A strain of myceliophthora and its culture method and use

Abstract

A new strain of Cordyceps cicadae, its culture method and use belong to the field of microbial technology and food production. The culture method of the new strain of Cordyceps cicadae comprises the following steps: 1) inoculating the new strain of Cordyceps cicadae into a culture medium composed of oats and buckwheat, and the inoculation amount is greater than or equal to 20%; 2) first dark culture for 5-7 days, and then light culture for 10-12 days, during which alternating blue light irradiation of 12-18 hours and darkness of 12-6 hours are adopted every day, and during the light culture, stage temperature difference culture is adopted, the light culture temperature is greater than the dark culture temperature, and the difference is greater than or equal to 4 DEG C. The above-mentioned new strain of Cordyceps cicadae, its culture method and use reduce the content of Beauvericin, increase the content of polysaccharide and N6-(2-hydroxyethyl) adenosine and other effective components, realize the unity of safety and nutrition, and provide solid technical support for the application of Cordyceps cicadae as food or functional food raw material.

Description

Technical Field

[0001] This application belongs to the field of edible and medicinal fungi production technology, specifically a strain of Cordyceps militaris, its cultivation method, and its uses. Background Technology

[0002] Cordyceps militaris ( Ophiocordyceps sobolifera ), former name Cordyceps sobolifera Cordyceps militaris, also known as cicada nymph or cicada flower, is a fungus-insect complex formed by the parasitism of cicada nymphs by a fungus belonging to the family Ophiocordycipitaceae in the phylum Ascomycota. It is mainly distributed in East my country, Central China, South China, and Southwest China. Cordyceps militaris has significant medicinal value; its fruiting bodies have been documented for improving sleep, boosting immunity, protecting the kidneys, fighting tumors, and lowering blood pressure, blood sugar, and cholesterol.

[0003] In 2020, the National Health Commission of China issued Announcement No. 9 of 2020, approving for the first time that artificially cultivated Cordyceps sinensis fruiting bodies can be used as a new food raw material, with broad development prospects. The announcement specifically mentioned the requirement that the beauvericin (BEA) content be ≤3mg / kg. Currently, there are no known technologies for cultivating Cordyceps sinensis to reduce BEA content. Therefore, there is a need to provide a Cordyceps sinensis strain and its cultivation method to achieve a beauvericin content ≤3mg / kg, and to increase the content of active ingredients such as polysaccharides and N6-(2-hydroxyethyl) adenosine (HEA). Summary of the Invention

[0004] To address the shortcomings of existing technologies, this application aims to provide a technical solution for a *Cicada nymph* strain, its cultivation method, and its uses. This solution reduces beauveria bassiana while increasing the content of polysaccharides and N6-(2-hydroxyethyl)adenosine, achieving a balance between safety and nutrition, and providing solid technical support for the application of *Cicada nymph* as a food ingredient.

[0005] To achieve the above objectives, this application adopts the following technical solution: This application provides a strain of Cordyceps militaris, which was deposited on December 29, 2016 at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO: 13572.

[0006] This application also provides a method for culturing the *Cicada Flower* strain, comprising the following steps: 1) Inoculate the *Cicada Flower* strain into a culture medium consisting of 75-85 parts by weight of oats and 15-25 parts by weight of buckwheat, with an inoculation amount ≥20%; 2) First, incubate in the dark for 5-7 days at a temperature of 16-20℃; then incubate in the light for 10-12 days. During the light incubation period, alternate between 12-18 hours of blue light irradiation and 12-6 hours of darkness per day. The temperature during the light incubation period should be 20-24℃, with the light incubation temperature being greater than the dark incubation temperature by a difference of ≥4℃. The blue light wavelength should be 450-495nm, and the blue light intensity should be 120-300Lux. The relative humidity during the dark incubation period should be 75%-85%, and the relative humidity during the light incubation period should be 80%-100%. The carbon dioxide concentration should be 501-1100PPM for the first 1 to 7 days of the light incubation period, and 1201-3000PPM for the 8th to 12th days of the light incubation period.

[0007] In the cultivation method of the *Cicada Flower* strain, step 1) involves a culture medium consisting of 80-82 parts oats and 20-18 parts buckwheat.

[0008] In the method for culturing the *Cicada Flower* strain, step 1) states that the thickness of the culture medium is 1.5-2.5 cm, preferably 2-2.2 cm, and the inoculation amount is 20-30%.

[0009] In the cultivation method of the *Cicada Flower* strain, step 2) involves a blue light wavelength of 465-480 nm and a blue light intensity of 180-240 Lux.

[0010] The application of the aforementioned *Cicada Flower* strain in the preparation of food or functional food ingredients.

[0011] This invention uses a higher inoculum size than traditional cultivation methods, enabling rapid mycelial coverage and reducing fermentation time by 1-2 days compared to traditional methods. The culture medium thickness is 1.5 to 2.5 cm, which is thinner than traditional media, ensuring sufficient nutrients for rapid mycelial coverage.

[0012] Compared with the prior art, the beneficial effects of this application are as follows: 1. This application is the first to apply Cordyceps militaris strains to the field of new food ingredients, ensuring their compliance with national safety standards for new food ingredients. The National Health Commission Announcement (No. 9 of 2020) approved artificially cultivated Cordyceps militaris fruiting bodies as new food ingredients, but explicitly required that the beauveria bassiana content be ≤3mg / kg. The cultivation method described in this application ensures that the beauveria bassiana content in the Cordyceps militaris strain is far below this limit, significantly improving its safety as a new food ingredient.

[0013] 2. In existing technologies, Cordyceps militaris is mainly used for medicinal purposes, with limited reports on its application in food. Most studies focus on its medicinal value, such as "anti-tumor, anti-inflammatory and analgesic effects, improvement of renal function, hypoglycemia, and hypotension." This application breaks through the traditional application scope by successfully applying Cordyceps militaris strains to the field of new food raw materials, expanding its application range. It can be used to develop health foods, functional foods, or general food ingredients, and has significant industrialization and commercial value.

[0014] 3. This application also focuses on the preservation and enhancement of nutritional components. While reducing beauveria bassiana, it increases the content of active ingredients such as polysaccharides and N6-(2-hydroxyethyl)adenosine, achieving a balance between safety and nutrition. This comprehensive quality improvement strategy has greater industrial value than single safety control and provides solid technical support for the application of Cordyceps sinensis as a food ingredient. Attached Figure Description

[0015] Figure 1 The growth of fruiting bodies under different light conditions was observed.

[0016] Figure 2 The BEA content of fruiting bodies cultured under different light conditions according to the present invention.

[0017] Figure 3 The BEA content of fruiting bodies cultured under blue light with different light intensities according to the present invention.

[0018] Figure 4 The polysaccharide content of fruiting bodies cultured under different light conditions was determined.

[0019] Figure 5 The HEA content of fruiting bodies cultured under different light conditions was determined. Detailed Implementation

[0020] To enable those skilled in the art to better understand the present application, the technical solutions in specific embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings.

[0021] A strain of *Cicada nymph* was isolated by the Laboratory of Plant Protection and Entomological Fungi, Zhejiang Subtropical Crops Research Institute, and deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 29, 2016, with accession number CGMCC NO: 13572. Example 1

[0022] 1) The *Cicada Flower* strain was inoculated into a culture medium consisting of 75 parts by weight of oats and 25 parts by weight of buckwheat, with an inoculation amount of 20%; 2) First, dark culture for 6 days at a temperature of 20℃ and a relative humidity of 85%; then, light culture at 24℃ for 10 days at a relative humidity of 100%. During the light culture period, the blue light irradiation and darkness were alternated for 12 hours each day. The light culture temperature was higher than the dark culture temperature by a difference of ≥4℃. The blue light wavelength was 450nm and the blue light intensity was 300Lux. The carbon dioxide concentration was 1100 PPM for the first 1 to 7 days of the light cultivation period, and 3000 PPM for the 8th to 10th days of the light cultivation process. This can uniformly regulate the growth nodes of mycelia and fruiting bodies, and facilitate the uniform harvesting of fruiting bodies in the future. Example 2

[0023] 1) The *Cicada Flower* strain was inoculated into a culture medium consisting of 80 parts by weight of oats and 20 parts by weight of buckwheat, with an inoculation amount of 25%. 2) First, dark culture for 7 days at a temperature of 18℃ and a relative humidity of 80%; then, light culture at 22℃ for 12 days at a relative humidity of 90%. During the light culture period, the blue light irradiation is alternated with 15 hours of darkness per day. The light culture temperature is greater than the dark culture temperature by a difference of ≥4℃. The blue light wavelength is 470nm and the blue light intensity is 260Lux. The carbon dioxide concentration was 700 PPM for the first 1 to 7 days of the light culture period, and 1500 PPM for the 8th to 12th days of the light culture period. Example 3

[0024] 1) The *Cicada Flower* strain was inoculated into a culture medium consisting of 85 parts by weight of oats and 15 parts by weight of buckwheat, with an inoculation amount of 30%; 2) First, dark culture for 5 days at a temperature of 21℃ and a relative humidity of 75%; then, light culture at 24℃ for 10 days at a relative humidity of 80%. During the light culture period, the blue light irradiation is alternated with 18 hours of light irradiation and 6 hours of darkness per day. The light culture temperature is greater than the dark culture temperature by a difference of ≥4℃. The blue light wavelength is 495nm and the blue light intensity is 180Lux. The carbon dioxide concentration was 501 PPM during the first 1 to 7 days of the light culture period, and 1201 PPM during the 8th to 10th days of the light culture period.

[0025] The following experiments further demonstrate the beneficial effects of the present invention.

[0026] The main instruments for the culture and result analysis of *Cicada Flower* strains include: CN-LQC50001 electronic balance, Shanghai Yaoxin Electronic Technology Co., Ltd.; MLS-3750 sterilizer, Sanyo Corporation, Japan; VS-1300L-U clean bench, Suzhou Antai Air Technology Co., Ltd., Sujing Group; SPH-2102 constant temperature incubator shaker, Shanghai Shiping Co., Ltd.; GLF5-DL-2 multi-functional liquid spawn quantitative inoculation machine for edible fungi, Beijunwang Equipment Co., Ltd.; CT-C-0 internal and external stainless steel hot air circulating oven, China Fanqun Drying Equipment Co., Ltd.; YXQ.WF22-0, 8SZ-00 horizontal rectangular pressure steam sterilizer, Hunan Gongchuang Medical Equipment Co., Ltd.; QE-300 high-speed pulverizer, Zhejiang Yili Industry and Trade Co., Ltd.

[0027] The system includes a QTRAP™ 5500 high-performance liquid chromatography-tandem mass spectrometer (equipped with an Exion LC liquid chromatography system and a QTRAP™ 5500 mass spectrometer, model HPLC-MS / MS, manufactured by AB Sciex, USA), a solid phase extraction (SPE) column (200mg, 3mL, manufactured by Waters, USA), a biosafety cabinet (manufactured by Beijing Dongfang Zhaosheng Co., Ltd.), a biochemical incubator, an analytical balance, an optical microscope, and a beat homogenizer (manufactured by Interscience, France).

[0028] RQ22-480J Ultrasonic Cleaner (manufacturer: Shanghai Rongwei Instrument Co., Ltd.); DKB-10 Refrigerated Circulating Water Bath (manufacturer: Changzhou Huapuda Teaching Instrument Co., Ltd.); ZNG50-1000 Vacuum Concentrator (manufacturer: Wenzhou Yunchi Technology Co., Ltd.); e2695 High Performance Liquid Chromatograph (manufacturer: Waters Corporation, USA); LC-20AR Semi-Preparative High Performance Liquid Chromatograph (manufacturer: Shimadzu Corporation, Japan).

[0029] The main reagents and materials used in the culture and analysis of *Cicada Flower* strains include: BEA standard (CAS: 26048-05-5), methanol, acetonitrile, and ammonium acetate were all chromatographically pure, and ultrapure water (prepared by a laboratory ultrapure water system). Methanol (chromatographically pure, manufacturer: Anhui Tiandi High Purity Solvent Co., Ltd.); N6-(2-hydroxyethyl)-adenosine standard, cordycepin standard, ammonium acetate, and formic acid (analytical grade, manufacturer: Beijing Solarbio Science & Technology Co., Ltd.). Oats, wheat, rice, sucrose, and potatoes were commercially available.

[0030] In the preliminary culture experiment, the culture medium used was a liquid culture medium, which included: 200g of 20% potato, 20g of 2% sucrose, and 1000mL of distilled water.

[0031] The experimental method includes the following steps: 1.2.1 Preparation of inoculated spore suspension The mycelia or conidia from the slant culture were inoculated into sterilized conical flasks under aseptic conditions and cultured in a shaker at 120 rpm and 25°C for 4 days.

[0032] 1.2.2 Effects of different light levels on Cordyceps militaris Steam the oats, weigh 1250g of oats and place them in a culture dish. Mix the oats thoroughly with gloved hands, seal with plastic film, and place in a 121℃ rectangular pressure steam sterilizer for 30 minutes. Prepare OW7 / 3 and OR7 / 3 culture media in the same way. Under aseptic conditions, pour the mycelium cultured on a shaker into a sterilized 1000mL filamentous bottle to prepare 3000mL. Connect the prepared inoculum suspension to the input end of a multi-functional quantitative inoculator, insert the inoculation needle into the center of the plastic film, and spray quantitatively. Place in a dark culture room for cultivation for no more than 10 days, maintaining 24℃ and 85% humidity. Set up five different light sources (white, red, yellow, green, and blue light). After the mycelium has spread, transfer to different light-lit culture rooms for further cultivation. Fruiting bodies can be collected and dried.

[0033] 1.2.3 Effects of different light intensities on Cordyceps militaris Based on the selection of the best light wavelength according to 2.2.2, the same culture conditions were used in the early stage. When switching to light culture, the light intensity was set to 60, 120, 180, 240 and 300 lux to cultivate the fruiting bodies. When the fruiting bodies matured, they were harvested for later use.

[0034] 1.2.4 Determination of the dry weight of fruiting bodies Weigh the collected fruiting bodies, dry them in an oven at 60°C, pack them into plastic bags, take out the fruiting bodies and put them into a grinder to grind them until they can pass through a 200-mesh sieve, collect 5g of the sieved fine powder into plastic bags, seal them with a sealing machine and number them, and wait for testing.

[0035] 1.2.5 Determination of Beauveria bassiana content The method used was as specified in the National Health Commission Announcement (No. 9 of 2020).

[0036] 1.2.6 Determination of Polysaccharide Content According to the industry standard NY / T 1676-2023 "Determination of Crude Polysaccharides in Edible Fungi - Spectrophotometric Method". Procedure: After hot water extraction and alcohol precipitation, the absorbance of the sample was measured using a spectrophotometer. The polysaccharide content was calculated using glucose as a standard.

[0037] 1.2.7 N 6 Determination of 2-hydroxyethyl adenosine content References: Ai Renli, Tan Aijuan, Lü Shiming. *Cicada Flower N* 6Optimization of extraction process and structural identification of N6-(2-hydroxyethyl)adenosine (HEA) [J]. Journal of Edible Fungi, 2023, 30(02):65-74.

[0038] The results and analysis are as follows: 2.1 Effect of inoculum size on the growth of Cordyceps militaris mycelium in automated production Under otherwise identical culture conditions, increasing the inoculum size is beneficial for mycelial growth and reduces contamination rates in automated production. As shown in Table 1, a 20% inoculum size results in mycelial coverage 1 day earlier than 10% and 2 days earlier than 5%, with a significantly reduced contamination rate (0% contamination rate). There is no significant difference between 30% and 20% inoculum sizes. Using an inoculum size of 20% or higher not only shortens the mycelial culture time but also increases yield by reducing the contamination rate. Since it is liquid inoculation, an inoculum size of 35% would result in excessive moisture, which is detrimental to solid-state fermentation, requiring 8 days for mycelial coverage. Therefore, an inoculum size of 20-30% is preferred.

[0039] Table 1. Effect of inoculum size on the mycelial growth of Cordyceps militaris.

[0040] Note: Each treatment consisted of 10 pots, repeated 3 times. P=0.01 (meaning that if the null hypothesis is true, the probability of observing the results shown in Table 1 with such or greater differences is only 1%).

[0041] 2.2 Effects of Culture Medium Formulation on the Growth of Cordyceps sinensis Fruiting Bodies in Automated Production Under otherwise identical culture conditions, adjusting the culture medium formulation is beneficial for inhibiting the production of BEA in fruiting bodies. Buckwheat is beneficial for increasing fruiting body yield but not for inhibiting BEA production. Considering all factors, an oat:buckwheat ratio of 4:1 is the optimal choice, resulting in high yield and low BEA content, as shown in Table 2.

[0042] Table 2 Effects of culture medium ratio on the growth of Cordyceps sinensis fruiting bodies

[0043] Note: Each treatment was repeated 3 times, P=0.01.

[0044] Both oats and buckwheat are common commercially available grains with low costs, which is conducive to industrial production. A specific combination of oats and buckwheat produces a synergistic effect, increasing yield while controlling BEA content. Furthermore, it provides the optimal nutrient ratio for mycelial growth and fruiting body development, promoting the accumulation of active ingredients.

[0045] 2.3 Effects of cultivation temperature and temperature difference on the growth of Cordyceps sinensis fruiting bodies in automated production Under otherwise identical culture conditions, a lower culture temperature is beneficial for inhibiting the production of BEA in Cordyceps sinensis fruiting bodies, and a greater temperature difference between dark and light culture is beneficial for reducing the BEA content of fruiting bodies and increasing the yield of fruiting bodies, as shown in Table 3.

[0046] Table 3. Effects of temperature on the growth of Cordyceps militaris

[0047] Note: Each treatment consisted of 10 pots, repeated 3 times, P=0.01.

[0048] This application employs a combination of "dark culture temperature of 16 to 20°C" and "light culture temperature of 20 to 24°C" to ensure a temperature difference of 2-4°C. Experiments show that the combination of 16°C dark culture and 20°C light culture yields the lowest BEA content and a higher fruiting body yield.

[0049] 2.4 Effects of different light levels on the yield and BEA content of Cordyceps militaris fruiting bodies in automated production Under identical culture conditions, no fruiting body differentiation was observed under red and yellow light (e.g.) Figure 1 As shown), the fruiting bodies obtained by culturing under other different lighting conditions (white light, blue light, and green light) (such as...) Figure 1 (As shown) The samples were dried, weighed, and tested for beauvericin (BEA). The results are shown in Table 4 and... Figure 2 As shown, the dry weight of fruiting bodies under blue light is significantly higher than that under green light and white light, while the content of beauveria bassiana is significantly lower than that under green light and white light. The dry weight of fruiting bodies under green light is the lowest, while the content of beauveria bassiana is the highest. The dry weight of fruiting bodies under white light is moderate, and the content of beauveria bassiana is moderate. Therefore, blue light is preferred.

[0050] Table 4. Dry weight and BEA content of fruiting bodies cultured under different light conditions.

[0051] 2.5 Effects of different blue light intensities on automated production of Cordyceps militaris The results under different blue light irradiation conditions are shown in Table 5 and... Figure 3As shown, the highest Beauveria bassiana content was observed under 60 lux light intensity; the content was slightly lower under 120 lux light intensity, already below the limit of 3 mg / kg; the content was even lower at 180 lux, at 1.574 ± 0.3 mg / kg, far below the limit of 3 mg / kg; the lowest content was observed under 240 lux light intensity, at 0.316 ± 0.137 mg / kg. There was no significant difference in Beauveria bassiana content between 300 lux and 240 lux light intensity. Therefore, 180 lux-300 lux is a suitable blue light intensity, and 180-240 lux is preferred to ensure the lowest BEA content while saving on electricity costs.

[0052] Table 5. BEA content (mg / kg) of fruiting bodies cultured under blue light with different light intensities.

[0053] 2.6 Blue light is beneficial for the accumulation of effective components in the fruiting bodies of Cordyceps militaris. This experiment also detected the polysaccharide and N6-(2-hydroxyethyl) adenosine content in fruiting bodies obtained under different light conditions. The results showed that there was no significant difference in polysaccharide content between blue and white light irradiation, but both were significantly higher than under green light. Under blue light irradiation, the N6-(2-hydroxyethyl) adenosine content in the fruiting bodies was significantly higher than under both green and white light irradiation. Polysaccharides and N6-(2-hydroxyethyl) adenosine are the main active ingredients of Cordyceps militaris, and both accumulate the most under blue light. Therefore, blue light was the preferred light source. The results are shown in Table 6. Figure 4 Table 7 and Figure 5 As shown.

[0054] Table 6. Polysaccharide content (mg / g) of fruiting bodies cultured under different light conditions

[0055] Table 7. N6-(2-hydroxyethyl)adenosine (HEA) content (mg / 100g) in fruiting bodies cultured under different light conditions

[0056] This application also provides the application of the above-described Cordyceps militaris strain in the preparation of food or functional food ingredients, such as powders, capsules, beverages, etc.

[0057] It should be noted that the terms "first," "second," and similar terms used in this application specification and claims do not indicate any order, quantity, or importance, but are merely used to distinguish different components. Similarly, "a" or "one," and similar terms do not indicate a quantity limitation, but rather indicate the presence of at least one. "A plurality" or "several" indicates at least two. Unless otherwise stated, terms such as "front," "back," "left," "right," "lower," and / or "upper" are for illustrative purposes only and are not limited to a location or spatial orientation. Terms such as "comprising" or "including" indicate that the elements or objects preceding "comprising" encompass the elements or objects listed following "comprising" or "including" and their equivalents, and do not exclude other elements or objects. Terms such as "connected" or "linked" are not limited to physical or mechanical connections, but can include electrical connections, whether direct or indirect.

[0058] The singular forms “a,” “the,” and “the” used in this application specification and appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0059] It should be understood that those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for culturing a *C. coli* strain, wherein the *C. coli* strain was deposited on December 29, 2016, at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC NO: 13572, characterized in that... The cultivation method includes the following steps: 1) Inoculate the *Cicada Flower* strain into a culture medium consisting of 75-85 parts by weight of oats and 15-25 parts by weight of buckwheat, with an inoculation amount ≥20%; 2) First, incubate in the dark for 5-7 days at a temperature of 16-20℃; then, incubate in light for 10-12 days. During the light incubation period, alternate between 12-18 hours of blue light irradiation and 12-6 hours of darkness daily. The temperature during the light incubation period should be 20-24℃, with the light incubation temperature being ≥4℃ higher than the dark incubation temperature. The blue light wavelength should be 450-495nm, and the blue light intensity should be 120-300 Lux. The relative humidity during the dark incubation period should be 75%-85%, and the relative humidity during the light incubation period should be 80%-100%. The carbon dioxide concentration was 501-1100 PPM during the first 1 to 7 days of the light culture process, and 1201-3000 PPM during the 8th to 12th days of the light culture process.

2. The method for culturing a *Cercis cicadae* strain as described in claim 1, characterized in that... In step 1): the culture medium consists of 80-82 parts oats and 20-18 parts buckwheat.

3. The method for culturing a *Cercis cicadae* strain as described in claim 1, characterized in that... In step 1): the thickness of the culture medium is 1.5-2.5 cm, and the inoculation amount is 20-30%.

4. The method for culturing the *Cicada Flower* strain as described in claim 1, characterized in that... In step 2): the blue light wavelength is 465-480nm and the blue light intensity is 180-240Lux.

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