Filamentous fungi and their use

Abstract

The present application belongs to the technical field of microorganism screening, and particularly relates to a Byssochlamys sp. and application thereof, wherein the Byssochlamys sp. has a preservation number of CGMCC No. 40543. When the Byssochlamys sp. is used for solid-state fermentation of pinellia, the digestion-assisting ability of pinellia powder can be improved, the toxicity of pinellia can be reduced, the content of active components such as amino acids, alkaloids and flavones for relieving cough can be increased, and the content of anticancer components can be increased. When the Byssochlamys sp. is used for liquid-state fermentation of pinellia, compared with traditional fermentation, the total acid and the soluble solids can be increased. Meanwhile, the Byssochlamys sp. is stable in fermentation, and can also be used for preparing feed containing a mint fragrance.

Description

Technical Field

[0001] This invention belongs to the field of microbial screening technology, specifically relating to a strain of Byssochlamys sp. and its applications. Background Technology

[0002] Pinellia ternata is a precious traditional Chinese medicine. After microbial fermentation, its organic acids and nucleosides are significantly increased, reducing oral irritation. Furthermore, fermentation enhances its flavor and digestive properties. The traditional fermentation method for Pinellia ternata is natural mixed-culture fermentation, requiring Shenqu (a type of starter culture) as an inoculum to initiate fermentation. The quality of Shenqu determines the fermentation cycle and quality of the Pinellia ternata starter culture. Currently, the quality of Shenqu on the market varies greatly, and the production process of Pinellia ternata starter culture relies heavily on it, often resulting in poor stability and a low pass rate. Moreover, due to the complexity and variability of the mixed fermentation system, quality monitoring and testing methods are difficult to establish, leading to inconsistent application effects of Pinellia ternata starter culture.

[0003] The safety of using Liushenqu has been verified over hundreds of years, and it has important application value in the production of Banxiaqu. Currently, multifunctional bacteria and fungi that can be used for Banxiaqu fermentation, including Bacillus subtilis, which produces high levels of amylase, Saccharomyces cerevisiae, Pichia pastoris, and Pichia bournei, have been isolated from Liushenqu. However, the role of these microorganisms in the fermentation process of Banxiaqu is still poorly understood.

[0004] Studies have shown that Bacillus subtilis, Aspergillus, and Paecilomyces in Pinellia ternata koji or Liushen koji are related to the promotion of active substances such as polysaccharides and organic acids. Some species have been isolated and purified for use in the pure fermentation of Pinellia ternata. Bacillus subtilis and Aspergillus are superior enzyme-producing bacteria and are commonly found in various fermentation koji, especially in Baijiu koji. In recent years, these bacteria have also been found in the analysis of the microbial community of traditional Chinese medicine koji. For example, Guo Jiajia et al. (Guo Jiajia, Su Mingsheng, Wang Liyuan, et al. Identification of dominant microorganisms in the processing of Pinellia ternata koji [J]. Chinese Journal of Traditional Chinese Medicine, 2016, 41(16)) isolated Saccharomyces giardi, Aspergillus niger, and Bacillus subtilis in Pinellia ternata koji, which are the main dominant bacteria in the fermentation of koji. Liang Qi (Liang Qi. Fermentation process of modified Pinellia ternata, isolation and identification of dominant strains and pure culture fermentation study [D]. Hunan Agricultural University, 2019.) also isolated Aspergillus niger with high enzyme activity from modified Pinellia ternata. Chen Yanlin et al. (Chen Yanlin, Wang Yunting, Guan Kaile, et al. Study on microbial community structure during fermentation of 6 Shenqu [J]. Chinese Journal of Traditional Chinese Medicine, 2020, 45(21):7) found that *Saccharomyces cerevisiae* and *Rhizopus oryzae* are the main sources of fermentation power in commercially available 6 Shenqu. To date, there is still a lack of understanding of the changes in the microbial community structure and fermentation characteristics of Pinellia ternata, and no strains with good performance have been isolated for pure culture fermentation of Pinellia ternata.

[0005] On the other hand, the quality of Liushenqu products varies from region to region and even from batch to batch from the same company, resulting in significant differences in the quality of Banxiaqu. Furthermore, Banxiaqu uses a natural mixed-culture inoculation method and open fermentation. As environmental factors such as airborne microorganisms, temperature, and humidity vary, the structure of the dominant microorganisms in the qu differs, which easily leads to fluctuations in the quality of different batches of products, making it difficult to achieve standardized production. Summary of the Invention

[0006] In order to obtain a superior strain of bacteria with reduced toxicity and enhanced efficacy for use in pure mycelial fermentation of Pinellia ternata and to improve the stability of Pinellia ternata products, this invention provides a strain of Byssochlamys sp. isolated from the fermentation process of Pinellia ternata in the workshop of Sichuan Ya'an Xunkang Pharmaceutical Co., Ltd.

[0007] The Byssochlamys sp. described in this invention has the accession number CGMCC No.40543 and was deposited on March 29, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0008] The physiological characteristics of the above-mentioned *Cryptomeria filamentosa* are as follows: after being cultured on PDA solid medium at 35°C for 48 hours, it forms a white hairy edge, a yellow circular center with a certain color difference from the surrounding color, and a dark brown back. It is not easy to pick up and has no obvious odor. The spores are elliptical, clustered in chains, with many branches around the main spore stalk. The branch spore stalks are curved, with only a bundle of small branches at the top.

[0009] The PDA solid culture medium formula is as follows: 200g potato, 20g glucose, 20g agar, 1L water, 0.1g chloramphenicol, sterilized at 121℃ for 15min.

[0010] The growth conditions of the above-mentioned *Cryptomeria filamentosa* are: temperature 35–38℃, pH 6.0–7.0; more preferably, the growth conditions are 35℃, pH 6.0–7.0. It can tolerate contamination by other microorganisms and can fully grow on the culture medium within 24–36 hours under suitable conditions.

[0011] The aforementioned *Hymenopterus* includes the following partially amplified IST1 and IST4 sequences:

[0012] SEQ ID NO:1: IST1

[0013] CACTTGCGTACGGATACGTCGAGGGACTGCAAGATGTCATTGGAGGGGGGGGGGGCCCCCCGCCGCCCCCAACAAAAAACGCGGTTGGCCGGAGGGGGGTTCAACCCCCCCGGCCCAAAACCGCCGAAGACCCCTGGAACGCTGCCTGGAAGGTTGCCGTCTGAGTATACAATCAATCAATTAAAACTTTCAACAACGGATCTCTTGGTTCCGGGATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCCCTGGCATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTAACCCTCCAGCCCGGCTGGTGTGTTGGGCCGCCGTCCCCCCTCCCCGGGGGACGGGCCCGAAAGGCAGCGGCGGCGTCGCGTCCGGTCCTCGAGCGTATGGGGCTCTGTCACACGCTTCAGTAGAACCGGCCGGCTTGCTGGCCATCACCTATATTTTTCTCTTAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAA。

[0014] SEQ ID NO:2:IST4

[0015] .

[0016] The above-mentioned application of *Cymbidium goeringii* in solid-state or liquid-state fermentation of traditional Chinese medicine.

[0017] Preferably, the traditional Chinese medicine is selected from at least one of Pinellia ternata and Pinellia ternata var. chinensis.

[0018] Preferably, the liquid fermentation conditions are: fermentation temperature of 33°C, inoculum amount of 6%, material-to-liquid ratio of 1:4, rotation speed of 60 r / min, and fermentation time of 72 h.

[0019] The above-mentioned application of *Cryptomeria filamentosa* in the preparation of Pinellia ternata and Shenqu (a traditional Chinese medicine formula).

[0020] The above-mentioned uses of *Veinium filamentum* in the fermentation production of feed.

[0021] Preferably, the feed ingredients include at least one of wheat bran, flour, and Polygonum hydropiper.

[0022] The above-mentioned application of *Hymenopterus xanthipes* in the fermentation preparation of a substance with a minty aroma, wherein the raw material for the substance with a minty aroma includes Polygonum hydropiper.

[0023] Beneficial effects: This invention screened and isolated a strain of Byssochlamys sp. from the fermentation workshop of Sichuan Ya'an Xunkang Pharmaceutical Co., Ltd., with the preservation number: CGMCC No.40543.

[0024] 1. The *Hymenopterus xylophilus* of this invention is a fungus isolated from the fermentation process of *Pinellia ternata*. It is well adapted to the environment of *Pinellia ternata* and can rapidly form a growth advantage in the fermentation substrate of *Pinellia ternata*. Vigorous mycelium visible to the naked eye can be formed within 48 hours, while the traditional fermentation method generally takes 72 hours.

[0025] 2. The *Ficus pumila* fungus of the present invention can enhance the digestive ability of *Pinellia ternata* koji. Compared with traditional mixed fermentation, the amylase activity of the fermented *Pinellia ternata* koji is as high as 26% to 41%, and the protease activity is as high as 15% to 23%.

[0026] 3. The fermentation of Pinellia ternata by *Cymbidium goeringii* of this invention can reduce the toxicity of Pinellia ternata. After fermentation, the calcium oxalate content decreases by 12% to 18%, reducing the irritation of the mucous membrane by calcium oxalate needle crystals in Pinellia ternata, thus achieving detoxification. The mannose content increases by 50%, and mannose can bind to the inflammatory agglutinin in Pinellia ternata (content between 2.6% and 4.1%), thereby reducing the toxicity of Pinellia ternata.

[0027] 4. The fermentation of Pinellia ternata by *Vitex negundo* of this invention significantly increases the content of amino acids, alkaloids, flavonoids, and other active components for sedation and cough relief, with a relative increase of more than 10 times; the content of antiemetic polysaccharides remains largely unchanged, while the total organic acid content increases by 20 times. It was also found that the content of other medicinally valuable components increased significantly, such as the anticancer component vitexin, which increased by more than 340 times. Fermentation using this fungus can effectively improve the quality of Pinellia ternata preparations.

[0028] 5. The *Synthia spp.* of this invention has good stability and is suitable for both solid-state and liquid-state fermentation environments. Moreover, its performance in liquid-state fermentation is superior to that in solid-state fermentation, which provides a possibility for the development of a modern pure-culture fermentation process for *Pinellia ternata*.

[0029] 6. The *Polygonum hydropiper* of the present invention can be used for fermentation production of feed, and when fermenting feed containing Polygonum hydropiper, it can produce a minty aroma. Attached Figure Description

[0030] Figure 1 The colony morphology of *Hymenopterus xylostella* produced by the present invention after being cultured on PDA solid medium at 35°C for 48 hours is shown.

[0031] Figure 2 This is a feedback diagram of calcium oxalate needle crystal stimulation of rabbit eye membrane in Example 2 of the present invention, where A: unfermented Pinellia ternata matrix; B: traditionally fermented Pinellia ternata starter; C: Pinellia ternata starter fermented with Hymenopseoincarnate.

[0032] Figure 3The substances that show significant differences after solid-state fermentation of Pinellia ternata by Hymenobacterium oxyphylla in Example 2 of this invention are shown in the figure. In the figure, hun refers to the fermentation substrate mixed with this strain, yuan refers to the raw material without inoculation with this strain, a is small peptide; b is fatty acid; c is flavonoid; d is organic acid; e is total sugar.

[0033] Figure 4 Example 4 of this invention uses a silk-coated mold starter made from wheat bran, flour, and Polygonum hydropiper;

[0034] Figure 5 Example 4 of this invention uses silk-coated mold starter made from pure wheat bran;

[0035] Figure 6 The silk-coated mold starter prepared using wheat bran and Polygonum hydropiper is described in Example 4 of this invention.

[0036] Preservation description of the *Byssochlamys* sp. of this invention: The *Byssochlamys* sp. of this invention, with accession number CGMCC No. 40543, was deposited on March 29, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. Detailed Implementation

[0037] The Ya'an Lushan Pinellia ternata planting area is the main production area for Pinellia ternata fermentation. Due to long-term domestication and selective enrichment, a unique fungal fermentation system has been formed, including fungal resources with special physiological functions. In this invention, a strain of *Byssochlamys* sp. was isolated from the fermentation process of Pinellia ternata in the fermentation workshop of Sichuan Ya'an XunKang Pharmaceutical Co., Ltd. Its preservation number is CGMCC No. 40543.

[0038] Compared with traditional mixed-culture fermentation, the *Vitex negundo* fungus of this invention can improve the digestive ability of *Pinellia ternata* koji. The fermented *Pinellia ternata* koji has an amylase activity of up to 26%–41% and a protease activity of up to 15%–23%. Fermentation of *Pinellia ternata* with *Vitex negundo* of this invention can reduce the toxicity of *Pinellia ternata*. After fermentation, the calcium oxalate content decreases by 12%–18%, reducing the irritation of the mucous membrane by calcium oxalate needle crystals in *Pinellia ternata*, thus achieving detoxification. The mannose content increases by 50%, and mannose can bind to the inflammatory agglutinin in *Pinellia ternata* (content between 2.6% and 4.1%), thereby reducing the toxicity of *Pinellia ternata*. Fermentation of *Pinellia ternata* with *Vitex negundo* of this invention can significantly increase the content of amino acids, alkaloids, flavonoids, and other sedative and antitussive active components, with a relative increase of more than 10 times; the content of antiemetic polysaccharides does not change much, while the total organic acid content increases by 20 times. It was also found that the content of other medicinally valuable components increased significantly, such as the anticancer component vitexin, which increased by more than 340 times. Fermentation using this fungus can effectively improve the quality of *Pinellia ternata* koji. The *Ficus pumila* strain of this invention exhibits good stability and is adaptable to both solid-state and liquid-state fermentation environments. Furthermore, its performance in liquid-state fermentation is superior to that in solid-state fermentation, thus providing a possibility for the development of a modern pure-culture fermentation process for *Pinellia ternata*.

[0039] More specifically, the *Polygonum aviculare* of the present invention can be used for fermentation production of feed, and when fermenting feed containing Polygonum hydropiper, it can produce a minty aroma.

[0040] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0041] The culture medium formulations used in the following examples are as follows:

[0042] The formula for PDA solid culture medium is: 200g potato, 20g glucose, 20g agar, 1L water, 0.1g chloramphenicol, sterilized at 121℃ for 15min.

[0043] PDA slant culture medium formula: 200g potato, 20g glucose, 20g agar, 1L water, 0.1g chloramphenicol, sterilized at 121℃ for 15min.

[0044] Example 1: Isolation, screening, and identification of *Cymbidium goeringii* according to the present invention

[0045] Pinellia ternata fermented for 24 hours was collected from the fermentation workshop of Sichuan Ya'an Xunkang Pharmaceutical Co., Ltd. The specific collection steps are as follows: Pinellia ternata, alum, and Shenqu (a type of fermented medicinal ingredient) were pulverized using a pulverizer and sieved through an 80-mesh sieve for later use. Ginger was weighed, and an equal weight of water was added to extract the juice. The ginger residue was filtered out, and the ginger juice was diluted with three times its weight of water for later use. A fermentation substrate was prepared by mixing Pinellia ternata, ginger juice, flour, alum, and Shenqu (a type of medicinal fermentation starter) in a mass ratio of 32:4:6.4:2:1. This substrate was loosely spread on a tray to a thickness of 1 cm. The fermentation temperature was 35–36℃, and the humidity was 80%–85%. Fermentation lasted 72 hours. Samples of Pinellia ternata fermentation were collected at 0h, 17h, 24h, 48h, and 71h using sample bags. A portion was stored at 4℃ for subsequent separation, and the remaining portion was frozen at -20℃ and sent to Paisennong Biotechnology Co., Ltd. for total DNA extraction. ITS1 and ITS4 sequences were amplified and sequenced. Sequences with a tail quality value below 20 and sequences below 50bp after quality control were filtered out. The sequences were then assembled, and sequences with a maximum mismatch ratio of 0.2 were screened for non-conforming sequences. Non-repetitive sequences were extracted from the optimized sequences. Usearch (vssession) was used to analyze the sequences. 7.1) Using the software platform (http: / / drive5.com / uparse / ), non-repetitive sequences (excluding single sequences) with a similarity of over 97% were grouped into one operational taxonomic unit (OTU) and clustered. Chimeras were removed during the clustering process to obtain representative sequences of the OTUs. All OTUs obtained from sequencing were compared with the corresponding sequences of type strains in Genbank (https: / / www.ncbi.nlm.nih.gov / ) to obtain the taxonomic units (including phylum, class, order, family, genus, and species) corresponding to the top 20 dominant species of OTUs and their corresponding abundance information. It was found that the abundance of *Cryptomyces hygroscopicus* exceeded 60% in the *Pinellia ternata* sample fermented for 24 hours, and *Cryptomyces hygroscopicus* was isolated from the sample.

[0046] Preparation of the isolation medium: One part of fermentation substrate was prepared by mixing Pinellia ternata, ginger juice, flour, alum, and Shenqu (medicated leaven) in a mass ratio of 32:4:6.4:2:1. Nine parts of water were added to this mixture, and the solution was sterilized by 0.22 μm filtration. The filtrate was aseptically collected for later use. PDA solid medium was sterilized at 121℃ for 20 min, cooled to 60℃, and then 5% of the above filtrate was added under aseptic conditions before plating to obtain the isolation medium.

[0047] Isolation and identification: A sample of *Pinellia ternata* fermented for 24 hours was directly dipped into an inoculation needle and inoculated onto an isolation medium. After incubation at 28°C for 36 hours, spores were picked and purified onto a new PDA solid medium. This process was repeated three times to obtain eight pure cultures of filamentous fungi. The colony and cell morphology of the eight fungi were observed. Comparison with the *Handbook of Fungal Identification* (by Wei Jingchao) revealed that three strains were highly similar to *Cryptomyces filamentosa*. DNA was extracted from these three strains at Shanghai Sangon Biotech, and their ITS1 and ITS4 sequences were amplified. After sequencing, the sequences were compared with the corresponding sequences of the type strain in Genbank (https: / / www.ncbi.nlm.nih.gov / ). Because this fungal strain could produce sporangiospores, while *Penicillium* generally produces chlamydospores and conidia, it was identified as *Cryptomyces filamentosa*. Figure 1 The colony morphology of asexual spores produced by *Hymenopterus xylostella* after culturing on PDA solid medium at 35°C for 48 hours is shown.

[0048] The identified strains were inoculated onto the above isolation medium, cultured at 28°C for 36 hours, and then stored at 4°C for 3 months. Alternatively, the sand tube storage method can be used for storage.

[0049] The slant culture was deposited with the China General Microbiological Culture Collection Center (CGMCC) on March 29, 2023, with accession number CGMCC No. 40543.

[0050] Example 2: Solid-state fermentation of Pinellia ternata using *Moldavobacterium oxyphylla* according to the present invention

[0051] 1. First, inoculate the preserved *Hymenopterus* strain into PDA solid medium using an inoculation needle and incubate at 30°C for 3 days. When the culture dish is completely covered and turns dark brown, collect mature spores using a sterile scraper. Then, prepare a solution with sterile water to a concentration of 1×10⁻⁶. 8 Prepare spore suspension at 1 spore / mL for later use.

[0052] The spore liquid of the above-mentioned *Pteris vittata* strain was inoculated into *Pinellia ternata* koji cake (obtained by uniformly mixing *Pinellia ternata*, ginger juice, alum, and flour in a ratio of 32:4:2:6.4), and simultaneously fermented with koji cake containing *Shenqu* as the inoculum (obtained by uniformly mixing *Pinellia ternata*, ginger juice, alum, flour, and *Shenqu* in a ratio of 32:4:2:6.4:1) for 72 hours. The fermentation process parameters were: fermentation temperature 30℃, material layer thickness 2cm, and moisture content 20%. Compared with traditional mixed fermentation of *Pinellia ternata* koji, the *Pinellia ternata* koji obtained by fermentation of *Pteris vittata* in this invention had a purer color, significantly reduced acidity, more uniform granulation, and a special aroma.

[0053] 2. Using pure *Hymenopsporum*, *Pinellia ternata* was fermented in solid state at 28℃ for 72 hours (fermentation conditions as above). After fermentation, the protease activity, amylase activity, and total acid of the samples were measured. The average protease activity of the three batches of fermentation substrates was 61.7±8.4 U / g, the average amylase activity was 123.6±7.5 U / g, and the total acid was 11.4±1.2 mg / g, as shown in Table 1. There were no significant differences in the various indicators of the three different batches of *Pinellia ternata* fermented using this fungus, indicating that the performance of this fungus is very stable.

[0054] Table 1. Validation results of the solid-state fermentation process of Pinellia ternata using *Moldobacterium oxyphylla*

[0055]

[0056]

[0057] Note: Superscripts with the same letter indicate that the indicators in the same row do not have significant differences.

[0058] 3. After solid-state fermentation of Pinellia ternata using the *Pteris vittata* strain of the present invention, the content of calcium oxalate needle crystals in the Pinellia ternata substrate was found to decrease from 0.205 g / kg to 0.175 g / kg.

[0059] 4. Rabbit experiment

[0060] Sample preparation: Pinellia ternata, ginger juice, flour, alum, and Shenqu (a type of fermented medicinal yeast) were mixed in a mass ratio of 32:4:6.4:2:1 to prepare fermentation raw material, which was then frozen for later use. The fermentation raw material was fermented at 28℃ for 72 hours to obtain traditional fermented Pinellia ternata starter, which was also frozen for later use. Pinellia ternata, ginger juice, flour, and alum were mixed in a mass ratio of 32:4:6.4:2 and inoculated with a 5% suspension of *Hymenopterus xanthipes* spores (concentration 1×10⁻⁶). 8 Fermentation of *Pteris vittata* (a type of fungus) at 28℃ for 72 hours yielded *Pteris vittata* koji, which was then frozen and stored for later use. After thawing, the above three samples were mixed with 9 times their weight of water by vortexing and centrifuging at 1000 rpm for 5 minutes to obtain three sample solutions. A control solution was prepared by mixing ginger juice, flour, and alum in a mass ratio of 4:6.4:2, adding 9 times their weight of water by vortexing, and centrifuging at 1000 rpm for 5 minutes.

[0061] Three rabbits were randomly selected, each for testing one type of sample solution. During the test, two drops of sample solution were instilled into the rabbit's right eye, and two drops of control solution into its left eye. The rabbit's upper and lower eyelids were then gently closed, followed by gentle rubbing of the eyes to ensure full contact between the solution and the conjunctiva, preventing leakage. After rubbing for 3 minutes, the rabbit's eyes were immediately rinsed with physiological saline until no drug residue was found and no abnormalities were observed. The rabbit's eyes were then observed every 0.5 hours, and scored according to the criteria shown in Table 2.

[0062] Table 2 Sensory rating table for corneal irritation in rabbits treated with Pinellia ternata.

[0063]

[0064] Figure 2 Table 3 shows the feedback diagram of calcium oxalate needle crystal irritation to the rabbit eye membrane. Table 3 illustrates the toxicity reduction effect of *Pinellia ternata* fermented with *Mycorrhiza glabra* according to this invention. As can be seen from Table 3, compared with the blank (Pinellia ternata raw material), the mucosal irritation caused by calcium oxalate needle crystals in both traditionally fermented and *Mycorrhiza glabra*-fermented Pinellia ternata was significantly reduced. Furthermore, the pure-culture fermented Pinellia ternata caused less irritation to the rabbit eye mucosa, achieving toxicity reduction. Simultaneously, the test revealed that the mannose content in the *Pinellia ternata* matrix after *Mycorrhiza glabra* fermentation increased by 50%. This mannose can bind more inflammatory agglutinin proteins in *Pinellia ternata*, reducing its toxicity.

[0065] Table 3. Rabbit eye membrane scoring results

[0066] congestion edema secretions Total Score Traditional fermented Pinellia ternata koji 8 25 30 63 Silk mold fermentation of Pinellia ternata koji 11 20 27 58 Pinellia raw materials 18 30 35 83

[0067] 5. After solid-state fermentation of Pinellia ternata using the *Hymenopsporum tobira* of this invention, tests revealed, as shown in Table 4, that it significantly increased the content of amino acids, alkaloids, flavonoids, and other active components for sedation and cough relief. For example, the relative content of norpseudoephedrine, which can relieve nasal congestion, increased by 8 times; the content of antiemetic polysaccharides remained relatively unchanged, while the total organic acid content increased by about 20 times. It was also found that the content of other medicinally valuable components increased significantly, such as the anticancer components DL-stachyine, scopolamine, and vitexin, which increased by 30 times, 90 times, and 342 times, respectively. This demonstrates that fermentation using this fungus can effectively improve the quality of Pinellia ternata fermentation.

[0068] Table 4. Effects of solid-state fermentation of Pinellia ternata by *Hymenopsinus* on the component content of Pinellia ternata.

[0069]

[0070]

[0071] 6. In this invention, after solid-state fermentation of Pinellia ternata by Hymenopsula ovata, the content of organic acids, fatty acids, and flavonoids is significantly increased, such as... Figure 3 As shown, the production of organic acids generally increased, such as 3-hydroxybutyric acid, which has neuroactive properties, which increased 21-fold, and the content of phosphatidylcholine, which has anti-inflammatory effects in fatty acids, which increased 13-fold. Figure 3 In this context, "hun" refers to the fermentation substrate mixed with the strain, while "yuan" refers to the raw material that was not inoculated with the strain, i.e., the fermented material of Pinellia ternata prepared according to the formula.

[0072] Example 3: Liquid fermentation of Pinellia ternata koji by *Synthia suspensa* according to the present invention

[0073] The applicant's preliminary experiments revealed that the effects of various process parameters on fermentation in liquid fermentation were as follows: fermentation temperature > inoculum size > solid-liquid ratio > rotation speed > fermentation time. Through orthogonal experimental design, the following process for liquid fermentation of Pinellia ternata using *Cymbopogon spp.* was determined: fermentation temperature 33℃, inoculum size 6%, solid-liquid ratio 1:4, rotation speed 60 r / min, and fermentation time 72 h. Three batches of Pinellia ternata were fermented using the above parameters, and the protease activity, total acid, and soluble solids in the samples were measured. The results are shown in Table 5. It can be seen that the process of liquid fermentation of Pinellia ternata using pure *Cymbopogon spp.* is stable. Compared with traditional fermentation, total acid increased slightly, soluble solids increased, and the product appeared purer and was easier to preserve.

[0074] Table 5. Liquid fermentation of Pinellia ternata by *Erythromyces cerevisiae*

[0075]

[0076] Example 4: Fermented feed of *Malus hygroscopicus* according to the present invention

[0077] Crush wheat bran, flour, and Polygonum hydropiper into powder, sift through an 80-mesh sieve, and mix thoroughly in a mass ratio of wheat bran: flour: Polygonum hydropiper powder 3:2:1. Sterilize the mixture, then add 3 times its weight of sterile water to moisten the culture medium until it can be formed into a ball when squeezed but crumbles easily when tossed. Inoculate the culture medium (wheat bran: flour: Polygonum hydropiper powder 3:2:1) from PDA slant culture medium using an inoculation needle. Incubate at 32℃ for 2 days. The resulting koji cake will have a grayish-green surface, a uniform color distribution on the cross-section, and robust, densely distributed white mycelium in the center. It will have a fragrant aroma with a hint of wheat bran, and a soft, dense texture. Figure 4 As shown. Its α-amylase activity reached as high as 43.85 U / g, about twice that of the peak fermentation of pure *Pinellia ternata*, but it had no obvious protease activity, possibly due to a lack of stimulating factors in the raw materials. Meanwhile, pure wheat bran was used ( Figure 5 ), bran + flour 2:1, bran + Polygonum hydropiper 5:1 ( Figure 6 The combination of these bacteria was fermented with pure *Cymbidium goeringii* at 32℃ for 48 hours, and the amylase activities were found to be 43.89 U / g, 41.52 U / g, and 43.15 U / g, respectively. The study found that the bacteria are suitable for the feed fermentation industry. After adding *Polygonum hydropiper*, the fermented cake produced a minty aroma.

Claims

1. Hymenopsporum aviculare ( Byssochlamys sp.), characterized by: The preservation number of the Trichothecium is CGMCC No. 40543.

2. The Trichothecium for use in solid or liquid fermentation of Pinellia ternate.

3. Use according to claim 2, wherein: The liquid fermentation conditions are as follows: fermentation temperature is 33℃, inoculation amount is 6%, solid-liquid ratio is 1:4, rotation speed is 60 r / min, and fermentation time is 72 h.

4. The Trichothecium for use in preparing Pinellia ternate koji or Sixian Qu.

5. The Trichothecium for use in fermentative production of feed.

6. Use according to claim 5, characterized in that: The feed raw material comprises at least one of bran, flour or polygonum hydropiper.

7. Use of the filamentous fungus of claim 1 for the fermentative preparation of substances producing a minty aroma, characterized in that: The mint-flavored aroma-producing material raw material comprises polygonum hydropiper.

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