Trametes versicolor, a microbial agent containing the fungus, a culture method and application thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NANJING NORMAL UNIVERSITY
- Filing Date
- 2025-06-30
- Publication Date
- 2026-06-02
Smart Images
Figure CN120758364B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to microbial fermentation technology, specifically to a fungus *Trametes versicolor*, a fungal agent containing the fungus, its cultivation method, and its application. Background Technology
[0002] Yunzhi fungus ( Trametes versicolor Belonging to the phylum Basidiomycota, class Agaricomycetes, order Polyporales, and family Polyporaceae, its mycelium and fruiting bodies are rich in polysaccharides, flavonoids, triterpenoids, and laccase, among other active ingredients. These components possess anti-tumor, immunomodulatory, and antioxidant properties, and are widely used in the pharmaceutical, food, and cosmetic fields. Currently, over-harvesting of wild *Trametes versicolor* resources has led to severe degradation of its natural habitat. Furthermore, traditional solid-state cultivation methods have long cycles (3-6 months) and low fruiting body yields (<5g / kg substrate), making it difficult to meet the demands of industrial production. Therefore, there is an urgent need to achieve sustainable resource utilization through artificial cultivation techniques.
[0003] Although liquid submerged fermentation technology can shorten the cultivation cycle of *Trametes versicolor*, existing strains generally have a biomass of less than 20 g / L during fermentation, and extremely low levels of active ingredients such as flavonoids and triterpenoids, often failing to achieve the efficacy of wild *Trametes versicolor*. Furthermore, traditional *Trametes versicolor* culture media rely on high-cost raw materials such as glucose and yeast extracts, limiting its large-scale production; and the parameters of the submerged fermentation process (such as dissolved oxygen and stirring rate) lack systematic optimization, resulting in low and highly volatile synthesis efficiency of active ingredients (fluctuation rate of approximately ±15%), making it difficult to stably supply high-purity products.
[0004] Therefore, developing new strains of *Trametes versicolor* that are adapted to low-cost, highly stable fermentation processes with high content of active ingredients and low volatility is of great value in promoting the application of *Trametes versicolor* in the pharmaceutical field. Summary of the Invention
[0005] The purpose of this invention is to overcome the problems of low biomass, low content of active ingredients, high fermentation cost, and poor stability of the existing technology of *Trametes versicolor* fermentation. This invention provides *Trametes versicolor*, a fungal agent containing the fungus, its cultivation method, and its application. The fermentation performance of this *Trametes versicolor* is stable, the cost is low, the biomass and mycelial content are high, and it is rich in a variety of flavonoids and terpenoids.
[0006] To achieve the above objectives, the first aspect of the present invention provides a fungus called *Trametes versicolor*, which is a fungus that... Trametes versicolor The accession number of the object is CCTCC NO: M2025762.
[0007] A second aspect of the present invention provides a microbial agent containing *Trametes versicolor* as described above.
[0008] Preferably, the microbial agent contains at least one of the following: live cells, dead cells, and fermentation products of the *Trametes versicolor*.
[0009] Preferably, the microbial agent is a liquid microbial agent or a solid microbial agent.
[0010] The third aspect of the present invention provides a method for culturing *Trametes versicolor*, the method comprising the following steps: sequentially subjecting *Trametes versicolor* or the aforementioned fungal agent to seed culture and fermentation culture.
[0011] Preferably, the seed culture medium used for seed culture contains corn cake powder, peanut cake powder, potassium dihydrogen phosphate, magnesium sulfate and trace element solution.
[0012] Preferably, the trace element solution in the seed culture medium contains: 0.05-0.1 g / L ferric sulfate heptahydrate, 0.02-0.05 g / L zinc sulfate heptahydrate, and 0.02-0.05 g / L copper sulfate pentahydrate.
[0013] Preferably, the seed culture medium contains: 30-50 g / L corn cake powder, 25-30 g / L peanut cake powder, 1.2-2 g / L potassium dihydrogen phosphate, 1.5-2.5 g / L magnesium sulfate heptahydrate, and 0.5-1.5 mL / L trace element solution.
[0014] Preferably, the fermentation culture medium used in the fermentation culture contains corn cake powder, peanut cake powder, yeast extract, corn steep liquor powder, potassium dihydrogen phosphate, magnesium sulfate and trace element solution.
[0015] Preferably, the trace element solution in the fermentation medium contains: 0.05-0.1 g / L ferric sulfate heptahydrate, 0.02-0.05 g / L zinc sulfate heptahydrate, and 0.02-0.05 g / L copper sulfate pentahydrate.
[0016] Preferably, the fermentation medium contains: 30-50 g / L corn cake powder, 15-25 g / L peanut cake powder, 3-8 g / L yeast extract, 2-5 g / L corn steep liquor powder, 1-2 g / L potassium dihydrogen phosphate, 0.3-0.5 g / L magnesium sulfate heptahydrate, and 0.5-1.5 mL / L trace element solution.
[0017] Preferably, the seed culture includes a primary seed culture and a secondary seed culture performed sequentially.
[0018] Preferably, the conditions for the primary seed culture include: an inoculum size of 1×10⁻⁶. 5 -1×10 6CFU / mL, initial pH 5.5-6.5, temperature 25-30℃, rotation speed 150-250 rpm, time 3-5 days.
[0019] Preferably, the conditions for the secondary seed culture include: an inoculum size of 1×10⁻⁶. 7 -5×10 7 CFU / mL, initial pH 5.5-6.5, temperature 25-30℃, rotation speed 150-250 rpm, time 6-8 days.
[0020] Preferably, the fermentation culture conditions include: an inoculum size of 10-20% by volume, a temperature of 25-30°C, a pH of 5.5-6.5, dissolved oxygen below 20%, and a time of 5-7 days.
[0021] The fourth aspect of the present invention provides the use of the aforementioned *Trametes versicolor*, the aforementioned microbial agent, or the aforementioned method in the fermentation production of active ingredients and / or the preparation of functional products, wherein the active ingredients include flavonoids and / or terpenoids.
[0022] Preferably, the flavonoid compound is selected from at least one of gallic acid, ginkgo biflavonoids, 2'-hydroxychalcone, and protocatechuic acid, with gallic acid and ginkgo biflavonoids being more preferred; the terpene compound is myrcene.
[0023] Preferably, the functional product is an antitumor drug and / or an immunomodulatory drug.
[0024] The beneficial effects of this invention through the above technical solution are as follows: the *Trametes versicolor* fermentation culture provided by this invention has stable performance, high biomass and mycelial content, and high synthesis efficiency and low fluctuation of active ingredients. The *Trametes versicolor* obtained from the culture is rich in various flavonoids and terpenoids, especially gallic acid, ginkgo biflavonoids and myrcene, which have higher medicinal value. At the same time, this *Trametes versicolor* uses inexpensive corn cake flour and peanut cake flour as the main carbon and nitrogen sources, resulting in low fermentation production costs and a short cycle, making it suitable for large-scale production of pharmaceutical raw materials.
[0025] Biological Preservation
[0026] The Yunzhi HW-119 provided by this invention ( Trametes versicolor HW-119 was deposited on April 11, 2025, at the China Center for Type Culture Collection (address: Wuhan University, Wuchang District, Wuhan, Hubei Province, 430072, abbreviated as CCTCC), accession number CCTCC NO: M2025762. Attached Figure Description
[0027] Figure 1 This is a colony morphology diagram of *Trametes versicolor* in Example 1;
[0028] Figure 2 This is a diagram of the mycelial state of *Trametes versicolor* in Example 1;
[0029] Figure 3 This is an NCBI sequence comparison image of *Trametes versicolor* from Example 1;
[0030] Figure 4 This is a phylogenetic analysis diagram of *Trametes versicolor* in Example 1;
[0031] Figure 5 This is a graph showing the content of flavonoids in the *Trametes versicolor* obtained by fermentation in Example 2;
[0032] Figure 6 This is a graph showing the content of terpenoid compounds in the *Trametes versicolor* obtained by fermentation in Example 2;
[0033] Figure 7 This is a graph showing the content of active ingredients in 10 batches of Example 3 and Comparative Example 1. Detailed Implementation
[0034] The endpoints and any values of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of the various ranges, the endpoint values of the various ranges and individual point values, and individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.
[0035] The first aspect of the present invention provides a fungus called *Trametes versicolor*, which is a fungus called *Trametes versicolor* (…). Trametes versicolor ) is Yunzhi HW-119 ( Trametes versicolor HW-119), with accession number CCTCC NO: M2025762.
[0036] The colonies of *Trametes versicolor* provided by this invention are white, covering the entire dish, with a flat surface, filamentous texture, and a light yellow reverse side. No exudate or soluble pigment is produced. The hyphae are intertwined and branched, septate, with smooth walls, 1.5-6 μm in diameter, and produce a large number of clamp connections.
[0037] A second aspect of the present invention provides a microbial agent containing *Trametes versicolor* as described above.
[0038] In this invention, there is no particular limitation on the concentration of *Trametes versicolor* in the fungal agent; the concentration can be selected according to specific circumstances.
[0039] According to the present invention, the microbial agent preferably contains at least one of the live mycelium, dead mycelium, and fermentation products of the *Trametes versicolor*, more preferably live mycelium. In the present invention, the term "fermentation product" refers to the metabolites produced by *Trametes versicolor* during fermentation or cultivation (e.g., products containing flavonoids and / or terpenoids isolated from the mycelium and / or fruiting bodies of *Trametes versicolor*).
[0040] According to the present invention, there are no particular limitations on the dosage form of the microbial agent. It can be prepared into different dosage forms according to different intended uses, and corresponding excipients and other components can be added. For example, the microbial agent can be a liquid microbial agent (e.g., a liquid culture of *Trametes versicolor*) and / or a solid microbial agent (e.g., a powder made from dried *Trametes versicolor* or a high-purity preparation prepared through separation and purification steps). The choice of which excipient to add to which dosage form of the microbial agent is well known to those skilled in the art and will not be described in detail here.
[0041] The third aspect of the present invention provides a method for culturing *Trametes versicolor*, the method comprising the following steps: sequentially subjecting *Trametes versicolor* or the aforementioned fungal agent to seed culture and fermentation culture.
[0042] In this invention, there are no particular restrictions on the seed culture and fermentation culture methods, as long as a large number of live mycelium and / or fermentation products of *Trametes versicolor* can be produced through seed culture and fermentation culture.
[0043] According to the present invention, preferably, the seed culture medium used for seed culture contains corn cake powder, peanut cake powder, potassium dihydrogen phosphate, magnesium sulfate, and trace element solution. The *Trametes versicolor* provided by the present invention can effectively utilize corn starch and peanut cake powder as carbon and nitrogen sources, and promotes the growth of *Trametes versicolor*, which is beneficial for improving biomass and the synthesis efficiency of active substances, and reducing production costs.
[0044] In this invention, the trace elements include iron, zinc and copper. Preferably, the trace element solution in the seed culture medium contains: 0.05-0.1 g / L ferric sulfate heptahydrate, 0.02-0.05 g / L zinc sulfate heptahydrate, and 0.02-0.05 g / L copper sulfate pentahydrate.
[0045] More preferably, the seed culture medium contains: 30-50 g / L corn cake powder, 25-30 g / L peanut cake powder, 1.2-2 g / L potassium dihydrogen phosphate, 1.5-2.5 g / L magnesium sulfate heptahydrate, and 0.5-1.5 mL / L trace element solution. Under this preferred embodiment, the activity of *Trametes versicolor* can be better enhanced, thereby improving the biomass and bioactive substance synthesis efficiency during the fermentation culture stage.
[0046] According to the present invention, preferably, the fermentation culture medium used in the fermentation culture contains corn cake powder, peanut cake powder, yeast extract, corn steep liquor powder, potassium dihydrogen phosphate, magnesium sulfate, and trace element solution. The *Trametes versicolor* provided by the present invention can effectively use corn starch and peanut cake powder as the main carbon and nitrogen sources, avoiding the use of large amounts of high-cost raw materials such as glucose and yeast extract, thus reducing production costs. Furthermore, under this fermentation culture medium formulation, *Trametes versicolor* exhibits rapid growth, high biomass, and high efficiency in the synthesis of active substances.
[0047] In this invention, the trace elements include iron, zinc and copper. Preferably, the trace element solution in the fermentation culture medium contains: 0.05-0.1 g / L ferric sulfate heptahydrate, 0.02-0.05 g / L zinc sulfate heptahydrate, and 0.02-0.05 g / L copper sulfate pentahydrate.
[0048] More preferably, the fermentation medium contains: 30-50 g / L corn cake powder, 15-25 g / L peanut cake powder, 3-8 g / L yeast extract, 2-5 g / L corn steep liquor powder, 1-2 g / L potassium dihydrogen phosphate, 0.3-0.5 g / L magnesium sulfate heptahydrate, and 0.5-1.5 mL / L trace element solution. Under this preferred embodiment, the growth of *Trametes versicolor* can be better promoted, and the biomass and bioactive substance synthesis efficiency can be improved.
[0049] In this invention, *Trametes versicolor* is generally cryopreserved. When cultivation is required, it is preferably activated before seed culture. Exemplarily, after the cryopreserved *Trametes versicolor* is removed, it is streaked onto a plate of solid activation medium for activation culture. Preferably, the solid activation medium is PDA medium, and the activation culture conditions include: a temperature of 25-30°C and a time of 3-5 days. After activation culture, the *Trametes versicolor* on the plate is inoculated for seed culture. Under this preferred embodiment, the viability of *Trametes versicolor* can be better enhanced, thereby improving the biomass and bioactive substance synthesis efficiency during the fermentation culture stage.
[0050] According to the present invention, preferably, the seed culture includes a primary seed culture and a secondary seed culture performed sequentially. Two-stage seed culture can effectively improve the fermentation performance of *Trametes versicolor*, promote its growth rate during the fermentation stage, increase biomass and mycelial content, and achieve high efficiency in the synthesis of active substances.
[0051] According to the present invention, preferably, the conditions for the primary seed culture include: an inoculum size of 1 × 10⁻⁶. 5 -1×10 6The inoculum concentration was CFU / mL, the initial pH was 5.5-6.5, the temperature was 25-30℃, the rotation speed was 150-250 rpm, and the time was 3-5 days. More preferably, the conditions for the secondary seed culture included: an inoculum size of 1×10⁻⁶ CFU / mL. 7 -5×10 7 The optimal fermentation method involves using CFU / mL, setting the initial pH at 5.5-6.5, maintaining a temperature of 25-30℃, rotating at 150-250 rpm, and allowing the fermentation time to be 6-8 days. This preferred method enhances the activity of *Trametes versicolor*, thereby improving biomass and the efficiency of bioactive substance synthesis during the fermentation stage.
[0052] The initial pH of primary and secondary seed cultures is adjusted to the required pH by adding hydrochloric acid or sodium hydroxide solution.
[0053] According to the present invention, preferably, the fermentation culture conditions include: an inoculum size of 10-20% by volume, a temperature of 25-30°C, a pH of 5.5-6.5, dissolved oxygen below 20%, and a time of 5-7 days. In this preferred embodiment, optimizing the dissolved oxygen control in conjunction with the components of the fermentation medium can further improve the biomass and yield of active ingredients of *Trametes versicolor*.
[0054] In this invention, "dissolved oxygen below 20%" refers to a dissolved oxygen concentration (DO) in the fermentation broth that is less than 20% of the saturated dissolved oxygen concentration. This saturation value is based on the dissolved oxygen concentration of pure water at 25°C and 101.3 kPa as 100%. Dissolved oxygen during fermentation can be controlled below 20% using conventional methods in the art. For example, dissolved oxygen is controlled by adjusting the stirring rate of the stirred fermenter to 200-300 rpm and the aeration rate to 1-2 vvm. The pH during fermentation is controlled at 5.5-6.5 by adding hydrochloric acid or sodium hydroxide solution.
[0055] As a relatively preferred embodiment of the cultivation method of *Trametes versicolor* in this invention, the cultivation method includes:
[0056] (1) Inoculate the frozen Trametes versicolor onto PDA medium plates and culture at 25-30℃ for 3-5 days;
[0057] (2) Inoculate the *Trametes versicolor* on the PDA culture medium plate with an inoculum size of 1×10⁻⁶. 5 -1×10 6 CFU / mL was inoculated into seed culture medium and cultured with shaking at an initial pH of 5.5-6.5, a temperature of 25-30℃, and a rotation speed of 150-250 rpm for 3-5 days to obtain primary seed culture.
[0058] (3) The primary seed culture was inoculated at a rate of 1×10⁻⁶. 7 -5×10 7 CFU / mL was transferred to seed culture medium and cultured with shaking at an initial pH of 5.5-6.5, a temperature of 25-30℃, and a rotation speed of 150-250 rpm for 6-8 days to obtain secondary seed culture.
[0059] (4) Inoculate the secondary seed culture into the fermentation medium at an inoculation rate of 10-20% by volume, and culture for 5-7 days at a temperature of 25-30℃, a pH of 5.5-6.5, and a dissolved oxygen value controlled below 20%.
[0060] The seed culture medium contains: 30-50 g / L corn cake powder, 25-30 g / L peanut cake powder, 1.2-2 g / L potassium dihydrogen phosphate, 1.5-2.5 g / L magnesium sulfate heptahydrate, and 0.5-1.5 mL / L trace element solution; the fermentation culture medium contains: 30-50 g / L corn cake powder, 15-25 g / L peanut cake powder, 3-8 g / L yeast extract, 2-5 g / L corn steep liquor powder, 1-2 g / L potassium dihydrogen phosphate, 0.3-0.5 g / L magnesium sulfate heptahydrate, and 0.5-1.5 mL / L trace element solution.
[0061] The trace element solution contains: 0.05-0.1 g / L ferric sulfate heptahydrate, 0.02-0.05 g / L zinc sulfate heptahydrate, and 0.02-0.05 g / L copper sulfate pentahydrate.
[0062] Based on the cultivation method provided above, the *Trametes versicolor* strain can better utilize the liquid submerged fermentation cultivation scheme compared to existing strains, exhibiting better fermentation performance, higher biomass and mycelial content, and higher yields of flavonoids and terpenoids.
[0063] The fourth aspect of the present invention provides the use of the aforementioned *Trametes versicolor*, the aforementioned microbial agent, or the aforementioned method in the fermentation production of active ingredients and / or the preparation of functional products, wherein the active ingredients include flavonoids and / or terpenoids.
[0064] According to the present invention, the total flavonoid content and total terpene content of the *Trametes versicolor* are increased after fermentation. Preferably, the flavonoids are selected from at least one of gallic acid, ginkgo biflavonoids, 2'-hydroxychalcone, and protocatechuic acid, and more preferably gallic acid and ginkgo biflavonoids; the terpene is myrcene.
[0065] According to the present invention, preferably, the functional product is an antitumor drug and / or an immunomodulatory drug. The mycelium and / or fruiting body of the *Trametes versicolor* can be used directly to prepare the functional product, or the portion containing the desired active ingredient (e.g., gallic acid, ginkgo biloba flavonoids, or myrcene, etc.) extracted or isolated from the *Trametes versicolor* can be used to prepare the functional product.
[0066] The present invention will be described in detail below through embodiments.
[0067] In the following examples, the preparation process of PDA culture medium is as follows: Weigh 200g of potatoes, cut them into small pieces, add water and boil until soft (boil for 25 minutes, until they can be pierced by a glass rod), filter with eight layers of gauze, heat, add 15g of agar, continue heating and stirring until well mixed, after the agar has dissolved, add 20g of glucose, stir well, cool slightly and then add water to make up to 1000ml, sterilize at 115℃ for 20 minutes;
[0068] Unless otherwise specified, all raw materials or reagents used are conventional commercial products.
[0069] Example 1
[0070] 1.1 The Yunzhi fungus provided by this invention ( Trametes versicolor The process of obtaining )
[0071] Naturally decayed deadwood was collected from the Changbai Mountain primeval forest system in Huadian City, Jilin City, Jilin Province. Under sterile conditions, internal tissue blocks of the deadwood were extracted, and after surface disinfection, they were placed in PDA medium for cultivation. Pure culture—strain HW-119—was obtained by repeatedly picking the hyphal tips.
[0072] 1.2 Physiological characteristics and molecular biological identification of the strain
[0073] The colonies of strain HW-119 obtained in 1.1 on PDA plates are shown as follows: Figure 1 As shown, the colonies are white, completely covering the entire dish, with a flat surface, filamentous texture, and a pale yellow reverse side. No exudate or soluble pigment is produced. The mycelium of strain HW-119 is observed under a microscope as follows. Figure 2 As shown, the hyphae are intertwined and branched, septate, with smooth walls, and a diameter of 1.5-6 μm, producing a large number of clamp connections.
[0074] DNA was extracted from strain HW-119 using a kit (TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver. 3.0). PCR amplification was performed using ITS1+ITS4 as primers. The PCR products were detected by electrophoresis and sequenced by Qingke (Nanjing) Biotechnology Co., Ltd. The ITS sequence is shown in SEQ ID NO. 1. BLAST analysis was performed on the obtained ITS sequence and existing sequences in the NCBI database. Phylogenetic trees were constructed using MEGA 7.0 software for strains with similar homology.
[0075] ITS sequence (SEQ ID NO. 1):
[0076] .
[0077] ITS sequence alignment on the NCBI database showed that strain HW-119 is related to *Trametes versicolor* (also known as *Trametes versicolor*). Trametes versicolorThe ITS sequences of the isolates are highly similar and relatively close in evolutionary distance. The NCBI sequence comparison diagram of this isolate is shown below. Figure 3 As shown, the phylogenetic tree is as follows: Figure 4 As shown, based on the physiological and biochemical characteristics of the strain, it was preliminarily identified as *Trametes versicolor* (Yun Zhi Jun). Trametes versicolor ).
[0078] The acclimatization process of strain HW-119: strain HW-119 was inoculated into acclimatization medium and passaged 10-15 times. The acclimatization medium formula was: glucose 20g / L, corn flour 10g / L, wheat bran 5g / L, peptone 3g / L, potassium dihydrogen phosphate 1.5g / L, magnesium sulfate 0.2g / L, and zinc sulfate 0.05g / L. The passage conditions were: temperature 26℃, shaking speed 120rpm, and each passage lasted for 6 days.
[0079] After the above-mentioned domestication culture of strain HW-119, it was used as Yunzhi HW-119 ( Trametes versicolor HW-119 was classified and named accordingly. It was deposited on April 11, 2025, at the China Center for Type Culture Collection (Address: Room 211, China Center for Type Culture Collection, Wuhan University, Wuchang District, Wuhan, Hubei Province, 430072, China; accession number: CCTCC NO: M2025762) and will be used for subsequent experimental research.
[0080] Example 2
[0081] The formula for the trace element solution is: 0.08 g / L ferric sulfate heptahydrate, 0.03 g / L zinc sulfate heptahydrate, and 0.03 g / L copper sulfate pentahydrate;
[0082] The seed culture medium formula is: 40 g / L corn cake powder, 28 g / L peanut cake powder, 1.5 g / L potassium dihydrogen phosphate, 2 g / L magnesium sulfate heptahydrate and 1 mL / L trace element solution;
[0083] The fermentation medium formula is as follows: 40 g / L corn cake powder, 20 g / L peanut cake powder, 5 g / L yeast extract, 3 g / L corn steep liquor powder, 1.5 g / L potassium dihydrogen phosphate, 0.4 g / L magnesium sulfate heptahydrate, and 1 mL / L trace element solution;
[0084] (1) The frozen strain HW-119 obtained in Example 1 was inoculated onto a PDA medium plate and cultured at 26°C for 4 days;
[0085] (2) Inoculate the *Trametes versicolor* on the PDA culture medium plate with an inoculum size of 5 × 10⁻⁶. 5CFU / mL was inoculated into the seed culture medium and cultured with shaking at an initial pH of 6, a temperature of 26℃, and a rotation speed of 200 rpm for 3-5 days to obtain the primary seed culture.
[0086] (3) The primary seed culture was inoculated at a rate of 3 × 10⁻⁶. 7 CFU / mL was transferred to seed culture medium and cultured with shaking at an initial pH of 6, a temperature of 26℃, and a rotation speed of 200 rpm for 6-8 days to obtain secondary seed culture.
[0087] (4) The secondary seed culture was inoculated into the fermentation medium at an inoculation rate of 15% (v / v) and cultured for 6 days at a temperature of 26℃, a pH of 6, and a dissolved oxygen value of approximately 18% (controlled by a stirring rate of 250 rpm and an aeration rate of 1.5 vvm).
[0088] Example 3
[0089] The formula for the trace element solution is: 0.1 g / L ferric sulfate heptahydrate, 0.02 g / L zinc sulfate heptahydrate, and 0.02 g / L copper sulfate pentahydrate;
[0090] The seed culture medium formula is: 30 g / L corn cake powder, 30 g / L peanut cake powder, 2 g / L potassium dihydrogen phosphate, 2.5 g / L magnesium sulfate heptahydrate and 1 mL / L trace element solution;
[0091] The fermentation medium formula is as follows: 30 g / L corn cake powder, 25 g / L peanut cake powder, 8 g / L yeast extract, 5 g / L corn steep liquor powder, 2 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate heptahydrate and 1 mL / L trace element solution;
[0092] (1) The frozen strain HW-119 obtained in Example 1 was inoculated onto a PDA medium plate and cultured at 25°C for 5 days;
[0093] (2) Inoculate the *Trametes versicolor* on the PDA culture medium plate with an inoculum size of 1×10⁻⁶. 5 CFU / mL was inoculated into the seed culture medium and cultured with shaking at an initial pH of 5.5, a temperature of 25℃, and a rotation speed of 250 rpm for 3 days to obtain the primary seed culture.
[0094] (3) The primary seed culture was inoculated at a rate of 1×10⁻⁶. 7 CFU / mL was transferred to seed culture medium and cultured with shaking at an initial pH of 5.5, a temperature of 25℃, and a rotation speed of 250 rpm for 6 days to obtain secondary seed culture;
[0095] (4) Inoculate the secondary seed culture into the fermentation medium at an inoculation rate of 10% (v / v) and culture for 5 days at a temperature of 25℃, a pH of 5.5, and a dissolved oxygen value of approximately 17% (controlled by a stirring rate of 300 rpm and an aeration rate of 2 vvm).
[0096] Example 4
[0097] The formula for the trace element solution is: 0.05 g / L ferric sulfate heptahydrate, 0.05 g / L zinc sulfate heptahydrate, and 0.05 g / L copper sulfate pentahydrate;
[0098] The seed culture medium formula is: 50 g / L corn cake powder, 25 g / L peanut cake powder, 1.2 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate heptahydrate and 1 mL / L trace element solution;
[0099] The fermentation medium formula is as follows: 50 g / L corn cake powder, 15 g / L peanut cake powder, 3 g / L yeast extract, 2 g / L corn steep liquor powder, 1 g / L potassium dihydrogen phosphate, 0.3 g / L magnesium sulfate heptahydrate and 1 mL / L trace element solution;
[0100] (1) The frozen strain HW-119 obtained in Example 1 was inoculated onto a PDA medium plate and cultured at 30°C for 3 days;
[0101] (2) Inoculate the *Trametes versicolor* on the PDA culture medium plate with an inoculum size of 1×10⁻⁶. 6 CFU / mL was inoculated into the seed culture medium and cultured with shaking at an initial pH of 6.5, a temperature of 30℃, and a rotation speed of 150 rpm for 5 days to obtain the primary seed culture.
[0102] (3) The primary seed culture was inoculated at a rate of 5 × 10⁻⁶. 7 CFU / mL was transferred to seed culture medium and cultured with shaking at an initial pH of 6.5, a temperature of 30℃, and a rotation speed of 150 rpm for 8 days to obtain secondary seed culture;
[0103] (4) The secondary seed culture was inoculated into the fermentation medium at an inoculation rate of 20% (v / v) and cultured for 7 days at a temperature of 30℃, a pH of 6.5, and a dissolved oxygen value of approximately 19% (controlled by a stirring rate of 200 rpm and an aeration rate of 1 vvm).
[0104] Example 5
[0105] The method of Example 3 was used to cultivate Ganoderma lucidum, except that the dissolved oxygen in step (4) was controlled at about 25% (by stirring at 100 rpm and aeration at 1 vvm).
[0106] Example 6
[0107] The method of Example 3 was used to cultivate Ganoderma lucidum, except that pH 6 in step (4) was replaced with pH 5.
[0108] Example 7
[0109] The *Trametes versicolor* was cultured according to the method in Example 3, except that the following ingredients were used: 50 g / L glucose, 15 g / L peanut cake powder, 3 g / L yeast extract, 2 g / L corn steep liquor powder, 1 g / L potassium dihydrogen phosphate, 0.1 g / L magnesium sulfate, and 1 mL / L trace element solution.
[0110] Example 8
[0111] The method of Example 3 was used to cultivate *Trametes versicolor*, except that the seed culture medium formula was replaced with: 50 g / L glucose, 25 g / L yeast extract, 1.2 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, and 1 mL / L trace element solution.
[0112] Comparative Example 1
[0113] The *Trametes versicolor* was cultured according to the method of Example 3, except that the strain HW-119 obtained in Example 1 was replaced with... Trametes versicolor (L.) Lloyd Yunzhi fungus (purchased from Beina Biotechnology, product number BNCC123332).
[0114] Test Example 1
[0115] In the fermentation broths obtained in Examples 2-8 and Comparative Example 1, the biomass of *Trametes versicolor* was obtained by the following method: 50 mL of fermentation broth was vacuum filtered, rinsed with distilled water, and then freeze-dried to constant weight. The results are shown in Table 2.
[0116] The flavonoid content of *Trametes versicolor* obtained by fermentation in Examples 2-8 and Comparative Example 1 was determined by the following methods:
[0117] 1) The fermentation broth after fermentation was filtered to separate the mycelium. The fruiting bodies of Ganoderma lucidum were freeze-dried at -80℃ for 48 hours, then taken out, crushed, dried, and then passed through a 60-mesh sieve. The lipids were removed by using 95% (v / v) ethanol to obtain a uniform powder.
[0118] 2) Mobile phase configuration
[0119] Organic phase: 0.3% formic acid-acetonitrile solution, obtained by adding 900 mL of chromatographic grade acetonitrile to a 1 L volumetric flask, adding 3 mL of formic acid, and diluting to 1 L with chromatographic grade acetonitrile, then inverting and mixing.
[0120] Inorganic phase: 0.3% formic acid-water solution, by adding 900mL of ultrapure water to a 1L volumetric flask, adding 3mL of formic acid, and making up to 1L with ultrapure water, and mixing by inverting.
[0121] 3) Weigh 5 mg of the powder sample obtained in step 1), add 5 mL of 1% hydrochloric acid-methanol solution, vortex for 1 min, and sonicate in an ice-water bath for 30 min;
[0122] 4) Centrifuge at 11000 rpm for 5 min and collect the supernatant;
[0123] 5) After thorough mixing, filter through a 0.22μm organic phase membrane and place in a -20℃ freezer for instrument testing;
[0124] 6) Liquid phase conditions
[0125] Column: Agilent ZORBAX Eclipse Plus C18 (3.5µm, 2.1m) 150mm);
[0126] Flow rate: 0.3 mL / min; Autosampler temperature: 4℃; Run time: 10 min; Column temperature: 35℃; Injection volume: 3 μL;
[0127] 7) Mobile phase: Phase A uses the 0.3% formic acid-water solution prepared in step 2), and Phase B uses the 0.3% formic acid-acetonitrile solution prepared in step 2). The gradient elution program is shown in Table 1.
[0128] The results of the detection of flavonoid components in *Trametes versicolor* obtained in each example and comparative example are shown in Tables 2 and 3. The results of the flavonoid components and their contents in *Trametes versicolor* obtained in Example 2 are shown in Table 3. Figure 5 .
[0129] Table 1
[0130]
[0131] The terpenoid content of *Trametes versicolor* obtained by fermentation in Examples 2-8 and Comparative Example 1 was determined by the following methods:
[0132] 1) Filter the fermentation broth after fermentation to separate the mycelium. Freeze-dry the fruiting bodies of *Trametes versicolor* at -80℃ for 48 hours, then remove, pulverize, dry, grind, and weigh 5g of powder sample. Add 5mL of methanol solution, vortex for 1min, and sonicate in an ice-water bath for 30min. Centrifuge at 11000rpm for 5min and collect the supernatant. Add 5mL of petroleum ether solution to the residue, vortex for 1min, and sonicate in an ice-water bath for 30min. Centrifuge at 11000rpm for 5min and collect the supernatant.
[0133] 2) Combine the two supernatants, dry them, filter them through a 0.22μm organic phase membrane, and place them in a -20℃ freezer for instrument testing;
[0134] 3) Gas chromatographic conditions for GC-MS analysis: The chromatographic column was a TG-5MS (30m×0.25mm×0.25μm) flexible quartz capillary column; the carrier gas was high-purity helium (purity 99.999%); the carrier gas flow rate was 1.0mL / min; splitless injection was used; the injection port temperature was 280℃; the temperature program was: initial temperature 40℃ held for 1 min, then increased to 300℃ at 10℃ / min and held for 6 min.
[0135] 4) Mass spectrometry conditions for GC-MS analysis: EI source, transfer line temperature: 280℃; ion source temperature: 300℃; electron energy: 70eV; scan range (m / z): 33-300amu, full scan acquisition mode;
[0136] 5) Plot a standard curve with the measured characteristic ion mass chromatographic peak external standard as the ordinate and the corresponding standard solution concentration as the abscissa, and find the regression equation and correlation coefficient.
[0137] The results of the detection of terpene components in the fermentation broth of *Trametes versicolor* obtained in each example and comparative example are shown in Tables 2 and 3. The results of the terpene components and their contents in *Trametes versicolor* obtained in Example 2 are shown in Table 3. Figure 6 .
[0138] Table 2
[0139]
[0140] Table 3
[0141]
[0142] Test Example 2
[0143] To observe the stability of the active ingredient production process of strain HW-119, the process of culturing *Trametes versicolor* in Example 3 was repeated 10 times. The content of active ingredients (gallic acid, ginkgo biflavonoids, 2'-hydroxychalcone, and myrcene) in the fermented *Trametes versicolor* mycelium of each of the 10 batches was measured, and the average value was calculated. Comparative Example 1 was repeated 10 times as a control. The results are shown in […]. Figure 7 .
[0144] It can be seen that the strain HW-119 provided by this invention not only has a good production capacity of active ingredients, but also, judging from the error bars of the 10 batches of data, it is significantly better than that in Comparative Example 1. Trametes versicolor (L.) Lloyd The content of active ingredients in the *Trametes versicolor* strain HW-119 fluctuated less, indicating that it has better stability in the production of active ingredients.
[0145] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.
Claims
1. A type of Yunzhi fungus ( Trametes versicolor ), characterized in that, The preservation number of this *Trametes versicolor* is CCTCCNO: M2025762.
2. An inoculant characterized in that, The microbial agent contains the *Trametes versicolor* as described in claim 1.
3. The bacterial agent of claim 2, wherein The microbial agent contains live cells of the *Trametes versicolor* and / or fermentation products; The bacterial agent can be a liquid bacterial agent or a solid bacterial agent.
4. A cultivation method of Coriolus versicolor, characterized by, The method includes the following steps: sequentially subjecting the *Trametes versicolor* of claim 1 or the fungal agent of claim 2 or 3 to seed culture and fermentation culture.
5. The method of claim 4, wherein, The seed culture medium used in the seed culture contains corn cake powder, peanut cake powder, potassium dihydrogen phosphate, magnesium sulfate and trace element solution; The trace element solution in the seed culture medium contains: ferric sulfate heptahydrate 0.05-0.1 g / L, zinc sulfate heptahydrate 0.02-0.05 g / L, and copper sulfate pentahydrate 0.02-0.05 g / L; The seed culture medium contains: 30-50 g / L corn cake powder, 25-30 g / L peanut cake powder, 1.2-2 g / L potassium dihydrogen phosphate, 1.5-2.5 g / L magnesium sulfate heptahydrate, and 0.5-1.5 mL / L trace element solution.
6. The method of claim 5, wherein, The fermentation culture medium used in the fermentation culture contains corn cake powder, peanut cake powder, yeast extract, corn steep liquor powder, potassium dihydrogen phosphate, magnesium sulfate and trace element solution. The trace element solution in the fermentation medium contains: ferric sulfate heptahydrate 0.05-0.1 g / L, zinc sulfate heptahydrate 0.02-0.05 g / L, and copper sulfate pentahydrate 0.02-0.05 g / L; The fermentation medium contains: 30-50 g / L corn cake powder, 15-25 g / L peanut cake powder, 3-8 g / L yeast extract, 2-5 g / L corn steep liquor powder, 1-2 g / L potassium dihydrogen phosphate, 0.3-0.5 g / L magnesium sulfate heptahydrate, and 0.5-1.5 mL / L trace element solution.
7. The method according to any one of claims 4 to 6, characterized in that, The seed culture includes a primary seed culture and a secondary seed culture performed sequentially. The conditions of the primary seed culture include: inoculation amount of 1×10 5 -1×10 6 CFU / mL, initial pH of 5.5-6.5, temperature of 25-30℃, rotation speed of 150-250rpm, and time of 3-5 days; The conditions of the secondary seed culture include: inoculation amount of 1×10 7 -5×10 7 CFU / mL, initial pH of 5.5-6.5, temperature of 25-30℃, rotation speed of 150-250rpm, and time of 6-8 days.
8. The method according to any one of claims 4 to 6, characterized in that, The fermentation culture conditions include: an inoculum size of 10-20% by volume, a temperature of 25-30℃, a pH of 5.5-6.5, dissolved oxygen below 20%, and a time of 5-7 days.
9. The use of the Trametes versicolor of claim 1 or the agent of claim 2 or 3 in the fermentation production of active ingredients; wherein, The active ingredient is a flavonoid and / or a terpene; the flavonoid is selected from at least one of gallic acid, ginkgo biflavonoid and 2'-hydroxychalcone, and the terpene is myrcene.
10. Use according to claim 9, characterized in that, The flavonoids mentioned are gallic acid and ginkgo biflavonoids.
11. Use of the process according to any one of claims 4 to 8 for the fermentative production of active ingredients; wherein, The active ingredient is a flavonoid and / or a terpene; the flavonoid is selected from at least one of gallic acid, ginkgo biflavonoid and 2'-hydroxychalcone, and the terpene is myrcene.
12. Use according to claim 11, characterized in that, The flavonoids mentioned are gallic acid and ginkgo biflavonoids.