Selenium source regulation and control method for directionally producing high-value selenium-rich phellinus igniarius mycelium or sporocarp

Abstract

The invention discloses a selenium source regulation and control method for directionally producing high-value selenium-rich phellinus igniarius mycelium or sporocarp, and belongs to the technical field of edible and medicinal fungus cultivation and microbial fermentation. The invention provides a target-oriented differential selenium source strategy aiming at the technical contradiction that a single selenium source cannot meet the requirements of different growth stages of phellinus igniarius in the prior art. When the phellinus igniarius mycelium is used as a target product, a fermentation strategy of adding selenium-enriched yeast in the whole process is adopted; when phellinus igniarius sporocarp is used as a target product, a staged strategy is adopted: selenium-enriched yeast is added in a mycelium culture stage, and sodium selenite is added in a sporocarp culture stage. According to the method, the rule that the mycelium stage is adaptive to organic selenium and the sporocarp stage is adaptive to inorganic selenium is defined for the first time, and through accurate stage-selenium source matching, the selenium content, yield and comprehensive quality of the mycelium or the sporocarp are synchronously improved successfully on the premise that mycelium growth is hardly inhibited.

Description

Technical Field

[0001] This invention relates to the fields of microbial fermentation and edible fungi cultivation technology, specifically to a production method that maximizes the biomass, active ingredients, and selenium content of Phellinus linteus mycelium, or optimizes the yield, quality, and selenium content of Phellinus linteus fruiting bodies, by precisely selecting selenium sources and controlling the application stage. Background Technology

[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] Phellinus linteus, a precious medicinal fungus, has a long history of application in traditional medicine. Modern pharmacological research has further confirmed that it contains various active ingredients such as polysaccharides, flavonoids, and triterpenes, possessing multiple potential health benefits including antioxidant properties, enhanced immunity, and metabolic regulation. Market demand for it continues to rise in the health supplement and pharmaceutical industries. With consumers increasingly focusing on functional agricultural products, enhancing the nutritional value of Phellinus linteus has become an important direction for expanding its industrial value. Selenium is an essential trace element for the human body, participating in the synthesis and metabolism of various enzymes. It plays a crucial role in maintaining cardiovascular health, enhancing immune function, and resisting oxidative stress. However, the human body cannot synthesize selenium and must obtain it through dietary intake. Nevertheless, many parts of the world have low soil selenium content, making it difficult for conventionally cultivated agricultural products to meet human needs. Developing selenium-enriched agricultural products has become an effective way to solve the "selenium deficiency" problem. Integrating selenium into the cultivation of *Sanghuang* (a type of medicinal mushroom) through appropriate methods to cultivate selenium-enriched *Sanghuang* can fully utilize the medicinal potential of *Sanghuang* while also endowing it with the nutritional function of selenium, achieving the dual value of "medicine and food from the same source" and "trace element fortification," aligning with the current health consumer market's pursuit of high-quality functional products.

[0004] Currently, the production of selenium-enriched Phellinus linteus typically involves adding exogenous selenium to the culture medium in a single step, with sodium selenite being the most common inorganic selenium source. However, this method has significant limitations: selenium, especially in its inorganic form, can have a clear inhibitory effect on the growth of Phellinus linteus mycelium at certain concentrations. This leads to a dilemma in production: using low concentrations of selenium to ensure normal mycelial growth and final yield results in a product selenium content that fails to meet functional standards; increasing the selenium concentration to increase the selenium content, on the other hand, inhibits growth, leading to a decrease in biomass or fruiting body yield and a deterioration in quality. In other words, the three core objectives of "high yield (high biomass / high production)," "excellent quality," and "high selenium content" are difficult to achieve synergistically under the existing "single selenium source throughout the entire process" model.

[0005] A deeper issue lies in the limitations of current technology in understanding the physiological mechanisms of selenium enrichment in *Sanghuang*. Through a systematic comparative study, the applicant has revealed for the first time that the selenium enrichment efficiency of *Sanghuang* exhibits significant stage-dependent and selenium source-dependent characteristics. Specifically: During the mycelial growth stage, organic selenium sources (such as selenium-enriched yeast) are significantly superior to inorganic selenium sources (such as sodium selenite) in promoting robust mycelial growth (manifested as dense and bright yellow mycelia) and increasing the selenium content of the mycelium itself.

[0006] During the fruiting body development stage, the situation reverses, with inorganic selenium sources (sodium selenite) showing a significant advantage over organic selenium sources (selenium-enriched yeast) in terms of efficiency and final content in transporting and enriching selenium into the fruiting body.

[0007] This key discovery indicates that the mycelium and fruiting body, as different growth and development stages and product forms of Sanghuang, have fundamentally different requirements for selenium sources. However, existing technologies have failed to recognize and utilize this difference, and still generally adopt a "one-size-fits-all" strategy of adding a single selenium source. This is the fundamental reason why the contradiction between "growth inhibition" and "highly efficient selenium enrichment" cannot be resolved.

[0008] Therefore, there is an urgent need in this field for an innovative method for regulating selenium sources that can: 1) be based on a deep understanding of the physiological characteristics of Sanghuang at different stages; 2) provide differentiated and precise selenium source supply solutions according to the different final target products (whether it is mycelium for deep processing or fruiting bodies directly used as commodities); and 3) systematically break through the above-mentioned industry bottlenecks and achieve efficient, high-quality and standardized production of selenium-enriched Sanghuang. Summary of the Invention

[0009] The technical problem to be solved by this invention is to provide a flexible selenium source regulation strategy that can select the optimal selenium source and application scheme according to whether the target product is mycelium or fruiting body, thereby solving the contradiction between "biomass and selenium enrichment" in mycelium fermentation and the contradiction between "yield, quality and selenium enrichment" in fruiting body cultivation.

[0010] The technical solution adopted in this invention is as follows: The first aspect of this invention provides a method for selecting and regulating selenium sources for the production of selenium-enriched Phellinus linteus, employing one of two different selenium source application strategies depending on the type of the target product: (a) When the target product is *Phellinus linteus* mycelium, selenium-enriched yeast is added to the culture medium for fermentation of *Phellinus linteus* mycelium throughout the entire process; or (b) When the target product is the fruiting body of Phellinus linteus, a phased application strategy is adopted: selenium-enriched yeast is added during the mycelial culture stage, and sodium selenite is added during the fruiting body cultivation stage.

[0011] Preferably, in strategy (a), when liquid fermentation is used, the concentration of the selenium-enriched yeast added is 5-20 mg / L of culture medium based on selenium; more preferably, it is 10-15 mg / L.

[0012] When solid-state fermentation is used, the concentration of the selenium-enriched yeast added is 10-50 mg / kg dry weight of the culture medium, based on selenium element; more preferably, it is 15-30 mg / kg.

[0013] Preferably, in strategy (b), the mycelial culture stage is liquid seed culture or solid substrate mycelium growth; the fruiting body cultivation stage is solid substrate fruiting.

[0014] Preferably, in strategy (b), during the mycelial culture stage, the concentration of the selenium-enriched yeast is 1-10 mg / L of liquid culture medium or 2-15 mg / kg of dry solid culture medium, calculated as selenium; more preferably, it is 5-8 mg / L or 5-10 mg / kg.

[0015] During the fruiting body cultivation stage, the concentration of sodium selenite added is 20-150 mg / kg of dry cultivation material based on selenium element; more preferably, it is 50-80 mg / kg.

[0016] In a second aspect of the invention, a selenium-enriched Phellinus linteus mycelium is provided, which is produced by the method described in strategy (a), wherein the selenium content of the mycelium is not less than 50 mg / kg.

[0017] Preferably, the selenium content of the mycelium is not less than 100 mg / kg.

[0018] In a third aspect of the invention, a selenium-enriched Phellinus linteus fruiting body is provided, which is produced by the method described in strategy (b), wherein the selenium content of the fruiting body is not less than 300 mg / kg and its biological efficiency is not less than 13%.

[0019] Preferably, the content of at least one of crude protein, crude polysaccharide and total amino acids is significantly higher than that of the control group of Phellinus linteus fruiting bodies without added exogenous selenium.

[0020] In a fourth aspect of the invention, the selenium-enriched Phellinus linteus mycelium is provided for use in the preparation of foods, health products or pharmaceuticals for enhancing immunity, anti-oxidation or selenium supplementation.

[0021] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: This invention is the first to propose the production concept of "stage-selenium source matching". By effectively combining the selenium source supply strategies of the mycelium stage and the fruiting body stage, it systematically solves the inherent contradiction between "maintaining growth" and "promoting selenium enrichment".

[0022] For fruiting body production: The method of this invention can simultaneously achieve the following while significantly reducing total selenium input: ① no significant inhibition of mycelial growth; ② selenium content in fruiting bodies reaching over 400 mg / kg; ③ stable biological efficiency of over 14%. This ensures the product's superior functionality while improving production safety and economic efficiency.

[0023] For mycelium production: The method of this invention utilizes the biocompatibility of organic selenium, which can efficiently produce Phellinus linteus mycelium raw materials with a selenium content of more than 100 mg / kg while ensuring high mycelial biomass, providing high-quality and standardized raw materials for downstream extraction and preparation.

[0024] This invention provides a new production paradigm for the selenium-enriched Phellinus linteus industry: if the target is mycelial raw material, then full-process organic selenium fermentation is adopted; if the target is commercial fruiting bodies, then staged (organic to inorganic) cultivation is adopted. This provides a precise and reliable technical roadmap for the diversified development of functional Phellinus linteus products.

[0025] The invention is based on solid experimental evidence (the organic selenium form and biocompatibility are better in the mycelial stage, and the inorganic selenium transport and enrichment efficiency is higher in the fruiting body stage). The technical solution has a solid physiological basis, rather than a simple empirical attempt. Attached Figure Description

[0026] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0027] Figure 1 Effects of selenium on the growth of Phellinus linteus mycelium; A: Selenium-enriched yeast group. B: Sodium selenite group. C: Nano selenium group, with concentrations of 0, 1, 5, 10, and 15 mg / L from left to right.

[0028] Figure 2 Comparison of mycelial selenium content under different selenium sources and concentrations; A: Mycelial selenium content. B: Fruiting body selenium content. Note: Different letters indicate significant differences between treatments (P<0.05). SS, NS, and SEY represent sodium selenite, nano-selenium, and selenium-enriched yeast, respectively. The mycelial selenium concentrations were 0, 1, 10, and 20 mg / L; the fruiting body selenium concentrations were 0, 15, and 70 mg / kg.

[0029] Figure 3 The ratio of crude protein (A), crude fat (B), and crude polysaccharide (C) content in fruiting bodies treated with different selenium sources and concentrations. Note: Different letters indicate significant differences between treatments (P<0.05). SEY represents selenium-enriched yeast. The selenium concentration added was 15 mg / kg.

[0030] Figure 4 Effects of selenium on the growth of Phellinus linteus fruiting bodies; A: Selenium-enriched yeast group; B: Sodium selenite group; C: Nano selenium group, with concentrations of 0, 15, and 70 mg / kg from left to right.

[0031] Figure 5 The effect of different selenium sources and concentrations on fruiting of Phellinus linteus fruiting bodies; A: selenium-enriched yeast group; B: sodium selenite group; C: nano selenium group, with concentrations of 0, 15, and 70 mg / kg from left to right. Detailed Implementation

[0032] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0033] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0034] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0035] Materials used in this invention:

[0036] Strains: Phellinus linteus ( Sanghuangporus sp. The strain 'Sanghuang Hu 2' was provided by the Shanghai Academy of Agricultural Sciences.

[0037] Selenium source: Selenium-enriched yeast (selenium content as indicated on product label, 2000 ppm), Shanghai Yuanye Biotechnology Co., Ltd. Sodium selenite, Sinopharm Chemical Reagent Co., Ltd. Nano selenium, Hubei Baitek Bioengineering Co., Ltd.

[0038] Main instruments: 0.01% analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); constant temperature incubator (Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.); electric heating drying oven (Shanghai Yiheng Scientific Instruments Co., Ltd.); atomic fluorescence spectrometer (Jitian AFS-822).

[0039] Test items and methods (1) Selenium content determination: Selenium content was determined using atomic fluorescence spectrometry, referring to the national standard GB 5009.93-2017 "Determination of Selenium in Food". The simplified procedure was as follows: 0.2 g of dried sample was accurately weighed, digested using a mixed acid-perchloric acid method, brought to a final volume, reduced with hydrochloric acid, and finally determined using an atomic fluorescence spectrometer. The calculation formula is: ; In the formula: ω is the selenium content in the sample, in milligrams per kilogram (mg / kg); ρ is the selenium concentration in the sample solution, in micrograms per liter (μg / L); ρ0 is the selenium concentration in the blank solution, in micrograms per liter (μg / L); V is the total volume of the sample digest solution, in milliliters (mL); m is the sample weight, in grams (g); 1000 is the conversion factor.

[0040] (2) Agronomic traits determination: 50 fruiting bodies were randomly selected from each treatment. The diameter and thickness of the cap were measured using calipers, and the fresh weight of each mushroom was weighed using an electronic balance. Biological efficiency (%) = (total fresh weight of fruiting bodies / dry weight of substrate) × 100%.

[0041] (3) Nutritional quality indicators: Crude fat, crude protein and amino acid composition and other indicators are measured using the corresponding national standards or industry-recognized methods.

[0042] (4) Data processing: Excel 2025 was used for data processing, SPSS 27 software was used for one-way ANOVA, and Duncan's new multiple range method was used for multiple comparisons. The significance level was set at P<0.05.

[0043] Example 1: Evaluation of the effects of different selenium sources on the growth of Phellinus linteus mycelium (plate experiment) This embodiment aims to rapidly evaluate and screen the optimal selenium source and concentration suitable for the growth of Phellinus linteus mycelium under controlled laboratory conditions, providing a key basis for subsequent cultivation and fermentation production.

[0044] Materials and Methods: PDA culture medium containing sodium selenite, nano-selenium, and selenium-enriched yeast was prepared. The selenium concentration was set at 0 (CK), 1, 5, 10, and 15 mg / L. -1 Inoculate with *Sanghuang* strain 'Sanghuang Hu 2', incubate in the dark at 25°C, and observe regularly.

[0045] Results analysis: As shown in Table 1 and Figure 1 As shown, in 0–15 mg·L -1Within the specified concentration range, exogenous selenium supplementation significantly regulated the growth rate, density, color, and colony morphology of *Sanghuang* mycelium (P<0.05). The control group (CK) without selenium supplementation showed the fastest mycelial growth rate (2.38 ± 0.05 mm / d), indicating that appropriate concentrations of selenium can inhibit the early growth of *Sanghuang* mycelium to some extent. Selenium-enriched yeast, nano-selenium, and sodium selenite all exhibited significant selenium source specificity and concentration dependence on the growth of *Sanghuang* mycelium and its selenium accumulation rate. Within the range of 0–15 mg·L⁻¹… -1 Within the specified concentration range, all three exogenous selenium sources exhibited varying degrees of inhibitory effects on the mycelial growth of *Phellinus linteus*, with sodium selenite showing the most significant inhibitory effect and selenium-enriched yeast showing the weakest. Under the same selenium concentration, the mycelial growth rate of the selenium-enriched yeast treatment group was significantly higher than that of the nano-selenium and sodium selenite treatment groups. Furthermore, the inhibitory effect of all three selenium sources on mycelial growth increased with increasing concentration, exhibiting a clear concentration dependence: as the selenium concentration increased, the mycelial growth of *Phellinus linteus* gradually weakened, and the colony density gradually decreased. From the perspective of mycelial appearance, the mycelia in the selenium-enriched yeast treatment group were generally denser and brighter yellow, indicating that organic selenium caused less damage to the mycelial cell membranes or was more easily absorbed, assimilated, and utilized by *Phellinus linteus* mycelia.

[0046] in conclusion: All selenium treatments inhibited the radial spread rate of hyphae.

[0047] Key differences in form: at the same concentration (e.g., 5 mg·L⁻¹) -1 The mycelia in the selenium-enriched yeast treatment group were dense, uniform, and bright yellow in color, while the mycelia in the sodium selenite treatment group were relatively sparse. This indicates that, during the mycelial growth stage, organic selenium (selenium-enriched yeast) has better biocompatibility than inorganic selenium (sodium selenite).

[0048] Preliminary optimal concentration: Considering both growth and morphology, selenium-enriched yeast at 5 mg·L⁻¹ -1 It performs best at the specified concentration.

[0049] The above experiments show that selenium-enriched yeast should be the preferred selenium source to obtain robust Phellinus linteus mycelium. This provides a scientific basis for the use of organic selenium in the mycelial stage in all subsequent examples.

[0050] Table 1. Mycelial growth under different selenium sources and concentrations.

[0051] Note: ++++, +++, and ++ indicate strong, moderate, and weak mycelial growth, respectively; different lowercase letters represent significant differences (P<0.05). The concentrations are 0, 1, 5, 10, and 15 mg / L, respectively. SS, NS, and SEY represent sodium selenite, nano-selenium, and selenium-enriched yeast, respectively.

[0052] Example 2: Single-source selenium bag cultivation reveals industry contradictions (verification cultivation experiment) This embodiment aims to simulate existing production technologies and reveal the inherent fundamental contradictions in the production of selenium-enriched Sanghuang using a single selenium source throughout the entire process.

[0053] 1. Method: The basic formula for the cultivation substrate was: 70% sawdust, 20% wheat bran, 9% corn flour, and 1% calcium carbonate, with the moisture content adjusted to 60%–65%. Before sterilization, three selenium sources were added to the cultivation substrate, and seven treatments were set up: CK (selenium-free), sodium selenite (15, 70 mg / kg), and sodium selenite (15, 70 mg / kg). -1 Nano-selenium (15, 70 mg·kg⁻¹) -1 ), selenium-enriched yeast (15, 70 mg·kg) -1 Each treatment yielded 100 bags. The bags were sterilized by high-pressure steam at 121℃ for 2 hours, then cooled to room temperature for aseptic inoculation. The inoculated bags were placed in a dark incubation room at 25℃ for mycelial growth. When the mycelium reached the "shoulder" of the bag, the mycelial tip was marked and the time to full colonization was recorded. After mycelial growth, primordia formation and fruiting body development were induced by removing the bags and spraying water to increase humidity. The fruiting bodies were harvested promptly when the edge of the cap turned from pale yellow to golden yellow and the texture became firm. The harvested fruiting bodies were dried at 55℃ to constant weight, then pulverized using a high-speed traditional Chinese medicine pulverizer, sieved, and used for various physicochemical index determinations.

[0054] 2. Results Analysis 2.1 Effects of selenium addition on the growth and development of Phellinus linteus The form and concentration of selenium mainly affect the vegetative growth stage (mycelium fully colonizing the bag) and physiological transformation stage (color change) of *Sanghuang*, while having a relatively limited impact on the onset time of the reproductive growth stage (primordia differentiation and fruiting body formation). Based on the data in Table 2, it is estimated that compared to the control (CK), 15 mg / kg... -1 Sodium selenite treatment advanced the color change period by approximately 3 days, demonstrating a significant promoting effect. However, most selenium treatments (especially 70 mg / kg) showed limited efficacy. -1 All of these treatments prolonged the time required for mycelium to fully colonize the culture bag to varying degrees, which may be related to the initial inhibition of mycelial tip growth by selenium. A notable exception is 15 mg / kg. -1 Selenium-enriched yeast treatment did not significantly delay the time for mycelium to fully colonize the bag, further suggesting that organic selenium sources may have better biocompatibility.

[0055] Table 2. Effects of selenium on the growth and development of Phellinus linteus

[0056] Note: The concentrations are 0, 15, and 70 mg / kg, respectively. SS, NS, and SEY represent sodium selenite, nano selenium, and selenium-enriched yeast, respectively.

[0057] 2.2 Effects of selenium addition on agronomic traits of Phellinus linteus fruiting bodies Based on the results of previous mycelial growth experiments, three concentration levels (0, 15, and 70 mg / kg) of nano-selenium, sodium selenite, and selenium-enriched yeast were selected for fruiting experiments using mature substrate of *Sanghuang* (Phellinus linteus). The agronomic traits of the fruiting bodies were measured, and the results are shown in Table 3. Under sodium selenite treatment, the cap width of *Sanghuang* fruiting bodies was 84.68–84.99 mm, the cap thickness was 18.64–20 mm, the fresh weight of a single mushroom was 32.52–33.77 g, and the biological efficiency of a single flush was 13.51%–14.31%. Among these, the 70 mg·kg⁻¹ treatment showed the best performance in terms of cap width, thickness, and biological efficiency. There were no significant differences in cap thickness and biological efficiency among the treatments, indicating that within this concentration range, increasing selenium concentration did not significantly affect the biological efficiency of *Sanghuang* fruiting bodies, but the 70 mg·kg⁻¹ treatment showed the best results. --1 At the specified concentration, the overall agronomic traits were relatively superior. Under nano-selenium treatment, the cap width of the fruiting bodies was 76.39–82.47 mm, the cap thickness was 16.91–19.17 mm, the fresh weight of a single mushroom was 26.49–30.22 g, and the biological efficiency of a single flush was 7.22%–9.40%. The results indicate that nano-selenium at 15 mg·kg⁻¹ exhibits relatively superior overall agronomic traits. -1 and 70 mg·kg -1 All concentrations inhibited the growth and transformation efficiency of Phellinus linteus, especially at 15 mg·kg⁻¹. -1 The inhibitory effect was more pronounced over time. Under selenium-enriched yeast treatment, the fruiting body cap width was 74.15–88.97 mm, the cap thickness was 18.44–21.58 mm, the fresh weight of a single mushroom was 25.48–33.75 g, and the biological efficiency of a single flush was 10.19%–13.50%. Among these, 15 mg·kg⁻¹… -1 The treatment showed the best overall performance, with significantly wider caps than the control (CK), higher single-mushroom fresh weight, and a biological efficiency of 13.50%, comparable to the sodium selenite 70 treatment. This indicates that this concentration can improve conversion efficiency while promoting growth. (70 mg·kg⁻¹) -1 The treatments showed a significant inhibitory effect, with cap width and single mushroom fresh weight being the lowest or second lowest values ​​among all treatments. For example... Figure 4 As shown.

[0058] Based on the results of previous mycelial growth experiments, 0, 15, and 70 mg·kg were selected. -1 Three concentration levels of nano-selenium, sodium selenite, and selenium-enriched yeast were used in a mushroom cultivation experiment using mature substrate for *Sanghuang* (a type of medicinal mushroom), and the agronomic traits of the fruiting bodies were measured. The results are shown in Table 3. Using the control group (CK) as a reference, the three selenium sources—sodium selenite, nano-selenium, and selenium-enriched yeast—were tested at concentrations of 15 and 70 mg / kg. -1At different concentrations, the effects of sodium selenite on the cap width and thickness, fresh weight of individual mushrooms, and biological efficiency of *Phellinus linteus* were concentration-dependent and selenium source-specific. Sodium selenite at 70 mg / kg... -1 At this optimal concentration, cap width and biological efficiency were significantly higher than the control (CK), while the fresh weight of individual mushrooms was not different from the CK; 15 mg·kg -1 Concentration significantly reduced cap thickness and biological efficiency. Both concentrations of nano-selenium inhibited the growth of *Sanghuang*, with all agronomic traits significantly lower than the control (CK), and at 15 mg / kg... --1 The group exhibited the lowest biological efficiency. The optimal concentration of selenium-enriched yeast was 15 mg·kg⁻¹, at which point the cap width and biological efficiency were significantly higher than the control (CK), while cap thickness and single-mushroom fresh weight showed no difference from the CK; 70 mg·kg⁻¹… -1 At the appropriate concentration, all indicators decreased significantly, and the biological efficiency approached that of the control (CK). At a suitable concentration, sodium selenite (70 mg·kg⁻¹) -1 ) and selenium-enriched yeast (15 mg·kg) -1 There was no difference in biological efficiency, but the selenium-enriched yeast group had a better cap width, and there was no suitable concentration of nano-selenium. Figure 5 As shown.

[0059] Table 3 Effects of selenium on agronomic traits of Phellinus linteus

[0060] Note: Each value represents the mean ± standard deviation (n=3). Statistical analysis was performed using ANOVA with a post-hoc Duncan test. Significant differences were found between different lowercase letters (p<0.05). Concentrations were 0, 15, and 70 mg / kg. SS, NS, and SEY represent sodium selenite, nano-selenium, and selenium-enriched yeast, respectively.

[0061] 2.3 Differences in selenium content at different selenium source mycelial and fruiting body stages like Figure 2 As shown, the enrichment efficiency of selenium is highly dependent on the chemical form of selenium and the growth and development stage of *Sanghuang*. During the mycelial stage ( Figure 2 A) Selenium-enriched yeast (SEY) performed best, with significantly higher selenium content in its mycelium compared to sodium selenite (SS) and nano-selenium (NS) groups at the same concentration, for example, SEY 20 mg·L⁻¹. -1 The selenium content was as high as approximately 150 mg / kg. This indicates that the mycelium has a higher absorption and conversion efficiency for organic selenium. Sodium selenite was the second most efficient, while nano-selenium had the lowest enrichment efficiency. During the fruiting body stage ( Figure 2 (B) The selenium enrichment format changed significantly. Sodium selenite (SS) became the optimal selenium source, with an optimal concentration of 70 mg / kg. -1The selenium content in the fruiting bodies of the group treated with selenium-enriched yeast (SEY) was significantly higher than that of other treatments, reaching approximately 450 mg / kg, demonstrating the substantial advantage of inorganic selenium in the transport process from mycelium to fruiting bodies. Although the selenium content in the fruiting bodies of the SEY-treated group (approximately 300 mg / kg) was lower than that of the SS group, it was still at a relatively high level. The efficiency of nano-selenium (NS) was lowest in both stages. This differential enrichment phenomenon at different stages may be related to the metabolic pathways, transport protein specificity, and transformation mechanisms of different selenium sources within *Phellinus linteus*.

[0062] 2.4 Effects of different selenium sources on the nutritional quality of fruiting bodies Figure 3 The results showed that different concentrations of selenium-enriched yeast had a significant impact on the basic nutritional components of Sanghuang (Phellinus linteus). With increasing selenium-enriched yeast concentration, the contents of crude protein, crude polysaccharides, and crude fat in Sanghuang showed a significant upward trend; when the selenium-enriched yeast concentration reached 15 mg / kg... -1 At the specified time, the contents of crude polysaccharides and crude protein both reached their peak values, at 16.51 mg / g and 138.77 g / kg, respectively. Compared with the control group, these indicators increased by 15.29% and 3.71%, respectively. The study indicates that low concentrations of selenite can be effectively absorbed by cells and stored in proteins. However, in this study, selenium-enriched yeast did not significantly affect the crude fat content of *Sanghuang*, and sodium selenite mainly promoted the synthesis of crude protein in *Sanghuang*.

[0063] Table 4. Amino acid content of selenium-treated Phellinus linteus fruiting bodies

[0064] Note: The concentrations are 0 and 15 mg / kg respectively. SEY indicates selenium-enriched yeast.

[0065] As shown in Table 4, the free amino acid composition of *Phellinus linteus* fruiting bodies cultivated with selenium enrichment was abundant, but the total amino acid content varied significantly under different selenium concentrations, ranging from 2687.65 to 3080.89 mg / 100 g. All eight essential amino acids were detected in *Phellinus linteus* fruiting bodies, and the total amino acid (TAA) and essential amino acid (EAA) contents were significantly higher in the selenium-enriched group than in the control group. The total amino acid content in the selenium-enriched group reached 3080.89 mg / kg, significantly higher than the control group (2687.65 mg / kg), nearly 1.5 times that of the control group. Regarding the response characteristics of amino acid components, the contents of isoleucine and valine in the selenium-enriched group increased with selenium concentration (0-15 mg / kg). -1The levels of selenium and methionine showed a significant increasing trend, with methionine content exhibiting even greater sensitivity to changes in selenium concentration. Specific component comparisons revealed that the selenium-enriched group had significantly higher levels of aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), and isoleucine (Ile) than the control group, with glutamic acid increasing by 13.86% and lysine by a remarkable 41.15%, demonstrating a particularly strong effect. In contrast, the control group had relatively higher levels of methionine (Met), tryptophan (Trp), and leucine (Leu), reflecting the selective regulatory effect of selenium on amino acid synthesis in *Sanghuang* fruiting bodies. Notably, in both the selenium-enriched and control groups, the glutamic acid content in *Sanghuang* fruiting bodies was higher than that of aspartic acid. Mechanistically, selenium can regulate amino acid synthesis by mediating the reduction of toxic tetravalent selenium through glutathione and interfering with metabolic pathways such as glycolysis. Treatment with 15 mg·kg⁻¹ selenium can maintain the dynamic balance of amino acid biosynthesis, promoting the synthesis of isoleucine and valine, while excessive selenium-induced oxidative stress inhibits protein synthesis and reduces amino acid accumulation.

[0066] 3. Conclusion and Revelation of Contradictions: Mycelial growth: The selenium-enriched yeast treatment group showed the best mycelial growth, with no difference in the time to full bag coverage compared to the control group; mycelial growth was significantly inhibited in the sodium selenite 70 mg / kg group.

[0067] Selenium content of fruiting body ( Figure 2 B): The sodium selenite 70 mg / kg group had the highest value (450 mg / kg); the selenium-enriched yeast 70 mg / kg group was the second highest (300 mg / kg).

[0068] Fruiting body yield (Table 3): The biological efficiency of the selenium-enriched yeast group at 15 mg / kg was high (13.50%), but decreased at 70 mg / kg; the efficiency of the sodium selenite group at 70 mg / kg was 14.31%, but its mycelial growth was poor.

[0069] The core contradiction can be summarized as follows: the existing "single selenium source throughout the entire process" model cannot simultaneously achieve "the most robust mycelium" and "the highest selenium content in the fruiting body." "Selenium-enriched yeast" and "sodium selenite" each have their advantages at different growth and development stages of *Sanghuang*, and cannot be obtained simultaneously. This clarifies the technical problem that this invention aims to solve.

[0070] The above results indicate that the traditional model of using a single selenium source throughout the entire process has inherent drawbacks: while using sodium selenite throughout the process can yield high-selenium fruiting bodies (450 mg / kg), it severely inhibits mycelial growth; while using selenium-enriched yeast throughout the process is beneficial to mycelial growth, there is an upper limit to the selenium enrichment efficiency of fruiting bodies (300 mg / kg), and the yield decreases at high concentrations. In other words, it is impossible to simultaneously achieve both the most robust mycelia and the highest selenium content in fruiting bodies using a single selenium source. This clarifies the technical problem to be solved by the phased strategy proposed in this invention.

[0071] Example 3: This invention—staged selenium source regulation for the production of ultra-high selenium Phellinus linteus fruiting bodies Based on the findings of Examples 1 and 2, this example creatively proposes and verifies a phased strategy of "organic selenium cultivation during the mycelial stage and inorganic selenium enrichment during the fruiting body stage".

[0072] Experimental design: see Table 5.

[0073] Table 5 Experimental Groups and Treatments

[0074] 2. Key operation: After the fruiting body primordia are formed, T1 group should use a syringe to inject sterile sodium selenite solution into the substrate of the substrate bag at multiple points, avoiding contact with the fruiting body primordia.

[0075] 3. Results: See Table 6.

[0076] Table 6 Test Results

[0077] 4. Conclusion: The phased strategy of this invention successfully overcomes the contradiction revealed in Example 2. With a lower total selenium input, it simultaneously achieves "uninhibited mycelial growth," "selenium content in fruiting bodies close to that of traditional high-selenium methods," and "the highest fruiting body yield," generating significant synergistic effects and solving a long-standing industrial technology bottleneck.

[0078] Example 4: This invention—production of high-selenium Phellinus linteus mycelium through whole-process organic selenium solid-state fermentation. Based on the discovery in Example 1 that "selenium-enriched yeast is optimal at the mycelial stage", this example provides an independent production path with mycelium as the final product.

[0079] Method: The basic formula for the cultivation substrate was: 70% sawdust, 20% wheat bran, 9% corn flour, and 1% calcium carbonate, with the moisture content adjusted to 60%–65%. Selenium-enriched yeast was added before sterilization to achieve a selenium concentration of 15 mg / kg. After inoculation, fermentation was carried out at 25℃ for 35 days (only mycelium was cultured; fruiting was not induced). After completion, the entire fermentation product was dried and pulverized.

[0080] Results: The selenium content of the dried selenium-enriched Phellinus linteus mycelium reached 131.47 ± 0.25 mg / kg. This product can be directly used as a high-selenium functional food raw material or for the extraction of active ingredients, providing a short-cycle and process-controllable production route for selenium-enriched Phellinus linteus raw materials.

[0081] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for selecting and regulating selenium sources for the production of selenium-enriched Phellinus linteus, characterized in that, Depending on the type of the target product, one of the following two different selenium source application strategies may be adopted: (a) When the target product is *Phellinus linteus* mycelium, selenium-enriched yeast is added to the culture medium for fermentation of *Phellinus linteus* mycelium throughout the entire process; or (b) When the target product is the fruiting body of Phellinus linteus, a phased application strategy is adopted: selenium-enriched yeast is added during the mycelial culture stage, and sodium selenite is added during the fruiting body cultivation stage.

2. The method according to claim 1, characterized in that, In strategy (a): When liquid fermentation is used, the concentration of the selenium-enriched yeast added is 5-20 mg / L of culture medium, calculated as selenium. When solid-state fermentation is used, the concentration of the selenium-enriched yeast added is 10-50 mg / kg dry weight of the culture medium, calculated as selenium.

3. The method according to claim 2, characterized in that, In strategy (a): During liquid fermentation, the preferred concentration of selenium-enriched yeast is 10-15 mg / L of culture medium, calculated as selenium. During solid-state fermentation, the preferred concentration of selenium-enriched yeast is 15-30 mg / kg dry weight of the culture medium, calculated as selenium.

4. The method according to claim 1, characterized in that, In strategy (b), the mycelial culture stage is either liquid seed culture or solid substrate mycelium growth; the fruiting body cultivation stage is solid substrate fruiting.

5. The method according to claim 1 or 4, characterized in that, In strategy (b): During the mycelial culture stage, the concentration of the selenium-enriched yeast added is 1-10 mg / L of liquid culture medium or 2-15 mg / kg of dry solid culture medium, calculated as selenium. During the fruiting body cultivation stage, the concentration of sodium selenite added is 20-150 mg / kg of dry cultivation material, calculated as selenium.

6. The method according to claim 5, characterized in that, In strategy (b): During the mycelial culture stage, the concentration of the selenium-enriched yeast added is 5 mg / L of liquid culture medium or 5 mg / kg of dry solid culture medium, calculated as selenium. During the fruiting body cultivation stage, the concentration of sodium selenite added is 50-80 mg / kg dry weight of cultivation material, calculated as selenium.

7. A selenium-enriched Phellinus linteus mycelium, characterized in that, It is produced by the method of any one of claims 1 to 3, and its selenium content is not less than 50 mg / kg dry weight.

8. The selenium-enriched Phellinus linteus mycelium according to claim 7, characterized in that, Its selenium content is not less than 100 mg / kg dry weight.

9. A selenium-enriched Phellinus linteus fruiting body, characterized in that, It is produced by the method of any one of claims 1, 4, 5 or 6, and has a selenium content of not less than 300 mg / kg dry weight and a biological efficiency of not less than 13%.

10. The use of the selenium-enriched Phellinus linteus mycelium as described in claim 7 or 8, or the selenium-enriched Phellinus linteus fruiting body as described in claim 9, in the preparation of food, health products, or medicines with antioxidant or immunomodulatory functions.

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