grow directly on the residues of Forsythia suspensa (Thunb.) Vahl and Lonicera japonica Thunb., its mycelium growth state is poor and it does not have the ability to produce high-yield Ganoderma lucidum polysaccharides. Based on this, it is necessary to optimize and innovate the culture medium formula of the residues of Forsythia suspensa (Thunb.) Vahl and Lonicera japonica Thunb. to facilitate the growth of Ganoderma lucidum mycelium and high-yield polysaccharides, but there is no relevant public report so far. Summary of the Invention

[0005] In view of the above situation, to overcome the defects of the prior art, the purpose of the present invention is to provide a culture medium for solid-state fermentation of the residues of Forsythia suspensa (Thunb.) Vahl and Lonicera japonica Thunb. with Ganoderma lucidum to produce high-yield Ganoderma lucidum polysaccharides, which can effectively solve the technical problems of the reuse of the residues of Forsythia suspensa (Thunb.) Vahl and Lonicera japonica Thunb. and the high-yield production of Ganoderma lucidum polysaccharides.

[0006] To achieve the above purpose, the technical solution solved by the present invention is that a culture medium for solid-state fermentation of the residues of Forsythia suspensa (Thunb.) Vahl and Lonicera japonica Thunb. with Ganoderma lucidum to produce high-yield Ganoderma lucidum polysaccharides is composed of the following components by weight percentage: 10-20% of the residues of Forsythia suspensa (Thunb.) Vahl and Lonicera japonica Thunb., 0.5-2% of wheat bran, 1-4% of soybean meal powder, 0.05-0.2% of urea, 0.05-0.3% of ammonium sulfate, and the balance is water, with a total of 100%; mix the components together and stir evenly, sterilize at 0.15 Mpa and 121 °C for 25 min, and cool to room temperature to obtain the product;

[0007] The residues of Forsythia suspensa (Thunb.) Vahl and Lonicera japonica Thunb. are the residues dried after industrial extraction of Forsythia suspensa (Thunb.) Vahl and Lonicera japonica Thunb. with a weight ratio of 1:2 (a well-known product, such as the residues of Forsythia suspensa (Thunb.) Vahl and Lonicera japonica Thunb. provided by Henan Fusen Pharmaceutical Co., Ltd.).

[0008] The Ganoderma lucidum is Ganoderma lucidum (Leyss. ex Fr.) Karst. (a well-known product, such as the Ganoderma lucidum strain preserved in the Key Laboratory of Microbiology of Henan Province, Institute of Biology, Henan Academy of Sciences).

[0009] The wheat bran is wheat bran, a by-product of wheat processing into flour, wheat-colored, flaky or powdery

[0010] The soybean meal powder described above is made from the first - stage pressed soybean meal after soybean oil extraction.

[0011] The present invention uses Coptis chinensis residue as the main raw material. By optimizing the formula of the residue culture medium, it is made more suitable for the growth of Ganoderma lucidum mycelium and can produce high - yield Ganoderma lucidum polysaccharide. It can not only greenly and efficiently solve the problem of Coptis chinensis residue waste, but also produce Ganoderma lucidum polysaccharide products with high added value, increase economic benefits, utilize waste, and reduce environmental pollution, with significant social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is a diagram of the solid - state culture of Ganoderma lucidum with Coptis chinensis residue blocks in the present invention.

[0013] Figure 2 It is a diagram of the state of Ganoderma lucidum cultured on a Petri dish with Coptis chinensis residue powder medium added with different nitrogen sources in the present invention.

[0014] Figure 3 It is a diagram of the Petri dish of dried Ganoderma lucidum with Coptis chinensis residue (A) and the peeled Ganoderma lucidum mycelium (B) in the present invention.

[0015] Figure 4 It is a diagram of the effect of adding different carbon sources to the Coptis chinensis residue medium on Ganoderma lucidum in the present invention.

[0016] Figure 5 It is a diagram of the effect of adding different nitrogen sources to the Coptis chinensis residue medium on Ganoderma lucidum in the present invention.

[0017] Figure 6 It is a diagram of the effect of adding different inorganic salts to the Coptis chinensis residue medium on Ganoderma lucidum in the present invention.

[0018] Figure 7 It is a diagram of the effect of different temperatures on the cultivation of Ganoderma lucidum with Coptis chinensis residue in the present invention.

[0019] Figure 8 It is a diagram of the effect of pH value on the cultivation of Ganoderma lucidum with Coptis chinensis residue in the present invention.

[0020] Figure 9 It is a diagram of the effect of single - factor addition amount experiment on the solid - state cultivation of Ganoderma lucidum with Coptis chinensis residue in the present invention.

[0021] Figure 10 It is a diagram of the relationship between the total content of Ganoderma lucidum mycelium polysaccharide and its growth vigor in the present invention.

[0022] Figure 11 It is a diagram of the comparison of the growth vigor of Ganoderma lucidum mycelium before and after the optimization of the Coptis chinensis residue medium in the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0023] The following detailed description of the specific implementation mode of the present invention is made with reference to the drawings and examples.

[0024] Example 1

[0025] A culture medium for solid fermentation of Ganoderma lucidum polysaccharide with high yield using Coptis chinensis residue is composed of the following components by weight percentage: Coptis chinensis residue 16.75%, wheat bran 2%, soybean meal powder 1%, urea 0.2%, ammonium sulfate 0.05% and water 80%. Mix all components evenly, sterilize at 0.15 Mpa and 121 °C for 25 min, and cool to room temperature to obtain the product.

[0026] Example 2

[0027] A culture medium for solid fermentation of Ganoderma lucidum polysaccharide with high yield using Coptis chinensis residue is composed of the following components by weight percentage: Coptis chinensis residue 12.3%, wheat bran 1%, soybean meal powder 1.5%, urea 0.1%, ammonium sulfate 0.1% and water 85%. The specific preparation method is the same as that in Example 1.

[0028] Example 3

[0029] A culture medium for solid fermentation of Ganoderma lucidum polysaccharide with high yield using Coptis chinensis residue is composed of the following components by weight percentage: Coptis chinensis residue 20%, wheat bran 0.5%, soybean meal powder 3%, urea 0.11%, ammonium sulfate 0.3% and water 76.09%. The specific preparation method is the same as that in Example 1.

[0030] Example 4

[0031] A culture medium for solid fermentation of Ganoderma lucidum polysaccharide with high yield using Coptis chinensis residue is composed of the following components by weight percentage: Coptis chinensis residue 12%, wheat bran 1.7%, soybean meal powder 1.2%, urea 0.05%, ammonium sulfate 0.05% and water 85%. The specific preparation method is the same as that in Example 1.

[0032] Example 5

[0033] A culture medium for solid fermentation of Ganoderma lucidum polysaccharide with high yield using Coptis chinensis residue is composed of the following components by weight percentage: Coptis chinensis residue 15%, wheat bran 2%, soybean meal powder 2%, urea 0.15%, ammonium sulfate 0.15% and water 80.7%. The specific preparation method is the same as that in Example 1.

[0034] The raw materials of the present invention are rich, and the preparation method is simple. It can be effectively used for fermentation culture of Ganoderma lucidum polysaccharide with high yield, which not only realizes the full utilization of resources, saves resources and prevents environmental pollution, but also can well meet the demand for Ganoderma lucidum polysaccharide. It is a major innovation in the reuse of Coptis chinensis residue and the production of Ganoderma lucidum polysaccharide with high yield. Through experiments and practices, very good technical effects have been achieved. The relevant experimental data are as follows:

[0035] I. Materials and Methods

[0036] 1.1 Experimental Materials

[0037] Bacterial strain: The bacterial strain used in the present invention is a Japanese Ganoderma lucidum strain (a well-known commercially available product) preserved by the Henan Academy of Sciences Biological Research Institute Co., Ltd. and the Henan Provincial Key Laboratory of Microbiology.

[0038] Jinlian medicinal residue: The medicinal residue used in the present invention is the Jinlian medicinal residue block and medicinal residue powder provided by Henan Fusen Pharmaceutical Co., Ltd.

[0039] 1.2 Preparation of culture medium

[0040] PDA solid medium: 200g peeled potatoes, cut into small pieces, add distilled water and boil for 1h, filter with four layers of gauze, then add 20g glucose and 20g agar, add distilled water to make up to 1000mL, and sterilize in an autoclave at 121℃ for 25min;

[0041] Jinlian medicine residue plate culture medium: weigh 15g of Jinlian medicine residue powder, 83ml of distilled water, add relevant test factors, 2g of agar, natural pH, put in a sterilizer, and sterilize at 121℃ for 25min.

[0042] It should be noted that agar was added to the Jinlian medicine residue culture medium in the experiment of the present invention because Ganoderma lucidum can directly attach and grow on the blocky Jinlian medicine residue to produce white Ganoderma lucidum hyphae (such as Figure 1 ), but the Ganoderma lucidum attached to the irregular block of medicinal residue cannot objectively quantify its growth, and the white mycelium cannot be accurately peeled off from the medicinal residue block, which is not conducive to the detection of the polysaccharide content of the Ganoderma lucidum mycelium. The medicinal residue plate made by grinding the Jinlian medicinal residue block into powder and mixing it with agar and water can solve the above problems well without affecting its growth. In other words, the addition of agar is only for the convenience of the experiment, and the culture medium of the present invention does not need to have an agar component. Ganoderma lucidum can grow round white mycelium on the surface of the Jinlian medicinal residue plate. The diameter of the round plaque can be measured to quantify the growth rate of Ganoderma lucidum, and the influence of different factors on the growth of Ganoderma lucidum can be studied (such as Figure 2 In addition, the dried Ganoderma lucidum plate can accurately peel off the white Ganoderma lucidum mycelium from the surface of the culture medium plate (such as Figure 3 ), used for the detection of polysaccharide content in Ganoderma mycelium.

[0043] 1.3 Bacterial activation and cultivation

[0044] The Ganoderma lucidum strains stored in the laboratory were inoculated onto PDA solid culture medium plates and cultured at 28°C for about 7 days. After the Ganoderma lucidum hyphae had grown all over the plate, the plates were placed at 4°C for later use.

[0045] Use a 1-cm-diameter hole puncher to obtain uniform-sized Ganoderma lucidum blocks from the activated Ganoderma lucidum plate, inoculate them in the central area of ​​the Jinlian medicinal residue culture medium plate, and culture them at 28°C for about 6 days. Measure the diameter of the Ganoderma lucidum plaques. The average value of each plaque diameter measured three times represents the growth rate of Ganoderma lucidum.

[0046] 1.4 Determination of Ganoderma lucidum polysaccharide content

[0047] The determination method of Ganoderma lucidum polysaccharide content adopts the phenol - sulfuric acid method, referring to the methods of Xu Zhixiang et al. and Zhang Zhijun et al. with slight modifications.

[0048] Preparation of the standard curve: Respectively pipette 0, 20, 40, 60, 80, 100, 120, 140, 160 μl of 0.1 mg / ml glucose standard solution into each 2 - ml centrifuge tube, and make up to 200 μl per tube with distilled water. Then add 100 μl of 5% phenol and 500 μl of concentrated sulfuric acid to each tube. After mixing, let it stand at room temperature for 5 min, then place it in a boiling water bath for 15 min. After cooling to room temperature, measure the absorbance value of the reaction solution in each tube at 489 nm with an enzyme - linked immunosorbent assay (ELISA) reader. Make a standard curve based on the absorbance values corresponding to different glucose concentrations, and obtain the curve equation for calculating the Ganoderma lucidum polysaccharide content.

[0049] Sample determination: Put the Ganoderma lucidum plate into an oven at 45 °C to dry, gently scrape the Ganoderma lucidum mycelium on the surface of the culture medium with a blade and weigh it. Take about 0.0100 g of Ganoderma lucidum mycelium into a 2 - ml centrifuge tube. Add 1 ml of 85% ethanol to every 0.0100 g of Ganoderma lucidum mycelium, place it in a water bath at 60 °C for 1 h, mix it every 10 min. Then centrifuge at 12000 g for 10 min and discard the supernatant. After drying the alcohol, add 1 ml of distilled water according to every 0.0100 g of mycelium, place it in a boiling water bath for extraction for 2 h, cool to room temperature, then centrifuge at 12000 g for 10 min. Take 40 μl of the supernatant and 160 μl of distilled water into a 2 - ml centrifuge tube and mix well. According to the operation method of making the standard curve, measure the absorbance value of the sample at 489 nm, calculate the percentage content of Ganoderma lucidum mycelium polysaccharide per unit mass according to the standard curve equation, and calculate its total polysaccharide content according to the total mass of Ganoderma lucidum mycelium.

[0050] II. Optimization of Jinlian medicinal residue medium

[0051] 2.1 Single - factor experiment

[0052] (1) Carbon source

[0053] Respectively add 1% of compound wheat bran and corn flour, 0.1% of single compounds soluble starch, sucrose and glucose as test factors into the Jinlian medicinal residue medium. Inoculate Ganoderma lucidum fungal blocks and measure the diameter of Ganoderma lucidum plaques after 6 - day culture. Each treatment is repeated three times to detect the influence of different carbon sources on the growth rate of Ganoderma lucidum mycelium.

[0054] (2) Nitrogen source

[0055] Three nitrogen sources with different properties, namely 1% complex soybean meal, 0.1% single compound urea, and 0.1% single compounds ammonium nitrate, ammonium sulfate, and ammonium chloride, were added as test factors to the Jinlian medicinal residue medium. After inoculating Ganoderma lucidum mycelium blocks and culturing for 6 days, the diameter of the Ganoderma lucidum plaque was measured. Each treatment was repeated three times to detect the effect of different nitrogen sources on the growth rate of Ganoderma lucidum mycelium.

[0056] (3) Inorganic salts

[0057] 0.05% of ferrous sulfate, calcium chloride, potassium dihydrogen phosphate, magnesium sulfate, and copper sulfate were added respectively as test factors to the Jinlian medicinal residue medium. After inoculating Ganoderma lucidum mycelium blocks and culturing for 6 days, the diameter of the Ganoderma lucidum plaque was measured. Each treatment was repeated three times to detect the effect of different inorganic salts on the growth rate of Ganoderma lucidum mycelium.

[0058] (4) Temperature

[0059] Ganoderma lucidum mycelium blocks were inoculated into the Jinlian medicinal residue medium without adding any factors and cultured at 24, 28, and 32 °C for 6 days. The diameter of the Ganoderma lucidum plaque was measured. Each treatment was repeated three times to detect the effect of different temperatures on the growth rate of Ganoderma lucidum mycelium.

[0060] (5) Different pH experiments

[0061] The pH of the Jinlian medicinal residue medium was adjusted to 4, 5, 6, 7, 8, 9, and 10 with hydrochloric acid or sodium hydroxide respectively, and then sealed and sterilized to prepare Jinlian medicinal residue media with different pH values. After inoculating Ganoderma lucidum mycelium blocks and culturing for 6 days, the diameter of the Ganoderma lucidum plaque was measured. Each treatment was repeated three times to detect the effect of different pH values on the growth rate of Ganoderma lucidum mycelium.

[0062] (6) Single-factor addition amount gradient test

[0063] According to the results of the single-factor test, factors with a significant promoting effect on the growth of Ganoderma lucidum were selected for the addition amount gradient test. Wheat bran, sucrose, soybean meal, ammonium sulfate, and urea were selected to prepare Jinlian medicinal residue medium plates respectively. The addition amounts of wheat bran and soybean meal were 0%, 0.5%, 1%, 1.5%, and 2% in sequence, and the addition amounts of sucrose, ammonium sulfate, and urea were 0%, 0.05%, 0.10%, 0.15%, and 0.20% in sequence. They were cultured at 28 °C for 6 days, and the diameter of the Ganoderma lucidum plaque was measured. Each treatment was repeated three times to detect the effect of different addition amounts of each factor on the growth rate of Ganoderma lucidum mycelium.

[0064] 2.2 Orthogonal test

[0065] According to the results of single-factor experiments, factors with obvious promoting effects on Ganoderma lucidum growth were selected for orthogonal experiments to determine the optimal formula of the multi-factor combination for the solid-state fermentation of Ganoderma lucidum on the Jinlian medicinal residue medium. According to the results of single-factor experiments, the optimal fermentation temperature was 28 °C. Since inorganic salts and the initial pH value had no significant promoting effect on Ganoderma lucidum growth, these two types of factors did not need to be considered. However, the addition of wheat bran, ammonium sulfate, soybean meal, and urea could significantly promote the growth of Ganoderma lucidum mycelium. Therefore, these four factors were selected for orthogonal experiments, and the plaque diameter was measured as the response value to determine the optimal formula of the Jinlian medicinal residue medium. The L9(34) orthogonal experimental design was adopted, and three concentration levels were set for each factor (see Table 1 below).

[0066] Table 1 Factor-level table of L9(34) orthogonal experiment

[0067]

[0068] In addition, by detecting the Ganoderma lucidum plaque diameter, total mycelium mass, polysaccharide percentage content, and total polysaccharide content of each treatment group in the orthogonal experiment respectively, the relationship between the polysaccharide content of Ganoderma lucidum mycelium and its growth trend was analyzed through Pearson correlation analysis.

[0069] 2.4 Statistical analysis

[0070] The significant difference in the growth rate of Ganoderma lucidum between each added factor and the CK control group (without added factors) was analyzed through independent t-tests. The Tukey HSD test in one-way ANOVA was used to analyze the significant differences in culture temperature and initial pH value among different treatment groups. The relationship between the polysaccharide content of Ganoderma lucidum mycelium and its growth trend was statistically analyzed through Pearson correlation analysis. The statistical analysis of the data in this invention was all completed using SPSS V22.0 software.

[0071] III. Results and analysis

[0072] 3.1 Carbon source

[0073] Adding different carbon sources to the Jinlian medicinal residue medium had different effects on the growth rate of Ganoderma lucidum (see Figure 4 ). Among them, 1% wheat bran and 0.1% glucose could extremely significantly (p < 0.01) promote the growth of Ganoderma lucidum mycelium, with a promotion rate of about 4.74%. 1% corn flour and 0.1% soluble starch had no promoting effect on Ganoderma lucidum growth (p > 0.05), while 0.1% sucrose had an extremely significant inhibitory effect on Ganoderma lucidum growth (p < 0.01).

[0074] 3.2 Nitrogen source

[0075] The 5 nitrogen sources selected in this invention all had extremely significant promoting effects on the growth of Ganoderma lucidum mycelium (p < 0.01) (seeFigure 5 ) Among them, the promoting effect of 0.1% urea on the growth of Ganoderma lucidum mycelium was extremely significantly higher than that of the other four nitrogen sources (t-test, p < 0.01), and the promotion rate of Ganoderma lucidum mycelium growth reached 21.6%. All five tested nitrogen sources could significantly promote the growth of Ganoderma lucidum mycelium. According to the properties of different nitrogen sources, urea (organic matter), ammonium sulfate (inorganic matter), and soybean meal (complex) were selected as the nitrogen sources for the medium.

[0076] 3.3 Inorganic salts

[0077] The effects of adding 0.05% of different kinds of inorganic salts on the growth of Ganoderma lucidum were studied. Magnesium sulfate and potassium dihydrogen phosphate had no promoting effect on the growth of Ganoderma lucidum (p > 0.05), while ferrous sulfate, calcium chloride, and copper sulfate had extremely significant inhibitory effects on the growth of Ganoderma lucidum (p < 0.01) (see Figure 6 ). The results showed that none of the tested inorganic salts promoted the growth of Ganoderma lucidum mycelium, and there was no need to add inorganic salts.

[0078] 3.4 Culture temperature

[0079] After inoculating Ganoderma lucidum on the Jinlian medicinal residue medium, it was placed at different temperatures to study the optimal culture temperature. As Figure 7 shown, the growth rate of Ganoderma lucidum mycelium at 28 and 32 °C was extremely significantly faster than that at 24 °C (p < 0.01). The growth rate of Ganoderma lucidum at 28 °C was slightly faster than that at 32 °C, but the difference was not significant (p = 0.506). The results showed that the optimal culture temperature for Ganoderma lucidum mycelium was 28 °C.

[0080] 3.5 Influence results of pH value

[0081] The effects of the initial pH on the growth of Ganoderma lucidum were studied by preparing Jinlian medicinal residue media with different pH values. As Figure 8 shown, except for the significant difference between the two treatments with pH values of 5 and 9 (p < 0.05), there was no significant difference among the other treatment groups (p > 0.05). The natural pH value of the medicinal residue medium was between 6 and 8, and there was no significant difference from the other pH value treatments. Therefore, the natural pH could be used for culturing Ganoderma lucidum with Jinlian medicinal residue, and there was no need to adjust the pH value of the medium additionally.

[0082] 3.6 Single-factor addition amount

[0083] Gradient tests of the addition amount were carried out on the factors that had a significant promoting effect on the growth of Ganoderma lucidum. As Figure 9 shown, the addition amount of wheat bran with the greatest promoting effect on the growth of Ganoderma lucidum was 1%, which was 17.33% higher than the treatment group without adding the factor. However, the promoting effect decreased with higher addition amounts ( Figure 9 A); the optimal addition amount of glucose was 0.10%, and the growth of Ganoderma lucidum increased by 8.10%. The promoting effect decreased with higher addition amounts ( Figure 9B); When adding wheat bran alone, the maximum plaque diameter is nearly 70 mm, while when adding glucose alone, the maximum is less than 64 mm. Therefore, the promoting effect of wheat bran is better than that of glucose. The promoting effect of soybean meal on the growth of Ganoderma lucidum gradually increases with the increase of the addition amount. When the addition amount is 2%, the promoting effect on the growth of Ganoderma lucidum reaches 25.24%( Figure 9 C); The optimal addition amount of urea is 0.15%, which increases the growth of Ganoderma lucidum by 24.25%( Figure 9 D); When the addition amounts of ammonium sulfate are 0.15% and 0.20%, the promoting effects on the growth of Ganoderma lucidum reach the highest, and the plaque diameters of Ganoderma lucidum can be increased by 25.42% and 25.99% respectively( Figure 9 E).

[0084] 3.7 Orthogonal experiment

[0085] According to the results of the single-factor experiment, four factors with the most obvious promoting effects on the growth of Ganoderma lucidum were selected. According to the three levels set in Table 1, an L9(34) orthogonal experiment was carried out to detect the total polysaccharide content of Ganoderma lucidum. The results are shown in Table 2. According to the magnitude of the range R value, the order of the influencing factors on the total polysaccharide content of Ganoderma lucidum mycelium is C > D > B > A. According to the results of the variance analysis in Table 3, the effects of the four addition factors on the total polysaccharide content of Ganoderma lucidum mycelium all reached an extremely significant level (p < 0.01), indicating that the four selected factors have an extremely significant promoting effect on the polysaccharide accumulation of Ganoderma lucidum mycelium and can be used as the main addition factors for optimizing the Jinlian medicinal residue medium.

[0086] Table 2 Results of orthogonal experiment

[0087]

[0088]

[0089] Table 3 Variance analysis of orthogonal experiment

[0090]

[0091] 3.8 Effects of medium optimization on the growth and polysaccharide content of Ganoderma lucidum

[0092] The plaque diameter, total mycelium mass, polysaccharide percentage content, and total polysaccharide amount of Ganoderma lucidum in each treatment group of the orthogonal experiment were detected respectively (Table 4), and the effects of medium optimization on the growth and polysaccharide content of Ganoderma lucidum mycelium were analyzed. In treatment group 1, no optimization factors were added, and it was the smallest in terms of the plaque diameter, total mycelium weight, and total polysaccharide amount of Ganoderma lucidum, while treatment groups 2 - 7 with added optimization factors were significantly improved. The highest plaque diameter could be expanded by 36.29%, the total mycelium weight was increased by 14.45 times, and the total polysaccharide amount was increased by 12.47 times. The scatter plot and trend line of the total Ganoderma lucidum polysaccharide content and the plaque diameter of Ganoderma lucidum showed a positive correlation between the total Ganoderma lucidum polysaccharide content and the plaque diameter (see Figure 10)。Through Pearson correlation analysis, there was a highly significant positive correlation between the total content of Ganoderma lucidum polysaccharides and the plaque diameter (r = 0.814, p < 0.01). In addition, there were also highly significant positive correlations between the total mass of mycelia and the Ganoderma lucidum plaque diameter (r = 0.849) and the total polysaccharide content (r = 0.973) (p < 0.01). However, there was no significant correlation between the polysaccharide percentage content of Ganoderma lucidum mycelia and the plaque diameter, the total mass of mycelia, and the total polysaccharide content (p > 0.05) (Table 5). The results showed that there was a significant positive correlation between the total amount of Ganoderma lucidum polysaccharides and its growth vigor, that is, the larger the Ganoderma lucidum plaque, the more the mycelia mass, and the higher the total amount of Ganoderma lucidum polysaccharides. The Jinlian medicinal residue medium of the present invention can significantly promote mycelial growth and increase the content of Ganoderma lucidum polysaccharides.

[0093] Table 1 Detection of Ganoderma lucidum mycelial growth parameters among different treatment groups

[0094]

[0095]

[0096] (Note: The data in the table are the mean ± standard deviation of three replicates)

[0097] Table 5 Pearson correlation analysis between the polysaccharide content of Ganoderma lucidum mycelia and its growth vigor

[0098]

[0099] (Note: The data in the table are the Pearson correlation coefficient r. A positive value indicates a positive correlation, and a negative value indicates a negative correlation. “**” indicates a highly significant correlation between the two (p < 0.01). “NS” indicates no significant correlation between the two (p > 0.05)).

[0100] In summary, although Ganoderma lucidum can grow directly on Jinlian medicinal residues, its growth state is not good. Based on this, the present invention uses Jinlian medicinal residues as the main raw material, optimizes the formula of the medicinal residue medium and regulates its physicochemical properties to explore its effects on the growth of Ganoderma lucidum mycelia and the accumulation of active ingredients (Ganoderma lucidum polysaccharides). Through single-factor experiments, the present invention studied the effects of different factors (carbon source, nitrogen source, inorganic salts, initial pH, culture temperature) on the growth of Ganoderma lucidum mycelia, screened out the factors that have a significant promoting effect on the growth of Ganoderma lucidum mycelia for the addition amount gradient experiment, and screened out the optimal medium formula under multi-factor and multi-level combinations through orthogonal experiments. The experimental results showed that the optimized Jinlian medicinal residue medium can significantly promote the growth of Ganoderma lucidum mycelia, with a larger plaque diameter and denser mycelia ( Figure 11), and comprehensively consider that when the addition amounts of wheat bran, ammonium sulfate, soybean meal, and urea in the Jinlian medicinal residue culture medium are 0.5-2%, 0.05-0.3%, 1-4%, and 0.05-0.2% respectively, the growth of Ganoderma lucidum mycelium is faster, the plaque diameter is larger, the mass of mycelium is more, and the total polysaccharide content is higher. The optimized Jinlian medicinal residue culture medium is more suitable for the growth of Ganoderma lucidum, the biomass of Ganoderma lucidum mycelium increases significantly, high-yield Ganoderma lucidum polysaccharide is produced, and high-value-added Ganoderma lucidum products are obtained. The experimental data show that the Jinlian medicinal residue culture medium of the present invention has extremely significant effects, and the polysaccharide content of Ganoderma lucidum mycelium can reach 4%-5%, which is generally higher than the reported data of Ganoderma lucidum mycelium polysaccharides (such as Yang Guangcheng et al., "Science and Technology of Food Industry", No. 05, 2001; Wu Xueqian et al., "Forest By-Products and Specialties in China", No. 05, 2009; Teng Liming et al., "Mycosystema", No. 07, 2021). At the same time, the culture medium of the present invention can promote the plaque diameter of Ganoderma lucidum to increase by at least 36.29%, the mycelium coverage area to expand by at least 85.72%, the biomass of mycelium to increase by at least 14.45 times, and the total polysaccharide content of Ganoderma lucidum to increase by at least 12.47 times. It not only realizes the green disposal of traditional Chinese medicine waste, but also produces high-value-added Ganoderma lucidum products in the mode of "turning waste into treasure", providing a theoretical basis and practical path for the ecological upgrading of the traditional Chinese medicine industrial chain, and having significant social and economic benefits for solving the healthy development of the traditional Chinese medicine industry.

[0101] It should be noted that the above are only the preferred embodiments of the present invention, and do not limit the present invention in any form or substance. It should be pointed out that for those of ordinary skill in the art in this technical field, without departing from the method of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention. Those skilled in the art, without departing from the spirit and scope of the present invention, when making some equivalent changes, modifications and evolutions using the technical content disclosed above, are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A culture medium for high-yield production of ganoderma polysaccharide by solid fermentation using forsythia residue, characterized in that, Made from the following by weight percentage: 10-20% of honeysuckle and forsythia residue, 0.5-2% of wheat bran, 1-4% of soybean meal powder, 0.05-0.2% of urea, 0.05-0.3% of ammonium sulfate, and the balance being water, with the total amount being 100%; mix all components together and stir evenly, sterilize at 0.15 Mpa and 121 °C for 25 min, and cool to room temperature to obtain; the honeysuckle and forsythia residue is the residue after industrial extraction and drying of honeysuckle and forsythia with a weight ratio of 1:2, and the Ganoderma lucidum is Ganoderma lucidum from Japan; the wheat bran is wheat bran, which is a by-product of wheat flour processing; the soybean meal powder is made from the first-class pressed soybean meal after soybean oil extraction.

2. The culture medium for high-yield Ganoderma lucidum polysaccharide by solid fermentation using honeysuckle residue according to claim 1, characterized in that Made from the following by weight percentage: 16.75% of honeysuckle and forsythia residue, 2% of wheat bran, 1% of soybean meal powder, 0.2% of urea, 0.05% of ammonium sulfate, and 80% of water.

3. The culture medium for high-yield Ganoderma lucidum polysaccharide by solid fermentation using Forsythia suspense residue according to claim 1, characterized in that, Made from the following by weight percentage: 12.3% of honeysuckle and forsythia residue, 1% of wheat bran, 1.5% of soybean meal powder, 0.1% of urea, 0.1% of ammonium sulfate, and 85% of water.

4. The culture medium for high-yield Ganoderma lucidum polysaccharide by solid fermentation using Forsythia suspense residue according to claim 1, wherein Made from the following by weight percentage: 20% of honeysuckle and forsythia residue, 0.5% of wheat bran, 3% of soybean meal powder, 0.11% of urea, 0.3% of ammonium sulfate, and 76.09% of water.

5. The culture medium for high-yield Ganoderma lucidum polysaccharide by solid fermentation using Forsythia suspense residue according to claim 1, wherein, Made from the following by weight percentage: 12% of honeysuckle and forsythia residue, 1.7% of wheat bran, 1.2% of soybean meal powder, 0.05% of urea, 0.05% of ammonium sulfate, and 85% of water.

6. The culture medium for high-yield Ganoderma lucidum polysaccharide by solid fermentation using Forsythia suspense residue according to claim 1, characterized in that Made from the following by weight percentage: 15% of honeysuckle and forsythia residue, 2% of wheat bran, 2% of soybean meal powder, 0.15% of urea, 0.15% of ammonium sulfate, and 80.7% of water.

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