Culture method of artificial hybrid alcohol peptide tricholoma matsutake strain

Abstract

The invention discloses a culture method of an artificial hybrid alcohol peptide tricholoma matsutake strain, and belongs to the technical field of strain culture. The invention discloses a culture method of an artificial hybrid alcohol peptide tricholoma matsutake strain, which comprises the following steps: culturing an artificial alcohol peptide tricholoma matsutake mother strain and a liquid strain according to a culture method from the mother strain to the liquid strain to a solid strain, and then inoculating the liquid strain on a solid culture medium to culture the solid strain. The method for culturing the artificial hybrid alcohol peptide tricholoma matsutake strain has the advantages of short culture period, high culture success rate, high purity and strong activity.

Description

Technical Field

[0001] The present invention relates to the technical field of strain cultivation, and more particularly to a cultivation method for artificially hybridized alcohol peptide Tricholoma matsutake strains. Background Art

[0002] Tricholoma matsutake, scientifically named Tricholoma matsutake, also known as pine mushroom, combined mushroom, and table mushroom, belongs to the Basidiomycetes subphylum and Tricholomataceae family. It is an ectomycorrhizal fungus growing on the outside of trees such as pine and oak. It has a unique strong fragrance and is a rare and precious natural medicinal mushroom in the world, known as the "king of mushrooms" and is a second-class endangered protected species in China.

[0003] Tricholoma matsutake contains 18 kinds of amino acids, 14 kinds of essential trace elements for the human body, 49 kinds of active nutrients, 5 kinds of unsaturated fatty acids, 8 kinds of vitamins, 2 kinds of glycoproteins, rich dietary fiber and various active enzymes. It also contains 3 kinds of precious active substances, namely double-stranded Tricholoma matsutake polysaccharide, Tricholoma matsutake polypeptide, and the world's unique anti-cancer substance - Tricholoma matsutake alcohol. It is the most precious natural medicinal mushroom in the world.

[0004] Tricholoma matsutake has extremely high nutritional and medicinal values. Modern medicine shows that Tricholoma matsutake has various effects such as enhancing immunity, anti-cancer and anti-tumor, treating diabetes and cardiovascular diseases, anti-aging and beautifying the skin, promoting the stomach and protecting the liver, etc. Therefore, it is widely used in the research and development of drugs, health products, and cosmetics globally.

[0005] Tricholoma matsutake is rare, precious, and has a low yield. Currently, it is still impossible to fully artificially cultivate wild Tricholoma matsutake globally. According to statistics, the annual global output of Tricholoma matsutake is only 2,000 tons, and due to climate change and unrestricted picking by people, the output is showing a decreasing trend year by year.

[0006] However, the demand for Tricholoma matsutake at home and abroad is still increasing continuously, resulting in a situation of supply falling short of demand in the Tricholoma matsutake industry. There is an urgent need to develop a new variety of Tricholoma matsutake to replace wild Tricholoma matsutake.

[0007] After more than 10 years of scientific research breakthroughs and overcoming numerous difficulties, the inventor selected wild Tricholoma matsutake from Shangri-La as the seed source, and through multiple artificial hybridization cultivations and domestication, and then through scientific strain cultivation processes such as liquid fermentation, the strain was inoculated into a special substrate and fertile and nutritious mushroom sticks. Simulating the growth environment of Tricholoma matsutake, under the intelligent and digital control of "constant temperature, constant humidity, constant oxygen, constant light" cultivation conditions and unique plant awakening technology, fresh Tricholoma matsutake that is clean, green, fresh, nourishing, and health-protecting was cultivated, and its nutritional components are comparable to those of wild Tricholoma matsutake. The successful cultivation of artificially hybridized alcohol peptide Tricholoma matsutake is the world's first breakthrough in the artificial cultivation technology of Tricholoma matsutake, filling a world gap.

[0008] The artificially hybridized alcohol peptide Tricholoma matsutake is obtained by selecting appropriate strains of wild Tricholoma matsutake in Yunnan and Tricholoma portentosum (commonly known as "old man's head") strains, separately isolating the mononuclear spores of Tricholoma matsutake and Tricholoma portentosum for pairing, culturing and germinating the hyphae on a PDA medium, and through screening, selecting the hybrid offspring with vigorous growth, whiteness and robustness. Through HPLC chromatogram analysis, the RT value of wild Tricholoma matsutake and the product of artificially hybridized alcohol peptide Tricholoma matsutake have a certain similarity, with the similarity approaching 35%. Due to the limited yield of wild Tricholoma matsutake restricted by climatic and seasonal conditions, the yield is increased by artificially cultivating alcohol peptide Tricholoma matsutake to meet people's needs. Based on this, there is an urgent need to provide an effective method for culturing strains from mother strains, liquid strains to solid strains.

[0009] Therefore, providing a method for culturing artificially hybridized alcohol peptide Tricholoma matsutake strains is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0010] In view of this, the present invention provides a method for culturing artificially hybridized alcohol peptide Tricholoma matsutake strains, which solves the problems of long culture period, low success rate, low activity and low purity of Tricholoma matsutake strains.

[0011] In order to achieve the above object, the present invention adopts the following technical scheme:

[0012] A method for culturing artificially hybridized alcohol peptide Tricholoma matsutake strains comprises the following steps:

[0013] (1) Inoculating the stipe tissue of artificially hybridized alcohol peptide Tricholoma matsutake on a mother strain medium for culture to obtain a mother strain;

[0014] The mother strain medium is composed of the following raw materials: 150 g / L - 200 g / L of potato, 15 g / L - 20 g / L of glucose, 20 g / L - 30 g / L of corn flour, 30 g / L - 50 g / L of wheat bran, 2 g / L - 5 g / L of peptone, 2 g / L - 3 g / L of yeast extract, 0.5 g / L - 1 g / L of magnesium sulfate, 1 g / L - 2 g / L of potassium dihydrogen phosphate, 30 g / L - 50 mg / L of VB 1 3 and 16 g / L - 20 g / L of agar powder;

[0015] (2) Taking 4 - 6 pieces of 5 - square - millimeter strain blocks of the mother strain obtained in step (1) and inoculating them into a liquid shake - flask medium for shake - flask culture. After the hyphae germinate and there is no contamination, carry out stirring culture to obtain a liquid shake - flask strain;

[0016] The liquid shake flask culture medium consists of the following raw materials: 150 g / L - 200 g / L of potatoes, 15 g / L - 20 g / L of glucose, 20 g / L - 30 g / L of corn flour, 30 g / L - 50 g / L of wheat bran, 3 g / L - 5 g / L of peptone, 2 g / L - 3 g / L of yeast extract, 0.5 g / L - 1 g / L of magnesium sulfate, 1 g / L - 2 g / L of potassium dihydrogen phosphate, VB 1 0.03 g / L - 0.05 g / L and 1 g / L - 2 g / L of agar powder;

[0017] (3) Inoculate the liquid shake flask strain obtained in step (2) onto the solid medium for cultivation to obtain a solid strain;

[0018] The solid medium consists of the following raw materials: 53.5% - 81.2% of wheat, 10% - 30% of oak sawdust or other broad-leaved hardwood miscellaneous sawdust, 5% - 10% of wheat bran, 3% - 5% of soybean meal, 0.5% - 1% of gypsum, and 0.3% - 0.5% of lime.

[0019] Furthermore, in step (1), inoculating the stipe tissue of artificially hybridized alcohol peptide Tricholoma matsutake on the mother culture medium for cultivation to obtain the mother culture, the specific steps are as follows: Select high-quality artificially hybridized (alcohol peptide) Tricholoma matsutake fruiting bodies, wipe the stipe of Tricholoma matsutake with a cotton ball containing 75% alcohol, use a scalpel to take a 2 - 3 mm mycelium tissue block from the middle part of the stipe and place it in a petri dish filled with the mother culture medium, and place it in an incubator at 22 - 26 °C for cultivation. When the mycelium fills the petri dish, the mother culture is obtained.

[0020] Furthermore, in step (2), the culture temperature for shake flask culture is 22 °C - 26 °C, and static culture is carried out for 2 - 3 days; the stirring culture is to place the shake flask on a magnetic stirrer for cultivation, the culture temperature for the stirring culture is 22 °C - 26 °C, and the culture time is 5 - 7 days.

[0021] Furthermore, in step (3), place the inoculated solid medium in a dark environment for fermentation culture for 20 - 30 days, and the culture temperature is 22 °C - 26 °C to obtain a solid strain.

[0022] Furthermore, the preparation method of the solid medium in step (3) is as follows:

[0023] Prepare each raw material according to the formula: 53.5% - 81.2% of wheat, 10% - 30% of oak sawdust or other miscellaneous sawdust, 5% - 10% of wheat bran, 3% - 5% of soybean meal, 0.5% - 1% of gypsum, and 0.3% - 0.5% of lime; soak the plump dry wheat, wash it, cook it until it is cooked but not mushy, and dry it in the sun and drain the water for later use; pre-wet the sawdust with water in advance, and then put the pre-wetted sawdust, wheat bran, soybean meal, gypsum, and lime into the wheat and mix them to obtain the solid medium; carry out high-pressure steam sterilization.

[0024] As can be seen from the above technical solutions, compared with the prior art, the present invention discloses a method for culturing an artificially hybridized alcohol peptide Tricholoma matsutake strain. The artificially hybridized alcohol peptide Tricholoma matsutake strain cultured by the method of the present invention has the advantages of short culture period, high culture success rate, high purity and strong activity; and has high contents of tricholoma alcohol and total polypeptides, and has an anti-aging effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0026] Figure 1 It is a sample diagram of artificially hybridized Tricholoma matsutake and wild Tricholoma matsutake; the upper figure is artificially hybridized Tricholoma matsutake, and the lower figure is wild Tricholoma matsutake;

[0027] Figure 2 It is the detection result of the core components of wild Tricholoma matsutake;

[0028] Figure 3 It is the detection result of the core components of artificially hybridized Tricholoma matsutake. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0029] The following will clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention.

[0030] Example 1

[0031] A method for culturing an artificially hybridized alcohol peptide Tricholoma matsutake strain includes the following steps:

[0032] 1) Inoculate the stipe tissue of the artificially hybridized alcohol peptide Tricholoma matsutake on a slant culture medium for cultivation to obtain a slant culture:

[0033] (1) Prepare each raw material according to the following formula to prepare a slant culture medium: 150 g / L of potato, 15 g / L of glucose, 20 g / L of corn flour, 30 g / L of wheat bran, 3 g / L of peptone, 2 g / L of yeast extract, 0.5 g / L of magnesium sulfate, 1 g / L of potassium dihydrogen phosphate, VB 1 0.03 g / L, 16 g / L of agar powder; preparation method:

[0034] Wash fresh potatoes, peel them, weigh them, cut them into thin slices and put them into a large beaker. Add a certain amount of water and boil them until the potatoes are cooked but not mushy. Filter to obtain the filtrate. At the same time, boil corn flour and bran and filter to obtain the filtrate. After mixing the two filtrates, add glucose, agar, peptone, yeast extract, magnesium sulfate, potassium dihydrogen phosphate, VB 1 , boil until the agar is completely dissolved, make up the volume to 1 liter, adjust the pH value to 4.0 - 6.0 to obtain the mother culture medium. Dispense the medium into Erlenmeyer flasks, sterilize at 121 °C for 30 minutes, and pour it into Petri dishes after cooling for use;

[0035] (2) Select high-quality artificially hybridized alcohol peptide Tricholoma matsutake fruit bodies. Wipe the stipe of Tricholoma matsutake with a cotton ball containing 75% alcohol. Use a scalpel to take a 2 - 3 mm piece of flesh tissue from the middle part of the stipe and place it in a Petri dish containing the mother culture medium. Incubate it in an incubator at 25 °C for 30 days until the mycelium fills the Petri dish to obtain the mother culture;

[0036] 2) Inoculate the mother culture into a liquid shake flask medium for shake flask culture. After the mycelium germinates and there is no contamination, carry out stirring culture to obtain a liquid shake flask strain:

[0037] Prepare each raw material according to the following formula to prepare the liquid shake flask medium: 150 g / L of potatoes, 15 g / L of glucose, 20 g / L of corn flour, 30 g / L of bran, 3 g / L of peptone, 2 g / L of yeast extract, 0.5 g / L of magnesium sulfate, 1 g / L of potassium dihydrogen phosphate, VB 1 0.03 g / L and 1 g / L of agar powder; The specific steps are as follows:

[0038] (1) Wash fresh potatoes, peel them, weigh them, cut them into thin slices and put them into a large beaker. Add a certain amount of water and boil them until the potatoes are cooked but not mushy. Filter to obtain the filtrate. At the same time, boil corn flour and bran and filter to obtain the filtrate. After mixing the two filtrates, add glucose, agar, peptone, yeast extract, magnesium sulfate, potassium dihydrogen phosphate, VB 1 , boil until the agar is completely dissolved, make up the volume to 1 liter to obtain the liquid medium. Dispense the medium into Erlenmeyer flasks, sterilize at 121 °C for 30 minutes, and reserve it after cooling;

[0039] (2) Inoculate the above mother culture into the liquid shake flask medium for shake flask culture. The culture temperature is 25 °C and static culture is carried out for 2 days. After the mycelium germinates and there is no contamination, place the shake flask on a magnetic stirrer for stirring culture. The culture temperature is 25 °C and the culture time is 5 days to obtain a liquid shake flask strain;

[0040] 3) Inoculate the above liquid shake flask strain onto the solid medium and culture it. The steps are as follows:

[0041] (1) Preparation of solid medium: Prepare the following raw materials according to the formula: wheat 53.5%, oak sawdust or other (broad-leaved hardwood) miscellaneous sawdust 30%, wheat bran 10%, soybean meal 5%, gypsum 1%, lime 0.5%; Soak the plump dry wheat, wash it, cook it until it is cooked but not mushy, and dry it in the sun to drain the water for later use; Pre-wet the sawdust with water in advance, and then put the pre-wet sawdust, wheat bran, soybean meal, gypsum and lime into the wheat and mix to obtain the solid medium. Pack the solid medium into polypropylene bags or bottles, and its water content is 50%;

[0042] (2) Sterilization: Perform high-pressure steam sterilization on the above solid medium. The sterilization pressure is 0.1 Mpa, the sterilization temperature is 121 °C, and the sterilization time is 1.5 h;

[0043] (3) Inoculation: Cool the sterilized solid medium to room temperature, and then inoculate the above liquid strain onto the solid medium at room temperature. The inoculation amount is 5%;

[0044] (4) Cultivation: Place the inoculated solid medium in a dark environment for cultivation. The cultivation temperature is 22 °C. Ventilate the cultivation environment twice a day, each time for 15 minutes, and cultivate for 30 days to obtain the artificial hybrid Tricholoma matsutake solid strain.

[0045] Example 2

[0046] A method for cultivating artificial hybrid alcohol peptide Tricholoma matsutake strain, comprising the following steps:

[0047] 1) Inoculate the stipe tissue of artificial hybrid alcohol peptide Tricholoma matsutake on the mother culture medium for cultivation to obtain the mother culture:

[0048] (1) Prepare the following raw materials according to the formula to prepare the mother culture medium: potato 200 g / L, glucose 20 g / L, corn flour 30 g / L, wheat bran 50 g / L, peptone 5 g / L, yeast extract 3 g / L, magnesium sulfate 1 g / L, potassium dihydrogen phosphate 2 g / L, VB 1 0.05 g / L, agar powder 20 g / L; Preparation method:

[0049] Wash, peel, weigh and cut the fresh potatoes into thin slices, put them into a large beaker, add a certain amount of water and boil them until the potatoes are cooked but not mushy, and filter to obtain the filtrate; At the same time, boil the corn flour and wheat bran and filter to obtain the filtrate; After mixing the two filtrates, add glucose, agar, peptone, yeast extract, magnesium sulfate, potassium dihydrogen phosphate, VB 1 , boil until the agar is completely melted, make up the volume to 1 liter, and adjust the pH value to 4.0 - 6.0 to obtain the mother culture medium; Dispense the medium into conical flasks, sterilize at 121 °C for 30 minutes, and pour it into petri dishes for use after cooling;

[0050] (2) Select high-quality artificially hybridized (alcohol peptide) matsutake fruit bodies. Wipe the stipe of the matsutake with a cotton ball containing 75% alcohol. Use a scalpel to take a 2-3 mm piece of flesh tissue from the middle part of the stipe and place it in a petri dish containing the mother culture medium. Incubate it in an incubator at 25°C for 30 days until the mycelium covers the petri dish to obtain the mother culture;

[0051] 2) Inoculate the mother culture into a liquid shake flask medium for shake flask culture. After the mycelium germinates and there is no contamination, carry out stirring culture to obtain the liquid shake flask strain:

[0052] Prepare each raw material according to the following formula to prepare the liquid shake flask medium: 200 g / L of potato, 20 g / L of glucose, 30 g / L of corn flour, 50 g / L of wheat bran, 3 g / L of peptone, 3 g / L of yeast extract, 1 g / L of magnesium sulfate, 2 g / L of potassium dihydrogen phosphate, VB 1 0.05 g / L and 2 g / L of agar powder; the specific steps are as follows:

[0053] (1) Wash, peel, weigh, and cut fresh potatoes into thin slices and put them into a large beaker. Add a certain amount of water and boil until the potatoes are cooked but not mushy. Filter to obtain the filtrate; at the same time, boil and filter the corn flour and wheat bran to obtain the filtrate; after mixing the two filtrates, add glucose, agar, peptone, yeast extract, magnesium sulfate, potassium dihydrogen phosphate, VB 1 , boil until the agar is completely melted, make up the volume to 1 liter, and adjust the pH value to 4.0 - 6.0 to obtain the liquid medium; divide the medium into Erlenmeyer flasks and sterilize at 121°C for 30 minutes. Wait until it cools and then reserve it for use;

[0054] (2) Inoculate the above-mentioned mother culture into the liquid shake flask medium for shake flask culture. The culture temperature is 25°C, and statically culture for 3 days. After the mycelium germinates and there is no contamination, place the shake flask on a magnetic stirrer for stirring culture. The culture temperature is 25°C, and the culture time is 7 days to obtain the liquid shake flask strain;

[0055] 3) Inoculate the above-mentioned liquid shake flask strain onto the solid medium and culture it. The steps are as follows:

[0056] (1) Prepare the solid medium: Prepare each raw material according to the following formula: 81.2% of wheat, 10% of oak sawdust or other (broad-leaved hardwood) miscellaneous sawdust, 5% of wheat bran, 3% of soybean meal, 0.5% of gypsum, 0.3% of lime; soak and wash the plump dry wheat until it is cooked but not mushy, and dry it in the sun to drain the water for later use; pre-wet the sawdust with water in advance, and then put the pre-wetted sawdust, wheat bran, soybean meal, gypsum, and lime into the wheat and mix them to obtain the solid medium. Pack the solid medium into polypropylene bags or bottles, and its water content is 50%;

[0057] (2) Sterilization: Perform high-pressure steam sterilization on the above solid medium. The sterilization pressure is 0.1 Mpa, the sterilization temperature is 121 °C, and the sterilization time is 1.5 h;

[0058] (3) Inoculation: Cool the sterilized solid medium to room temperature, and then inoculate the above liquid strain onto the solid medium at room temperature. The inoculation amount is 10%;

[0059] (4) Cultivation: Place the inoculated solid medium in a dark environment for cultivation. The cultivation temperature is 25 °C. Ventilate the cultivation environment 2 times a day, 15 minutes each time, and cultivate for 20 days to obtain the artificial hybrid Tricholoma matsutake solid strain.

[0060] The sample diagrams of artificial hybrid Tricholoma matsutake and wild-type Tricholoma matsutake are shown in Figure 1 .

[0061] Detect the artificial hybrid Tricholoma matsutake and wild-type Tricholoma matsutake cultivated in Example 2. The detection results of the core components of wild Tricholoma matsutake are shown in Figure 2 ; The detection results of the core components of artificial hybrid Tricholoma matsutake are shown in Figure 3 .

[0062] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the broadest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for culturing an artificial hybrid alcohol peptide Tricholoma matsutake strain, characterized in that: The following steps are involved: (1) inoculating the stipe tissue of the artificial hybrid peptide Tricholoma matsutake on a mother culture medium for culturing to obtain a mother culture; The mother seed culture medium is composed of the following raw materials: 150g / L-200g / L potato, 15g / L-20g / L glucose, 20g / L-30g / L corn flour, 30g / L-50g / L bran, 2g / L-5g / L peptone, 2g / L-3g / L yeast extract, 0.5g / L-1g / L magnesium sulfate, 1g / L-2g / L potassium dihydrogen phosphate, 30g / L-50mg / L VB1 and 16g / L-20g / L agar powder; (2) taking 4-6 5 mm2 seed blocks of the mother culture obtained in step (1) and inoculating them into a liquid shake flask culture medium for shake flask culture, stirring and culturing after the mycelium germinates and is free of contamination, to obtain a liquid shake flask culture; The liquid shake flask culture medium is composed of the following raw materials: 150g / L-200g / L potato, 15g / L-20g / L glucose, 20g / L-30g / L corn flour, 30g / L-50g / L bran, 3g / L-5g / L peptone, 2g / L-3g / L yeast extract, 0.5g / L-1g / L magnesium sulfate, 1g / L-2g / L potassium dihydrogen phosphate, 0.03g / L-0.05g / L VB1 and 1g / L-2g / L agar powder; (3) inoculating the liquid shake flask strain obtained in step (2) onto a solid culture medium for culturing to obtain a solid strain; The solid culture medium is composed of the following raw materials: 53.5%-81.2% of wheat, 10%-30% of oak wood chips or other broad-leaved hardwood wood chips, 5%-10% of bran, 3%-5% of soybean meal, 0.5%-1% of gypsum and 0.3%-0.5% of lime.

2. The method for culturing an artificial hybrid alcohol peptide Tricholoma matsutake strain according to claim 1, characterized in that: In step (1), the stipe tissue of the artificial hybrid alcohol peptide matsutake is inoculated on the mother culture medium for cultivation to obtain the mother culture. The specific steps are as follows: select artificial hybrid (alcohol peptide) matsutake fruiting bodies of good quality, wipe the matsutake stipe with a cotton ball containing 75% alcohol, use a scalpel to take a 2-3 mm mushroom meat tissue block in the middle part of the stipe and place it in a culture dish filled with the mother culture medium, place it in an incubator at 22-26°C for cultivation, and the mycelium fills the culture dish to obtain the mother culture.

3. The method for culturing an artificial hybrid alcohol peptide Tricholoma matsutake strain according to claim 1, characterized in that: The culture temperature of the shake flask culture in step (2) is 22°C-26°C, and the static culture is 2-3 days; the stirring culture is to place the shake flask on a magnetic stirrer for culture, the culture temperature of the stirring culture is 22°C-26°C, and the culture time is 5-7 days.

4. The method for culturing an artificial hybrid alcohol peptide Tricholoma matsutake strain according to claim 1, characterized in that: Step (3) placing the inoculated solid culture medium in a dark environment for fermentation culture for 20-30 days at a culture temperature of 22° C.-26° C. to obtain a solid bacterial strain.

5. The method for culturing an artificial hybrid alcohol peptide Tricholoma matsutake strain according to claim 1, characterized in that: The preparation method of the solid culture medium in step (3) is as follows: Prepare the raw materials according to the formula: 53.5%-81.2% wheat, 10%-30% oak wood chips or other miscellaneous wood chips, 5%-10% bran, 3%-5% soybean meal, 0.5%-1% gypsum, and 0.3%-0.5% lime; soak and wash the plump dry wheat, then cook it until it is cooked but not mushy, dry it in the sun and drain the water for later use; pre-wet the wood chips with water in advance, then separately put the pre-wet wood chips, bran, soybean meal, gypsum and lime into the wheat and mix them to obtain a solid culture medium; perform high-pressure steam sterilization.

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