Method for relieving cadmium stress of ganoderma lucidum mycelia

Abstract

The invention discloses a method for relieving cadmium stress of ganoderma lucidum mycelia, which comprises the following specific steps: inoculating a ganoderma lucidum strain into a basic solid culture medium, and culturing the ganoderma lucidum strain at room temperature; melatonin (MT) is added into the culture medium, so that the concentration of the Melatonin in the basic solid culture medium is at least 100 [mu] mol / L or above, and cadmium stress caused by Cd < 2 + > in the culture medium to ganoderma lucidum mycelia can be relieved. The method shows that the melatonin can relieve Cd stress through a multi-way synergistic effect (hypha growth speed, accumulation of metabolites and an anti-oxidation system), a safe and environment-friendly solution is provided for efficient cultivation of ganoderma lucidum in the cadmium-polluted environment, and meanwhile, a theoretical basis is provided for application of the melatonin in the field of stress resistance of other edible and medicinal fungi.

Description

Technical Field

[0001] The invention relates to the technical field of edible fungus cultivation, and in particular to a method for alleviating cadmium stress on Ganoderma lucidum mycelium. Background Art

[0002] Lingzhi (Ganoderma lucidum) is an important large fungus used for both medicine and food, with a history spanning over 2,000 years. As early as the Shennong's Herbal Classic, it's mentioned as sweet and warm in nature. It can help with hearing loss, improve joint function, nourish the spirit and improve essence, strengthen bones and tendons, and improve complexion. Long-term consumption can also promote weight loss and longevity. Due to its health-promoting and longevity-promoting properties, it's known as the "Ganoderma" or "Immortal Grass," and possesses significant economic and social value.

[0003] Cd is a highly toxic element with a high transfer rate from soil to plants. Compared to plants, edible fungi are more capable of accumulating Cd, resulting in higher concentrations of the heavy metal. With the rapid rise and development of industry and agriculture in my country, Cd pollution has become increasingly severe in recent years, becoming the primary heavy metal pollutant. The main sources of Cd are the development of the mining industry, the expansion of the electroplating industry, the application of fertilizers and pesticides, and the discharge of industrial and domestic wastewater. Ganoderma lucidum has a strong Cd accumulation capacity. Cd accumulates in Ganoderma lucidum and enters the human body through the food chain. Once in the body, it can damage tissues and organs and is difficult to excrete. In severe cases, it can even cause cell mutations and cancer, thus affecting human health.

[0004] Existing research on cadmium stress in Ganoderma lucidum focuses on hydrogen fumigation or the mechanisms by which Cd damages Ganoderma lucidum cells. However, this hydrogen fumigation method is not only difficult to operate and costly, but also difficult to promote and apply. Furthermore, existing technologies for treating cadmium stress in Ganoderma lucidum can only address low cadmium concentrations, and lack a better treatment method for environments with higher cadmium concentrations. Summary of the Invention

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a method for alleviating cadmium stress in Ganoderma lucidum mycelium, so as to solve the problems in the prior art of treating Ganoderma lucidum cadmium stress, such as high difficulty in operation, high cost, difficulty in practical promotion, and lack of treatment methods in environments with high cadmium concentrations.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A method for alleviating cadmium stress on Ganoderma lucidum mycelium, comprising the following steps:

[0008] inoculating the Ganoderma lucidum strain into a solid culture medium and culturing it at room temperature;

[0009] Adding melatonin to the solid culture medium to make the concentration of melatonin in the culture medium at least 100 μmol / L can alleviate the Cd 2+ Cadmium stress on Ganoderma lucidum mycelium.

[0010] Preferably, the solid culture medium is a mixture of peeled potato extract, sucrose, potassium dihydrogen phosphate, magnesium sulfate, agar powder and water; wherein the peeled potato extract is 200 g / L, the sucrose is 20 g / L, the potassium dihydrogen phosphate is 1.5 g / L, the magnesium sulfate is 0.5 g / L, and the agar powder is 20 g / L.

[0011] Preferably, the concentration of melatonin in the basic solid culture medium is 100-800 μmol / L.

[0012] Preferably, the Cd 2+ The concentration is 600μmol / L.

[0013] Preferably, the Ganoderma lucidum strain is Ganoderma lucidum Du585 or GSD strain.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 1. The present invention found that in an environment with relatively high cadmium concentration, the introduction of melatonin can promote the 2+ Ganoderma lucidum mycelial growth and metabolite accumulation under stress, melatonin increases Cd 2+ The growth rate of Ganoderma lucidum mycelium, the content of polysaccharides, triterpenes and soluble proteins in mycelium under Cd stress effectively alleviated 2+ Toxicity; the polysaccharide and triterpenoid contents of the Cd+MT treatment group were significantly higher than those of the Cd stress treatment. This may be related to the fact that melatonin, an emerging plant hormone, reduces the degree of oxidative damage in plants by regulating the substance levels and enzyme activities of the antioxidant system, thereby increasing the metabolites in the Ganoderma mycelium. The present invention found that by adding a certain amount of melatonin, not only can the damage to the mycelium be slowed down, but the polysaccharide and triterpenoid contents of the mycelium can also be significantly increased to approach the normal level without stress. In addition, Cd+MT treatment can increase the soluble protein content of Du585 and GSD Ganoderma mycelium under Cd stress, indicating that MT can increase the content of metabolites in Ganoderma mycelium under Cd stress, so as to resist the damage to Ganoderma mycelium caused by cadmium stress and play a mitigating role.

[0016] 2, The application also finds that applying melatonin can effectively improve the ROS scavenging capacity of the Ganoderma lucidum antioxidant system. When the Ganoderma lucidum mycelium is subjected to stress, applying melatonin can significantly improve the proline and soluble protein content of the mycelium of the strain under Cd stress, indicating that melatonin can remove active oxygen by accumulating osmotic adjustment substances such as proline and soluble protein (particularly for Cd-resistant strains), directly remove free radicals and regulate the enzymatic antioxidant system to resist the damage caused by stress to the Ganoderma lucidum mycelium. The application also finds that the introduction of melatonin can trigger the activation of antioxidant enzyme systems such as APX, CAT and POD in the Ganoderma lucidum mycelium, and improve antioxidant enzyme activity to alleviate Cd toxicity to the Ganoderma lucidum mycelium. At the same time, the application also finds that the addition of melatonin can improve the content of the antioxidant molecule GSH under Cd stress, and alleviate the Cd toxicity to the Ganoderma lucidum mycelium. In addition, the content of malondialdehyde (MDA) can reflect the degree of cell damage to a certain extent, and in the application, melatonin can significantly reduce the content of MDA in the mycelium of the Ganoderma lucidum strains Du585 and GSD, indicating that the addition of MT in the Ganoderma lucidum culture medium under Cd stress can remove ROS through a comprehensive approach of antioxidant enzyme systems, antioxidant molecules and osmotic adjustment substances to alleviate Cd toxicity to Ganoderma lucidum.

[0017] 3, The application selects 100 μmol / L as the optimal melatonin concentration by introducing melatonin in the Ganoderma lucidum culture process, which is effective for both Ganoderma lucidum strains (Cd-resistant Du585 and sensitive GSD), indicating that melatonin has broad-spectrum applicability in different genetic backgrounds, and its effect is derived from the regulation of Cd enrichment capacity or the regulation of cell signaling pathways.

[0018] 4, The method described in the application alleviates Cd stress through multiple pathways (metabolic regulation, antioxidant defense and osmotic balance), provides a safe and environmentally friendly solution for efficient cultivation of Ganoderma lucidum in a cadmium-polluted environment, and provides a theoretical basis for the application of melatonin in other medicinal fungus stress resistance fields. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Figure 1 is a graph of the effect of melatonin on the growth rate of Du585 and GSD strains under Cd stress in the application.

[0020] Figure 2 Figure 2 is a graph of the effect of different concentrations of melatonin on the growth of Du585 and GSD under Cd stress in the application.

[0021] Figure 3 Figure 3 is a graph of the growth radius of Du585 and GSD at different culture periods in the application.

[0022] Figure 4 Figure 4 is a graph of the growth of Du585 and GSD at different culture periods in the application.

[0023] Figure 5 This is a graph showing the effect of melatonin MT on the polysaccharide content in the mycelium of cadmium-stressed Du585 and GSD strains in the present invention.

[0024] Figure 6 This is a graph showing the effect of MT on the triterpenoid content in the mycelium of Du585 and GSD strains under cadmium stress.

[0025] Figure 7 This is a diagram showing the effect of MT on the mycelial protein content of cadmium-stressed Du585 and GSD strains in the present invention.

[0026] Figure 8 This is a diagram showing the effect of MT on the Pro content in the mycelium of the cadmium-stressed Du585 and GSD strains in the present invention.

[0027] Figure 9 This is a diagram showing the effect of MT on the MDA content of mycelium of Du585 and GSD strains under cadmium stress in the present invention.

[0028] Figure 10 These are images of the APX, CAT, and POD enzyme activities of MT on the mycelia of Du585 and GSD strains under cadmium stress in the present invention; wherein A represents APX, B represents CAT, and C represents POD. DETAILED DESCRIPTION

[0029] The present invention will be described clearly and completely with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments derived by persons of ordinary skill in the art based on the present invention are within the scope of protection of the present invention.

[0030] Unless otherwise indicated in specific cases, the numerical ranges listed herein include the upper and lower limits, and all integers and fractions within the range, and are not limited to the specific values ​​listed when defining the range.

[0031] 1. A method for alleviating cadmium stress on Ganoderma lucidum mycelium, comprising the following steps:

[0032] inoculating the Ganoderma lucidum strain into a solid culture medium and culturing it at room temperature;

[0033] Adding melatonin to the solid culture medium to make the concentration of melatonin in the solid culture medium at least above 100 μmol / L can alleviate the Cd 2+ Cadmium stress on Ganoderma lucidum mycelium.

[0034] The present invention found that heavy metal Cd is a strong competitive ion for oxygen donors when studying the cadmium stress of Ganoderma lucidum. When Cd accumulates excessively in the body, Cd will inhibit the antioxidant system in the body, thereby generating a large amount of active oxygen, such as superoxide anions (O2 - ), hydrogen peroxide (H2O2), hydroxyl radical (-OH), alkoxyl radical (RO - ), alkyl peroxy (ROO - ), organic hydroperoxides (ROOH), etc., which can cause a certain degree of cell damage, thereby affecting the health of Ganoderma mycelium during growth. To this end, the present invention considers introducing melatonin to alleviate cadmium stress during cultivation. However, the cadmium ion concentration in the existing research on relieving cadmium stress is not high. Therefore, the present invention hopes to introduce melatonin to enable Ganoderma mycelium to resist high concentrations of cadmium stress.

[0035] In some embodiments of the present invention, the basic solid culture medium is a mixture of potato peel extract, sucrose, potassium dihydrogen phosphate, magnesium sulfate and agar powder; wherein the potato peel extract is 200 g / L, the sucrose is 20 g / L, the potassium dihydrogen phosphate is 1.5 g / L, the magnesium sulfate is 0.5 g / L, and the agar powder is 20 g / L.

[0036] In some embodiments of the present invention, the concentration of melatonin in the basic solid culture medium is 100-800 μmol / L.

[0037] In some embodiments of the present invention, the Cd 2+ The concentration does not exceed 600μmol / L.

[0038] In some embodiments of the present invention, the Ganoderma lucidum strain is Ganoderma lucidum Du585 or GSD strain.

[0039] 2. Experimental situation

[0040] 1. Selection of Ganoderma lucidum strains

[0041] In order to verify the broad spectrum of the method of the present invention, the cadmium-resistant Ganoderma lucidum (G.lingzhi) Du585 strain and the cadmium-sensitive Ganoderma lucidum (G.tsugae) GSD strain were used as test strains. The strains were obtained from the Edible Fungus Cultivation Laboratory of Yangtze Normal University.

[0042] 2. Culture medium

[0043] Basic solid culture medium: potato peel extract 200g / L, sucrose 20g / L, potassium dihydrogen phosphate 1.5g / L, magnesium sulfate 0.5g / L, agar powder 20g / L, natural pH.

[0044] 3. Cultivation methods

[0045] The Ganoderma lucidum strains were inoculated into a solid culture medium and cultured in an incubator at 25°C.

[0046] 4. Effect of melatonin concentration on alleviating cadmium stress in Ganoderma lucidum mycelium

[0047] Cd in the culture medium 2+ The concentration was set to 600 μmol / L, and the melatonin (MT) concentration was set to 9 levels of 0, 100, 200, 300, 400, 500, 600, 700, and 800 μmol / L. 2+ Analytically pure CdCl2 and MT were added, respectively, using analytically pure CdCl2 and MT speciation. The required CdCl2 and MT stock solution volumes were calculated according to the formula C1V1=C2V2. The inoculated strains were cultured in a 25°C incubator with six replicates. Mycelial growth rate was used as the measurement metric to identify the optimal concentration of exogenous melatonin for alleviating cadmium stress in Ganoderma lucidum mycelia. The average daily mycelial growth rate (cm / d) was calculated as colony radius (cm) / number of days mycelial growth (d). A blank culture without Cd and MT was used as the control (CK).

[0048] Depend on Figure 1 It can be seen that in the absence of Cd stress (CK group), the growth rate of both strains was fast. When Cd stress was added, the growth rate of the two strains decreased significantly, indicating that Cd had a significant inhibitory effect on the growth of the strains. Compared with the CK treatment group, Cd stress significantly reduced the mycelial growth rate, among which the growth rate of the cadmium-resistant strain Du585 decreased by 17.3% under Cd stress; the growth rate of the cadmium-sensitive strain GSD decreased by 74.4% under Cd stress. It can be seen that even under the same Cd stress, the growth rate of the two strains decreased significantly. 2+ In an environment with high concentrations of cadmium, the effects of cadmium stress on different Ganoderma lucidum strains are completely different.

[0049] In addition, with the increase of MT concentration, the growth rate of the two strains generally showed a trend of first increasing and then decreasing. When the MT concentration was 100 μmol / L, the growth rate of the two strains was significantly higher than that of the Cd stress group, indicating that at this concentration, MT can alleviate the inhibitory effect of Cd on the mycelial growth of the strains. When melatonin was 100 μmol / L, the mycelial growth rate of the cadmium-sensitive GSD strain was significantly higher than that of the cadmium stress treatment, and the growth rate increased by 64.3%, but it was still significantly lower than that of CK; the mycelial growth rate of the cadmium-resistant strain Du585 was also significantly higher than that of the cadmium stress treatment, and the growth rate increased by 14.1%. Figure 2It can also be seen that the mycelial growth rates of strains Du585 and GSD under cadmium stress differ significantly. The addition of melatonin has a certain mitigating effect on the growth of Ganoderma lucidum mycelia under cadmium stress. Under cadmium stress, the growth rate of strain Du585 is generally higher than that of strain GSD, indicating that strain Du585 is more tolerant to cadmium stress. The mycelial growth rate of strain GSD increases even more significantly after the addition of MT, indicating that strain GSD is more sensitive to MT.

[0050] In summary, melatonin can significantly alleviate the inhibitory effect of Cd on the growth of Ganoderma lucidum strains within a certain concentration range. This indicates that melatonin may have a potential mechanism to alleviate Cd toxicity, providing a new protection strategy for the growth of Ganoderma lucidum strains in Cd-contaminated environments. At the same time, it was also found that the MT concentration at 100 μmol / L had the most significant effect on promoting strain growth. This finding provides a scientific basis for the use of melatonin concentrations in practical applications, avoiding the adverse effects of too low or too high concentrations. There are significant differences in the responses of the two strains to Cd stress and melatonin. The Du585 strain shows stronger tolerance, while the GSD strain responds sensitively. This result provides a new direction for subsequent research and may help to screen Ganoderma lucidum strains that are more suitable for growth in Cd-contaminated environments.

[0051] 5. Analysis of the alleviating effect of melatonin on cadmium stress in Ganoderma lucidum mycelium

[0052] Melatonin alleviates cadmium stress in Ganoderma lucidum mycelium. Two treatments, Cd and Cd+MT, were set up, with blanks without Cd and MT as controls (CK). 2+ The concentration was set at 600 μmol / L, and Cd in Cd+MT treatment 2+ The concentration of Cd and MT was set at 600 μmol / L and 100 μmol / L, respectively. Activated Du585 and GSD strains were inoculated into CK, Cd-treated, and Cd+MT-treated culture media using a 0.5 cm borer and cultured in a 25°C incubator. Mycelia were stripped from the culture media on the 6th, 9th, and 12th day of culture, immediately frozen in liquid nitrogen, and then stored in a -80°C freezer for subsequent analysis.

[0053] Figure 3 is the growth radius of strain Du585 and strain GSD at different culture periods, Figure 4 The growth of Du585 and strain GSD at different culture periods. Figure 4The left side of the figure shows strain Du585, and the right side shows strain GSD. Using these as test materials, starting from the 6th day of culture, samples of Du585 and GSD mycelia were taken every three days to measure their growth rate, metabolites, and antioxidant-related indicators. Since strain GSD was too small to be sampled on the 6th day, there is no relevant data for strain GSD on the 6th day. Figure 3 It can be seen that the growth radius of the cadmium-resistant strain Du585 under Cd+MT treatment was significantly higher than that under Cd treatment except at the 6th day of culture, and had no significant difference with the CK group in the rest of the culture periods; the growth radius of the cadmium-sensitive strain GSD under Cd+MT treatment was significantly higher than that under Cd treatment except at the 6th day of culture, and had no significant difference with the Cd treatment in the rest of the culture periods, and was significantly lower than that under CK.

[0054] from Figure 3 and Figure 4 As can be seen, strains Du585 and GSD both exhibited stable growth trends under the CK treatment, with their growth radius gradually increasing with prolonged culture time. The growth radius of strain Du585 was slightly higher than that of strain GSD at all time points, indicating that Du585 grew faster under normal conditions. Under Cd treatment, the growth radius of both strains was significantly lower than that of the CK treatment, indicating that cadmium stress significantly inhibited mycelial growth. The growth radius of strain Du585 under Cd treatment was slightly higher than that of strain GSD, suggesting that Du585 may be slightly more tolerant to cadmium stress than GSD. The growth radius of strain Du585 under the Cd+MT treatment was significantly higher than that of the Cd treatment at all other culture periods, except at day 6, when there was no significant difference between the two treatments. The growth radius of strain GSD under the Cd+MT treatment was significantly higher than that of the Cd treatment at all other culture periods, except at day 6, when there was no significant difference between the two treatments.

[0055] Under Cd treatment, mycelial growth was significantly inhibited, but after the addition of melatonin, mycelial growth rate recovered significantly, approaching or even exceeding that of the control at certain stages. This suggests that melatonin may mitigate the toxic effects of cadmium on mycelia through various mechanisms, such as antioxidant activity and metabolic regulation. Strain Du585 and strain GSD showed different growth performance under different treatments, with strain Du585 showing a significantly higher growth rate under Cd+MT treatment. This may indicate that different strains have different response mechanisms to melatonin, with strain Du585 exhibiting greater tolerance to cadmium. The growth inhibition under Cd treatment did not significantly increase with prolonged incubation time. Instead, the growth radius under Cd+MT treatment gradually approached that of the control. This suggests that melatonin not only alleviates short-term cadmium stress but may also have a sustained mitigating effect on long-term cadmium stress.

[0056] 6. Analysis of polysaccharide content in mycelium at different culture periods

[0057] Ganoderma lucidum polysaccharides were extracted by water immersion method, and the polysaccharide content was determined by phenol-sulfuric acid method. 0.1 g of mycelium was accurately weighed using an electronic balance and placed in a 10 mL centrifuge tube. 3 mL of distilled water was added and extracted in a 95°C water bath for 1 hour. The sample was then centrifuged at 5500 r / min for 10 minutes. The supernatant was collected and the residue was extracted twice according to the above method. Finally, the supernatants of the three extractions were combined and the volume was adjusted to 10 mL with ultrapure water to obtain the total extract. 1 mL of the water extract was taken and placed in a 10 mL centrifuge tube. 4 times the volume of 95% ethanol was added and the sample was allowed to stand at 4°C for 24 hours. The sample was then centrifuged at 5000 r / min for 15 minutes and the supernatant was discarded. The precipitate was dissolved in ultrapure water and the volume was adjusted to 10 mL to obtain the mycelial polysaccharide sample solution. Three replicates were set for each sample solution. The absorbance at 490 nm was measured using a spectrophotometer and the average value was taken. The polysaccharide content of the sample solution was calculated according to the linear regression equation obtained from the standard curve:

[0058]

[0059] Where: C is the polysaccharide concentration calculated according to the standard curve; V is the total volume of the water extract; n is the dilution multiple; M is the sample mass.

[0060] Depend on Figure 5 As shown, the polysaccharide content of strain Du585 was higher than that of GSD in all treatments. With prolonged culture time, the polysaccharide content of strains Du585 and GSD increased across treatments, reaching its peak on day 12, with the CK group containing 11.74±0.24 mg / g and 3.16±0.21 mg / g, respectively. The polysaccharide content of strain Du585 under Cd+MT treatment was significantly higher than that under Cd treatment at all other times except day 6, when there was no significant difference between the two treatments. At day 9, there was even no significant difference between the Cd+MT treatment and the CK. For strain GSD, the polysaccharide content under Cd+MT treatment was significantly higher than that under Cd treatment at both days 9 and 12, but lower than that under CK.

[0061] It can be seen that the polysaccharide content increases with the extension of culture time. Whether in the control group (CK), cadmium stress group (Cd), or melatonin treatment group (Cd+MT), the polysaccharide content of Ganoderma mycelium increases with the extension of culture time. This shows that Ganoderma mycelium can continuously accumulate polysaccharides during growth. Under cadmium stress conditions (Cd), the polysaccharide content of Ganoderma mycelium was significantly lower than that of the control group (CK), indicating that cadmium stress has an inhibitory effect on the accumulation of polysaccharides in Ganoderma mycelium. However, after adding melatonin (Cd+MT) under cadmium stress, the polysaccharide content of Ganoderma mycelium was significantly higher than that of the cadmium stress group (Cd) and close to that of the control group (CK), indicating that melatonin can alleviate the inhibitory effect of cadmium stress on the accumulation of polysaccharides in Ganoderma mycelium.Figure 5 Figure 2 shows the changes in polysaccharide content of two strains (Du585 and GSD) under different treatment conditions. It can be seen that the two strains differ in polysaccharide accumulation, especially under cadmium stress and melatonin treatment, which also indicates that the different strains have different responses to cadmium stress and melatonin.

[0062] Melatonin significantly promotes polysaccharide accumulation in Ganoderma mycelium under cadmium stress. Under cadmium stress, the polysaccharide content of Ganoderma mycelium decreased significantly, but after adding melatonin, the polysaccharide content was restored to the level of the control group. This suggests that melatonin has strong potential in alleviating cadmium stress and can promote the growth and polysaccharide accumulation of Ganoderma mycelium through multiple mechanisms (such as antioxidant and metabolic regulation). At the same time, there were significant differences in the responses of different strains to cadmium stress and melatonin treatment, which also suggests that different strains may have different response mechanisms to cadmium stress and melatonin.

[0063] 7. Analysis of triterpenoid content in mycelium at different culture periods

[0064] Triterpenes from Ganoderma lucidum mycelia were extracted using an ultrasonic-assisted alcohol extraction method, and the triterpenoid content was determined using a vanillin-glacial acetic acid-spectrophotometric method. 0.1 g of mycelia were accurately weighed using an electronic balance and placed in a 10 ml centrifuge tube. 1 ml of 70% ethanol solution was added and the tube was ultrasonicated at 50°C for 1 hour using a sonicator at 47% power (225.6 W). The tube was then centrifuged at 5500 rpm for 15 minutes. The supernatant was collected and the precipitate was extracted twice as described above. The supernatants from the three extractions were combined and the volume was adjusted to 5 ml with 70% ethanol to obtain an alcohol extract. 0.1 ml of the extract was transferred to a test tube and placed in a drying oven to evaporate the solvent. After complete drying, 0.2 ml of 5% vanillin-glacial acetic acid solution and 0.5 ml of perchloric acid were added and shaken. The tube was heated in a 60°C water bath for 20 minutes, immediately cooled in an ice bath, and 5 ml of glacial acetic acid was added and shaken. The absorbance at 550 nm was measured using a multifunctional microplate reader. The triterpene content of the sample solution was calculated according to the equation obtained from the standard curve.

[0065]

[0066] In the formula: C is the concentration converted from the standard curve; V1 is the volume of the extract; V2 is the volume used for determination; m is the mass of the sample weighed.

[0067] Depend on Figure 6As shown, under cadmium stress (Cd treatment), the triterpene content of both strains was lower than that of the control group (CK), indicating that cadmium stress inhibits triterpene synthesis. When melatonin was added (Cd+MT treatment), triterpene content increased significantly in the late culture period, approaching the level of the control group, indicating that melatonin can alleviate the inhibitory effect of cadmium stress on triterpene synthesis. Under Cd+MT treatment, the triterpene content of both strains in the late culture period was significantly higher than that of the Cd treatment group, indicating that melatonin not only alleviates cadmium stress but also promotes triterpene accumulation.

[0068] Melatonin significantly increased triterpene content in mycelia under cadmium stress, bringing the triterpene content close to that in the control group, suggesting its potential application in alleviating cadmium stress. Melatonin not only alleviates the inhibitory effects of cadmium stress on triterpene synthesis but also promotes triterpene accumulation through other mechanisms, providing new insights into the mechanisms of action of melatonin.

[0069] 8. Analysis of soluble protein content in mycelium at different culture periods

[0070] Soluble protein content was determined using a soluble protein assay kit (Ruixin). The assay was performed according to the kit instructions. The absorbance was measured at 600 nm using a multifunctional microplate reader, and the soluble protein content of the mycelium was calculated using the formula. Soluble protein is expressed in mg / g.

[0071] Depend on Figure 7 Under cadmium stress (Cd), the protein content of both strains was lower than that of the control group (CK), indicating that cadmium stress inhibits mycelial protein synthesis. After the addition of melatonin (Cd+MT), the protein content increased significantly, approaching or reaching the level of the control group, indicating that melatonin can alleviate the inhibitory effect of cadmium stress on protein synthesis. Under cadmium stress, the addition of melatonin not only restored the protein content, but even showed no significant difference from the control group, suggesting that melatonin may have a role in promoting protein synthesis. The protein content changes in the two strains (Du585 and GSD) under cadmium stress and melatonin treatment were similar, indicating that different strains may have different responses to cadmium stress and melatonin. Melatonin can not only alleviate the inhibitory effect of cadmium stress on protein synthesis, but may also have a direct role in promoting protein synthesis. This finding may provide new ideas for future research on the application of melatonin in other organisms.

[0072] 9. Effects of melatonin on related indicators of antioxidant system in Ganoderma lucidum mycelium under cadmium stress

[0073] Free proline (Pro), malondialdehyde (MDA), catalase (CAT) activity, peroxidase (POD) enzyme activity and ascorbate peroxidase (APX) enzyme activity were measured using a free proline (Pro) content detection kit (Solaibao), a malondialdehyde (MDA) content detection kit (Suzhou Gres), a catalase (CAT) enzyme activity detection kit (Suzhou Gres) and an ascorbate peroxidase (APX) enzyme activity detection kit (Solaibao) according to the instructions, respectively.

[0074] (1) Effect on free proline (Pro) content

[0075] Depend on Figure 8 As shown, with the extension of culture time, the Pro content of strain Du585 in all three treatments showed a dynamic trend of first increasing and then decreasing. The Pro content of strain GSD in the three different treatment groups also showed an upward trend. The Pro content in the mycelium of strain Du585 under Cd+MT treatment was significantly higher than that in the Cd treatment and the CK at all other times, except at 9 days of culture, when there was no significant difference between the Cd treatment and the Cd treatment. At 12 days of culture, there was no significant difference between the Cd+MT treatment and the Cd treatment in strain GSD, but it was significantly higher than that in the Cd treatment and the CK at all other times. Thus, under Cd treatment, the proline content was significantly higher than that in the control group (CK), indicating that cadmium stress leads to proline accumulation in Ganoderma lucidum mycelium, which is a common response of plants and fungi to heavy metal stress. The proline content in the Cd+MT treatment was significantly lower than that in the Cd treatment group, indicating that melatonin can significantly alleviate cadmium stress and reduce proline accumulation in Ganoderma lucidum mycelium. This result was unexpected because, normally, adverse stress leads to a large accumulation of proline, while the addition of melatonin can effectively inhibit this accumulation, showing its strong potential in alleviating cadmium stress. Under Cd+MT treatment, the reduction in proline content may mean that the growth and metabolic activity of Ganoderma mycelium have been restored or promoted. The accumulation of proline is usually associated with growth inhibition and metabolic disorders, while the addition of melatonin reduces the accumulation of proline by regulating the metabolic pathways in the cells, thereby promoting the normal growth and metabolism of Ganoderma mycelium. This technical effect is of great significance for the cultivation and production of Ganoderma lucidum, because in actual production, heavy metal pollution is a common problem. The application of melatonin can effectively alleviate heavy metal stress and improve the yield and quality of Ganoderma lucidum.

[0076] (2) Effect on malondialdehyde (MDA) content

[0077] Figure 9The effect of melatonin (MT) on the content of malondialdehyde (MDA) in Ganoderma lucidum mycelium under cadmium (Cd) stress is demonstrated. MDA is a product of lipid peroxidation, and its content can reflect the degree of cell membrane damage. Cd stress leads to a significant increase in MDA content, and in the Cd-treated group, the MDA content in both strain Du585 and strain GSD mycelium is significantly higher than that in the control group (CK), indicating that Cd stress leads to cell membrane damage and increased lipid peroxidation. In the Cd+MT treatment group with added melatonin, the MDA content is significantly lower than that in the Cd treatment group, even close to the control group (CK). This indicates that melatonin can effectively reduce lipid peroxidation caused by Cd stress and protect the cell membrane from damage. In addition, different strains respond differently to melatonin, with strain Du585 and strain GSD showing different trends in MDA content under Cd+MT treatment. The MDA content of strain Du585 under Cd+MT treatment is close to the control group, while the MDA content of strain GSD has decreased but is still higher than the control group. This indicates that different strains respond differently to melatonin, which may be related to their physiological characteristics and metabolic pathways. The protective effect of melatonin continues to increase with the extension of the culture time: on the 6th, 9th, and 12th days of culture, the MDA content of the Cd+MT treatment group is significantly lower than that of the Cd treatment group, and this difference becomes more pronounced with the extension of the culture time. This indicates that the protective effect of melatonin is time-dependent, and its protective effect becomes more significant with the extension of the culture time.

[0078] In summary, this demonstrates that melatonin has a significant protective effect on Ganoderma lucidum mycelium under cadmium stress, effectively reducing lipid peroxidation and cell membrane damage caused by cadmium stress, providing protection for the growth and metabolism of Ganoderma lucidum mycelium in cadmium-contaminated environments.

[0079] (3) Effect on antioxidant active enzymes

[0080] Figure 10 The effect of melatonin (MT) on the antioxidant enzyme activity of Ganoderma lucidum mycelium (strains Du585 and GSD) under cadmium (Cd) stress is demonstrated. By comparing the APX, CAT, and POD enzyme activities of different treatment groups (CK, Cd, Cd+MT) at different culture days (6, 9, 12 days), the protective effect of melatonin on Ganoderma lucidum mycelium under cadmium stress can be observed.

[0081] 1) APX (ascorbate peroxidase) activity

[0082] APX activity decreased significantly in the Cd-treated group at 6 and 9 days, indicating a significant inhibitory effect of cadmium stress on APX activity. APX activity in the Cd+MT-treated group at 6 and 9 days was significantly higher than that in the Cd-treated group, approaching the level in the CK group, indicating that melatonin significantly alleviates the inhibitory effect of cadmium stress on APX activity. Melatonin not only restored APX activity, but at 9 days, APX activity in the Cd+MT-treated group even exceeded that in the CK group, demonstrating a significant enhancement of APX activity in response to cadmium stress.

[0083] 2) CAT (catalase) activity

[0084] CAT activity decreased significantly in the Cd-treated group at 6 and 9 days, indicating a significant inhibitory effect of cadmium stress on CAT activity. CAT activity in the Cd+MT-treated group at 6 and 9 days was significantly higher than that in the Cd-treated group, approaching or exceeding the levels in the CK group, indicating that melatonin can significantly alleviate the inhibitory effects of cadmium stress on CAT activity. Melatonin not only restored CAT activity, but also, at 9 days, CAT activity in the Cd+MT-treated group was not significantly different from that in the CK group, demonstrating that melatonin significantly enhances CAT activity under cadmium stress.

[0085] 3) POD (peroxidase) activity

[0086] POD activity decreased significantly in the Cd-treated group at 6 and 9 days, indicating a significant inhibitory effect of cadmium stress on POD activity. POD activity in the Cd+MT-treated group was significantly higher at 6 and 9 days than in the Cd-treated group, approaching that of the CK group, indicating that melatonin significantly alleviates the inhibitory effect of cadmium stress on POD activity. Melatonin not only restored POD activity, but also, at 9 days, the POD activity in the Cd+MT-treated group approached that of the CK group, demonstrating a significant enhancement of POD activity under cadmium stress.

[0087] These results indicate that melatonin significantly protects and enhances the antioxidant enzyme activities of Ganoderma mycelium under cadmium stress. At 6 and 9 days, melatonin not only restored the APX, CAT, and POD activities that were suppressed by cadmium stress, but in some cases, their activities were very close to those of the non-cadmium stress control group. This result demonstrates that melatonin has an unexpectedly potent effect on the antioxidant defense system of Ganoderma mycelium under cadmium stress, providing a new strategy for the growth and protection of Ganoderma mycelium in heavy metal-contaminated environments.

[0088] 10. Summary

[0089] Cd stress can affect the growth of edible fungi mycelium and the activity of their metabolites, resulting in poor quality or reduced yield. The present invention screened and determined that the optimal MT concentration for alleviating Cd stress in Ganoderma mycelium strains Du585 and GSD was 100 μmol / L, demonstrating that MT can effectively alleviate Cd stress. Inconsistent MT application concentrations may be due to varying Cd accumulation capacities among different test materials or inconsistent Cd concentrations set during the experiments.

[0090] Ganoderma lucidum polysaccharides and triterpenes are the main active ingredients of Ganoderma lucidum and have different functions. Polysaccharides have immunomodulatory, anti-tumor, and blood sugar-lowering effects, while triterpenes have anti-inflammatory, hepatoprotective, detoxifying, and antioxidant effects. In the present invention, the polysaccharide content of Ganoderma lucidum mycelia of strains Du585 and GSD was significantly reduced under Cd stress, while the addition of MT under Cd stress significantly increased the polysaccharide content of Ganoderma lucidum mycelia of strains Du585 and GSD. This indicates that the MT treatment group helps alleviate the reduction in polysaccharide content in strains Du585 and GSD caused by Cd stress during cultivation, thereby achieving a mitigating effect. Furthermore, Cd treatment also significantly reduced triterpenoid content in the mycelia of Ganoderma lucidum strains Du585 and GSD. Analysis showed that Cd+MT treatment significantly affected triterpenoid content in Du585 in the late culture period, while the effect on GSD was less pronounced. However, triterpenoid content in both Du585 and GSD treated with Cd+MT was significantly higher than that in the Cd treatment. This suggests that MT can alleviate the reduction in triterpenoid content in Du585 and GSD caused by Cd stress during cultivation, thereby alleviating Cd stress. Soluble protein is an important metabolite and osmotic regulator in Ganoderma lucidum. When plants are subjected to adverse stress, soluble protein rapidly accumulates, reducing cellular osmotic potential, increasing water absorption and retention, and protecting against damage caused by stress. The Cd+MT treatment in the present invention can increase the soluble protein content of the mycelia of strain Du585 and strain GSD under Cd stress, indicating that MT can increase the content of metabolites in the mycelia of Ganoderma lucidum under Cd stress, thereby resisting the damage caused by cadmium stress to the mycelia of Ganoderma lucidum and playing a mitigating role.

[0091] Free proline is a non-essential amino acid in Ganoderma lucidum mycelium and is the most water-soluble amino acid. It has multiple important functions within cells, not only participating in protein synthesis but also playing a key role in regulating cellular osmotic pressure, stabilizing the structure of biomacromolecules, and participating in intracellular energy metabolism. Furthermore, the proline content in heavy metal-tolerant organisms is generally higher than that in non-heavy metal-tolerant organisms. In the present invention, Cd+MT treatment significantly increased the proline content of the mycelia of strains Du585 and GSD under Cd stress, with the proline content in both the cadmium-tolerant strain Du585 being significantly higher than that in the cadmium-sensitive strain GSD. This suggests that when Ganoderma lucidum mycelium is subjected to adverse stress, its proline accumulates to resist damage caused by the stress.

[0092] Malondialdehyde (MDA) is one of the end products of lipid peroxidation. When cells are damaged by oxidation, polyunsaturated fatty acids in the cell membrane undergo peroxidation, thereby generating MDA. By detecting the MDA content, the degree of cell damage can be reflected to a certain extent. In the present invention, Cd treatment can significantly increase the MDA content of the mycelium of Ganoderma lucidum strain Du585 and strain GSD, indicating that Ganoderma lucidum mycelium cells suffer a certain degree of damage under Cd stress, while Cd+MT treatment can significantly reduce the MDA content of the mycelium of Ganoderma lucidum strain Du585 and strain GSD, indicating that the addition of MT can enhance the free radical scavenging ability of Ganoderma lucidum mycelium under Cd stress, reduce the accumulation of reactive oxygen species, and alleviate the degree of membrane lipid peroxidation.

[0093] APX, CAT, and POD are important antioxidant enzymes in the enzymatic scavenging system and play a key role in maintaining ROS metabolic balance. Cd treatment significantly reduced the APX, CAT, and POD contents in the mycelia of Ganoderma lucidum strains Du585 and GSD. Cd+MT treatment significantly increased the APX, CAT, and POD enzyme activities in the mycelia of Ganoderma lucidum strains Du585 and GSD, thereby enhancing the antioxidant capacity of these strains. This suggests that MT can alleviate Cd stress in Ganoderma lucidum mycelia by increasing antioxidant enzyme activity.

[0094] The present invention adds different concentrations of MT to culture the cadmium-resistant strain Du585 and the cadmium-sensitive strain GSD obtained in the early screening on a plate, and uses the mycelial growth rate as an indicator to screen the MT concentration that best alleviates the cadmium stress of Ganoderma lucidum mycelium. Subsequently, the strains Du585 and GSD are cultured at this concentration, and their metabolites and antioxidant-related indicators are measured. The results showed that the MT concentration that best alleviates the Cd stress of Ganoderma lucidum mycelium is 100 μmol / L; compared with the Cd treatment, MT can increase the content of polysaccharides, triterpenes, soluble proteins, and proline, reduce the content of MDA, and at the same time increase the enzyme activities of APX, CAT, and POD. In summary, MT can alleviate the Cd stress of Ganoderma lucidum mycelium by promoting mycelial growth, the accumulation of metabolites, and increasing the activity of antioxidant enzymes.

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the technical solutions. Those skilled in the art should understand that modifications or equivalent replacements of the technical solutions of the present invention that do not depart from the purpose and scope of the technical solutions of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for alleviating cadmium stress on Ganoderma lucidum mycelium, characterized in that: The specific steps are as follows: Inoculating the Ganoderma lucidum strain into a basic solid culture medium and culturing it at room temperature; Adding melatonin to the basic solid culture medium to make the concentration of melatonin in the culture medium above 100 μmol / L can alleviate the Cd 2+ Cadmium stress on Ganoderma lucidum mycelium.

2. The method according to claim 1, characterized in that The basic solid culture medium is a mixture of potato peel extract, sucrose, potassium dihydrogen phosphate, magnesium sulfate, agar powder and water; wherein the potato peel extract is 200g / L, the sucrose is 20g / L, the potassium dihydrogen phosphate is 1.5g / L, the magnesium sulfate is 0.5g / L, and the agar powder is 20g / L.

3. The method according to claim 2, characterized in that The concentration of melatonin (MT) in the basic solid culture medium is 100-800 μmol / L.

4. The method according to claim 1, characterized in that The Cd 2+ The concentration does not exceed 600μmol / L.

5. The method according to claim 1, characterized in that: The Ganoderma lucidum strain is Ganoderma lucidum Du585 or GSD strain.

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