A method for obtaining auricularia auricula-judae fruiting bodies through mixed strains

Abstract

This invention provides a method for obtaining *Auricularia auricula-judae* fruiting bodies through a mixed culture. The method includes: (1) culturing yeast-like spores; (2) culturing *Trichoderma harzianum*; and (3) preparing a mixed culture solution. Auricularia auricula-judae spores cultured on a culture medium mixed with *Trichoderma harzianum* produce fruiting bodies in an agaric culture. The mixed culture solution produces fruiting bodies in 18-20 days in a cultivation bag and in 23-25 ​​days in a primary culture bottle, with a high fruiting rate and good quality. This method allows for direct inoculation of the liquid culture onto the culture medium. After fruiting, the F1 generation is obtained. Using the F1 generation as a primary culture for transfer can solve the subculturing problem, satisfying both the requirements for single-use pure liquid inoculation cultivation and the source of culture for solid-state spawn production.

Description

Technical Field

[0001] This invention belongs to the field of edible fungus cultivation technology, specifically relating to a method for obtaining auricularia auricula-judae fruiting bodies through mixed strains. Background Technology

[0002] Golden ear fungus, also known as brain-type silver ear or golden silver ear, belongs to the genus Tremella of the family Tremellaceae in the phylum Basidiomycota. It is a precious edible and medicinal fungus resource, mainly distributed in southwestern my country. The fruiting body of golden ear fungus has a brain-like appearance, a golden color, and is rich in gelatinous substance, with a smooth and refreshing texture. Clinical studies have found that golden ear fungus polysaccharides have effects such as lowering blood sugar and blood lipids, enhancing immunity, anticoagulation, and anti-tumor properties. Golden ear fungus is a delicacy among edible fungi, containing abundant fat, protein, and various trace elements such as sulfur, phosphorus, manganese, and iron. It is also a health and beauty product with broad development prospects.

[0003] Due to the extreme scarcity of wild golden ear fungus resources, the supply cannot meet market demand. To ensure the sustainable utilization of this precious resource, scientists began exploring artificial cultivation of golden ear fungus in the 1980s. However, the complex life cycle of golden ear fungus, including a large cycle of one sexual generation and several smaller cycles of asexual generations, along with the interaction of the parasitic fungus *Trichoderma*, makes cultivation difficult to control artificially. Its unique physiological and seed production characteristics mean that the fungal strain is currently unstable during cultivation, making it susceptible to contamination by other fungi in the early stages of primordia formation. This leads to fungal rot and uneven emergence, severely impacting yield and bioconversion rate, and hindering the large-scale promotion of substrate-based golden ear fungus cultivation.

[0004] Currently, most auricularia auricula cultivation methods are based on a liquid + solid model, which involves separately cultivating the liquid spawn and the auricularia auricula fruiting bodies. The primary spawn cultivation of auricularia auricula fruiting bodies requires 30-40 days, which is time-consuming and requires 4-5 times the labor and 9-10 times the glassware (mainly spawn bottles and conical flasks) compared to liquid spawn preparation. Furthermore, the primary spawn preparation method for fruiting bodies has strict requirements for subculturing; multiple subculturings may lead to reduced spawn quality, resulting in poor resistance and reduced yield (see...). Figure 1 );

[0005] Therefore, it is necessary to provide a method that can satisfy both the requirements for single-use pure liquid inoculation and the requirements for solid-state spawn production. After long-term experimental research, our team has developed a method for obtaining *Auricularia auricula-judae* fruiting bodies through mixed spawn. This method allows for direct inoculation of the liquid spawn onto the cultivation substrate, yielding F1 generation (first generation) spawn after fruiting. The F1 generation is then used as a transfer strain for primary spawn preparation, thus solving the subculturing problem (see...). Figure 2 It also solves the existing problems of high manpower consumption, long time consumption, and high material consumption. Summary of the Invention

[0006] The purpose of this invention is to provide a method for obtaining fruiting bodies of *Auricularia auricula-judae* through mixed strains.

[0007] This invention is achieved through the following technical solution:

[0008] The present invention discloses a method for obtaining auricularia auricula-judae fruiting bodies through mixed strains, comprising the following steps:

[0009] (1) Culture of yeast-like spores: Pick yeast-like spores on PDA medium and culture them in the dark at 23-26℃ for 5-12 days. Then transfer them to a 1000mL Erlenmeyer flask containing a 500mL culture medium formula and culture them at 23-26℃ and 140-160r / min for 4-8 days. When the flask shows a white suspension, it is ready for use.

[0010] (2) Culture of *Trichoderma truncatum*: Pick *Trichoderma truncatum* onto PDA medium to activate it. When the hyphae grow to 2 / 3 of the culture dish, transfer it to a 1000mL Erlenmeyer flask containing a 500mL culture medium formula. Culture at 23-26℃ and 140-160r / min for 4-8 days. When the bacterial solution in the shake flask turns from white to red, take it out for use.

[0011] (3) Preparation of mixed bacterial solution: Mix the above-mentioned cultured spores and *Trichoderma harzianum* bacterial solutions at a volume ratio of 1:1 in a clean bench, place them on a shaker at 23-26℃ and 140-160r / min overnight, and they can be used for inoculation of cultivation bags and original culture bottles.

[0012] (4) Ear emergence: Inoculate the cultured mixed strain into the original culture bottle or cultivation bag at an inoculation rate of 0.2-0.4% of the dry weight of the culture medium. The young ears will appear after culturing at a temperature of 19-23℃ for 23-25 ​​days and 18-20 days respectively.

[0013] The method of the present invention includes the following steps:

[0014] (1) Culture of yeast-like spores: Pick yeast-like spores on PDA medium and culture them in the dark at 25°C for 7-10 days. Then transfer them to a 1000mL Erlenmeyer flask containing a 500mL culture medium formula and culture them at 25°C and 150r / min for 5-7 days. When the flask shows a white suspension, it is ready for use.

[0015] (2) Culture of *Trichoderma truncatum*: Pick *Trichoderma truncatum* onto PDA medium to activate it. When the hyphae grow to 2 / 3 of the culture dish, transfer it to a 1000mL Erlenmeyer flask containing a 500mL culture medium formula. Culture at 25℃ and 150r / min for 5-7 days. When the bacterial solution in the shake flask turns from white to red, take it out for use.

[0016] (3) Preparation of mixed bacterial solution: Mix the above-mentioned cultured spores and *Trichoderma harzianum* bacterial solutions in a clean bench and place them on a shaker at 25°C and 150 r / min overnight. The mixture can then be used to inoculate cultivation bags and original culture bottles.

[0017] (4) Ear emergence: Inoculate the cultured mixed strain into the original culture bottle or cultivation bag at an inoculation rate of 0.2-0.4% of the dry weight of the culture medium. The young ears will appear after culturing at a temperature of 19-23℃ for 23 days and 18 days respectively.

[0018] The formula of the exclusive culture medium described in this invention is as follows: 18-28g glucose, 1-4g peptone, 1-4g yeast extract, 1-4g potassium dihydrogen phosphate, 0.5-2.0g magnesium sulfate, and 1L distilled water.

[0019] Preferably, the formula of the exclusive culture medium of the present invention is as follows: 20-26g glucose, 1-3.5g peptone, 1-3g yeast extract, 1-3g potassium dihydrogen phosphate, 0.6-1.5g magnesium sulfate, and 1L distilled water.

[0020] A further preferred embodiment of the culture medium of the present invention is as follows: 24-26g glucose, 2.5-3.5g peptone, 2-3g yeast extract, 1-2g potassium dihydrogen phosphate, 0.6-1.0g magnesium sulfate, and 1L distilled water.

[0021] More preferably, the formula of the exclusive culture medium of the present invention is as follows: 26g glucose, 3.5g peptone, 2g yeast extract, 1g potassium dihydrogen phosphate, 0.6g magnesium sulfate, and 1L pure water.

[0022] In this invention, the activation temperature in step (2) is 18-28℃ and the activation time is 4-10 days.

[0023] Preferably, the activation temperature in step (2) of the present invention is 20-25°C, and the activation time is 5-8 days.

[0024] In this invention, the mixing in step (3) is: mixing at a volume ratio of 1-2:1.

[0025] Preferably, the mixing in step (3) of the present invention is: mixing at a volume ratio of 1:1.

[0026] The beneficial effects of this invention are:

[0027] 1. The method of the present invention mixes the buds of Auricularia auricula-judae cultured in the culture medium with the fungal strain of Mycorrhiza pubescens. The mixed fungal solution can produce fruiting bodies in 18-20 days in the cultivation bag and in 23-25 ​​days in the original culture bottle. The fruiting body production rate is high and the quality of the obtained Auricularia auricula-judae fruiting bodies is good.

[0028] 2. This method allows direct inoculation of liquid spawn onto the culture medium. After fruiting, F1 generation (first generation) spawn is obtained. The F1 generation is then used as the primary strain for transfer in spawn preparation. This not only solves the problems of strict requirements for subculturing, numerous subculturing cycles, and potential reduction in spawn quality leading to poor resistance and yield loss, but also addresses the issues of high labor, time, and material costs associated with existing methods. It satisfies both the requirements for single-use pure liquid inoculation and the need for a suitable spawn source for solid-state spawn production.

[0029] 3. The optimal culture medium formula selected by the method of this invention is: 26g glucose, 3.5g peptone, 2g yeast extract, 1g potassium dihydrogen phosphate, 0.6g magnesium sulfate, and 1L pure water. This results in intact fruiting bodies of *Auricularia auricula-judae* with a pure color; moderate mycelium content; rapid growth rate; no obvious yellow secretions on the surface of the culture medium around the fruiting bodies; and a fruiting body survival rate of 96%. Attached Figure Description

[0030] Figure 1 The effect of the number of subcultures on strain quality (from left to right: 1 subculture, 2 subcultures, 5 subcultures, 6 subcultures)

[0031] Figure 2 Chart showing the change in the number of days from the time of transfer of liquid seed larvae to the original seed bottle (from left to right: 7 days after larvae emergence, 14 days after larvae emergence, 21 days after larvae emergence, 28 days after larvae emergence, 35 days after larvae emergence).

[0032] Figure 3 : Liquid seed inoculated into the original seed bottle (24 days) Seedling emergence diagram

[0033] Figure 4 : Liquid seed grafting into the cultivation bag (20 days) - Ear emergence diagram

[0034] Figure 5 Image of mixed hyphae under a microscope

[0035] Figure 6 Morphological diagram of mixed bacterial strains under a microscope

[0036] Figure 7 Image of the culture after the liquid culture of *Bacillus auricula-judae* spores and *Trichoderma harzianum* was completed, mixed at a 1:1 ratio and incubated overnight.

[0037] Figure 8 Macroscopic morphology diagram of liquid bacterial strains Detailed Implementation

[0038] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0039] Example 1: Method for obtaining fruiting bodies of *Auricularia auricula-judae* through mixed strains

[0040] (1) Culture of yeast-like spores: Pick yeast-like spores on PDA medium and culture them in the dark at 25°C for 8 days. Then transfer them to a 1000mL Erlenmeyer flask containing a 500mL culture medium formula and culture them at 25°C and 150r / min for 6 days. When the flask shows a white suspension, it is ready for use.

[0041] (2) Culture of *Trichoderma truncatum*: *Trichoderma truncatum* was picked and activated on PDA medium. It was activated at 23°C for 7 days. When the mycelium grew to 2 / 3 of the culture dish, it was transferred to a 1000mL Erlenmeyer flask containing a 500mL culture medium formula. It was cultured at 25°C and 150r / min for 6 days. When the bacterial solution in the shake flask turned from white to red, it was taken out for use.

[0042] The above-mentioned specific culture medium formula is as follows: 26g glucose, 3.5g peptone, 2g yeast extract, 1g potassium dihydrogen phosphate, 0.6g magnesium sulfate, and 1L distilled water;

[0043] (3) Preparation of mixed bacterial solution: The above-cultured spores and *Trichoderma harzianum* bacterial solutions were mixed in a volume ratio of 1:1 in a clean bench and placed on a shaker at 25℃ and 150r / min overnight.

[0044] (4) Ear emergence: Inoculate the cultured mixed strain into the original culture bottle or cultivation bag at an inoculation rate of 0.3% of the dry weight of the culture medium. The young ears will appear after culturing at 21℃ for 23 days and 18 days respectively.

[0045] Example 2: Method for obtaining auricularia auricula-judae fruiting bodies through mixed strains

[0046] (1) Culture of yeast-like spores: Pick yeast-like spores on PDA medium and culture them in the dark at 25°C for 7 days. Then transfer them to a 1000mL Erlenmeyer flask containing a 500mL culture medium formula and culture them at 25°C and 150r / min for 5 days. When the flask shows a white suspension, it is ready for use.

[0047] (2) Culture of *Trichoderma truncatum*: *Trichoderma truncatum* was picked and activated on PDA medium. It was activated at 20°C for 5 days. When the mycelium grew to 2 / 3 of the culture dish, it was transferred to a 1000mL Erlenmeyer flask containing a 500mL culture medium formula. It was cultured at 25°C and 150r / min for 5 days. When the bacterial solution in the shake flask turned from white to red, it was taken out for use.

[0048] The above-mentioned specific culture medium formula is as follows: 26g glucose, 3.5g peptone, 2g yeast extract, 1g potassium dihydrogen phosphate, 0.6g magnesium sulfate, and 1L distilled water;

[0049] (3) Preparation of mixed bacterial solution: The above-cultured spores and *Trichoderma harzianum* bacterial solutions were mixed in a volume ratio of 1:1 in a clean bench and placed on a shaker at 25℃ and 150r / min overnight.

[0050] (4) Ear emergence: Inoculate the cultured mixed strain into the original culture bottle or cultivation bag at an inoculation rate of 0.2-0.4% of the dry weight of the culture medium. The young ears will appear after culturing at 19℃ for 23 days and 18 days respectively.

[0051] Example 3: Method for obtaining auricularia auricula fruiting bodies through mixed strains

[0052] (1) Culture of yeast-like spores: Pick yeast-like spores on PDA medium and culture them in the dark at 25°C for 10 days. Then transfer them to a 1000mL Erlenmeyer flask containing a 500mL culture medium formula and culture them at 25°C and 150r / min for 7 days. When the flask shows a white suspension, it is ready for use.

[0053] (2) Culture of *Trichoderma truncatum*: *Trichoderma truncatum* was picked and activated on PDA medium. It was activated at 25°C for 8 days. When the mycelium grew to 2 / 3 of the culture dish, it was transferred to a 1000mL Erlenmeyer flask containing a 500mL culture medium formula. It was cultured at 25°C and 150r / min for 7 days. When the bacterial solution in the shake flask turned from white to red, it was taken out for use.

[0054] The above-mentioned specific culture medium formula is as follows: 26g glucose, 3.5g peptone, 2g yeast extract, 1g potassium dihydrogen phosphate, 0.6g magnesium sulfate, and 1L distilled water;

[0055] (3) Preparation of mixed bacterial solution: The above-cultured spores and *Trichoderma harzianum* bacterial solutions were mixed in a volume ratio of 1:1 in a clean bench and placed on a shaker at 25℃ and 150r / min overnight.

[0056] (4) Ear emergence: Inoculate the cultured mixed strain into the original culture bottle or cultivation bag at an inoculation rate of 0.4% of the dry weight of the culture medium. The young ears will appear after culturing at 23℃ for 23 days and 18 days respectively.

[0057] Example 4: Method for obtaining auricularia auricula-judae fruiting bodies through mixed strains

[0058] (1) Culture of yeast-like spores: Pick yeast-like spores on PDA medium and culture them in the dark at 23°C for 5 days. Then transfer them to a 1000mL Erlenmeyer flask containing a 500mL culture medium formula and culture them at 23°C and 140r / min for 4 days. When the flask shows a white suspension, it is ready for use.

[0059] (2) Culture of *Trichoderma truncatum*: *Trichoderma truncatum* was picked and activated on PDA medium. It was activated at 18°C ​​for 10 days. When the mycelium grew to 2 / 3 of the culture dish, it was transferred to a 1000mL Erlenmeyer flask containing a 500mL culture medium formula. It was cultured at 23°C and 140r / min for 4 days. When the bacterial solution in the shake flask changed from white to red, it was taken out for use.

[0060] The above-mentioned specific culture medium formula is as follows: 18g glucose, 1g peptone, 1g yeast extract, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate, and 1L distilled water;

[0061] (3) Preparation of mixed bacterial solution: The above-cultured spores and *Trichoderma harzianum* bacterial solutions were mixed at a volume ratio of 1:1 in a clean bench and placed on a shaker at 23°C and 160 r / min overnight.

[0062] (4) Ear emergence: Inoculate the cultured mixed strain into the original culture bottle or cultivation bag at an inoculation rate of 0.2% of the dry weight of the culture medium. The young ears will appear after culturing at 19℃ for 23 days and 18 days respectively.

[0063] Example 5: Method for obtaining auricularia auricula-judae fruiting bodies through mixed strains

[0064] (1) Culture of yeast-like spores: Pick yeast-like spores on PDA medium and culture them in the dark at 26℃ for 12 days. Then transfer them to a 1000mL Erlenmeyer flask containing a 500mL culture medium formula and culture them at 26℃ and 160r / min for 8 days. When the flask shows a white suspension, it is ready for use.

[0065] (2) Culture of *Trichoderma truncatum*: *Trichoderma truncatum* was picked and activated on PDA medium. It was activated at 28°C for 4 days. When the mycelium grew to 2 / 3 of the culture dish, it was transferred to a 1000mL Erlenmeyer flask containing a 500mL culture medium formula. It was cultured at 26°C and 160r / min for 8 days. When the bacterial solution in the shake flask changed from white to red, it was taken out for use.

[0066] The above-mentioned proprietary culture medium formula is as follows: 28g glucose, 4g peptone, 4g yeast extract, 4g potassium dihydrogen phosphate, 2g magnesium sulfate, and 1L distilled water;

[0067] (3) Preparation of mixed bacterial solution: The above-cultured spores and *Trichoderma harzianum* bacterial solutions were mixed at a volume ratio of 2:1 in a clean bench and placed on a shaker at 26℃ and 160r / min overnight.

[0068] (4) Ear emergence: Inoculate the cultured mixed strain into the original culture bottle or cultivation bag at an inoculation rate of 0.4% of the dry weight of the culture medium. The young ears will appear after culturing at 23℃ for 23 days and 18 days respectively.

[0069] Example 6: Method for obtaining auricularia auricula fruiting bodies through mixed strains

[0070] (1) Culture of yeast-like spores: Pick yeast-like spores on PDA medium and culture them in the dark at 24℃ for 6 days. Then transfer them to a 1000mL Erlenmeyer flask containing a 500mL culture medium formula and culture them at 24℃ and 150r / min for 6 days. When the flask shows a white suspension, it is ready for use.

[0071] (2) Culture of *Trichoderma truncatum*: *Trichoderma truncatum* was picked and activated on PDA medium. It was activated at 25°C for 6 days. When the mycelium grew to 2 / 3 of the culture dish, it was transferred to a 1000mL Erlenmeyer flask containing a 500mL culture medium formula. It was cultured at 24°C and 150r / min for 6 days. When the bacterial solution in the shake flask changed from white to red, it was taken out for use.

[0072] The specific culture medium formula is as follows: 24g glucose, 1.5g peptone, 2g yeast extract, 2g potassium dihydrogen phosphate, 1.0g magnesium sulfate, and 1L distilled water;

[0073] (3) Preparation of mixed bacterial solution: The above-cultured spores and Trichoderma truncatum bacterial solutions were mixed in a volume ratio of 1:1 in a clean bench and placed on a shaker at 24℃ and 150r / min overnight.

[0074] (4) Ear emergence: Inoculate the cultured mixed strain into the original culture bottle or cultivation bag at an inoculation rate of 0.3% of the dry weight of the culture medium. The young ears will appear after culturing at 22℃ for 23 days and 18 days respectively.

[0075] Example 7: Method for obtaining auricularia auricula fruiting bodies through mixed strains

[0076] (1) Culture of yeast-like spores: Pick yeast-like spores on PDA medium and culture them in the dark at 25°C for 8 days. Then transfer them to a 1000mL Erlenmeyer flask containing a 500mL culture medium formula and culture them at 25°C and 150r / min for 6 days. When the flask shows a white suspension, it is ready for use.

[0077] (2) Culture of *Trichoderma truncatum*: *Trichoderma truncatum* was picked and activated on PDA medium. It was activated at 23°C for 7 days. When the mycelium grew to 2 / 3 of the culture dish, it was transferred to a 1000mL Erlenmeyer flask containing a 500mL culture medium formula. It was cultured at 25°C and 150r / min for 6 days. When the bacterial solution in the shake flask turned from white to red, it was taken out for use.

[0078] The specific culture medium formula is as follows: 24g glucose, 2.5g peptone, 2g yeast extract, 1g potassium dihydrogen phosphate, 1.4g magnesium sulfate, and 1L distilled water;

[0079] (3) Preparation of mixed bacterial solution: The above-cultured spores and *Trichoderma harzianum* bacterial solutions were mixed in a volume ratio of 1:1 in a clean bench and placed on a shaker at 25℃ and 150r / min overnight.

[0080] (4) Ear emergence: Inoculate the cultured mixed strain into the original culture bottle or cultivation bag at an inoculation rate of 0.3% of the dry weight of the culture medium. The young ears will appear after culturing at 20℃ for 23 days and 18 days respectively.

[0081] Example 8: Method for obtaining auricularia auricula-judae fruiting bodies through mixed strains

[0082] (1) Culture of yeast-like spores: Pick yeast-like spores on PDA medium and culture them in the dark at 25°C for 8 days. Then transfer them to a 1000mL Erlenmeyer flask containing a 500mL culture medium formula and culture them at 25°C and 150r / min for 6 days. When the flask shows a white suspension, it is ready for use.

[0083] (2) Culture of *Trichoderma truncatum*: *Trichoderma truncatum* was picked and activated on PDA medium. It was activated at 23°C for 7 days. When the mycelium grew to 2 / 3 of the culture dish, it was transferred to a 1000mL Erlenmeyer flask containing a 500mL culture medium formula. It was cultured at 25°C and 150r / min for 6 days. When the bacterial solution in the shake flask turned from white to red, it was taken out for use.

[0084] The specific culture medium formula is as follows: 25g glucose, 3g peptone, 2g yeast extract, 1g potassium dihydrogen phosphate, 1g magnesium sulfate, and 1L distilled water;

[0085] (3) Preparation of mixed bacterial solution: The above-cultured spores and *Trichoderma harzianum* bacterial solutions were mixed in a volume ratio of 1:1 in a clean bench and placed on a shaker at 25℃ and 150r / min overnight.

[0086] (4) Ear emergence: Inoculate the cultured mixed strain into the original culture bottle or cultivation bag at an inoculation rate of 0.3% of the dry weight of the culture medium. The young ears will appear after culturing at 20℃ for 23 days and 18 days respectively.

[0087] Example 9: Method for obtaining auricularia auricula-judae fruiting bodies through mixed strains

[0088] (1) Culture of yeast-like spores: Pick yeast-like spores on PDA medium and culture them in the dark at 25°C for 8 days. Then transfer them to a 1000mL Erlenmeyer flask containing a 500mL culture medium formula and culture them at 25°C and 150r / min for 6 days. When the flask shows a white suspension, it is ready for use.

[0089] (2) Culture of *Trichoderma truncatum*: *Trichoderma truncatum* was picked and activated on PDA medium. It was activated at 23°C for 7 days. When the mycelium grew to 2 / 3 of the culture dish, it was transferred to a 1000mL Erlenmeyer flask containing a 500mL culture medium formula. It was cultured at 25°C and 150r / min for 6 days. When the bacterial solution in the shake flask turned from white to red, it was taken out for use.

[0090] The specific culture medium formula is as follows: 24g glucose, 3.5g peptone, 3g yeast extract, 1g potassium dihydrogen phosphate, 0.6g magnesium sulfate, and 1L distilled water.

[0091] (3) Preparation of mixed bacterial solution: The above-cultured spores and *Trichoderma harzianum* bacterial solutions were mixed in a volume ratio of 1:1 in a clean bench and placed on a shaker at 25℃ and 150r / min overnight.

[0092] (4) Ear emergence: Inoculate the cultured mixed strain into the original culture bottle or cultivation bag at an inoculation rate of 0.3% of the dry weight of the culture medium. The young ears will appear after culturing at 20℃ for 23 days and 18 days respectively.

[0093] Example 10: Method for obtaining auricularia auricula-judae fruiting bodies through mixed strains

[0094] (1) Culture of yeast-like spores: Pick yeast-like spores on PDA medium and culture them in the dark at 25°C for 8 days. Then transfer them to a 1000mL Erlenmeyer flask containing a 500mL culture medium formula and culture them at 25°C and 150r / min for 6 days. When the flask shows a white suspension, it is ready for use.

[0095] (2) Culture of *Trichoderma truncatum*: *Trichoderma truncatum* was picked and activated on PDA medium. It was activated at 23°C for 7 days. When the mycelium grew to 2 / 3 of the culture dish, it was transferred to a 1000mL Erlenmeyer flask containing a 500mL culture medium formula. It was cultured at 25°C and 150r / min for 6 days. When the bacterial solution in the shake flask turned from white to red, it was taken out for use.

[0096] The specific culture medium formula is as follows: 26g glucose, 2.5g peptone, 1g yeast extract, 1g potassium dihydrogen phosphate, 1.4g magnesium sulfate, and 1L distilled water;

[0097] (3) Preparation of mixed bacterial solution: The above-cultured spores and *Trichoderma harzianum* bacterial solutions were mixed in a volume ratio of 1:1 in a clean bench and placed on a shaker at 25℃ and 150r / min overnight.

[0098] (4) Ear emergence: Inoculate the cultured mixed strain into the original culture bottle or cultivation bag at an inoculation rate of 0.3% of the dry weight of the culture medium. The young ears will appear after culturing at 20℃ for 23 days and 18 days respectively.

[0099] To further verify the feasibility of the present invention, the inventors conducted a series of experiments, as follows:

[0100] I. Experimental Section

[0101] 1. Experimental Materials and Instruments

[0102] 1.1 Main experimental equipment: clean bench, constant temperature incubator, shaker;

[0103] 1.2 Experimental materials: Company-owned *Trichoderma harzianum* strains and spores, stored at 4℃;

[0104] 1.3 Experimental reagents: glucose, peptone, yeast extract, potassium dihydrogen phosphate, magnesium sulfate (all purchased from Best Biotech Co., Ltd.).

[0105] 2. Experimental methods:

[0106] 2.1 Formulation Screening Test of Dedicated Liquid Culture Medium

[0107] 2.1.1 Experiment 1:

[0108] The component selection experiment included five formulations, as shown in Table 1.

[0109] Table 1. Screening and Results of Special Liquid Culture Medium Formulations

[0110]

[0111]

[0112] As a result, the mixed strain cultured with formula 4 performed the best, with the best fruiting quality, good fruiting body elasticity, pure color, and a survival rate of 88%.

[0113] 2.1.2 Experiment 2:

[0114] Based on Experiment 1, Formula 4 was optimized, as shown in Table 2.

[0115] Table 2. Formulation of Special Liquid Culture Medium 4. Optimization, Screening and Results

[0116]

[0117]

[0118] As a result, formula C showed the best overall performance.

[0119] 2.2 Culture of yeast-like spores

[0120] Pick yeast-like spores onto PDA medium and culture them in the dark at 25°C for 7-10 days. Then transfer them to a 1000mL Erlenmeyer flask containing the above formula (500mL). Culture them at 25°C and 150r / min for 5-7 days until a white suspension appears in the flask.

[0121] 2.3 Culture of *Trichoderma harzianum*

[0122] Pick the *Trichoderma harzianum* onto PDA medium for activation. When the mycelium has grown to 2 / 3 of the culture dish, transfer it to a 1000mL Erlenmeyer flask containing the above formula (500mL). Incubate at 25℃ and 150r / min for 5-7 days. When the bacterial solution in the shake flask turns from white to red, remove it for later use.

[0123] 2.4 Preparation of mixed bacterial culture

[0124] Mix the two bacterial solutions at a 1:1 (V / V) ratio in a clean bench and incubate overnight on a shaker at 25°C and 150 rpm. This mixture is then ready for use in inoculating cultivation bags and primary culture bottles. (See...) Figures 7-8 )

[0125] 2.5 Results

[0126] The above method allows the mixed bacterial solution to develop ear-like structures in the original culture bottle after 23-25 ​​days (see...). Figure 3 Ears will appear in the cultivation bag after 18-20 days (see...). Figure 4 The fruiting bodies are intact and of pure color; the mycelium is moderately vigorous, and the growth rate is fast, with no obvious yellow secretions on the surface of the culture medium around the fruiting bodies; the fruiting rate is high, the survival rate of fruiting bodies is 96%, and the quality of the obtained golden ear fruiting bodies is good; it can be used after being cultured in the culture medium at 19-21℃ for 5 days; under a microscope, the mixed strain can be seen to germinate spores, which are tadpole-shaped and interspersed in the mycelium (see...). Figure 5 The prepared mixed culture was inoculated into the original culture bottle or cultivation bag at an inoculation rate of 0.2-0.4% of the dry weight of the culture medium (see...). Figure 6 Young ears can appear after culturing at temperatures of 19-23℃ for 23 days and 18 days, respectively.

[0127] Although the present invention has been described in detail above with general descriptions, specific embodiments and experiments, some modifications or improvements can be made to it based on the present invention, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. A method for obtaining *Auricularia auricula-judae* fruiting bodies through mixed strains, characterized in that, Includes the following steps: (1) Culture of yeast-like spores: Pick yeast-like spores on PDA medium and culture them in the dark at 25°C for 7-10 days. Then transfer them to a 1000mL Erlenmeyer flask containing a 500mL culture medium formula and culture them at 25°C and 150r / min for 5-7 days. When the flask shows a white suspension, it is ready for use. (2) Culture of *Trichoderma truncatum*: Pick *Trichoderma truncatum* onto PDA medium and activate it at 20-25℃ for 5-8 days. When the mycelium grows to 2 / 3 of the culture dish, transfer it to a 1000mL Erlenmeyer flask containing a 500mL culture medium formula and culture at 25℃ and 150r / min for 5-7 days. When the bacterial solution in the shake flask turns from white to red, remove it for later use. (3) Preparation of mixed bacterial solution: The above-cultured spores and *Trichoderma harzianum* bacterial solutions were mixed in a volume ratio of 1:1 in a clean bench and placed on a shaker at 25℃ and 150r / min overnight. (4) Ear emergence: Inoculate the cultured mixed strain into the original culture bottle or cultivation bag at an inoculation rate of 0.2-0.4% of the dry weight of the culture medium. The young ears will appear after culturing at a temperature of 19-23℃ for 23 days and 18 days respectively. The specific culture medium formula is as follows: 26g glucose, 3.5g peptone, 2g yeast extract, 1g potassium dihydrogen phosphate, 0.6g magnesium sulfate, and 1L pure water.

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