Method for reducing cadmium content in stropharia rugoso-annulata hyphae and increasing mineral element content at same time

Abstract

The invention discloses a method for reducing the content of cadmium in stropharia rugoso-annulata hyphae and increasing the content of mineral elements at the same time. By adding more than 100 mg / L of zinc ions into the stropharia rugoso-annulata culture medium, the cadmium content in stropharia rugoso-annulata hyphae is reduced, the contents of mineral elements such as zinc, magnesium, iron and calcium are increased, in addition, activation of an antioxidant system in the hyphae can be accelerated, oxidative stress induced by cadmium is relieved, normal growth of the hyphae is recovered, and the yield of the stropharia rugoso-annulata is increased. And the method can be widely applied to large-scale production.

Description

Technical Field

[0001] The invention belongs to the field of edible fungus cultivation and relates to a method for reducing the cadmium content in Stropharia rugosus mycelium while increasing the mineral element content. Background Art

[0002] Studies have found that most large fungi, such as shiitake mushrooms, oyster mushrooms, morels, and giant puffball mushrooms, have the ability to accumulate heavy metals, even exceeding that of green plants. The main sources of heavy metal pollution in edible fungi are soil, atmosphere, water, and cultivation substrates. The sources of raw materials for the cultivation of edible fungi are very wide, and the main cultivation materials are usually some agricultural and forestry by-products, such as sawdust, cottonseed hulls, corn cobs, straw, bran, etc. Plants have a certain absorption, accumulation, and transformation effect on heavy metal elements and cannot release them into the environment in a short period of time. When the cultivation environment of edible fungi is contaminated by heavy metals, or when the cultivation raw materials themselves contain heavy metal elements, the mycelium of edible fungi has a biological adsorption effect on heavy metals, and through the growth and transformation of edible fungi, the heavy metals are enriched in the fruiting bodies, causing great harm to people's health.

[0003] Cadmium (Cd), a heavy metal widely present in the environment, has become a global environmental problem due to its high toxicity and persistence. The accumulation of Cd in soil not only affects plant growth and development but also threatens human health through its transmission through the food chain. Studies have shown that Cd contamination can lead to reduced crop yields and quality, and even cause food safety issues. The main hazards of Cd to the human body are its carcinogenicity, nephrotoxicity, and bone disease.

[0004] Stropharia rugosoannulata Farl. ex Murrill, also known as the wrinkled ball cap mushroom and the wine-red ball cap mushroom, is one of the edible mushrooms recommended for cultivation by the Food and Agriculture Organization of the United Nations for developing countries. In recent years, Stropharia rugosoannulata has rapidly become a popular new edible mushroom, with strong industry development momentum. Stropharia rugosoannulata contains a variety of nutrients, amino acids, and bioactive compounds such as polysaccharides, flavonoids, sterols, and phenols that are beneficial to the human body. These bioactive compounds not only provide health benefits such as free radical scavenging, antioxidant protection, and blood sugar lowering, but also possess anti-tumor, osteoclast inhibition, and Alzheimer's disease prevention properties, making it a promising raw material for future biopharmaceuticals. In recent years, with the unprecedented emphasis on improving immunity, sales of edible mushrooms such as Stropharia rugosoannulata have continued to increase, demonstrating significant market potential. This has also brought economic benefits to mushroom farmers and businesses, promoting the vigorous development of the edible mushroom industry.

[0005] Stropharia mushrooms primarily utilize agricultural waste such as rice straw, stalks, and wheat straw as their cultivation substrate. They feature simple cultivation techniques, easy scalability, strong resistance, and high yields. Currently, most regions have adopted a "rice-mushroom rotation" cultivation model. This involves using rice straw in rice fields after the rice harvest, turning waste into valuable resources and significantly reducing the pollution caused by straw burning. The rice straw decomposes during the production of Stropharia mushrooms, and after harvesting, the mushroom residue is returned to the fields, further improving the soil. Stropharia mushrooms are particularly effective at transforming agricultural waste. While producing mushrooms, they also address environmental pollution issues. They possess high ecological value and are gradually becoming a key project for adjusting crop production structures and achieving circular agriculture.

[0006] In the "rice-mushroom rotation" cultivation model, Stropharia officinalis is grown on rice straw and requires soil covering to stimulate fruiting. Because both rice and edible fungi are known to accumulate heavy metals, the use of rice straw in the cultivation cycle of Stropharia officinalis presents certain safety risks. Research has shown that rice straw generally accumulates Cd more than leaves and grains. Other crops in the grass family, such as wheat, corn, and barley, also accumulate heavy metals. Using these plant straws as the primary cultivation material for Stropharia officinalis will also lead to secondary accumulation of heavy metals, posing a certain threat to the safety of the mushrooms. Research on Stropharia rugosa showed that after 21 days of cultivation, Cd concentrations in mycelium under 0.2 mg / L and 2 mg / L Cd stress reached 0.19 mg / kg and 71.09 mg / kg, respectively. At 10 mg / kg Cd, the Cd concentration in Stropharia rugosa fruiting bodies reached 8.10 mg / kg, with an enrichment coefficient of 0.81, indicating that Stropharia rugosa has a certain ability to accumulate Cd. Therefore, excessive Cd levels in Stropharia rugosa are prone to occur, and excessive Cd levels have become a key factor hindering the development of related industries.

[0007] Preventing or controlling heavy metal accumulation during edible fungi production, or minimizing the risk of accumulation, is an important technical measure for ensuring food safety. This can typically be achieved by selecting low-heavy metal accumulators or by employing physical or chemical methods to reduce the incorporation of heavy metals into the fruiting bodies of edible fungi, thereby reducing heavy metal content in the fungi. Compared to selective breeding, physical or chemical methods are more convenient and effective in reducing cadmium in the fruiting bodies of edible fungi. Heavy metals in edible fungi are first absorbed from the culture medium through the mycelium and then enter the fruiting bodies through specific transport pathways. Studies have reported that the addition of compounds such as activated carbon, salicylic acid, zeolite, gypsum, and chitosan can reduce cadmium content in shiitake mushroom fruiting bodies. Furthermore, the addition of beneficial elements to the culture medium can inhibit heavy metal absorption by edible fungi through ion competition. For example, the addition of magnesium and zinc can effectively reduce cadmium content in shiitake mushrooms, and the addition of selenium can inhibit lead absorption by Stropharia rugosa. Research on improving the mineral content of edible fungi has primarily focused on the effects of different light qualities during supplemental lighting periods. Currently, there are no reports on simultaneously increasing mineral content while reducing cadmium content. Summary of the Invention

[0008] The invention provides a method for reducing the cadmium content in Stropharia rugosus mycelium and simultaneously increasing the mineral element content.

[0009] The technical solutions of the present invention are as follows:

[0010] The method for reducing the cadmium content in the mycelium of Stropharia rugosa and increasing the content of mineral elements is as follows: adding zinc ions (Zn 2+ ), wherein the added concentration of zinc ions is above 100 mg / L.

[0011] Furthermore, the Stropharia capillaris culture medium of the present invention is a culture medium commonly used for Stropharia capillaris, such as PDA culture medium, PDB culture medium, etc.

[0012] Furthermore, the zinc ions are added in the form of zinc sulfate, zinc chloride, etc. In the specific embodiment of the present invention, zinc sulfate is taken as an example.

[0013] Compared with the prior art, the present invention has the following advantages:

[0014] The present invention effectively reduces the cadmium concentration entering the mycelium by adding a certain concentration of zinc ions to the culture medium of Stropharia rugosodium, while simultaneously increasing the content of mineral elements such as zinc, magnesium, iron, and calcium. Furthermore, the method accelerates the activation of the mycelium's antioxidant system, alleviates cadmium-induced oxidative stress, restores normal mycelial growth, and significantly improves the nutritional value of the edible fungus. The method is simple to operate, low in cost, and effective, making it suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1The effect of different Cd concentrations on the mycelial growth rate of Stropharia chaoyangensis strain;

[0016] Figure 2 is the Cd concentration in the mycelium of Stropharia chaoyangensis strain under different Cd concentration treatments;

[0017] Figure 3 The growth of mycelium of Stropharia chaoyangensis strain under different concentrations of Cd / Zn treatments;

[0018] Figure 4 The effect of different concentrations of Cd / Zn on the mycelial growth rate of Stropharia chaoyangensis strain;

[0019] Figure 5 is the Cd concentration in the mycelium of Stropharia chaoyangensis strain under different Cd / Zn concentration treatments;

[0020] Figure 6 is the Zn concentration in the mycelium of Stropharia chaoyangensis strain under different Cd / Zn concentration treatments;

[0021] Figure 7 is the Mg concentration in the mycelium of Stropharia chaoyangensis strain under different Cd / Zn concentration treatments;

[0022] Figure 8 is the Fe concentration in the mycelium of Stropharia chaoyangensis strain under different Cd / Zn concentration treatments;

[0023] Figure 9 is the Ca concentration in the mycelium of Stropharia chaoyangensis strain under different Cd / Zn concentration treatments;

[0024] Figure 10 The H2O2 content in the mycelium of Stropharia chaoyangensis strain under different Cd / Zn concentrations;

[0025] Figure 11 is the GSH content in the mycelium of Stropharia chaoyangensis strain under different Cd / Zn concentrations;

[0026] Figure 12 is the GSSG content in the mycelium of Stropharia chaoyangensis strain under different Cd / Zn concentration treatments;

[0027] Figure 13 The GR activity in the mycelium of Stropharia chaoyangensis strain under different Cd / Zn concentrations;

[0028] In the figure, 0 / 0: represents the treatment concentration of Chaoyang strain at 0 mg / L Cd + 0 mg / L Zn; 1 / 0: represents the treatment concentration of Chaoyang strain at 1 mg / L Cd + 0 mg / L Zn; 1-50: represents the treatment concentration of Chaoyang strain at 1 mg / L Cd + 50 mg / L Zn; 1-100: represents the treatment concentration of Chaoyang strain at 1 mg / L Cd + 100 mg / L Zn. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with specific examples and accompanying drawings. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention in any way. Various modifications of the present invention beyond those shown and described herein will be apparent to those skilled in the art through the foregoing description and the following examples and are within the scope of the appended claims.

[0030] Unless otherwise defined, all professional and scientific terms have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0031] In the following examples, the sources of the materials are as follows:

[0032] Test strain: Stropharia rugosus Chaoyang strain, from the National Edible Fungi Engineering Technology Research Center;

[0033] PDA culture medium: Difco TM Potato Dextrose Agar;

[0034] PDB culture medium: Difco TM Potato Dextrose Broth;

[0035] Test water: Purified water produced by Watsons;

[0036] Cadmium chloride: Cadmium chloride reagent (CdCl2·2.5H2O) produced by Sinopharm Group is used, with a purity of more than 99%;

[0037] Zinc sulfate: Use zinc sulfate reagent (ZnSO4·7H2O) produced by Sinopharm Group, with a purity of more than 99%.

[0038] Example 1: Effects of different cadmium contents in the culture medium on mycelial growth and cadmium content of the Chaoyang strain

[0039] 1. Test strain: Stropharia rugosodium chinense Chaoyang strain.

[0040] 2. Cadmium treatment concentrations: 0 mg / L, 0.5 mg / L, 1 mg / L, and 2 mg / L.

[0041] 3. Test method:

[0042] (1) The Chaoyang strain of Stropharia officinalis was used as the test strain. Different concentrations of cadmium chloride were added to the PDA culture medium to make the cadmium ion concentration in the PDA reach 0.5 mg / L, 1 mg / L, and 2 mg / L, respectively. The PDA culture medium without cadmium was used as the control. After the PDA culture medium solidified, the same size of the fungus block was inoculated into the center of the culture dish. After the mycelium germinated, the plate streak method was used to determine the growth rate of the mycelium.

[0043] (2) The Chaoyang strain of Stropharia capillaris was used as the test strain. Different concentrations of cadmium chloride were added to the PDB culture medium to make the cadmium ion concentration in PDB reach 0.5 mg / L, 1 mg / L, and 2 mg / L, respectively. The PDB culture medium without cadmium was used as the control. After 21 days of culture, the mycelium was filtered out with a non-woven fabric and soaked in an EDTA-Na2 solution for 15 minutes to remove the cadmium chloride adsorbed on the surface of the mycelium. The mycelium was washed with deionized water and freeze-dried. The cadmium content in the mycelium of Stropharia capillaris was determined using the cadmium determination method in the national standard (GB 5009.15-2023).

[0044] 4. Results Analysis

[0045] from Figure 1 It can be seen that with the increase of cadmium concentration in the culture medium, the growth rate of Stropharia rugosus mycelium decreased significantly and the growth was inhibited, indicating that cadmium in the growth environment had a serious stress effect on the growth of mycelium.

[0046] from Figure 2 It can be seen that with the increase of cadmium concentration in the culture medium, the cadmium concentration in the mycelium of Stropharia rugosa increased significantly, indicating that the cadmium content in the growth environment directly affects the cadmium content in the mycelium of Stropharia rugosa.

[0047] Example 2: Optimization of the method for reducing cadmium content in mycelium

[0048] 1. Test strain: Stropharia rugosodium chinense Chaoyang strain.

[0049] 2. Cadmium treatment concentration: 0 mg / L, 1 mg / L.

[0050] 3. Additives to reduce cadmium concentration: ZnSO4·7H2O; added concentrations: 0 mg / L, 50 mg / L, 100 mg / L.

[0051] 4. Test method:

[0052] (1) Using the Chaoyang strain of Stropharia rugosodium as the test strain, different concentrations of cadmium chloride were added to the PDA culture medium to increase the cadmium ion concentration to 1 mg / L. ZnSO4·7H2O was then added to the culture medium to increase the zinc ion concentration to 0 mg / L, 50 mg / L, and 100 mg / L, respectively. A PDA culture medium without cadmium and zinc was used as a control. After the PDA culture medium solidified, a bacterial block of the same size was inoculated into the center of the culture dish. After mycelium germinated, the mycelial growth rate was measured by the plate streak method.

[0053] (2) The Chaoyang strain of Stropharia capillaris was used as the test strain. Different concentrations of cadmium chloride were added to the PDB culture medium to make the cadmium ion concentration in PDB reach 0.5 mg / L, 1 mg / L, and 2 mg / L, respectively. The PDB culture medium without cadmium was used as the control. After 21 days of culture, the mycelium was filtered out with a non-woven fabric and soaked in EDTA-Na2 solution for 15 minutes to remove the cadmium chloride adsorbed on the surface of the mycelium. The mycelium was washed with deionized water and freeze-dried. The contents of cadmium, zinc, magnesium, iron, and calcium in the mycelium of Stropharia capillaris were respectively determined according to the national standards (GB 5009.15-2023, GB 5009.14-2017, GB 5009.241-2017, GB 5009.90-2016, GB 5009.92-2016), and the contents of H2O2 (hydrogen peroxide), glutathione (GSH), oxidized glutathione (GSSG), and glutathione reductase (GR) were determined using kits produced by Suzhou Keming Biotechnology Co., Ltd.

[0054] 5. Results Analysis

[0055] from Figure 3 、 Figure 4 、 Figure 5 It can be seen that when the cadmium concentration in the culture medium is 1 mg / L, adding 50-100 mg / L zinc to the culture medium can significantly reduce the cadmium concentration in the mycelium of Stropharia rugosus; when the zinc addition concentration is 50 mg / L, the cadmium concentration in the mycelium is 80.79 mg / kg, which is 50.82% lower than the treatment without zinc addition (164.25 mg / kg). At this time, the growth rate of the mycelium is significantly increased compared with the treatment without zinc addition; when the zinc addition concentration is 100 mg / L, the cadmium concentration in the mycelium is 94.16 mg / kg, which is 42.67% lower than the treatment without zinc addition, and there is no significant difference with the 50 mg / L zinc treatment. At this time, the mycelium growth rate is further increased and returned to the control (no cadmium, zinc addition) level.

[0056] from Figure 6 、 Figure 7 、 Figure 8 、 Figure 9As can be seen from the results, when the cadmium concentration in the culture medium was 1 mg / L, adding 100 mg / L of zinc to the culture medium effectively increased the concentrations of zinc, magnesium, iron, and calcium in Stropharia mycelia. In particular, when the zinc concentration was 100 mg / L, the concentrations of zinc, magnesium, iron, and calcium increased by 1825.17%, 234.06%, 21.65%, and 77.90%, respectively, compared to the treatment without zinc addition, and by 2961.58%, 409.85%, 272.95%, and 107.96%, respectively, compared to the control (no cadmium, zinc addition), significantly increasing the content of mineral elements.

[0057] from Figure 10 、 Figure 11 、 Figure 12 、 Figure 13 It can be seen that compared with the control (no cadmium and zinc addition), the H2O2 content in the mycelium of Stropharia rugosus under 1 mg / L cadmium treatment increased significantly, indicating that cadmium stress would cause oxidative damage to the mycelium, resulting in growth inhibition. At this time, the antioxidant system of the mycelium has been activated, and the contents of antioxidants GSH and GSSG and the activity of antioxidant enzyme GR have increased significantly. When 1 mg / L cadmium and 50 mg / L zinc were added to the culture medium, the GSSG content and GR activity continued to increase to maintain the normal physiological function of the mycelium. At the same time, the oxidative damage induced by Cd was effectively alleviated, and the H2O2 level was significantly reduced. At this time, the degree of mycelial growth inhibition was significantly alleviated. When 1 mg / L cadmium and 100 mg / L zinc were added to the culture medium, the H2O2, GSH, GSSG contents and GR activity all returned to the control levels, indicating that 100 mg / L zinc can completely alleviate the oxidative damage induced by cadmium. At the same time, zinc has a synergistic effect with magnesium, iron and calcium ions, which improves the nutritional level of mycelium and also helps its resistance to cadmium stress. Therefore, the growth rate of this mycelium is not significantly different from that of the control.

[0058] In summary, when cadmium stress exists in the growth environment of Stropharia mycelium, the cadmium concentration in the mycelium increases with the increase of cadmium concentration in the environment because the mycelium has an enrichment effect on cadmium in the environment. Adding zinc ions to the culture medium can effectively reduce the cadmium concentration in the mycelium, thereby reducing the mycelium's enrichment of cadmium in the growth medium. As can be seen from the examples, adding 100 mg / L zinc ions can effectively reduce the cadmium concentration in Stropharia mycelium, increase the content of mineral elements such as zinc, magnesium, iron, and calcium in the mycelium, accelerate the activation of the antioxidant system in the mycelium, alleviate cadmium-induced oxidative stress, and restore normal mycelial growth. This method can reduce the cadmium content in Stropharia mycelium fruiting bodies while improving their nutritional value. It is simple to operate, low-cost, and effective, and has broad application prospects for large-scale production.

Claims

1. A method for reducing the cadmium content in Stropharia rugosus mycelium while increasing the mineral element content, characterized in that: Specifically, zinc ions are added to the culture medium of Stropharia rugosa, and the added concentration of zinc ions is above 100 mg / L.

2. The method according to claim 1, characterized in that The culture medium for Stropharia rugosa is PDA medium or PDB medium.

3. The method according to claim 1, characterized in that The zinc ion is added in the form of zinc sulfate or zinc chloride.

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