Method for promoting high yield of cordyceps militaris sporocarp and application thereof

Abstract

The invention relates to the technical field of bioengineering, in particular to a method for promoting high yield of cordyceps militaris sporocarp and application of the method. The invention provides a method for promoting high yield of cordyceps militaris sporocarps, which comprises the following steps of: in a cordyceps militaris mycelium culture stage, spraying hydrophobin CmHYD1 on the surface of a culture medium or mycelium after mycelium is subjected to mycelium stimulation treatment under light. According to the method, hydrophobin CmHYD1 with a specific concentration is exogenously sprayed, so that the growth and development of cordyceps militaris sporocarps can be remarkably promoted, and the yield is effectively increased. Experimental data show that when 400 g of CmHYD1 is sprayed, the number of entities in a single bottle of cordyceps militaris is increased by 231.07% compared with that of a control group, the yield and the biological conversion rate are also synchronously increased by 72.70%, and it is proved that the method can achieve high yield of cordyceps militaris by remarkably increasing the number of the fruiting bodies.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a method for promoting high yield of Cordyceps militaris fruiting bodies and its application. Background Technology

[0002] Cordyceps militaris is an edible and medicinal fungus with high medicinal and nutritional value. Its fruiting bodies contain abundant active ingredients, such as cordycepin, polysaccharides, and adenosine, which possess various physiological activities including anti-tumor, immunomodulatory, and antioxidant effects, leading to increasing market demand. Currently, Cordyceps militaris is mainly produced on a large scale through liquid culture. However, the formation and development of its fruiting bodies are regulated by multiple factors, and improving yield and quality remains a key issue in the industry.

[0003] In the artificial cultivation of Cordyceps militaris, the induction and development of fruiting bodies are often limited by factors such as strain characteristics, environmental conditions, and nutrient regulation, leading to problems such as unstable yield and low bioconversion rate. Currently, common methods to increase yield include optimizing culture medium formulation, adjusting light and temperature, and using plant growth regulators. However, these methods often have limited effectiveness or pose safety risks, making it difficult to achieve stable and efficient yield increases.

[0004] Hydrophobins are a class of small proteins secreted by fungi. They possess the ability to self-assemble into amphiphilic membranes and are non-cytotoxic and non-immunogenic, making them widely used in the medical, industrial, and food industries. For example, hydrophobins can stabilize oil droplet structures and act as emulsifiers in food processing, liposome applications, and essential oil extraction. Furthermore, hydrophobins play an indispensable role in the growth and development of filamentous fungi, influencing processes such as hyphal aggregation, attachment, and the formation of aerial tissue structures. Previous studies have shown that certain hydrophobins can promote the formation and development of edible fungal fruiting bodies. In Cordyceps militaris, the hydrophobin CmHYD1 has been shown to participate in the positive regulation of fruiting body development; however, it is usually expressed as an endogenous protein within the fungus, and systematic research on exogenous addition methods and their effects on fruiting body yield and accumulation of active ingredients remains relatively lacking.

[0005] Currently, research on directly regulating the growth and metabolism of Cordyceps militaris fruiting bodies through exogenous spraying of hydrophobic proteins is scarce, especially lacking systematic screening and evaluation of the effects of different spraying concentration gradients on fruiting body morphology, yield, and polysaccharide content. Therefore, developing a safe, efficient, and easy-to-operate method for exogenous protein spraying and determining its optimal concentration is of significant application value and research importance for achieving high-yield and high-quality cultivation of Cordyceps militaris. Summary of the Invention

[0006] This invention aims to provide a method for promoting high-yield and high-quality Cordyceps militaris fruiting bodies by exogenously spraying the hydrophobic protein CmHYD1. Specifically, the hydrophobic protein CmHYD1 is obtained by exogenous expression using a yeast system, and is prepared into solutions with different concentration gradients. After the Cordyceps militaris mycelia are exposed to light and scratched, the solutions are sprayed onto the surface of the culture medium. By systematically comparing the morphology, number, fresh weight, biological conversion rate, and polysaccharide content of the fruiting bodies in each treatment group, the optimal CmHYD1 spraying concentration that can significantly improve the yield and accumulation of active ingredients in Cordyceps militaris is screened.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for promoting high yield of Cordyceps militaris fruiting bodies. During the Cordyceps militaris mycelium culture stage, after the mycelium is treated with light-induced mycelial irradiation, hydrophobic protein CmHYD1 is sprayed onto the surface of the culture medium or the mycelium.

[0008] Preferably, the application rate of the hydrophobic protein CmHYD1 is 100-500 µg per culture flask.

[0009] Preferably, the culture flask has the following specifications: height 90~110mm, bottom diameter 60~80mm.

[0010] Preferably, the hydrophobic protein CmHYD1 is obtained by exogenous expression in Pichia pastoris.

[0011] This invention provides the use of the hydrophobic protein CmHYD1 in promoting the development of Cordyceps militaris fruiting bodies and / or increasing the yield of Cordyceps militaris fruiting bodies.

[0012] This invention provides the use of hydrophobic protein CmHYD1 in increasing the polysaccharide content of Cordyceps militaris fruiting bodies.

[0013] This invention provides a method for producing Cordyceps militaris fruiting bodies, which involves cultivating the fruiting bodies until they mature and are then harvested.

[0014] This invention provides a spray formulation for Cordyceps militaris cultivation, comprising the hydrophobic protein CmHYD1 and an agriculturally acceptable carrier or diluent.

[0015] Compared with the prior art, the present invention has the following beneficial effects: Firstly, by exogenously spraying a specific concentration of the hydrophobic protein CmHYD1, the growth and development of Cordyceps militaris fruiting bodies can be significantly promoted, effectively increasing yield. Experimental data show that when 400 µg of CmHYD1 is sprayed, the number of Cordyceps militaris fruits per bottle increases by 231.07% compared to the control group, and the yield and biological conversion rate also increase by 72.70% simultaneously, confirming that this method can achieve high yield of Cordyceps militaris by significantly increasing the number of fruiting bodies.

[0016] Secondly, this method not only increases yield but also improves the quality of Cordyceps militaris fruiting bodies. Polysaccharide content testing results showed that after spraying with 200 µg, 300 µg, and 400 µg CmHYD1, the polysaccharide content of the fruiting bodies increased by an average of 93.85% compared to the control group. The 400 µg treatment group showed the best overall effect, indicating that at this concentration, Cordyceps militaris achieved optimal levels in both yield and accumulation of active substances. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0018] Figure 1 The development of fruiting bodies of Cordyceps militaris after exogenous spraying with different amounts of hydrophobic protein CmHYD1; A: Growth of fruiting bodies of Cordyceps militaris after exogenous spraying with different amounts of hydrophobic protein CmHYD1; B: Development data of fruiting bodies of Cordyceps militaris after exogenous spraying with different amounts of hydrophobic protein CmHYD1.

[0019] Figure 2 The polysaccharide content of fruiting bodies of Cordyceps militaris was determined by different amounts of hydrophobic protein CmHYD1 added through exogenous spraying. Detailed Implementation

[0020] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0021] Example 1

[0022] (1) Development of fruiting bodies of Cordyceps militaris with different amounts of hydrophobic protein CmHYD1 added by exogenous spraying

[0023] CmHYD1 was expressed exogenously in Pichia pastoris (the active substance was obtained from the literature: Li Xiao, “Study on the function and regulatory mechanism of hydrophobic protein family genes in Cordyceps militaris”).

[0024] The cultured Cordyceps militaris liquid spawn was transferred to sterilized wheat culture medium, 5 mL per bottle, and incubated at 20℃ in the dark for 4 days. Then, it was placed in an artificial climate chamber and incubated for another 4 days under 20℃-12 h light and 18℃-12 h darkness. After scratching treatment, 700 µL of CmHYD1 containing different amounts (100, 200, 300, 400, 500 µg) was sprayed onto the surface of the culture medium. The untreated group served as the blank control (CK), and the group sprayed with 700 µL of H2O served as the negative control. After the fruiting bodies matured, they were harvested uniformly, and individual bottles (107 mm in height) of fruiting bodies were sorted. (Base diameter 70 mm.) Statistical analysis was performed on the number, height, fresh weight, and biological conversion rate of fruiting bodies.

[0025] The results showed that, compared with the control (no treatment) and the H2O spraying group, the exogenous spraying of CmHYD1 400 µg resulted in the highest number of Cordyceps militaris bodies per bottle, a year-on-year increase of 242.20%, and also the highest yield and biological conversion rate, with year-on-year increases of 72.41% and 82.33%, respectively. Figure 1 This indicates that CmHYD1 increases the yield of Cordyceps militaris by increasing the number of fruiting bodies.

[0026] (2) The content of polysaccharides in the fruiting bodies of Cordyceps militaris after different amounts of hydrophobic protein CmHYD1 were obtained from exogenous spraying.

[0027] The polysaccharide content of Cordyceps militaris fruiting bodies cultured with different amounts of CmHYD1 was detected by the phenol-sulfuric acid method. The results showed that, compared with the control group and the H2O spraying treatment group, the polysaccharide content of Cordyceps militaris fruiting bodies cultured with exogenous spraying of 200 µg and 300 µg of CmHYD1 was higher, with a year-on-year increase of 90%. Figure 2 When CmHYD1 400 µg was applied exogenously, the polysaccharide content of the fruiting body was the second highest, with a year-on-year increase of 67.57%.

[0028] Considering the development of Cordyceps militaris fruiting bodies and polysaccharide content, the optimal yield and polysaccharide content of Cordyceps militaris were achieved when 400 µg of hydrophobic protein CmHYD1 was applied exogenously.

[0029] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for promoting high yield of Cordyceps militaris fruiting bodies, characterized in that, During the Cordyceps militaris mycelium culture stage, after the mycelium is treated with light-induced mycelial irrigation, hydrophobic protein CmHYD1 is sprayed onto the surface of the culture medium or the mycelium.

2. The method according to claim 1, characterized in that, The application rate of the hydrophobic protein CmHYD1 is 100-500 µg per culture flask.

3. The method according to claim 1, characterized in that, The specifications of the culture flask are: height 90~110mm, bottom diameter 60~80mm.

4. The method according to claim 1, characterized in that, The hydrophobic protein CmHYD1 was obtained through exogenous expression in Pichia pastoris.

5. Use of hydrophobic protein CmHYD1 in promoting the development of Cordyceps militaris fruiting bodies and / or increasing the yield of Cordyceps militaris fruiting bodies.

6. The use of hydrophobic protein CmHYD1 in increasing the polysaccharide content of Cordyceps militaris fruiting bodies.

7. A method for producing Cordyceps militaris fruiting bodies, characterized in that, Cultivate using the method described in any one of claims 1 to 4 until the fruiting bodies mature and are then harvested.

8. A spray formulation for Cordyceps militaris cultivation, characterized in that, It contains the hydrophobic protein CmHYD1, as well as an agriculturally acceptable carrier or diluent.

Images