A tricholoma caligatum cultivation strain and a preparation process thereof

Abstract

The application discloses a kind of mountain fungus cultivation strain and its preparation process, it utilizes Coprinus radians N2 as mother seed, after liquid medium in conical flask is shaken bed culture, it is carried out in fermentation tank and is enlarged culture, the secondary liquid strain obtained is directly connected into cultivation medium in indoor bacteria cultivation, and mountain fungus cultivation strain is obtained.The application optimizes cultivation medium ratio and preparation process, ensures that upper and lower humidity is uniform, strain grows well, seed is transferred into ground and bacteria is transferred fast and durable, provides high-quality and durable growth nutrition for the growth key link during mountain fungus seed inoculation, germination, growth germination and leaf expansion;Meanwhile, the method for liquid inoculation and preparation of bacteria is adopted, the transfer generation is reduced, the strain activity is high, inoculation is fast and saves labor and material, infection rate is less, easy to survive, and grows fast.The strain cultivated by the method of the application, the strain nutrition supply is more than 90 days, the cultivation strain inactivation is 0%, the low bacteria infection is low, the loss is low, the cost is low, and the growth time is 40-50 days earlier than the current solid strain.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a *Cremastra appendiculata* cultivation strain and a preparation process thereof. Background Art

[0002] Cremastra appendiculata, an orchidaceous plant Cremastra appendiculata (D. Don)Makino> processed medicinal material is commonly known as Maocigu. Its pseudobulb is processed for medicinal use, and the medicinal material "Shancigu (Cremastra appendiculata)" is sweet and slightly pungent in taste, cool in nature, and has the effects of clearing heat and removing toxicity, resolving carbuncle and dissipating stagnation. It is commonly used for carbuncle, swelling, furuncle, scrofula, phlegm nodule, snake and insect bite, and abdominal mass. Modern literature studies have found that the extract of Cremastra appendiculata can inhibit angiogenesis, and this mechanism of action has a good prospect in anti-tumor. It is clinically used for treating liver cirrhosis, esophageal and cardiac cancer obstruction, cervical cancer and other diseases in modern times. Abundant polyphenolic components in Cremastra appendiculata, especially pleionol, bibenzyl components such as pleionin C, pleionin D and pleionol, as well as other active substances such as cremastrine and hexadecanone-3-ol, constitute its strong biological activity basis. Clinical application is mainly focused on resolving phlegm and detoxifying, and it also has anti-tumor, antihypertensive and other effects, which is widely used in treating various diseases. The dosage absorbed by pharmaceutical enterprises as feeding materials is increasing day by day, and the dosage for medicinal formulas in large-scale traditional Chinese medicine hospitals and private hospitals is also increasing. The yield decreases year by year while the dosage increases year by year, and the supply shortage is intensifying.

[0003] Cremastra appendiculata is an orchidaceous plant, whose seeds are very small and large in quantity, with 20,000 to 30,000 seeds in a pod. However, the seeds have no endosperm, leading to an extremely low germination rate in nature. Wild resources are limited while the market demand is strong, resulting in a sharp decline in wild population. At present, no public strain for Cremastra appendiculata cultivation has been disclosed, and the main propagation technologies are limited to two types: the first is cultivation after aseptic germination of Cremastra appendiculata seeds into seedlings; the second is vegetative propagation via pseudobulbs. The first type requires cultivating seedlings in an aseptic environment, and the obtained seedlings need procedures such as acclimatization before planting, which has high technical requirements, high cost, few surviving seedlings, and is not suitable for large-scale cultivation; the second type belongs to vegetative propagation, only one pseudobulb is produced per plant every year, so the propagation efficiency is extremely low. It is understood that the production of Cremastra appendiculata strains in the market is mostly in the experimental stage. At present, most productions adopt the traditional solid strain production mode of first-class strains, second-class strains and third-class strains. The growth period of first-class strains is mostly 10 to 15 days, that of second-class strains is 26 to 27 days, and that of third-class solid strains is about 70 to 80 days. Material mixing requires 5 to 6 workers per 10,000 bags, sterilization requires 2 to 3 workers per 10,000 bags, bagging requires 12 to 15 workers per 10,000 bags, and inoculation requires 34 to 36 workers per 10,000 bags. There are problems such as large labor consumption, increased cost due to the need of plastic bottles for second-class strain production, and long growth cycle. In addition, for traditional solid strains, 5% to 6% of strains at the bag opening are inactivated after sterilization, and mixed bacteria infection accounts for 3% to 4%, with an average of 4% to 5%. There are problems such as easy infection and inactivation, which cause bag damage and increase losses. Summary of the Invention

[0004] The purpose of this invention is to provide a process for preparing a *Cremastra appendiculata* cultivar that is fast, labor-saving, and material-saving, as well as the *Cremastra appendiculata* cultivar prepared using this process.

[0005] To achieve the above objectives, the preparation process of the *Cremastra appendiculata* cultivar spawn used in this invention includes the following steps: Step 1: Place the liquid culture medium in an Erlenmeyer flask, autoclave, cool to room temperature, and inoculate with *Coprinus radiata* (a type of variegated cap). Coprinus radians N2 was cultured on a shaker until a large number of white flocculent hyphae or spherical bacterial balls appeared in the culture medium in the conical flask. After standing, the culture medium became clear without obvious stratification. Under aseptic conditions, no bacterial infection was observed under a microscope. It was then used as a liquid culture.

[0006] Step 2: Place the expansion culture medium in the fermenter, sterilize it under high temperature and high pressure, and then cool it to room temperature. Pour the liquid inoculum from the conical flask in Step 1 into the expansion culture medium in the fermenter for expansion culture. Cultivate until a large number of white flocculent hyphae or spherical mycelia appear in the fermenter. After standing, the culture medium becomes clear, with no obvious layering or floating matter. There is no sour or foul smell when the gas is exhausted, and no bacterial infection is observed under a microscope under aseptic conditions. This is considered a secondary liquid inoculum.

[0007] Step 3: Fill the cultivation substrate into a high-temperature resistant and non-toxic plastic bag using a bagging machine, filling it to 3-6 cm from the bag opening. Insert a 2-3 mm diameter forming tube into the center, attach a buckle, seal the bag with a high-temperature and high-pressure resistant cap, sterilize it under high temperature and high pressure, and then cool it to room temperature.

[0008] Step 4: The bagged culture substrate cooled to room temperature in Step 3 is sent to the inoculation room via conveyor belt. Under sterile conditions, the cap is removed, the plastic bag is opened, the forming tube is pulled out, and the secondary liquid inoculum from the fermenter in Step 2 is injected into the culture substrate through the channel formed by the forming tube using an inoculation gun. The bag is then sealed with the cap.

[0009] Step 5: The bagged cultivation substrate inoculated with the spawn in Step 4 is output from the inoculation room via an automatic conveyor belt, placed in a basket and sent into a dark room with an environment of 15-25℃ and 50%-60% humidity for cultivation. After 5-7 days of cultivation, the basket is turned upside down and then cultivated for another 12-15 days to obtain the spawn of *Cremastra appendiculata*.

[0010] In step 1 above, the liquid culture medium is prepared by adding 2.5-3.5g of potato starch, 1.5-2.5g of soybean flour, 0.8-1.2g of corn flour, 2.5-3.5g of peptone, 15-25g of glucose monohydrate, 0.7-0.75g of potassium dihydrogen phosphate, 1.5-2.5g of magnesium sulfate, and 2.5-3.5g of yeast extract to every 1000mL of purified water, with a pH of 7.5-8.

[0011] In step 1 above, the high-temperature and high-pressure sterilization temperature is 120-125℃, the pressure is 0.12-0.15MPa, and the sterilization time is 30-40min.

[0012] In step 1 above, the radially radiated gibbon ( Coprinus radians N2 was deposited on November 2, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 40970.

[0013] Furthermore, in step 1 above, the radially radiated gromwell ( Coprinus radians The inoculum size of N2 is 2.5% to 3% of the volume of liquid culture medium. The culture temperature on a shaker is 20 to 25°C, and the culture time is 36 to 72 hours.

[0014] In step 2 above, the expanded culture medium is prepared by adding 580-620g of potato starch, 580-620g of soybean flour, 180-220g of corn flour, 580-620g of peptone, 3800-4200g of glucose monohydrate, 130-150g of high-purity potassium dihydrogen phosphate, 380-420g of magnesium sulfate, and 580-620g of yeast extract to every 200L of purified water, with a pH of 7.5-8.

[0015] In step 2 above, the high-temperature and high-pressure sterilization temperature is 120-125℃, the pressure is 0.12-0.15MPa, and the sterilization time is 70-80min.

[0016] Furthermore, in step 2 above, the amount of liquid bacterial culture added is 0.3% to 0.6% of the volume of the culture medium, the temperature of the expansion culture is 20 to 25°C, and the culture time is greater than 48 to 72 hours.

[0017] In step 3 above, the cultivation substrate is a mixture of substrate and water at a mass ratio of 1:1.1-1.5, with a pH of 6.5-7. The substrate's mass percentage composition is: 50%-60% sawdust, 20%-28% cottonseed hull particles, 17%-20% wheat bran, 1%-1.5% gypsum, 1%-1.5% white sugar, and 0.3%-0.6% glucose. The cultivation substrate is prepared by mixing sawdust, cottonseed hull particles, wheat bran, and gypsum according to the substrate composition; dissolving white sugar and glucose in water; and then mixing the two together thoroughly. The diameter of the sawdust is 0.8-1.2 cm.

[0018] In step 3 above, the high-temperature and high-pressure sterilization temperature is 120-125℃, the pressure is 0.12-0.15MPa, and the sterilization time is 150-200min.

[0019] In step 3 above, the high-temperature resistant and non-toxic plastic bag is a special bag for edible fungi made of polypropylene or polyethylene, and the high-temperature and high-pressure resistant cap is a cap made of polypropylene containing air filter cotton.

[0020] In step 4 above, the amount of secondary liquid inoculum injected is 0.01% to 0.02% of the weight of the cultivation substrate.

[0021] The beneficial effects of this invention are as follows: This invention utilizes the radiated hair of the small ghost umbrella ( Coprinus radians N2 is used as the mother culture. After being cultured in a shaker with liquid culture medium in an Erlenmeyer flask, it is expanded in a fermenter. The resulting secondary liquid spawn is inoculated into the cultivation substrate and cultured indoors to obtain the *Cremastra appendiculata* spawn. This invention optimizes the cultivation substrate ratio and preparation process to ensure uniform humidity, good spawn growth, and rapid and long-lasting inoculation in the ground. It provides high-quality and long-lasting nutrients for the key growth stages of *Cremastra appendiculata* seeds during inoculation, germination, growth, and leaf expansion. At the same time, the liquid inoculation method, which involves primary liquid culture, secondary expansion culture, and direct inoculation culture, reduces the number of subculturing generations. The spawn has high viability, fast inoculation, saves labor and materials, has a low contamination rate, and the spawn is easy to survive and grows quickly. Compared to traditional solid spawn, the process of this invention requires 2-3 man-days per 10,000 bags for mixing, 2-3 man-days per 10,000 bags for sterilization, 12-15 man-days per 10,000 bags for bagging, and 11-18 man-days per 10,000 bags for inoculation, saving half the labor. Cultivation at 20-25℃ and 50-60% humidity is 40-50 days shorter than with traditional spawn. The spawn cultivated using this method has a nutrient supply period of over 90 days, is rich in nutrients, and has a 0% inactivation rate, with a contamination rate of 0.3%-0.5% (average 0.3%-0.5%), reducing losses by 10% and saving 18-20 man-days per 10,000 bags per batch. Cultivation at 20-25℃ and 50-60% humidity shortens the growth time by 40-50 days compared to traditional solid spawn. Attached Figure Description

[0022] Figure 1 This is a photo of the traditional solid spawn of *Cremastra appendiculata*.

[0023] Figure 2 This is a photograph taken after the secondary liquid bacterial strain in Example 1 has been cultured in a dark room for 3 days.

[0024] Figure 3 This is a photograph taken after the secondary liquid bacterial strain in Example 1 has been cultured in a dark room for 11 days.

[0025] Figure 4 This is a photograph taken after the secondary liquid bacterial strain in Example 1 has been cultured in a dark room for 13 days.

[0026] Figure 5This is a photograph taken after the secondary liquid bacterial strain in Example 1 has been cultured in a dark room for 14 days.

[0027] Figure 6 The image shows a one-year-old *Cremastra appendiculata* grown using the strain cultivated in Example 1. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to these embodiments. Example

[0029] Step 1: Transfer 600 mL of liquid culture medium into an 800 mL Erlenmeyer flask and sterilize at 125°C and 0.1 MPa–0.15 MPa for 40 min (after venting the autoclave for 20 min, start timing for 40 min when the pressure reaches 0.1 MPa, maintaining the pressure between 0.1 MPa and 0.15 MPa). After sterilization, cool to room temperature and inoculate with *Coprinus radiata* (a type of variegated cap). Coprinus radians N2, with an inoculum size of 2.5%–3% of the liquid culture medium volume, is incubated on a shaker at 22°C for 2–3 days, and the bacterial activity is observed. When a large amount of white flocculent hyphae appear in the culture medium in the conical flask, and the culture medium becomes clear after standing without obvious stratification, and when a sample is taken under aseptic conditions and observed under a 1600x microscope without bacterial infection, it is considered a liquid inoculum. The liquid culture medium is prepared by adding 3g of potato starch, 3g of soybean flour, 1g of corn flour, 3g of peptone, 20g of glucose monohydrate, 0.72g of potassium dihydrogen phosphate, 2g of magnesium sulfate, and 3g of yeast extract per 1000mL of purified water, with a pH of 8; the *Coprinus comatus* (*Coprinus comatus*) is used as the inoculum. Coprinus radians N2 was deposited on November 2, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 40970.

[0030] Step 2: 200L of expansion medium was placed in a 220L fermenter and sterilized at 125℃ and 0.13MPa for 75 minutes. After sterilization, it was cooled to room temperature. The liquid culture from the conical flask in Step 1 was poured into the expansion medium in the fermenter. After expansion culture at 22℃ for 2-3 days, the liquid flow rate was observed to slow down, and a large number of white flocculent mycelia appeared in the fermenter. After standing, the culture solution became clear, with no obvious stratification or floating matter. No sour or foul odor was detected by venting, and after aseptic sampling and observation under a 1600x microscope, no bacterial infection was found. This was then used as the secondary liquid culture. The expansion medium was prepared by adding 600g of potato starch, 600g of soybean flour, 200g of corn flour, 600g of peptone, 4000g of glucose monohydrate, 140g of high-purity potassium dihydrogen phosphate, 400g of magnesium sulfate, and 600g of yeast extract to every 200L of purified water, with a pH of 8.

[0031] Step 3: According to the mass percentage composition of the substrate: 55% sawdust, 24.3% cottonseed hull particles, 18% wheat bran, 1.3% gypsum, 1% white sugar, and 0.4% glucose, and the substrate and water are mixed at a mass ratio of 1:1.3, with sawdust, cottonseed hull particles, wheat bran, and gypsum (0.8-1.2 cm in diameter) mixed evenly. The white sugar and glucose are dissolved in water, and then the two are mixed evenly to obtain the cultivation substrate with a pH of 6.8. The cultivation substrate is then filled into 16.5 cm diameter polypropylene edible mushroom plastic bags using a bagging machine, filling to 5 cm from the bag opening. A 2.5 mm diameter forming tube is inserted into the center, a buckle is attached, and the bags are sealed with polypropylene caps containing air filter cotton. The bags are then sterilized at 125℃ and 0.13 MPa for 180 minutes, and then cooled to room temperature.

[0032] Step 4: The bagged cultivation substrate cooled to room temperature in Step 3 is sent to the inoculation room via conveyor belt. Under sterile conditions, the cap is removed, the plastic bag is opened, the forming tube is pulled out, and the secondary liquid inoculum from the fermenter in Step 2 is injected into the cultivation substrate through the channel formed by the forming tube using an inoculation gun. The amount of secondary liquid inoculum injected is 0.01% to 0.02% of the weight of the cultivation substrate. The bag is then sealed with the cap.

[0033] Step 5: The bagged cultivation substrate inoculated in Step 4 is automatically conveyed out of the inoculation room, placed in baskets, and placed in a dark room at a temperature of 20℃ and a humidity of 50%–60% for incubation. After 3 days of incubation, white mycelium is observed at the bag opening (see...). Figure 2 After culturing for 7 days, the basket was turned upside down, and culturing continued for another 11 days. Mycelia were observed to begin changing color (see...). Figure 3 After 13 days of cultivation, the inoculum has fully grown (see...). Figure 4 After 14 days of cultivation, the bacterial culture had fully grown and almost all of it had changed color (see...). Figure 5 ), and obtained the cultivation strain of Pseudobulbus Cremastrae.

[0034] Using the *Cremastra appendiculata* spawn from Example 1 above, mature *Cremastra appendiculata* (also known as *Cremastra appendiculata*) pods were sown outdoors in early July. Two months later, the *Cremastra appendiculata* seeds germinated and grew into seedlings 1.5–2.5 cm tall (see Example 1). Figure 6 (Left) After one year, the seedlings of *Sagittaria sagittifolia* can reach a height of 15-25cm, with pseudobulbs approximately 3.4cm wide and 2.4cm high (see left). Figure 6 right).

Claims

1. A preparation process for a *Cremastra appendiculata* cultivar strain, characterized in that, Includes the following steps: Step 1: Place the liquid culture medium in an Erlenmeyer flask, autoclave, cool to room temperature, and inoculate with *Coprinus radiata* (a type of variegated cap). Coprinus radians N2 was cultured on a shaker until a large number of white flocculent hyphae or spherical bacterial balls appeared in the culture medium in the conical flask. After standing, the culture medium became clear without obvious stratification. Under aseptic conditions, no bacterial infection was observed under a microscope. It was then used as a liquid culture. The liquid culture medium is prepared by adding 2.5-3.5g of potato starch, 1.5-2.5g of soybean flour, 0.8-1.2g of corn flour, 2.5-3.5g of peptone, 15-25g of glucose monohydrate, 0.7-0.75g of potassium dihydrogen phosphate, 1.5-2.5g of magnesium sulfate, and 2.5-3.5g of yeast extract to every 1000mL of purified water, with a pH of 7.5-8. The radiated little ghost umbrella ( Coprinus radians N2 was deposited on November 2, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO: 40970; the described *Coprinus radiata* ( Coprinus radians The inoculum size of N2 is 2.5% to 3% of the volume of liquid culture medium. The temperature for incubation on a shaker is 20 to 25°C, and the incubation time is 36 to 72 hours. Step 2: Place the expansion culture medium in a fermenter, autoclave it at high temperature and pressure, and then cool it to room temperature. Pour the liquid culture from the conical flask in Step 1 into the fermenter for expansion culture. Cultivate until a large number of white flocculent hyphae or spherical mycelia appear in the fermenter. After standing, the culture medium becomes clear, with no obvious layering or floating matter. There is no sour or foul smell when the gas is exhausted, and no bacterial infection is observed under a microscope under aseptic conditions. This is used as a secondary liquid culture. The expanded culture medium is prepared by adding 580-620g of potato starch, 580-620g of soybean flour, 180-220g of corn flour, 580-620g of peptone, 3800-4200g of glucose monohydrate, 130-150g of high-purity potassium dihydrogen phosphate, 380-420g of magnesium sulfate, and 580-620g of yeast extract to every 200L of purified water, with a pH of 7.5-8; the amount of liquid inoculum added is 0.3%-0.6% of the volume of the expanded culture medium, the temperature of the expanded culture is 20-25℃, and the culture time is greater than 72 hours; Step 3: Fill the cultivation substrate into a high-temperature resistant and non-toxic plastic bag using a bagging machine. Fill the bag to a depth of 3-6 cm from the opening. Insert a 2-3 mm diameter forming tube into the center, attach a buckle, seal the bag with a high-temperature and high-pressure resistant cap, sterilize it under high temperature and high pressure, and then cool it to room temperature. The cultivation substrate is a mixture of substrate and water at a mass ratio of 1:1.1 to 1.5, with a pH of 6.5 to 7; the mass percentage composition of the substrate is: 50% to 60% sawdust, 20% to 28% cottonseed hull particles, 17% to 20% wheat bran, 1% to 1.5% gypsum, 1% to 1.5% white sugar, and 0.3% to 0.6% glucose; Step 4: The bagged cultivation substrate cooled to room temperature in Step 3 is transported to the inoculation room via conveyor belt. Under sterile conditions, the cap is removed, the plastic bag is opened, the forming tube is pulled out, and the secondary liquid inoculum from the fermenter in Step 2 is injected into the cultivation substrate through the channel formed by the forming tube using an inoculation gun. The bag is then sealed with the cap. The amount of secondary liquid inoculum injected is 0.01% to 0.02% of the weight of the cultivation substrate. Step 5: The bagged cultivation substrate inoculated with the spawn in Step 4 is output from the inoculation room via an automatic conveyor belt, placed in baskets and sent into a dark room with an environment of 15-25℃ and 50%-60% humidity for cultivation. After 5-7 days, the baskets are turned upside down and the cultivation continues for another 12-15 days to obtain the spawn for the cultivation of Cremastra appendiculata.

2. The preparation process of the *Cremastra appendiculata* cultivar according to claim 1, characterized in that: In step 1, the high-temperature and high-pressure sterilization is carried out at a temperature of 120-125°C, a pressure of 0.12-0.15 MPa, and a sterilization time of 30-40 min.

3. The preparation process of the *Cremastra appendiculata* cultivation strain according to claim 1, characterized in that: In step 2, the high-temperature and high-pressure sterilization is carried out at a temperature of 120-125°C, a pressure of 0.12-0.15 MPa, and a sterilization time of 70-80 min.

4. The preparation process of the *Cremastra appendiculata* cultivation strain according to claim 1, characterized in that: In step 3, the high-temperature and high-pressure sterilization is carried out at a temperature of 120-125°C, a pressure of 0.12-0.15 MPa, and a sterilization time of 150-200 min.

5. The preparation process of the *Cremastra appendiculata* cultivation strain according to claim 1, characterized in that: In step 3, according to the composition of the cultivation substrate, sawdust, cottonseed hull particles, bran, and gypsum are mixed evenly, white sugar and glucose are dissolved in water, and then the two are mixed evenly to obtain the cultivation substrate; wherein the diameter of the sawdust is 0.8-1.2 cm.

6. The preparation process of the *Cremastra appendiculata* cultivation strain according to claim 1, characterized in that: In step 3, the high-temperature resistant and non-toxic plastic bag is a special bag for edible fungi made of polypropylene or polyethylene, and the high-temperature resistant and high-pressure cap is a cap made of polypropylene containing air filter cotton.

7. The *Cremastra appendiculata* spawn prepared by any one of the processes described in claims 1 to 6.

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