Mutant strain of high selenium-tolerant and selenium-enriched polyporus versicolor and application thereof

Abstract

The present application relates to a high selenium-tolerant tree tongue mutant strain and its application, belonging to the technical field of fungal breeding. It discloses a high selenium-tolerant and selenium-rich tree tongue mutant strain YL108, which has been preserved in China Center for Type Culture Collection (CCTCC) in Wuhan University, Wuhan, China, classified as Ganoderma applanatum YL108, with the preservation number CCTCC NO: M 20221347 and the preservation date of August 29, 2022. The strain can maintain normal growth in a culture medium with a selenium content of 90 mg / L, and the mycelium obtained has no red selenium phenomenon, and the amino acid form selenium content in the mycelium protein is up to 511 mg / kg, accounting for about 55% of the total protein selenium. The above-mentioned high selenium-tolerant tree tongue mutant strain Ganoderma applanatum YL108 is more suitable for cultivation in a high-selenium-content medium, and high-quality selenium-containing protein with high amino acid form selenium is obtained, which is a high-quality germplasm resource for preparing fungal selenium-rich products.

Description

Technical Field

[0001] This invention relates to a highly selenium-tolerant tree tongue mutant strain and its application, specifically to a tree tongue mutant obtained by mutagenesis breeding that can maintain a high growth rate under high selenium concentration conditions and is not prone to the "red selenium phenomenon," and its application, belonging to the field of fungal breeding technology. Background Technology

[0002] Selenium is one of the trace elements required by the human body. Due to the poor solubility of selenium compounds and the localized concentration of selenium on Earth, most people in the world cannot fully meet their selenium needs from normal food. Fungi have the characteristic of accumulating selenium, and selenium-enriched fungi are one of the effective ways for people to obtain selenium. In addition to helping people obtain selenium, selenium-enriched fungi also have special applications in anti-cancer health care. Studies have shown that some fungal selenium-containing polysaccharides have the effect of inducing tumor cell apoptosis (Shang DJ, Li QW, Cui Q, et al. A novelpolusaccharide from Se-riched Ganoderma lucidum induces apoptosis of human breast cancer cells. Oncology Reports, 2011, 25(1):267-272.). Some selenium-containing proteins have free radical scavenging activity (Du Ming, Zhao Lei, Li Chaorui. Purification, properties and free radical scavenging activity of a new selenium-containing protein in selenium-enriched Ganoderma lucidum. Journal of Chemical Research in Chinese Universities, 2007, 28(1):75-78.).

[0003] Enriching fungi with selenium through mycelial culture is a convenient method for obtaining selenium-enriched fungi. While fungi can sometimes accumulate large amounts of selenium in high-selenium media, this accumulation often leads to over-enrichment, resulting in red mycelia, a phenomenon known as "red selenium phenomenon." Fungal mycelia exhibiting this phenomenon typically contain high levels of elemental selenium and other forms of inorganic selenium. The selenium supplementation effect from such fungi is often unsatisfactory; sometimes, elemental selenium is excreted in urine, causing red urine. Therefore, avoiding the red selenium phenomenon is a crucial factor to consider when preparing selenium-enriched fungal products to improve their quality.

[0004] Ganoderma applanatum is a type of large fruiting body fungus that grows saprophytically on dead broadleaf tree wood in nature. It belongs to the same genus (Ganoderma) as Ganoderma, and is therefore sometimes called tree-tongue Ganoderma. In traditional Chinese medicine, Ganoderma applanatum is generally considered an edible fungus used for medicinal or health-promoting purposes, and is used in the treatment of pharyngitis, esophageal cancer, and nasopharyngeal carcinoma.

[0005] In the preliminary work for this patent, the project team collected fungi from the roots of dead trees near a selenium mine in Lichuan City, Hubei Province. After isolating the mycelium, they studied its selenium-enriching capacity. The study found that the mycelium of one *Gynostemma pentaphyllum* strain had a higher selenium tolerance than other fungi and was less prone to red selenium formation when cultured in a high-selenium medium. Considering the special significance of *Gynostemma pentaphyllum* in anti-cancer and health-promoting aspects, developing selenium-enriched *Gynostemma pentaphyllum* strains would undoubtedly be of great significance in further enriching my country's selenium-enriched fungal germplasm resources.

[0006] To date, there is a lack of research on selenium-enriched *Gnaphalium affine* both domestically and internationally. There is a lack of both operational methods and superior germplasm resources for selenium enrichment using *Gnaphalium affine*. This patent discloses a *Gnaphalium affine* germplasm resource with high selenium tolerance and selenium enrichment capacity, a method for obtaining it, and a method for cultivating selenium-enriched mycelium using this *Gnaphalium affine* strain. Summary of the Invention

[0007] The purpose of this invention is to overcome the above-mentioned shortcomings and provide a highly selenium-tolerant and selenium-enriched tree tongue mutant strain and its application. This tree tongue strain has a high selenium tolerance and can grow at a high selenium concentration environment. The mycelium obtained under high selenium culture conditions is less likely to exhibit the "red selenium phenomenon". The selenium content of the mycelium protein is significantly higher than that of the mycelium obtained under normal conditions. The selenium content of amino acid form in the selenium-containing protein reaches about 55%.

[0008] The technical solution of this invention is a highly selenium-tolerant and selenium-enriched Ganoderma applanatum mutant strain YL108, which has been deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China. It is classified and named Ganoderma applanatum YL108, with accession number CCTCC NO: M 20221347 and deposit date of August 29, 2022.

[0009] The ITS sequence of the highly selenium-tolerant selenium-enriched tree tongue mutant strain YL108 is shown in SEQ ID No. 1.

[0010] The highly selenium-tolerant selenium-enriched tree tongue mutant strain YL108 grows rapidly and is less prone to red selenium formation in liquid PDA medium with a selenium content of 90 mg / L.

[0011] The screening method for the highly selenium-tolerant selenium-enriched tree tongue mutant strain YL108 includes the following steps:

[0012] (1) Collection and isolation culture of selenium-enriched fungal fruiting bodies: Select fungal fruiting bodies and isolate and culture mycelium; take the robust part of the root of the above-mentioned fruiting body at the junction with the white mycelial cord, clean it, take a small amount of tissue, inoculate it with PDA solid medium and culture for 1 to 4 weeks to obtain large fungal mycelial culture;

[0013] (2) Screening of selenium-tolerant fungi: The mycelial culture of macrofungi was inoculated in selenium-containing PDA liquid medium and cultured. The strains that did not show red selenium phenomenon were selected and sequenced to obtain the tree tongue Ganoderma applanatum XQ20115 strain.

[0014] (3) Mutagenesis of tree tongue and screening of high selenium-tolerant tree tongue mutants: After inoculating and culturing strain XQ20115, collect the blocky fruiting bodies, and then collect the spores after further culturing; after mutagenesis, further culture and screening were carried out to obtain the high selenium-tolerant selenium-enriched tree tongue mutant strain YL108, which has a fast growth rate and is not prone to red selenium phenomenon.

[0015] Furthermore, the selenium content in the selenium-containing PDA liquid culture medium in step (2) is 100 mg / L.

[0016] Further, in step (2), the strains obtained from the preliminary screening are further inoculated into liquid PDA medium at concentrations of 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, 60 mg / L, 70 mg / L and 80 mg / L, and strains that do not show red selenium in the secondary screening are selected.

[0017] Further, in step (2), the strains after the second screening were inoculated into PDA liquid medium with a selenium content of 100 mg / L, and after the third screening, the Ganoderma applanatum XQ20115 strain was obtained.

[0018] Furthermore, when the XQ20115 strain was inoculated in liquid PDA medium with a selenium content exceeding 120 mg / L, the bacterial growth rate decreased significantly. In liquid medium with a selenium content of 200 mg / L, the growth of XQ20115 was extremely slow. When XQ20115 was inoculated on solid PDA plates, the strain showed high selenium tolerance, and when the selenium content reached 600 mg / L, almost no signs of growth were observed.

[0019] Furthermore, the specific process of mutagenesis of the tree tongue and screening of highly selenium-tolerant tree tongue mutants in step (3) is as follows:

[0020] a. Spore Collection: Mycelium of strain XQ20115 was inoculated onto a special tree branch; cultured in special liquid medium No. 1 for 5-7 months, resulting in several block-shaped fruiting bodies; the branch with fruiting bodies was removed and inoculated into a special spore-forming medium, and cultured continuously while carefully observing the aluminum foil beneath the fruiting bodies daily; a small amount of spore powder could be seen on the aluminum foil beneath some of the cultured tree ligule fruiting bodies; the spore powder was collected under aseptic conditions, suspended in physiological saline, and stored at -70℃. The collected spore powder was centrifuged and then resuspended in 10 mL of physiological saline.

[0021] b. Mutagenesis: The suspended spore powder is subjected to Co... 60 Irradiation was performed at a dose controlled between 0.2 and 0.4 kGy. The mutagenized spore suspension was transferred to ordinary PDA liquid medium and cultured in shake flasks at 25°C for 1 day. 10 mL of the culture solution was inoculated into 200 mL of high-selenium PDA liquid medium with a selenium content of 240 mg / L and cultured for 12 days. The culture solution was diluted and spread onto solid PDA medium containing 600 mg / L selenium. Colonies of mycelium with weak growth on PDA medium were selected and transferred to new high-selenium PDA plates with a selenium content of 600 mg / L. After 4-6 days of culture, the mycelial colonies were inoculated into high-selenium PDA liquid medium with a selenium content of 200 mg / L and cultured in shake flasks at 30°C and 120 r / min. Starting from 48 hours, samples were taken at 24-hour intervals to detect the dry weight of the mycelium. Finally, 11 selenium-tolerant mutant strains were obtained.

[0022] c. Screening: The above 11 strains were cultured in a high-selenium fermentation medium with a selenium content of 200 mg / L and the cell mass and selenium content were detected. The strains were cultured in a medium with a selenium content of 90 mg / L, and finally the high selenium-tolerant selenium-enriched tree tongue mutant strain YL108 was obtained.

[0023] Another technical objective of this invention is to apply the highly selenium-tolerant and selenium-enriched tree tongue mutant strain YL108 to prepare fungal selenium-enriched products and obtain high-quality selenium-containing proteins with high amino acid selenium content.

[0024] The beneficial effects of this invention are as follows: Compared with the mycelial culture of the original strain, the mutant *Ganoderma applanatum* YL108 obtained by screening in this invention can maintain normal growth in a culture medium with a selenium content of 90 mg / L, while the mycelium of the original strain grows slowly in the same fermentation culture medium. The mycelial protein obtained by culturing YL108 in the above-mentioned culture medium has an amino acid selenium content of 511 mg / kg, accounting for 55% of the total protein selenium. YL108 is an excellent strain that can produce high-quality protein with high amino acid selenium content through mycelial fermentation.

[0025] Biological material sample preservation: A highly selenium-tolerant and selenium-enriched Ganoderma applanatum mutant strain YL108 has been deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China. It is classified and named Ganoderma applanatum YL108, with accession number CCTCC NO: M 20221347, and the deposit date is August 29, 2022. Attached Figure Description

[0026] Figure 1 Growth curve of *Syngonium argentea* in fermentation medium containing 60 mg / L selenium.

[0027] Figure 2 Growth curve of *Syngonium argentea* in fermentation medium containing 90 mg / L selenium. Detailed Implementation

[0028] The invention will be further illustrated by means of embodiments, which will not limit the scope of the invention in any way.

[0029] Extraction method of mycelial protein of Ganoderma lucidum: Same as the literature: Yang Menglian, Shan Yilan, Shen Wei, et al. Cloning and expression of β-1,3-glucanase of fibrous microorganisms and its hydrolytic effect on Ganoderma lucidum cell wall [J]. Food and Fermentation Industries, 2023, 49(1): 10-18. The mycelial protein was extracted by directly using ultrasonic disruption without the use of cell-wall disrupting enzymes as described in the literature.

[0030] Detection method of different forms of selenium in selenoproteins: Ye Mei, Yu Ruipeng, Cong Xin, et al. Speciation analysis and identification of selenium compounds in selenium-enriched Corydalis leaf spp. [J]. Journal of Analytical and Testing Technology, 2022, 41(01):100-107. Different forms of selenium were detected using the same instruments and methods as described in the literature.

[0031] Materials and methods:

[0032] Basic PDA medium: Wash and peel 400g of potatoes, cut them into small pieces about 3mm thick, add water to 1L, heat to boiling for 10 minutes, and filter out the solid portion with gauze. Take the filtrate and add water to 700mL. Add glucose at a rate of 20g / L of final medium to obtain basic PDA liquid medium; add agar powder at a rate of 15g / L of final medium to obtain basic solid PDA medium. The above mediums are generally prepared in 1L Erlenmeyer flasks. Before preparation, measure 1L of water into the Erlenmeyer flask using a graduated cylinder and accurately mark the 1L volume level. After the above ingredients are prepared, sterilize at 110℃ for 10 minutes.

[0033] In practical use, PDA media with different selenium contents are prepared by adding sodium selenite solution and water to the sterilized basal medium according to the preparation method below. The medium with only water added and no sodium selenite solution added is generally referred to as PDA medium or solid PDA medium.

[0034] High-selenium culture medium: Prepare a 66.67 g / L aqueous solution of sodium selenite pentahydrate, and sterilize it by filtration using a bacterial filter to obtain the sodium selenite solution. When preparing PDA culture medium or fermentation medium, add different volumes of the above sodium selenite solution after sterilization to obtain the corresponding concentration of selenium-containing medium. The molecular weight of sodium selenite pentahydrate is 262, while the atomic weight of selenium is 79. The selenium content in sodium selenite pentahydrate is 30%, so the selenium content in the above solution is 20 g / L.

[0035] In Example 1, the 200 mg / L high-selenium culture medium was generally prepared by mixing 700 mL of pre-sterilized culture medium with 10 mL of the aforementioned sodium selenite solution, and then adding 290 mL of sterile water to obtain 1 L of high-selenium culture medium with a selenium content of 200 mg / L. In actual operation, because the sterilization process causes changes in the volume of the culture medium, the final amount of sterile water added is based on the pre-marked 1 L water level line. Other concentrations of high-selenium culture media were prepared in the same manner.

[0036] Fermentation medium: 40g glucose, 0.2g potassium dihydrogen phosphate, 1g urea, 0.1g anhydrous calcium chloride, 0.05g anhydrous magnesium chloride, 10g malt extract powder, and 5g yeast powder. Generally, take 700mL of water, add the above ingredients, mix and dissolve to obtain the initial medium. After preparation, sterilize at 110℃ for 15min, and prepare sterile water simultaneously. When using the above medium, if preparing a selenium-free ordinary medium, simply add water to 1L to obtain a total volume of 1L fermentation medium. If preparing a selenium-containing fermentation medium, add the corresponding sodium selenite solution to the above initial medium, and further add water to 1L to obtain the selenium-containing fermentation medium. See the high-selenium medium section for specific operating methods.

[0037] Special culture medium No. 1: Take 700mL of tap water, add 0.1g of potassium dihydrogen phosphate, 0.5g of sodium nitrate, 0.05g of anhydrous calcium chloride, and 0.1g of anhydrous magnesium chloride, add water to 1L, and filter to sterilize.

[0038] First, place a specially selected tree branch into a 1L wide-mouthed Erlenmeyer flask. The branch should be about 10cm long, ensuring it stands upright in the flask and can be easily removed with large forceps. Seal the flask with sealing film and sterilize at 121°C for 30 minutes. When inoculation is needed, carefully remove the branch and use sterile, pointed forceps to carefully insert pre-cultured mycelium from a plate into the small hole in the branch. Place the inoculated branch back into the Erlenmeyer flask and add approximately 200mL of the above-mentioned culture medium (No. 1). Seal with sterile sealing film. Incubate at room temperature. When the volume of the special culture medium in the flask falls below 50mL due to evaporation, add water to bring the volume back to approximately 200mL.

[0039] The aforementioned special branches were dried willow branches purchased from farmers in Junzhang Mountain, Wuxi, with a diameter controlled to be around 5cm. Before use, holes of about 5mm in diameter were drilled into the branches using an impact drill.

[0040] Sporosome culture medium: Take an oak wood block about 1 cm in size and an equal volume of pebbles about 1 cm in size, and place them in a 1L Erlenmeyer flask, making a thickness of about 3 cm. Add water until the wood block is moist. Cover the flask with two layers of aluminum foil with a central perforation. Seal with heat-resistant tissue culture sealing film (Beekman Company) and sterilize at 121°C for 30 minutes.

[0041] Generally, after the mycelium of *Gnaphalium affine* growing on the branch in the No. 1 special culture medium forms a large fruiting body, the entire branch is transferred to an Erlenmeyer flask containing the above-mentioned spore-forming culture medium. The branch is inserted into the lower layer of culture medium through the opening in the center of the aluminum foil. The flask is then incubated statically, and the aluminum foil is observed daily for yellow spores that have fallen from the *Gnaphalium affine* fruiting body. Once a significant amount of yellow powder (i.e., spores) is observed, the branch is removed, and then the upper layer of aluminum foil is removed to collect the spores.

[0042] Determination of cell dry weight: Same as method 10 in the literature [Shen Wei, Wang Bingbo, Zhu Jincun et al. An improved pullulanase chimera and a high-yielding Pichia pastoris mutant strain of the chimera. Patent No.: 201610306855.6].

[0043] Chromosomal DNA extraction and ITS gene sequence analysis methods: The chromosome extraction method is the same as that in the literature (Zhou Xiaoling, Shen Wei, Rao Zhiming, Wang Zhengxiang, Zhuge Jian. A rapid method for extracting fungal chromosomal DNA [J]. Bulletin of Microbiology, 2004, 31(4):89-92); The method for further ITS gene amplification and sequence analysis after chromosome extraction is the same as that in the literature (Chen Yuanyuan, Shen Wei, Shi Guiyang, Wang Zhengxiang. Comparison of two molecular markers in yeast molecular identification [J]. Science and Technology of Food Industry, 2011, 32(2):175-177) for ITS gene identification.

[0044] Example 1: Breeding of YL108, a high-selenium-tolerant, selenium-enriched tree tongue mutant strain

[0045] (1) Collection and isolation culture of selenium-enriched fungal fruiting bodies: Fungal fruiting bodies were collected from severely decayed tree roots and branches in the soil near abandoned selenium mines in Lichuan County, Hubei Province, my country. After sampling, mycelial culture was performed in the laboratory. The robust portion of the fruiting body at the junction of the root and the white mycelial cord was taken, rinsed with tap water for an extended period, then washed with a large amount of sterile water. Finally, the tissue at the junction of the fruiting body and the mycelial cord was carefully dissected, and a small amount of the central tissue was inoculated onto PDA solid medium and cultured for 1–4 weeks. If mycelial growth occurred, a small amount of mycelium was transferred to new PDA solid medium. Culture was continued for 6 weeks. If spores of various colors were visible to the naked eye, the culture was identified as mold or contaminated with mold and was not used. Further microscopic observation was performed on the remaining cultures. If only mycelia were present and no spherical cells (potentially spores) were observed, the culture was identified as mycelium of macrofungi and used for subsequent experiments. A total of 222 macrofungi mycelial cultures were obtained using the above method. The mycelial slant culture was deposited in the strain bank of Wuxi Xianqin Biotechnology Co., Ltd., with accession numbers XQ20101~XQ20222.

[0046] (2) Screening of selenium-tolerant fungi: The mycelia were inoculated into PDA liquid medium with a selenium content of 100 mg / L and cultured at 25°C. Growth and color changes were observed. The results showed that 36 strains could form a large amount of mycelial culture after 7 days. Further, the 36 strains were inoculated into PDA liquid medium with selenium contents of 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, 60 mg / L, 70 mg / L, and 80 mg / L. Among them, 17 strains were white in color when cultured in medium with a selenium content of 60 mg / L or lower, without showing red selenium. Further, the mycelia preserved on the slant were inoculated into high-selenium liquid medium with a selenium content of 100 mg / L. Samples were taken and centrifuged every 3 days to observe the cell volume. The results showed that strain XQ20115 had a significantly larger cell volume after 3 days of culture.

[0047] Molecular identification of the ITS gene in XQ20115 was performed. Its ITS sequence is listed in SEQ ID NO:1. The sequence was submitted to the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov) and sequence alignment was performed using BLAST software. The results showed that its ITS sequence was identical to that of the Ganoderma applanatum strain collected by the center. Therefore, this strain was named Ganoderma applanatum XQ20115, or Ganoderma applanatum XQ20115.

[0048] The XQ20115 strain was inoculated into liquid PDA media with different selenium concentrations. The results showed that the bacterial growth rate decreased significantly when inoculated in liquid PDA media with a selenium concentration exceeding 120 mg / L, and the growth of XQ20115 was extremely slow in liquid media with a selenium concentration of 200 mg / L. When XQ20115 was inoculated onto solid PDA plates, the strain exhibited high selenium tolerance; at a selenium concentration of 600 mg / L, almost no growth was observed.

[0049] (3) Mutagenesis of the tree tongue and screening of highly selenium-tolerant tree tongue mutants:

[0050] a. Spore Collection: Mycelium of strain XQ20115 was inoculated onto a specialized tree branch approximately 5 cm in diameter and 10 cm in length. After approximately 6 months of cultivation in specialized liquid medium (No. 1), 11 fruiting bodies approximately 4 cm in size were formed. The branch containing the fruiting bodies was then removed and inoculated into a special spore-forming medium. The cultivation continued, with careful daily observation of the aluminum foil beneath the fruiting bodies. A small amount of spore powder was visible on the aluminum foil beneath some of the cultured tree ligule fruiting bodies. The spore powder was aseptically collected, suspended in physiological saline, and stored at -70°C. The collected spore powder was centrifuged and then resuspended in approximately 10 mL of physiological saline.

[0051] b. Mutagenesis: The suspended spore powder is subjected to Co... 60 Irradiation was performed, with the irradiation dose controlled at approximately 0.3 kGy. The mutagenized spore suspension was transferred to ordinary PDA liquid medium and incubated in shake flasks at 25°C for 1 day. During incubation, the Erlenmeyer flasks were wrapped with aluminum foil to prevent light exposure. 10 mL of the above culture solution was inoculated into 200 mL of high-selenium PDA liquid medium with a selenium content of 240 mg / L and incubated for 12 days. After diluting the culture solution, it was spread onto solid PDA medium containing 600 mg / L selenium, totaling approximately 5000 plates. After approximately one month of incubation, a total of 312 weakly growing mycelial colonies formed on approximately 131 of the PDA plates. These mycelia were then transferred to new high-selenium PDA plates with a selenium content of 600 mg / L. After approximately 5 days of incubation, approximately 120 mycelial colonies were obtained. The mycelia were inoculated into high-selenium PDA liquid medium with a selenium content of 200 mg / L and cultured in shake flasks at 30℃ and 120 r / min. Starting from 48 hours, samples were taken at 24-hour intervals to determine the dry weight of the mycelium. The results showed that 11 strains had a significantly faster growth rate than the other strains, and were identified as selenium-resistant mutants. These 11 strains were named *Ganoderma applanatum* YL101–YL111 and are all deposited in the strain bank of Wuxi Xianqin Biotechnology Co., Ltd., with accession numbers XQ20223–XQ20233, respectively.

[0052] The 11 strains were further cultured in a high-selenium fermentation medium with a selenium concentration of 200 mg / L, and the cell mass and selenium content were measured. The results showed that all 11 strains reached their maximum cell mass after 8 days of culture, with strains YL102, YL108, and YL110 showing significantly higher cell masses than the other strains. The 11 strains were then inoculated into liquid PDA medium with a selenium concentration of 80–120 mg / L. Shake-flask fermentation results showed that YL102, YL108, YL112, and YL116 exhibited white cells when cultured in a medium with a selenium concentration of 90 mg / L, while the remaining strains showed red selenium. Further culture of the strains in a medium with a selenium concentration of 90 mg / L showed that the cell mass of YL108 was more than 10% higher than the other strains, while its selenium content was comparable. Clearly, strain YL108 is a selenium-tolerant and highly selenium-enriched mutant strain that grows rapidly in a culture medium with a selenium content of 90 mg / L and is less prone to red selenium formation.

[0053] ITS gene sequence analysis of the YL108 strain showed that its ITS sequence was completely identical to that of XQ20115, and the specific sequence is shown in SEQ ID NO: 1.

[0054] The aforementioned Ganoderma applanatum mutant strain YL108 has been deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, and is classified as Ganoderma applanatum YL108, with accession number CCTCC NO: M20221347 and deposit date of August 29, 2022.

[0055] The starting strain Ganoderma applanatum XQ20115, which was screened from nature and used in this invention, has been formally deposited at the China University Industrial Microbial Resource Platform of Jiangnan University, with strain number CICIM F7096.

[0056] Example 2: Stability of selenium-enriched strain YL108

[0057] The starting strain XQ20115 and the mutant YL108 were inoculated into the fermentation medium described in the specific implementation method and cultured at 30°C for 7 days. A 1 / 100 volume transfer to new fermentation medium was then performed, and after 5 days of culture, another 1 / 100 volume transfer to new fermentation medium was performed. This process was repeated 10 times.

[0058] The original strain XQ20115 and mutant strain YL108, as well as the original strain and mutant strain (named XQ20115b and YL108b respectively for ease of description) that were transferred through the above shake flasks, were inoculated into fermentation medium and high selenium fermentation medium with a selenium content of 200 mg / L, respectively.

[0059] The results showed that the growth rates of the four strains in the fermentation medium without added selenium were basically the same, with no significant differences. After inoculation with a 1% inoculum, the time to reach the maximum cell count in shake-flask fermentation was 7 days, and the maximum cell count reached was basically the same.

[0060] In a high-selenium fermentation medium with a selenium content of 200 mg / L, both YL108 and YL108b reached their peak cell count in 8 days, with essentially the same peak cell count. This was one day longer than the time taken to reach peak cell count in a selenium-free fermentation medium, with both reaching a peak cell count of approximately 10 g / L.

[0061] The same experiment was also conducted on XQ20115. In a high-selenium fermentation medium with a selenium content of 200 mg / L, the growth of XQ20115 was extremely slow. After 8 days of culture, the cell count reached less than 1 g, and after a longer period of culture, the cell count did not exceed 1.2 g.

[0062] As can be seen from the above experiments, YL108 did not show significant performance degradation after multiple passages in selenium-free medium, and is a genetically stable selenium-tolerant mutant.

[0063] Example 3: Fermentation performance of the mutant strain Ganoderma applanatum YL108

[0064] Mycelial cultures of the genus XQ20115 and the mutant strain YL108 were inoculated into PDA liquid medium. After culturing for 5 days, fermentation medium containing 60 mg / L and 90 mg / L selenium was inoculated at 1 / 100 volume respectively for comparison of fermentation performance.

[0065] The growth curves of XQ20115 and YL108 in a fermentation medium containing 60 mg / L selenium are as follows: Figure 1 As shown in the figure, the experimental data in this figure is the average data of 5 parallel experiments, and the data error of the parallel experiments is within 10%. The experimental results of the starting strain XQ20115 are not significantly different from those of YL108, and the bacterial cell color of both is white. After 7 days of bacterial culture, the selenium content and amino acid selenium content of the bacterial protein were detected, and the results are shown in Table 1.

[0066] The growth curves of the starting strain XQ20115 and the mutant YL108 in 90 mg / L fermentation medium are as follows: Figure 2As shown in the figure, the experimental data in this figure is the average data of 5 parallel experiments, and the data error of the parallel experiments is within 10%. The growth curve of YL108 in fermentation medium with a selenium content of 90 mg / L is similar to that in fermentation medium with a selenium content of 60 mg / L. The growth rate of the starting strain XQ20115 is significantly slower than that of YL108, but it can still reach the maximum cell mass on day 8. The cell mass is significantly smaller than that of YL108, but the difference is not significant. From the observation, the biggest difference between the two is that the cells of YL108 are always white, while the cells of XQ20115 are obviously red, showing the phenomenon of red selenium. Further, the selenium content was measured after extracting protein from the cells that reached the maximum cell mass, and the results are shown in Table 1.

[0067] Table 1 Comparison of fermentation performance between YL108 and the starting strain XQ20115

[0068]

[0069]

[0070] Note: Selenium in amino acid form refers to the total amount of selenium detected in selenomethionine, selenocystine, and methylselenocystine.

[0071] As shown in Table 1, in a fermentation medium with a selenium content of 60 mg / L, there was no significant difference in the growth of the mutant strain YL108 and the original strain XQ20115, but there were differences in fermentation performance. The total selenium content and the content of amino acid selenium in the cell protein of strain YL108 were higher than those of XQ20115, but the difference was not significant.

[0072] In a culture medium with a selenium concentration of 90 mg / L, YL108 grew significantly faster than XQ20115, and its cells remained white throughout. Under the same conditions, XQ20115 grew significantly slower and exhibited red selenium formation. The final cell mass of the two strains differed, but not drastically. The total selenium content in the final bacterial protein was actually higher in XQ20115, but the biggest difference lay in the content of selenium in amino acid forms. YL108 contained 511 mg / L of selenium in all three amino acid forms, while XQ20115 contained only 152 mg / L. This indicates that in YL108 cells, selenium primarily exists in the form of amino acids, while in XQ20115, most of it has changed and exists in other forms within the protein. Visually, both types of *Gnaphalium* cultured in a medium with a selenium concentration of 60 mg / L produced white cells, and the extracted protein was pale yellow. The mycelia of the two bacteria cultured in a medium containing 90 mg / L selenium showed significant differences. YL108 mycelia remained white, and the extracted protein remained pale yellow, while XQ20115 mycelia appeared red, and the extracted protein was dark brownish-red. The bacterial protein obtained from XQ20115 cultured in a medium containing 90 mg / L selenium contained a large amount of selenium in non-amino acid forms.

[0073] Based on the comparison of the results of cell color, selenoprotein color and the content of different forms of selenium in the protein, YL108 is a strain that is more suitable for obtaining proteins with high amino acid selenium content through fermentation. It is suitable for preparing high-quality selenoprotein products and is also conducive to obtaining materials with special health care or therapeutic functions such as selenopeptides through moderate hydrolysis.

Claims

1. A highly selenium-tolerant, selenium-enriched *Ganoderma lucidum* mutant strain, YL108, has been deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, and has been classified and named *Ganoderma lucidum* (Ganoderma lucidum). Ganoderma applanatum YL108, accession number CCTCC NO: M 20221347, accession date is August 29, 2022.

2. The highly selenium-tolerant selenium-enriched tree tongue mutant strain YL108 according to claim 1, characterized in that: The ITS sequence is shown in SEQ ID No.

1.

3. The application of the highly selenium-tolerant selenium-enriched tree tongue mutant strain YL108 according to claim 1, characterized in that: It was used to prepare selenium-enriched fungal products to obtain selenium-containing proteins with high levels of selenium in amino acid forms.

4. The application of the highly selenium-tolerant selenium-enriched tree tongue mutant strain YL108 as described in claim 3, characterized in that: The strain was cultured in liquid PDA medium containing 90 mg / L of selenium.

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