CN115039635A - Liquid culture medium for preparing morchella stock culture in high altitude area and stock culture preparation method - Google Patents
Liquid culture medium for preparing morchella stock culture in high altitude area and stock culture preparation method Download PDFInfo
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- CN115039635A CN115039635A CN202210640226.2A CN202210640226A CN115039635A CN 115039635 A CN115039635 A CN 115039635A CN 202210640226 A CN202210640226 A CN 202210640226A CN 115039635 A CN115039635 A CN 115039635A
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01G18/00—Cultivation of mushrooms
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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Abstract
The invention provides a liquid culture medium for preparing a morchella stock culture in a high-altitude area, which consists of a primary liquid strain liquid culture medium and a secondary liquid strain liquid culture medium; the formula of the first-stage liquid strain liquid culture medium is as follows: the culture medium contains 10-15g of corn starch, 2.4g of yeast powder, 46-60g of bran, 15-25g of glucose, 2.5g of peptone, 0.5g of magnesium sulfate and 1g of monopotassium phosphate according to the proportion per liter, and the pH value is 5.5; the second-stage liquid strain liquid culture medium is prepared by adding 0.9 wt% of sodium carboxymethylcellulose into the first-stage liquid strain liquid culture medium. The invention also provides a corresponding preparation method of the morchella stock. The method can obtain the stock of the morchella esculenta within 10-11 days, can obtain the cultivated species within 30-35 days, and can obtain 10.8-14.9g of hypha per milliliter, and the diameter of the mycelium pellet is below 2 mm. The method is suitable for rapid propagation of morchella in high altitude areas.
Description
Technical Field
The invention relates to a liquid culture medium for preparing a morchella stock culture in a high altitude area and a stock culture preparation method.
Background
Morchella esculenta is generally prepared by three steps of mother strain activation, stock strain preparation and cultivar propagation, and a solid culture medium is usually adopted for stock strain preparation in the prior art. The morchella mycelium has a higher growth rate than other edible fungi, but is easily aged and degraded. At present, the toadstool needs 25-30 days from mother strain activation to original strain, the cultivated strain needs 45-50 days, and the production efficiency is low.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a preparation method of morchella esculenta in a high altitude area. The invention changes the traditional stock seed preparation steps from using a solid culture medium to using a liquid culture medium, changes the stock seed into a liquid strain, can effectively reduce the cost and shorten the seed production period: the method of the invention can obtain the stock strains of the morchella within 10-11 days and the cultivated strains within 30-35 days, thereby greatly improving the production efficiency. The original strains of the morchella esculenta prepared by the method have consistent ages and strong hypha activity.
The invention provides a liquid culture medium for preparing a morchella stock culture in a high-altitude area, which consists of a primary liquid strain liquid culture medium and a secondary liquid strain liquid culture medium;
the formula of the primary liquid strain liquid culture medium is as follows: according to the proportion, each liter of culture medium contains 10-15g of corn starch, 2.4g of yeast powder, 46-60g of bran, 15-25g of glucose, 2.5g of peptone, 0.5g of magnesium sulfate, 1g of monopotassium phosphate and pH 5.5;
the second-stage liquid strain liquid culture medium is prepared by adding 0.9 wt% of sodium carboxymethylcellulose into the first-stage liquid strain liquid culture medium.
Preferably, the formula of the primary liquid strain liquid culture medium is as follows: according to the proportion, each liter of culture medium contains 10g of corn starch, 2.4g of yeast powder, 60g of bran, 25g of glucose, 2.5g of peptone, 0.5g of magnesium sulfate, 1g of monopotassium phosphate and pH 5.5.
Preferably, the formula of the primary liquid strain liquid culture medium is as follows: the culture medium contains 15g of corn starch, 2.4g of yeast powder, 50g of bran, 15g of glucose, 2.5g of peptone, 0.5g of magnesium sulfate, 1g of monopotassium phosphate and pH5.5 in proportion per liter.
Preferably, the formula of the primary liquid strain liquid culture medium is as follows: the culture medium contains 15g of corn starch, 2.4g of yeast powder, 46g of bran, 25g of glucose, 2.5g of peptone, 0.5g of magnesium sulfate, 1g of monopotassium phosphate and pH5.5 in proportion per liter.
The invention also provides a preparation method of the original strains of morchella esculenta in high altitude areas, which comprises the following steps:
(1) obtaining a first-level liquid strain of morchella esculenta: inoculating three Morchella mother strains with the diameter of 4.8-5.2mm to each 100ml of the primary liquid strain liquid culture medium, and culturing for 3-5 days at 18 ℃ of a shaking table and 200 revolutions per minute at 180 ℃ to obtain Morchella primary liquid strains;
(2) obtaining a second-level liquid strain of morchella esculenta: inoculating the first-stage liquid strain into a fermentation tank containing the second-stage liquid strain liquid culture medium according to the inoculation amount of 10-15%, standing and culturing in the dark for 1 day after inoculation, and then culturing for 5-6 days under the conditions of the ventilation volume of 3.5-4.0L/min, natural pH, the rotation speed of 300-350 r/min and the temperature of 15-18 ℃ to obtain the second-stage liquid strain of the morchella esculenta.
Preferably, the method comprises the following steps:
(1) obtaining a first-level liquid strain of morchella esculenta: inoculating three Morchella mother strains with the diameter of 5mm to each 100ml of the primary liquid strain liquid culture medium, and culturing for 5 days at the temperature of 18 ℃ in a shaking table at 180 r/min to obtain Morchella primary liquid strains;
(2) obtaining secondary liquid strains of morchella esculenta: inoculating the first-stage liquid strain into a fermentation tank containing the second-stage liquid strain liquid culture medium according to the inoculation amount of 10%, standing and culturing for 1 day in the dark after inoculation, and then culturing for 5 days under the conditions of ventilation capacity of 4.0L/min, natural pH, rotation speed of 300 r/min and 18 ℃ to obtain the morchella second-stage liquid strain.
The invention also provides a preparation method of the morchella esculenta in the high-altitude area, which comprises the preparation method of the original morchella esculenta in the high-altitude area.
Preferably, the altitude is 1900-2300 meters.
The invention provides a liquid culture medium aiming at the optimal growth condition of morchella and a stock seed preparation method, by using the method for propagation, the stock seed of morchella can be obtained within 10-11 days, the cultivated species can be obtained within 30-35 days, the hypha per milliliter can reach 10.8-14.9g, and the diameter of a mycelium pellet is less than 2 mm. The method is suitable for rapid propagation of morchella in high altitude areas.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 shows the stock Morchella strains of the present invention.
FIG. 2 shows the primary fermentation of the inventive Morchella stock.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemicals, unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
According to the method, original seed preparation steps in the preparation process of the morchella strain are optimized, a solid culture medium is changed into a liquid culture medium, the cost is effectively reduced, the production efficiency is improved, and the seed preparation period is shortened, the original morchella strain can be prepared by expanding propagation for 10-11 days, and morchella cultivars can be obtained in 30-35 days.
The formula of the liquid culture medium used in the stock seed preparation step in the preparation process of the morchella strain is as follows:
first-stage liquid strain liquid culture medium: according to the proportion, each liter of culture medium contains 10-15g of corn starch, 2.4g of yeast powder, 46-60g of bran, 15-25g of glucose, 2.5g of peptone, 0.5g of magnesium sulfate, 1g of monopotassium phosphate and pH 5.5.
Liquid culture medium of secondary liquid strain: adding 0.5 wt% sodium carboxymethylcellulose (C) into the first-stage liquid strain liquid culture medium 6 H 7 O 2 (OH) 2 OCH 2 COONa] n 。
Cellulose after hydrolysis of the sodium carboxymethyl cellulose is a main component of cell walls, and can provide a carbon source, effectively increase the activity and accelerate the growth of cells.
The invention mainly adjusts the C/N of the formula by changing the proportion of corn starch, bran and glucose, and the C/N of the formula is about 20-30: 1, meeting the nutritional requirement of morchella growth.
The invention adjusts the dosage of corn starch, bran and glucose by controlling the C/N of the formula, and the C/N sets three gradients: 20: 1. 25: 1.③ 30: 1; the formula is as follows:
10g of corn starch, 2.4g of yeast powder, 60g of bran, 25g of glucose, 2.5g of peptone, 0.5g of magnesium sulfate and 1g of monopotassium phosphate, and the pH value is 5.5.
② 15g of corn starch, 2.4g of yeast powder, 50g of bran, 15g of glucose, 2.5g of peptone, 0.5g of magnesium sulfate, 1g of monopotassium phosphate and pH 5.5.
③ 15g of corn starch, 2.4g of yeast powder, 46g of bran, 25g of glucose, 2.5g of peptone, 0.5g of magnesium sulfate, 1g of monopotassium phosphate and pH 5.5.
The preparation method of the liquid culture medium used in the stock seed preparation step in the preparation process of the morchella strain comprises the following steps:
firstly, weighing bran, wrapping the bran by two layers of gauze, boiling the bran in boiling water for 20 minutes, and filtering the bran to obtain filtrate for later use;
dissolving corn starch with distilled water, adding the dissolved corn starch into bran filtrate while stirring, boiling the mixture for 5 minutes with soft fire, filtering the mixture twice by 4 layers of gauze, and reserving filtrate for later use;
dissolving yeast powder, glucose, peptone, magnesium sulfate and potassium dihydrogen phosphate (the second-stage liquid strain liquid culture medium also comprises sodium carboxymethyl cellulose) with distilled water, adding into the filtrate in the previous step, stirring, heating with slow fire, decocting for 1 min, and adding deionized water to reach sufficient amount;
fourthly, detecting by using precise pH test paper, wherein the pH value is about 5.5, otherwise, adjusting by using 1 mol/L NaOH solution or HCl solution;
subpackaging and sterilizing at 115 ℃ for 30 minutes.
The method for preparing the stock spawn of the morchella by using the liquid culture medium comprises the following steps:
1. obtaining a first-stage liquid strain: inoculating three Morchella mother strains with the diameter of about 5mm to each 100ml of the primary liquid strain liquid culture medium, and culturing for 3-5 days at 18 ℃ and 180 revolutions per minute of a shaking table to obtain the Morchella primary liquid strain.
The morchella is a low-temperature bacterium, the culture temperature is too high, the growth speed of hyphae is extremely high, but the hyphae are thin and weak, and the production is not facilitated.
2. Obtaining secondary liquid strains: inoculating the first-stage liquid strain into a fermentation tank containing a second-stage liquid strain liquid culture medium according to the inoculation amount of 10-15%, standing and culturing in the dark for 1 day after inoculation, and then culturing for 5-6 days under the conditions of the ventilation volume of 3.5-4.0L/min, natural pH, the rotation speed of 300-.
The dry weight of morchella mycelium obtained under the above conditions can reach 10.8-14.9gmg/ml, and the diameter of mycelium pellet is below 2 mm.
The method for calculating the dry weight of the hyphae comprises the following steps: taking 10ml of uniformly fermented morchella stock, taking a sample, repeating the steps for 3 times, centrifuging the sample for 5 minutes at 3000 revolutions per minute by using a centrifuge, discarding supernatant, washing the sample by using distilled water, centrifuging the sample again, washing the sample again, and repeating the steps for 5 times; drying at 65 deg.C to constant weight, weighing, and converting.
Example 1
The test is carried out in laboratories of Linxia Hui autonomous State academy of agricultural sciences and Hezhou Yungu agro-Tech Co., Ltd, and the altitude is 2010-2035 m.
The invention relates to a preparation method of morchella esculenta in a high altitude area, which comprises the following steps:
morchella activation
1. Activating culture medium formula: peeled potato 200g, glucose 20g, peptone 3g, potassium dihydrogen phosphate 3g, magnesium sulfate 1g, agar powder 20g, pH 7, and water 1000 ml.
2. Preparation of an activation medium:
cleaning and peeling potato, weighing 200g, and cutting into 1cm 3 Boiling the square blocks with the size of 300ml of distilled water, adding the potato blocks, boiling for about 20 minutes with soft fire until the potato blocks can be poked but are not scattered by a glass rod, filtering twice by four layers of gauze, and reserving filtrate for later use;
weighing 20g of glucose, 3g of peptone, 3g of monopotassium phosphate and 1g of magnesium sulfate, dissolving with 200ml of distilled water, adding into the filtrate in the previous step while stirring for later use;
③ weighing 20g of agar powder, dissolving with 200ml of distilled water, adding into the filtrate in the previous step, stirring while adding, heating with soft fire after uniform, turning off the fire after boiling for 1 minute, replenishing water to 1000ml, adjusting the pH to 7, stirring uniformly, and subpackaging; sterilizing at 115 deg.C for 30 min;
preparing a flat plate: sterilizing a glass culture dish with the diameter of 10cm in advance for later use, and carrying out ultraviolet irradiation on an ultra-clean workbench for 30 minutes for later use; after the sterilization of the culture medium is finished, the culture medium is rapidly transferred to a clean bench, an operator can clean hands with soap, the culture medium is stretched into the clean bench and then wiped with alcohol cotton for sterilization, an alcohol lamp is ignited after alcohol is dried, the plate is poured, about 15ml of the culture medium is poured on each plate, namely, the thickness of the culture medium is about 0.5cm, the culture dish is placed in the clean bench for cooling after pouring, and ultraviolet irradiation is carried out for 30 minutes again.
3. And (3) activation: taking Morchella strain 0.5cm 2 Placing the agar blocks with mycelium in the step 2The prepared activated medium is inverted on a flat plate in the center, and is cultured in a constant temperature incubator at 18 ℃ for 5 days to obtain an activated morchella stock culture.
Second, preparation of original strain of Morchella (liquid culture)
1. The formula of the liquid culture medium is as follows:
first-stage liquid strain liquid culture medium: the culture medium contains 15g of corn starch, 2.4g of yeast powder, 46g of bran, 25g of glucose, 2.5g of peptone, 0.5g of magnesium sulfate, 1g of monopotassium phosphate and pH5.5 in proportion per liter.
Secondary liquid strain liquid culture medium: adding 0.5 wt% sodium carboxymethylcellulose (C) into the first-stage liquid strain liquid culture medium 6 H 7 O 2 (OH) 2 OCH 2 COONa] n 。
2. Preparation of liquid medium:
firstly, weighing bran, wrapping the bran by two layers of gauze, boiling the bran in boiling water for 20 minutes, and filtering the bran to obtain filtrate for later use;
dissolving corn starch with distilled water, adding the dissolved corn starch into bran filtrate while stirring, boiling the mixture for 5 minutes with soft fire, filtering the mixture twice by 4 layers of gauze, and reserving filtrate for later use;
dissolving yeast powder, glucose, peptone, magnesium sulfate and potassium dihydrogen phosphate (the second-stage liquid strain liquid culture medium also comprises sodium carboxymethyl cellulose) with distilled water, adding into the filtrate in the previous step, stirring, heating with slow fire, decocting for 1 min, and adding deionized water to reach sufficient amount;
detecting with precise pH test paper until the pH value is about 5.5, otherwise, adjusting with 1 mol/L NaOH solution or HCl solution;
fifthly, subpackaging and sterilizing at 115 ℃ for 30 minutes.
3. Obtaining a first-stage liquid strain: inoculating three Morchella mother strains with diameter of 5mm per 100ml of liquid culture medium, and culturing at 18 ℃ on a shaking table and 180 r/min for 5 days to obtain the first-stage liquid strains of Morchella.
4. Obtaining secondary liquid strains: inoculating the first-stage liquid strain into a fermentation tank containing a second-stage liquid strain liquid culture medium according to the inoculation amount of 10%, standing and culturing for 1 day in the dark after inoculation, and then culturing for 5 days under the conditions of ventilation capacity of 4.0L/min, natural pH, rotation speed of 300 r/min and 18 ℃ to obtain the morchella second-stage liquid strain.
The Morchella stock culture obtained under the above conditions has dry weight of Morchella mycelium of 14.9mg/ml, and diameter of mycelium pellet below 1.5 mm.
FIG. 1 shows the stock Morchella strains of the present invention.
FIG. 2 shows the primary fermentation of the inventive Morchella stock.
Thirdly, inoculating and culturing morchella cultivars
Inoculating Morchella secondary liquid strain 3.5ml per 500g of cultivation material, and culturing at 18 deg.C for 10-20 days.
The cultivation material can be selected from commercially available Morchella cultivation material.
Example 2
The present embodiment is different from embodiment 1 in that: in the second step, the liquid culture medium of the original strain of morchella is prepared from 10g of corn starch, 2.4g of yeast powder, 60g of bran, 25g of glucose, 2.5g of peptone, 0.5g of magnesium sulfate, 1g of monopotassium phosphate and pH 5.5.
The dry weight of the mycelium of the original Morchella esculenta strain obtained under the above conditions can reach 10.8mg/ml, and the diameter of the mycelium pellet is below 2 mm.
Example 3
The present embodiment is different from embodiment 1 in that: in the second step, the liquid medium of morchella strain has different raw material ratios, and the raw material ratios of the medium in the embodiment are 15g of corn starch, 2.4g of yeast powder, 50g of bran, 15g of glucose, 2.5g of peptone, 0.5g of magnesium sulfate, 1g of potassium dihydrogen phosphate and pH 5.5.
The dry weight of the original strain mycelium of morchella obtained under the conditions can reach 12.2mg/ml, and the diameter of the mycelium pellet is less than 1.5 mm.
Comparative example
This comparative example differs from example 1 in that: in the second step, the liquid culture medium of the second-stage liquid strain of the morchella is different in raw material ratio, and the secondary fermentation culture medium in the comparative example is prepared from the following raw materials in percentage by weight: 15g of corn starch, 2.4g of yeast powder, 60g of bran, 25g of glucose, 2.5g of peptone, 0.5g of magnesium sulfate, 1g of monopotassium phosphate and pH 5.5. The remaining steps and parameters were the same as in example 1. Tests show that the effect of single carbon source fermentation is not as good as that of a composite carbon source, and the hypha biomass is only 10.3g/ml after secondary fermentation when no sodium carboxymethyl cellulose is added.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described above, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A liquid culture medium for preparing original strains of morchella in high altitude areas is characterized in that: the liquid culture medium consists of a primary liquid strain liquid culture medium and a secondary liquid strain liquid culture medium;
the formula of the primary liquid strain liquid culture medium is as follows: the culture medium contains 10-15g of corn starch, 2.4g of yeast powder, 46-60g of bran, 15-25g of glucose, 2.5g of peptone, 0.5g of magnesium sulfate and 1g of monopotassium phosphate according to the proportion per liter, and the pH value is 5.5;
the second-stage liquid strain liquid culture medium is prepared by adding 0.9 wt% of sodium carboxymethylcellulose into the first-stage liquid strain liquid culture medium.
2. The liquid culture medium for preparing the morchella stock culture in the high-altitude area according to claim 1, wherein the liquid culture medium comprises: the formula of the primary liquid strain liquid culture medium is as follows: according to the proportion, each liter of culture medium contains 10g of corn starch, 2.4g of yeast powder, 60g of bran, 25g of glucose, 2.5g of peptone, 0.5g of magnesium sulfate, 1g of monopotassium phosphate and pH 5.5.
3. The liquid culture medium for preparing the morchella stock culture in the high-altitude area according to claim 1, wherein the liquid culture medium comprises: the formula of the primary liquid strain liquid culture medium is as follows: the culture medium contains 15g of corn starch, 2.4g of yeast powder, 50g of bran, 15g of glucose, 2.5g of peptone, 0.5g of magnesium sulfate, 1g of monopotassium phosphate and pH5.5 in proportion per liter.
4. The liquid culture medium for preparing the morchella stock culture in the high-altitude area according to claim 1, wherein the liquid culture medium comprises: the formula of the primary liquid strain liquid culture medium is as follows: the culture medium contains 15g of corn starch, 2.4g of yeast powder, 46g of bran, 25g of glucose, 2.5g of peptone, 0.5g of magnesium sulfate, 1g of monopotassium phosphate and pH5.5 in proportion per liter.
5. A preparation method of original strains of morchella in high altitude areas is characterized by comprising the following steps: the method comprises the following steps:
(1) obtaining a morchella primary liquid strain: inoculating three morchella mother strains with the diameter of 4.8-5.2mm to each 100ml of the primary liquid strain liquid culture medium as defined in any one of claims 1-4, and culturing for 3-5 days at 18 ℃ of a shaking table and 200 revolutions per minute at 180 ℃ to obtain a morchella primary liquid strain;
(2) obtaining a second-level liquid strain of morchella esculenta: inoculating the primary liquid strain into a fermentation tank containing the secondary liquid strain liquid culture medium as claimed in any one of claims 1 to 4 according to the inoculation amount of 10 to 15 percent, standing and culturing in the dark for 1 to 2 days after inoculation, and then culturing for 5 to 6 days under the conditions of the ventilation volume of 3.5 to 4.0L/min, the natural pH value, the rotation speed of 300 to 350 revolutions per minute and the temperature of 15 to 18 ℃ to obtain the secondary liquid strain of the morchella esculenta.
6. The method for preparing the morchella stock culture in the high-altitude area according to claim 5, wherein the method comprises the following steps: the method comprises the following steps:
(1) obtaining a first-level liquid strain of morchella esculenta: inoculating three morchella mother strains with the diameter of 5mm into each 100ml of the primary liquid strain liquid culture medium as defined in any one of claims 1 to 4, and culturing for 5 days at the temperature of 18 ℃ and 180 r/min by a shaking table to obtain a morchella primary liquid strain;
(2) obtaining a second-level liquid strain of morchella esculenta: inoculating the primary liquid strain into a fermentation tank containing the secondary liquid strain liquid culture medium of any one of claims 1-4 according to the inoculation amount of 10%, standing and culturing in dark for 1 day after inoculation, and then culturing for 5 days under the conditions of ventilation capacity of 4.0L/min, natural pH, rotation speed of 300 r/min and 18 ℃ to obtain the secondary liquid strain of morchella esculenta.
7. A preparation method of morchella esculenta in high altitude areas comprises the preparation method of original morchella esculenta strains in high altitude areas of claim 5 or 6.
8. The preparation method of morchella esculenta in high altitude areas according to claim 7, wherein the method comprises the following steps: the altitude is 1900 and 2300 meters.
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