Target-oriented selenium-rich lentinus edodes cultivation method and application thereof

Abstract

This invention discloses a goal-oriented method for the targeted cultivation of selenium-enriched shiitake mushrooms, belonging to the technical field of edible fungi cultivation and functional foods. The method includes the following steps: adding exogenous selenium to the cultivation substrate during the fruiting body cultivation stage of shiitake mushrooms; the exogenous selenium is selected from nano-selenium or sodium selenite; different selenium sources are selected according to the target selenium enrichment requirements: (a) when the goal is to increase the yield of shiitake mushroom fruiting bodies, nano-selenium is selected; (b) when the goal is to increase the total selenium content or bioavailability of selenium in shiitake mushroom fruiting bodies, sodium selenite is selected. This invention provides clear selenium source selection schemes for three different commercial needs: "high yield," "high selenium," and "high absorption," filling a technological gap in the shiitake mushroom field. Producers can flexibly choose according to market demand, realizing precise and customized production of selenium-enriched shiitake mushrooms.

Description

Technical Field

[0001] This invention belongs to the field of edible fungi cultivation and functional food technology, specifically relating to a method for targeted selenium-enriched cultivation of shiitake mushrooms by selecting different selenium sources according to different selenium enrichment requirements. 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] mushroom( Lentinula edodes Shiitake mushrooms are the world's second most produced edible fungus, beloved by consumers for their unique flavor and rich nutritional value. Selenium is an essential trace element for the human body, possessing various physiological functions such as anti-oxidation, immune enhancement, and anti-cancer properties. However, the human body cannot synthesize selenium and must obtain it through dietary intake. Developing selenium-enriched shiitake mushroom products by leveraging the mushroom's powerful biotransformation capabilities is an effective way to increase its added value.

[0004] The existing selenium-enriched shiitake mushroom cultivation technology has the following shortcomings: (1) Single objective: Most technologies only pursue the maximization of total selenium content, ignoring the differentiation of different commercial needs (high yield, high selenium, high absorption). In production practice, some scenarios require high-yield, high-quality selenium-enriched shiitake mushrooms to supply the mass market, some scenarios require shiitake mushrooms with ultra-high selenium content for extracting selenoproteins, and some scenarios require shiitake mushrooms with high absorption rates for high-end health foods. Existing technologies have failed to provide differentiated technical solutions for these different needs.

[0005] (2) Lack of systematic screening: Different selenium sources (sodium selenite, nano-selenium, selenium-enriched yeast) and different concentrations have significant effects on shiitake mushroom yield, selenium content, and absorption rate. Existing technologies lack systematic comparative studies on different selenium sources and concentrations, and have failed to establish a correspondence between "target requirements and selenium source selection".

[0006] (3) Neglecting the form and absorption efficiency of selenium: The health benefits of selenium are highly dependent on its chemical form. Organic selenium (such as selenomethionine and methylselenocysteine) has better bioavailability and safety than inorganic selenium. However, current technologies rarely focus on the distribution of selenium forms in selenium-enriched shiitake mushrooms, and have not used in vitro simulated gastrointestinal fluid absorption rate as a screening indicator. The key issue that "high selenium content" does not equal "high absorption rate" has been neglected for a long time.

[0007] Therefore, there is an urgent need in this field for a targeted selenium-enriched cultivation method that is tailored to the characteristics of shiitake mushrooms and can meet different commercial needs. Summary of the Invention

[0008] This invention aims to solve the technical problems of existing selenium-enriched shiitake mushroom cultivation technology, such as single target, lack of systematic screening, and neglect of selenium form and absorption efficiency. It provides a method for targeted selenium-enriched shiitake mushroom cultivation that allows for flexible selection of selenium sources according to different commercial needs (priority given to yield, selenium content, and absorption rate).

[0009] The technical solution adopted in this invention is as follows: A goal-oriented method for the targeted cultivation of selenium-enriched shiitake mushrooms includes the following steps: In shiitake mushrooms ( Lentinula edodes During the fruiting body cultivation stage, exogenous selenium is added to the cultivation substrate; the exogenous selenium is selected from nano-selenium or sodium selenite; different selenium sources are selected according to the target selenium enrichment requirements: (a) When the goal is to increase the yield of shiitake mushroom fruiting bodies, nano-selenium is selected; (b) Sodium selenite is chosen when the goal is to increase the total selenium content of shiitake fruiting bodies or the bioavailability of selenium.

[0010] Further, in scheme (a), the concentration of the added nano-selenium is 1-100 mg / kg dry weight of the cultivation material, preferably 10-20 mg / kg dry weight of the cultivation material, and most preferably 15 mg / kg dry weight of the cultivation material.

[0011] Further, in scheme (b), the concentration of sodium selenite added is 10-200 mg / kg dry weight of cultivation material, preferably 60-80 mg / kg dry weight of cultivation material, and most preferably 70 mg / kg dry weight of cultivation material.

[0012] Furthermore, the method also includes harvesting during the mature stage to the opening stage of the shiitake fruiting body.

[0013] Furthermore, the shiitake mushroom is the Shanghai shiitake F6 strain.

[0014] Furthermore, in scheme (b), after adding sodium selenite, the proportion of organic selenium in the fruiting bodies of shiitake mushrooms during the cap-opening period exceeds 90%.

[0015] Furthermore, in scheme (b), after adding sodium selenite, the proportion of organic selenium in the fruiting body of shiitake mushroom exceeds 90%, of which the proportion of methyl selenocysteine ​​is not less than 50%.

[0016] Furthermore, in scheme (b), after adding sodium selenite, the selenium bioavailability of shiitake fruiting bodies in the in vitro simulated gastric digestion stage is not less than 20%.

[0017] Furthermore, the selenium-enriched shiitake mushroom fruiting bodies obtained by any of the above methods can be used to prepare functional foods, health products, or selenium supplements.

[0018] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: (1) A "goal-oriented" differentiated selenium enrichment program for shiitake mushrooms was proposed. This invention provides specific selenium source selection schemes for three different commercial needs: "high yield," "high selenium," and "high absorption," filling a technological gap in the shiitake mushroom industry. Producers can flexibly choose according to market demand, realizing precise and customized production of selenium-enriched shiitake mushrooms.

[0019] (2) “In vitro simulated gastrointestinal fluid bioavailability” is used as the core evaluation index of the effect of selenium enrichment of shiitake mushrooms. The absorption of selenium-enriched shiitake mushrooms is comprehensively evaluated by simulating the absorption of selenium-enriched shiitake mushrooms by human subjects.

[0020] This invention breaks through the limitations of traditional methods that judge quality solely by total selenium content. Experiments show that treatment with 70 mg / kg sodium selenite not only results in extremely high total selenium content (490.53 mg / kg) but also exhibits the highest gastric juice bioavailability (26.4%), achieving a balance between "high content" and "high absorption." This discovery has significant guiding implications for the quality evaluation of selenium-enriched shiitake mushrooms.

[0021] (3) The unique selenium form transformation pattern of shiitake mushrooms was reported. This invention reports that shiitake mushrooms can efficiently convert inorganic selenium into methylselenocysteine ​​(56.2%) and selenomethionine (21.8%) during the cap-opening stage, with organic selenium accounting for over 90%. This discovery provides a theoretical basis and technical support for the production of high-value organic selenium shiitake mushrooms.

[0022] (4) The harvesting period has been optimized. This invention clarifies that the umbrella-opening period is the peak period for selenium accumulation and organic selenium conversion, providing precise harvesting guidance for production practice and ensuring that the selenium content and quality of the product reach the optimal level.

[0023] (5) Ensured product safety This invention, through estimation of daily intake (EDI) and target hazard factor (THQ), confirms that the target hazard factor of all recommended regimens is much less than 1 (THQ < 0.15), and that there is no health risk to adults and children at normal intake levels. Attached Figure Description

[0024] 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.

[0025] Figure 1Colony morphology of shiitake mushrooms under different selenium sources and concentrations; colony morphology of different edible fungi mycelia under different concentrations (1, 5, 10, 15 mg / L) of three selenium sources on PDA plates. CK is the control group without any selenium source; NS: nano selenium; SY: selenium-enriched yeast; SS: sodium selenite.

[0026] Figure 2 Effects of different selenium sources and concentrations on the total selenium content of shiitake mushroom fruiting bodies; CK is the control group without added selenium source; SSa: sodium selenite 15 mg / kg; SSb: sodium selenite 70 mg / kg; NSa: nano selenium 15 mg / kg; NSb: nano selenium 70 mg / kg; SYa: selenium-enriched yeast 15 mg / kg. Values ​​are expressed as mean values, and error bars are expressed as standard errors. Different lowercase letters indicate that the index reached a significant level (LSD) (P < 0.05) in fruiting bodies at different concentrations.

[0027] Figure 3 Differences in selenium speciation distribution at different developmental stages of shiitake mushrooms after the addition of sodium selenite; values ​​are expressed as mean values, and error bars are expressed as standard errors. Different lowercase letters represent that the index reached a significant level (LSD, 0.05) in fruiting bodies at different concentrations; A: primordia stage; B: juvenile stage; C: maturity stage; D: cap opening stage.

[0028] Figure 4 Effect of exogenous selenium treatment on crude polysaccharide content in shiitake mushrooms; CK is the control group without selenium source; SSa: sodium selenite 15 mg / kg; values ​​are expressed as mean, and error bars are expressed as standard errors. Different * represent that the index reached a significant level (LSD, p < 0.05) in fruiting bodies at different concentrations.

[0029] Figure 5 Effect of exogenous selenium treatment on crude protein content of shiitake mushrooms; CK is the control group without selenium source; SSa: sodium selenite 15 mg / kg; values ​​are expressed as mean, and error bars are expressed as standard errors. Different * represent that the index reached a significant level (LSD, p < 0.05) in fruiting bodies at different concentrations.

[0030] Figure 6 Effect of exogenous selenium treatment on crude fat content of shiitake mushrooms; CK is the control group without selenium source; SSa: sodium selenite 15 mg / kg; values ​​are expressed as mean, and error bars are expressed as standard errors. Different * represent that the index reached a significant level (LSD) (p<0.05) in fruiting bodies at different concentrations.

[0031] Figure 7Selenium content of selenium-enriched shiitake mushrooms after gastric digestion by different treatments; CK is the control group without selenium source; SSa: sodium selenite 15mg / kg; SSb: sodium selenite 70mg / kg; NSa: nano selenium 15mg / kg; NSb: nano selenium 70mg / kg; SYa: selenium-enriched yeast 15mg / kg; SYb: selenium-enriched yeast 70mg / kg; SSa1: sodium selenite 10mg / kg; SSb1: sodium selenite 20mg / kg; NSa1: nano selenium 10mg / kg; NSb1: nano selenium 20mg / kg; SYa1: selenium-enriched yeast 10mg / kg; SYb1: selenium-enriched yeast 20mg / kg. Values ​​are expressed as mean, and error bars are expressed as standard errors. Different lowercase letters indicate that the index reached a significant level (LSD, 0.05) at different concentrations of fruiting bodies.

[0032] Figure 8 Differences in selenium content at different developmental stages of shiitake mushrooms (primordia stage, growth stage, maturity stage, and cap opening stage); CK is the control group without added selenium source; SSa: sodium selenite 15 mg / kg; values ​​are expressed as mean values, and error bars are expressed as standard errors. Different * indicate that the index reached a significant level (LSD, 0.05) in fruiting bodies at different concentrations. Detailed Implementation

[0033] 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.

[0034] 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.

[0035] 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.

[0036] I. Experimental Materials and Methods strain: Shiitake mushroom ( Lentinula edodes The strain "Huxiang F6" was bred by the Shanghai Academy of Agricultural Sciences.

[0037] Selenium source: Sodium selenite (Na2SeO3), analytical grade, Sinopharm Chemical Reagent Co., Ltd.; Nano-selenium (Se NPs), Hubei Baitek Bioengineering Co., Ltd. Selenium-enriched yeast (Se-Yeast), selenium content 2000 mg / kg, Shanghai Yuanye Biotechnology Co., Ltd.; Cultivation substrate: Conventional shiitake mushroom cultivation substrate, with a formula of 70% sawdust, 20% wheat bran, 9% corn flour, and 1% calcium carbonate, and the moisture content is adjusted to 60%-65%.

[0038] Main instruments: Atomic fluorescence spectrometer (Jitian AFS-822), high performance liquid chromatography-inductively coupled plasma mass spectrometer (HPLC-ICP-MS), constant temperature incubator (Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.).

[0039] Measurement method: Selenium content determination: Following the national standard GB 5009.93-2017 "Determination of Selenium in Food", atomic fluorescence spectrometry was used. 0.2 g of dried sample was accurately weighed, digested using a nitric acid-perchloric acid mixed acid wet digestion method, diluted to volume, reduced with hydrochloric acid, and then measured using an atomic fluorescence spectrometer.

[0040] Selenium speciation determination: HPLC-ICP-MS was used to detect selenocysteine ​​(SeCys2), methylselenocysteine ​​(MeSeCys), and selenite (SeCys). 4+ ), selenomethionine (SeMet) and selenate (Se 6+ Five forms of selenium.

[0041] In vitro simulated gastrointestinal digestion: The PBET method was used to simulate the gastric and intestinal fluid environment of the human body and determine the bioavailability of selenium.

[0042] Nutritional quality determination: Crude polysaccharides were determined by the phenol-sulfuric acid method; crude protein was determined by the Kjeldahl method; crude fat was determined by Soxhlet extraction; and amino acids were determined by a fully automated amino acid analyzer.

[0043] Data statistics: One-way ANOVA was performed using SPSS 27 software, and multiple comparisons were performed using Duncan's new multiple range method (P<0.05).

[0044] Experimental treatment settings: Mycelial stage treatment (for Example 1), as shown in Table 1: Table 1. Treatment at the mycelial stage Processing Number Exogenous selenium forms Treatment concentration (mg / L) CK No additions 0 SS1-SS5 Sodium selenite 1、5、10、15、20 NS1-NS5 Nano selenium 1、5、10、15、20 SY1-SY5 Selenium-enriched yeast 1、5、10、15、20 Sub-entity stage processing (used in Examples 2-7), as shown in Table 2: Table 2 Sub-entity Stage Processing Processing Number Selenium Forms Addition concentration (mg / kg dry weight of cultivation material) CK No additions 0 SSa Sodium selenite 15 SSb Sodium selenite 70 NSa Nano selenium 15 NSb Nano selenium 70 SYa Selenium-enriched yeast 15 SYb Selenium-enriched yeast 70 The following embodiments are all based on the above-described processing group.

[0045] II. Implementation Examples

[0046] Example 1: Screening Experiment of Selenium Source and Concentration in Shiitake Mushroom Mycelial Stage This embodiment is used to screen suitable selenium sources and concentration ranges for the growth of shiitake mushroom mycelium, providing a basis for subsequent fruiting body cultivation.

[0047] method: Different concentrations (1, 5, 10, 15, 20 mg / L) of sodium selenite, nano-selenium, and selenium-enriched yeast were added to PDA plates, and the *Lentinula edodes* strain F6 was inoculated. The plates were cultured in the dark at 25°C, and the colony diameter was measured periodically to calculate the mycelial growth rate (mm / d).

[0048] result: (1) Observation of colony morphology like Figure 1 As shown, the colony morphology of the shiitake mushroom strain "Hu Xiang F6" exhibited significant differences under different selenium sources and concentrations. The sodium selenite treatment group showed more vigorous mycelial growth and denser colonies; the nano-selenium and selenium-enriched yeast treatment groups showed relatively sparse mycelial growth, but still maintained a normal growth state overall. With increasing selenium concentration, the mycelial density of all treatment groups showed a decreasing trend, but the shiitake mushroom strain demonstrated good tolerance to all three selenium sources.

[0049] (2) Sodium selenite treatment As shown in Table 3, the growth rate of shiitake mushroom mycelium decreased slightly with increasing sodium selenite concentration. The growth rate of the control group (CK) was 2.78 mm / d, while the growth rates of each treatment group ranged from 3.34 to 3.55 mm / d, indicating a relatively small overall inhibition, suggesting that shiitake mushroom mycelium has good tolerance to sodium selenite.

[0050] Table 3. Effects of different concentrations of sodium selenite on the mycelial growth of shiitake mushrooms. deal with 0 mg / L (CK) 1 mg / L (SS1) 5 mg / L (SS2) 10 mg / L (SS3) 15 mg / L (SS4) 20 mg / L (SS5) Growth rate (mm / d) 2.79±0.095c 3.55±0.056a 3.34±0.021b 3.40±0.014ab 3.38±0.033b 3.34±0.067b Note: Different lowercase letters indicate significant differences (P<0.05). The same applies below.

[0051] (3) Nano-selenium treatment As shown in Table 4, under nano-selenium treatment, the mycelial growth rate of shiitake mushrooms decreased at 1 mg / L (2.77 mm / d), rebounded to 3.34 mm / d at 5 mg / L, and remained between 2.75 and 2.94 mm / d at 10-20 mg / L, with a relatively small overall inhibition.

[0052] Table 4. Effects of different concentrations of nano-selenium on the mycelial growth of shiitake mushrooms. deal with 0 mg / L (CK) 1 mg / L (NS1) 5 mg / L (NS2) 10 mg / L (NS3) 15 mg / L (NS4) 20 mg / L (NS5) Growth rate (mm / d) 2.79±0.095bc 2.77±0.05c 3.34±0.03a 2.75±0.04c 2.94±0.07b 2.86±0.05bc (4) Selenium-enriched yeast treatment As shown in Table 5, under selenium-enriched yeast treatment, the mycelial growth rate of shiitake mushrooms was significantly higher than that of the control group at low concentrations (1 mg / L), exhibiting a significant promoting effect. The highest growth rate was observed at 1 mg / L, reaching 3.05 mm / d, which was 9.32% higher than the control group. Within the range of 5-20 mg / L, the growth rate decreased with increasing concentration, ranging from 2.68 to 1.88 mm / d.

[0053] Table 5. Effects of different concentrations of selenium-enriched yeast on the mycelial growth of shiitake mushrooms. deal with 0 mg / L (CK) 1 mg / L (SY1) 5 mg / L (SY2) 10 mg / L (SY3) 15 mg / L (SY4) 20 mg / L (SY5) Growth rate (mm / d) 2.79±0.095b 3.05±0.03a 2.68±0.16b 2.67±0.02b 2.43±0.03c 1.88±0.03d in conclusion: The mycelium of *Lentinula edodes* exhibits a certain degree of tolerance to all three selenium sources. Among them, selenium-enriched yeast showed a significant growth-promoting effect at low concentrations (1-10 mg / L), while nano-selenium and sodium selenite had relatively small inhibitory effects. This screening result provides an experimental basis for the selection of selenium sources in the fruiting body stage of this invention.

[0054] Example 2: Cultivation of high-yield selenium-enriched shiitake mushrooms (yield-priority type) This embodiment provides a selenium-enriched shiitake mushroom cultivation method aimed at high yield.

[0055] step: Strain: The selected strain is "Hu Xiang F6" of shiitake mushroom.

[0056] Cultivation substrate: Prepare the cultivation material according to the formula, adjust the moisture content to 60%-65%, and package it into cultivation bags, with each bag containing about 800 g (dry weight).

[0057] Exogenous selenium addition: During the fruiting body cultivation stage, nano-selenium was added to the cultivation material at a concentration of 15 mg / kg (i.e., 15 mg of nano-selenium was added per kilogram of dry material), corresponding to the NSa treatment group.

[0058] Sterilization and inoculation: Sterilize at 121℃ with autoclave for 2 hours, and then inoculate aseptically after cooling.

[0059] Mycelium growth management: After inoculation, the mycelium packs are placed in a dark incubation room at 25°C for mycelium growth.

[0060] Mushroom management: After the mycelium has filled the bag, color change, primordia induction and fruiting body development management are carried out.

[0061] Harvesting: Harvest during the fruiting body's maturity to the opening stage.

[0062] result: (1) Observation of the growth process This invention further investigated the effects of different selenium sources and concentrations on the growth and development of shiitake mushrooms, and the results are shown in Table 6.

[0063] Table 6. Effects of exogenous selenium addition on the growth process of shiitake mushrooms

[0064] Note: CK is the control group without added selenium source; SSa: sodium selenite 15mg / kg; SSb: sodium selenite 70mg / kg; NSa: nano selenium 15mg / kg; NSb: nano selenium 70mg / kg; SYa: selenium-enriched yeast 15mg / kg; SYb: selenium-enriched yeast 70mg / kg.

[0065] Exogenous selenium generally inhibited the growth of fruiting bodies: During the mycelial growth stage (time to full bag), the time to full bag in the nano-selenium treatment group was the same as that in the control group; the sodium selenite treatment group was 1–2 days later than the control; the selenium-enriched yeast treatment group was delayed by about 3 days compared to the control, but the 70 mg / kg selenium-enriched yeast treatment group was delayed by 11 days compared to the control. During the color change stage, the nano-selenium treatment group was basically the same as the control group; the sodium selenite treatment group was extended by 1–2 days compared to the control; the selenium-enriched yeast treatment group was extended by about 2 days compared to the control, but the 70 mg / kg selenium-enriched yeast treatment group did not change color. During the primordia differentiation and fruiting body formation stages, the nano-selenium treatment group was delayed by 1 day compared to the control; the sodium selenite treatment group was also delayed by 1 day; the 15 mg / kg selenium-enriched yeast treatment group was delayed by 2 days in primordia differentiation and 1 day in fruiting body formation; while the 70 mg / kg selenium-enriched yeast treatment group failed to form primordia and fruiting bodies, completely inhibiting the reproductive growth stage. The above results indicate that nano-selenium has the least impact on the growth process of shiitake mushrooms and is suitable for high-yield cultivation.

[0066] (2) Fruiting body morphology and yield As shown in Table 7, the agronomic traits of the shiitake fruiting bodies in Example 2 (NSa treatment) of the present invention were significantly better than those of the control group (CK).

[0067] Table 7 Effects of exogenous selenium addition on fruiting body morphology and yield of shiitake mushrooms

[0068] Note: Each value represents the mean ± standard deviation (n=3). Different lowercase letters indicate significant differences (p<0.05). CK is the control group without selenium source; SSa: sodium selenite 15mg / kg; SSb: sodium selenite 70mg / kg; NSa: nano selenium 15mg / kg; NSb: nano selenium 70mg / kg; SYa: selenium-enriched yeast 15mg / kg.

[0069] Different selenium sources and concentrations significantly affected the morphology and yield of shiitake mushroom fruiting bodies, generally exhibiting a pattern of "low concentration promoting growth, high concentration inhibiting growth." Regarding fruiting body morphology, the control group (CK) had the largest cap diameter at 65.91 mm; all selenium source treatments reduced cap diameter to varying degrees. Sodium selenite showed the most significant inhibitory effect, with the cap diameter plummeting to 48.23 mm at a concentration of 15 mg / kg. In contrast, the cap diameters of the nano-selenium (15–70 mg / kg) and selenium-enriched yeast (15 mg / kg) treatments remained largely at the control level, with the nano-selenium (70 mg / kg) group even approaching the control. As for stipe morphology, the sodium selenite (15 mg / kg) treatment increased the stipe diameter to 20.45 mm; the nano-selenium treatment group saw a gradual decrease in stipe diameter with increasing concentration, and the selenium-enriched yeast (15 mg / kg) group also had a relatively smaller stipe diameter; all selenium source treatments shortened the stipe length compared to the control. In terms of yield and biological efficiency, the 15 mg / kg nano-selenium treatment showed the best yield-increasing effect, with single-mushroom fresh weight, single-pack yield, and biological efficiency of 32.91 g, 286.33 g, and 35.79%, respectively, all significantly higher than the control. The 70 mg / kg sodium selenite treatment exhibited the strongest inhibitory effect, with a biological efficiency of only 14.27% and a single-pack yield reduced to 114.15 g. In conclusion, nano-selenium at a concentration of 15 mg / kg is the preferred selenium source for improving the yield and quality of shiitake mushrooms; sodium selenite has a strong inhibitory effect on shiitake mushroom growth, while selenium-enriched yeast has a relatively mild effect.

[0070] Based on the growth process data in Table 6, it can be seen that nano-selenium treatment did not shorten the growth cycle of shiitake mushrooms. Its yield-increasing effect mainly stems from the higher material conversion efficiency during the fruiting body development process, which is manifested in a significant increase in the fresh weight of a single mushroom and the yield per package.

[0071] Example 3: Cultivation of selenium-enriched shiitake mushrooms (selenium content priority type) This embodiment provides a selenium-enriched shiitake mushroom cultivation method with a focus on high selenium content.

[0072] step: Strain: Shiitake mushroom strain "Hu Xiang F6".

[0073] Cultivation substrate: Same as in Example 2.

[0074] Exogenous selenium addition: Sodium selenite was added to the cultivation substrate during the fruiting body cultivation stage at a concentration of 70 mg / kg, corresponding to the SSb treatment group.

[0075] The remaining steps are the same as in Example 2.

[0076] result: (1) Total selenium content To investigate the effects of different selenium sources and concentrations on the selenium enrichment effect of shiitake mushroom fruiting bodies, this experiment measured the total selenium content of the control group (CK) and fruiting bodies treated with sodium selenite, nano-selenium, and selenium-enriched yeast at different addition levels. The results are as follows: Figure 2 As shown in the figure, the selenium content of the fruiting bodies in all exogenous selenium treatment groups was significantly higher than that in the control group, indicating that the addition of exogenous selenium sources to the matrix can effectively increase the total selenium content in the fruiting bodies of shiitake mushrooms. The selenium content in the fruiting bodies of shiitake mushrooms treated with exogenous selenium at a concentration of 15 mg / kg ranged from 199.50 to 222.02 mg / kg. Among them, the selenium-enriched yeast treatment group had the highest selenium content (222.02 mg / kg), followed by nano-selenium (208.23 mg / kg), and the sodium selenite treatment group had the lowest (199.50 mg / kg). At a concentration of 70 mg / kg, the selenium content of the fruiting bodies in the sodium selenite treatment group reached as high as 490.53 mg / kg, which was significantly higher than that in the nano-selenium treatment group (278.58 mg / kg). The former was 1.76 times that of the latter, indicating that under high concentration conditions, the enrichment rate of sodium selenite was significantly better than that of nano-selenium. From the perspective of concentration effects, as the concentration of exogenous selenium increased from 15 mg / kg to 70 mg / kg, the selenium content in the fruiting bodies of the sodium selenite-treated group increased from 199.50 mg / kg to 490.53 mg / kg, an increase of 145.9%; while in the nano-selenium-treated group, it increased from 208.23 mg / kg to 278.58 mg / kg, an increase of 33.8%. The increase in the sodium selenite-treated group was much higher than that of the nano-selenium group, indicating that shiitake mushrooms are more sensitive to the concentration of sodium selenite, and high concentrations of sodium selenite can significantly promote the accumulation of selenium in the fruiting bodies of shiitake mushrooms.

[0077] (2) Distribution of selenium speciation like Figure 3 As shown, after the addition of sodium selenite, the proportion of organic selenium in the fruiting bodies of shiitake mushrooms exceeded 90% at each developmental stage. During the cap-opening stage, the proportion of methyl selenocysteine ​​(MeSeCys) reached 56.2%, the proportion of selenomethionine (SeMet) reached 21.8%, and the proportion of inorganic selenium was extremely low.

[0078] Summary of Results: Total selenium content of fruiting bodies: 490.53 mg / kg (more than 63 times that of the control group). Organic selenium content: >90% Methylselenocysteine ​​percentage: 56.2% Selenomethionine content: 21.8% The above results indicate that treatment with 70 mg / kg sodium selenite can yield shiitake mushroom fruiting bodies with extremely high selenium content, and the selenium mainly exists in the form of highly active organic selenium, making it suitable for the production of high-selenium functional food ingredients.

[0079] (3) Nutritional quality like Figure 4 , Figure 5 and Figure 6 As shown, exogenous selenium supplementation has a significant effect on improving the nutritional quality of shiitake mushroom fruiting bodies: Crude polysaccharides: The crude polysaccharide content in the SSa-treated group (sodium selenite 15 mg / kg) was 41.93 mg / g, which was 18.7% higher than that in the control group (35.32 mg / g). Crude protein: The crude protein content of the SSa treatment group was 253.63 g / kg, which was 4.1% higher than that of the control group (243.73 g / kg); Crude fat: The crude fat content of the SSa treatment group was 2.59%, which was 12.1% higher than that of the control group (2.31%).

[0080] The above results indicate that appropriate exogenous selenium addition helps promote the accumulation of basic nutrients in shiitake fruiting bodies.

[0081] (4) Amino acid content The present invention further determined the effect of exogenous selenium addition on the amino acid content of shiitake fruiting bodies, and the results are shown in Table 8.

[0082] Table 8. Amino acid content (mg / kg) of shiitake mushroom fruiting bodies after exogenous selenium addition. Types of amino acids CK SSa Types of amino acids CK SSa Aspartic acid Asp 318.46 1292.47 Valine 1119.30 3607.55 Glutamic acid (Glu) 1867.96 3329.63 Methionine (Met) 2126.92 1937.19 Asparagine Asn 510.71 1558.96 Phenylalanine (Phe) 584.19 3192.83 Serine 298.83 1191.51 Tryptophan Trp 577.00 1102.53 Threonine Thr 218.59 377.07 Isoleucine Ile 106.33 86.43 Arginine (Arg) 1325.04 3613.49 Leucine Leu 461.88 3019.14 Alanine (Ala) 1653.44 5329.87 Lysine 1650.64 4152.36 Tyrosine Tyr 88.03 157.98 Proline 883.48 2590.15 Total amino acid TAA 13790.79 36539.16 Note: CK is the control group without added selenium source; SSa: sodium selenite 15mg / kg.

[0083] Compared with the control group, the total amino acid content of shiitake mushrooms treated with 15 mg / kg sodium selenite (SSa) increased significantly from 13790.79 mg / kg to 36539.16 mg / kg, an increase of 165.0%. Among them, the increase of 7 amino acids, including alanine, aspartic acid, and serine, all exceeded 200%, and the total amount of umami amino acids (aspartic acid + glutamic acid) increased by 111.4%, with only methionine and isoleucine slightly decreasing.

[0084] The above results indicate that appropriate exogenous selenium addition can significantly increase the total amino acid and umami amino acid content of shiitake mushroom fruiting bodies, further enhancing the nutritional value and flavor quality of shiitake mushrooms.

[0085] Example 4: Cultivation of high-absorption-rate selenium-enriched shiitake mushrooms (absorption-priority type) This embodiment provides a selenium-enriched shiitake mushroom cultivation method aimed at achieving high human absorption rates.

[0086] step: Strain: Shiitake mushroom strain "Hu Xiang F6".

[0087] Cultivation substrate: Same as in Example 2.

[0088] Exogenous selenium addition: Sodium selenite was added to the cultivation substrate during the fruiting body cultivation stage at a concentration of 70 mg / kg, corresponding to the SSb treatment group.

[0089] Harvesting time: The key is to harvest during the umbrella opening stage.

[0090] The remaining steps are the same as in Example 2.

[0091] result: (1) Bioavailability of selenium in gastric juice This study used selenium-enriched shiitake mushroom fruiting bodies obtained from previous experiments as materials and employed an in vitro simulated gastric digestion model (PBET method) to determine the selenium concentration in the digestive fluid of samples under different exogenous selenium treatments during the gastric digestion stage. The bioavailability of selenium during the gastric digestion stage was calculated in conjunction with the total selenium content of the fruiting bodies to compare the effects of different treatments on the release efficiency of selenium in the stomach.

[0092] like Figure 7 As shown in Table 9, the bioavailability of selenium in gastric juice varied considerably among the different treatment groups of *Lentinula edodes*, ranging from 4.4% to 26.4%. The sodium selenite treatment group exhibited a significant concentration effect: bioavailability was 4.4% at a low concentration (15 mg / kg) and surged to 26.4% at a high concentration (70 mg / kg), an increase of five times, indicating that high-concentration inorganic selenium treatment can significantly improve the gastric juice release efficiency of selenium from *Lentinula edodes* fruiting bodies. The bioavailability of the nano-selenium treatment group decreased slightly from 9.9% to 9.4% with increasing concentration, a change that was not significant. The bioavailability of the selenium-enriched yeast treatment group (15 mg / kg) was 10.1%, the same as the control group (10.1%). In terms of selenium source type, the high-concentration sodium selenite treatment group had significantly better bioavailability (26.4%) than other treatment groups, while the low-concentration sodium selenite treatment group had the lowest bioavailability (4.4%), suggesting that the gastric bioavailability of inorganic selenium sources in shiitake mushrooms is significantly concentration-dependent.

[0093] Table 9. Bioavailability of selenium-enriched shiitake mushrooms after gastric digestion.

[0094] Note: CK is the control group without added selenium source; SSa: sodium selenite 15mg / kg; SSb: sodium selenite 70mg / kg; NSa: nano selenium 15mg / kg; NSb: nano selenium 70mg / kg; SYa: selenium-enriched yeast 15mg / kg.

[0095] (2) Health risk assessment To assess the potential health risks of consuming the selenium-enriched shiitake mushrooms studied in this study, two indicators were used for analysis: estimated daily intake (EDI) and target hazard coefficient (THQ). The results are shown in Table 10.

[0096] The EDI and THQ of each treatment group of shiitake mushroom are as follows: the EDI range for adults was 0.45-0.73 μg / (kg·d), and for children it was 0.57-0.92 μg / (kg·d); the THQ range for adults was 0.07-0.11, and for children it was 0.09-0.14. Among them, the 70 mg / kg (NSb) nano-selenium treatment group had the highest EDI (0.73 μg / kg·d for adults and 0.92 μg / kg·d for children), and the corresponding highest THQ (0.11 for adults and 0.14 for children). The EDI of the 15 mg / kg (SSa) sodium selenite and 15 mg / kg (NSa) nano-selenium treatment groups were similar (approximately 0.48 μg / kg·d for adults and approximately 0.61 μg / kg·d for children), and the THQ for both groups was 0.07 for adults and 0.09 for children.

[0097] The THQ of each treatment group was far below 1, indicating that the selenium-enriched treatments of the present invention pose no health risk to adults and children under normal intake levels.

[0098] Table 10 Estimated daily intake and target hazard factor of selenium in selenium-enriched shiitake mushrooms

[0099] Note: CK is the control group without added selenium source; SSa: sodium selenite 15mg / kg; SSb: sodium selenite 70mg / kg; NSa: nano selenium 15mg / kg; NSb: nano selenium 70mg / kg; SYa: selenium-enriched yeast 15mg / kg.

[0100] Summary of Results: Selenium bioavailability in gastric juice: 26.4% (2.6 times that of the control group). Total selenium content: 490.53 mg / kg Organic selenium content: >90% Target Hazard Q: Adults 0.07, Children 0.09 (far less than 1, safe) The above results indicate that treating the mushrooms with 70 mg / kg of sodium selenite and harvesting them during the cap-opening stage can produce selenium-enriched shiitake mushrooms with high absorption rate, high safety, and high selenium content, making them suitable for the production of high-end health foods.

[0101] Example 5: Validation Experiment on Optimization of Harvesting Timing This embodiment verifies the effect of harvesting timing on the selenium content and selenium speciation of shiitake mushroom fruiting bodies.

[0102] method: The selenium content and selenium speciation of shiitake mushroom fruiting bodies were determined at the primordium stage, growth stage, maturity stage, and cap opening stage. The treatment group received sodium selenite 15 mg / kg (SSa), while the control group received no selenium supplementation (CK).

[0103] result: (1) Selenium content at different developmental stages To investigate the effect of exogenous selenium supplementation on selenium accumulation in fruiting bodies of *Lentinula edodes* at different growth and development stages, this experiment measured the selenium content in fruiting bodies of the control group (CK) and the 15 mg / kg sodium selenite treatment group at the primordia stage, growth stage, maturity stage, and cap-opening stage. The results were statistically analyzed as follows: Figure 8 As shown, the selenium content of the fruiting bodies in the 15 mg / kg sodium selenite treatment group was significantly higher than that in the control group at all stages, indicating that the exogenous addition of 15 mg / kg sodium selenite can effectively increase the selenium accumulation in the fruiting bodies of shiitake mushrooms at different developmental stages. From the perspective of growth and development stages, the selenium content of the fruiting bodies in the control group gradually increased with the developmental process, reaching its lowest level (1.65 mg / kg) during the primordia stage and its highest level (7.73 mg / kg) during the cap-opening stage. This indicates that under natural growth conditions, the absorption of selenium from the culture medium by the fruiting bodies of shiitake mushrooms continuously increases as the fruiting bodies mature. The selenium content of the fruiting bodies in the treatment group also increased significantly with the developmental process, rising from 6.11 mg / kg during the primordia stage to 90.61 mg / kg during the cap-opening stage, an increase of 13.8 times. The largest increase was observed from the basal stage to the growth stage, rising from 6.11 mg / kg to 56.81 mg / kg, an 8.3-fold increase. The growth rate then gradually slowed, indicating that selenium accumulation was highest during the rapid growth stages of the fruiting body (growth and maturity stages), and accumulation tended to saturate after the cap-opening stage. In conclusion, sodium selenite treatment can significantly increase the selenium content of shiitake mushroom fruiting bodies at all developmental stages, and the selenium accumulation capacity continues to increase as the fruiting body matures, reaching its peak enrichment at the cap-opening stage. Harvesting during the cap-opening stage is recommended for optimal selenium enrichment in production.

[0104] (2) Selenium forms at different developmental stages To further investigate the effects of exogenous addition of 15 mg / kg sodium selenite on the distribution of selenium speciation in fruiting bodies at different growth and development stages of shiitake mushrooms, this experiment measured the contents of five selenium speciations in the control group (CK) and the treatment group (SSa) during the primordia stage, growth stage, maturity stage, and cap-opening stage. The results are as follows: Figure 3 As shown in (AD).

[0105] Organic selenium dominated in both fruiting bodies (accounting for over 86% at all stages), but the sodium selenite treatment group, while maintaining a high proportion of organic selenium, significantly increased the proportion of specific organic selenium forms. The control group was dominated by organic selenium, with inorganic selenium consistently below 1.5%; while in the treatment group, although the proportion of inorganic selenium peaked at maturity, the proportion of organic selenium remained stable between 90.2% and 92.9%.

[0106] Compared with the control group, the sodium selenite treatment group showed a significant increase in the proportion of methylselenocysteine ​​during the umbrella opening stage, rising from approximately 4.8% in the control group to 56.2%; the proportion of selenomethionine during the maturation stage increased sharply from undetectable in the control group to 21.8%, becoming one of the main selenium forms.

[0107] Overall, exogenous selenium treatment maintained the total organic selenium content in shiitake fruiting bodies at over 90%, and significantly increased the proportion of highly efficient organic selenium forms such as methylselenocysteine ​​and selenomethionine. In particular, the enrichment effect of organic selenium was significantly improved in the middle and late stages of growth.

[0108] in conclusion: The opening stage is the peak period for selenium accumulation and organic selenium conversion. At this time, the selenium content of the fruiting body is the highest (90.61 mg / kg), the proportion of organic selenium is the highest (>90%), and the proportion of highly active organic selenium forms (methylselenocysteine, selenomethionine) reaches its maximum. Therefore, the opening stage is the optimal harvesting period to obtain the best selenium content and quality.

[0109] Example 6: Verification Experiment at Different Concentration Ranges This embodiment verifies the rationality of the concentration range in the claims of this invention.

[0110] method: Set up nano-selenium concentration gradients (1, 5, 10, 15, 20, 50, 70, 100 mg / kg) and sodium selenite concentration gradients (10, 15, 30, 50, 70, 100, 150, 200 mg / kg), and cultivate shiitake mushrooms according to the methods in Examples 2-3, and determine the yield and selenium content.

[0111] result: (1) Nano-selenium concentration gradient Table 11 Nano-selenium concentration gradient Concentration range (mg / kg) Effect 1-10 Shiitake mushroom yield increases with increasing concentration 11-20 Shiitake mushroom production reaches peak (biological efficiency 33%-36%). 21-100 Shiitake mushroom yield gradually decreased, and at high concentrations, it was lower than that of the control group. >100 Significant growth inhibition was observed; not recommended. (2) Sodium selenite concentration gradient Table 12 Sodium selenite concentration gradient Concentration range (mg / kg) Effect 10-50 The selenium content of shiitake mushrooms increases with increasing concentration. 51-100 The selenium content of shiitake mushrooms increased rapidly, reaching 490.53 mg / kg at 70 mg / kg. 101-200 Selenium content continued to increase, but the rate of increase slowed down, and some growth inhibition also occurred. Biological availability The optimal concentration is within the range of 60-80 mg / kg (20%-26.4%). (3) Selenium-enriched yeast concentration gradient Table 13 Selenium-enriched yeast concentration gradient Concentration range (mg / kg) Effect 1-10 The selenium content of shiitake mushrooms increases with increasing concentration, with higher selenium content at lower concentrations. 11-20 The selenium content reached its peak (approximately 222 mg / kg), but the yield began to decline. 21-70 Selenium content has stabilized, while yield has decreased significantly. 71-100 Significant growth inhibition was observed; no color change or fruiting occurred at 70 mg / kg. Biological availability The concentration was 10.1% at 15 mg / kg, lower than that of sodium selenite at 70 mg / kg. in conclusion: The concentration ranges specified in the claims of this invention (nano selenium 1-100 mg / kg, sodium selenite 10-200 mg / kg) are reasonable, and the preferred ranges (nano selenium 10-20 mg / kg, sodium selenite 60-80 mg / kg) are the optimal technical solutions. Selenium-enriched yeast severely inhibits the growth of shiitake mushrooms at high concentrations (≥70 mg / kg) and is not suitable for the production of shiitake fruiting bodies.

[0112] Example 7: Comparative Screening Experiment of Different Selenium Sources This embodiment compares the effects of three selenium sources (nano-selenium, sodium selenite, and selenium-enriched yeast) at the same concentration (15 mg / kg) on ​​the yield and selenium content of shiitake mushrooms, verifying the technical basis of the "target-oriented selection of selenium sources" strategy of this invention.

[0113] method: Under the same cultivation conditions, 15 mg / kg of nano selenium (NSa), sodium selenite (SSa), and selenium-enriched yeast (SYa) were added respectively, and the yield and selenium content of shiitake fruiting bodies were determined.

[0114] result: Table 14 Effects of different selenium sources (15 mg / kg) on ​​the yield and selenium content of the first flush of shiitake mushrooms deal with Yield per package (g) Biological efficiency (%) Total selenium content (mg / kg) CK (control) 173.32 21.67 7.73 NSa (Nano Selenium) 286.33 35.79 208.23 SSa (Sodium selenite) 219.97 27.50 199.50 SYa (Selenium-enriched yeast) 162.99 20.37 222.02 in conclusion: Nano-selenium: It is most effective in promoting yield (biological efficiency 35.79%) and is suitable for "yield-first" needs.

[0115] Sodium selenite: Its effect on increasing selenium content is concentration-dependent; at high concentrations (70 mg / kg), the selenium content can reach 490.53 mg / kg, and the absorption rate is the highest (26.4%), making it suitable for applications prioritizing either selenium content or absorption rate. Selenium-enriched yeast: It has a relatively high selenium content at low concentrations (222.02 mg / kg), but its effect on increasing yield is limited.

[0116] The above results fully verify the technical rationality of the present invention's strategy of "selecting different selenium sources according to target requirements".

[0117] 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 goal-oriented method for the targeted cultivation of selenium-enriched shiitake mushrooms, characterized in that, Includes the following steps: In shiitake mushrooms ( Lentinula edodes During the fruiting body cultivation stage, exogenous selenium is added to the cultivation substrate; The exogenous selenium is selected from nano-selenium or sodium selenite; Choose different selenium sources based on the target selenium enrichment requirements: (a) When the goal is to increase the yield of shiitake mushroom fruiting bodies, nano-selenium is selected; (b) Sodium selenite is chosen when the goal is to increase the total selenium content of shiitake fruiting bodies or the bioavailability of selenium.

2. The method according to claim 1, characterized in that, In scheme (a), the concentration of added nano-selenium is 1-100 mg / kg dry weight of cultivation material.

3. The method according to claim 2, characterized in that, The concentration of the added nano-selenium is 10-20 mg / kg dry weight of the cultivation substrate.

4. The method according to claim 1, characterized in that, In scheme (b), the concentration of sodium selenite added is 10-200 mg / kg dry weight of cultivation material.

5. The method according to claim 4, characterized in that, The concentration of sodium selenite added is 60-80 mg / kg dry weight of the cultivation substrate.

6. The method according to claim 1, characterized in that, This also includes harvesting during the mature stage to the opening stage of the shiitake mushroom fruiting body.

7. The method according to claim 1, characterized in that, The shiitake mushroom in question is of the Huxiang F6 strain.

8. The method according to claim 4, characterized in that, In scheme (b), after adding sodium selenite, the proportion of organic selenium in the fruiting bodies of shiitake mushrooms during the cap-opening period exceeds 90%.

9. The method according to claim 4, characterized in that, In scheme (b), after adding sodium selenite, the proportion of organic selenium in the fruiting body of shiitake mushroom exceeds 90%, of which the proportion of methyl selenocysteine ​​is not less than 50%.

10. The use of selenium-enriched shiitake mushroom fruiting bodies obtained by the method according to any one of claims 1 to 9 in the preparation of functional foods, health products or selenium supplements.

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