CN111642326A - Mother culture method of pleurotus eryngii liquid strain - Google Patents
Mother culture method of pleurotus eryngii liquid strain Download PDFInfo
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- CN111642326A CN111642326A CN202010653976.4A CN202010653976A CN111642326A CN 111642326 A CN111642326 A CN 111642326A CN 202010653976 A CN202010653976 A CN 202010653976A CN 111642326 A CN111642326 A CN 111642326A
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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
- A01G18/00—Cultivation of mushrooms
- A01G18/40—Cultivation of spawn
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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
- A01G18/00—Cultivation of mushrooms
- A01G18/20—Culture media, e.g. compost
Abstract
The invention relates to the technical field of pleurotus eryngii liquid strains, in particular to a mother strain culture method of pleurotus eryngii liquid strains, which comprises the following steps: s1: activating the PSA slant strain of the pleurotus eryngii, inoculating the activated strain into an active culture medium, and culturing the activated strain into a flat plate mother strain of the pleurotus eryngii; s2: carrying out aeration treatment on tap water at 10-15 ℃ by using an aeration instrument, and stopping aeration when the oxygen content in the tap water is 15-20 mg/L; s3: mixing the tap water subjected to oxygen exposure treatment with an active culture medium according to the mass ratio of 1: 2, placing the mixture into a shake flask and mixing the mixture into slurry, and then placing the shake flask filled with the slurry into a sterilization pot for sterilization treatment; s4: adding the flat mother seeds of the pleurotus eryngii cultured in the S1 into a homogenizer after sterilization treatment for homogenizing treatment; s5: inoculating the serous fluid in the S4 into a shake flask of S3, standing for 24h, transferring to a constant temperature shaking table of 25 ℃ for culturing for 36-48h, and preparing a mother strain of the pleurotus eryngii liquid strain; the mother seeds prepared by the method have uneven seed growing speed, better disease resistance and suitability for further popularization and application.
Description
Technical Field
The invention relates to the technical field of pleurotus eryngii liquid strains, and particularly relates to a mother strain culture method of pleurotus eryngii liquid strains.
Background
Lentinus edodes belongs to Basidiomycetes (Basidaomyetes), Agaricales (Agaricales), Tricholomataceae (Tricholomataceae), Lentinus (Lentinus) and Lentinusedodes (Lentinus) with the academic name of Lentinusedodes, which originates from China, is the second largest mushroom in the world and is also a rare edible fungus which is famous for a long time in China.
The traditional mushroom production generally adopts solid strains for inoculation culture, the strains mainly comprise conventional solid strains such as wood chip strains, branch strains and the like, the production period of the method is long, the method generally needs 55-60 days, the process is complex, and the production method has low efficiency; the production process of the strain needs a large space, needs a large amount of manpower and material resources, and has high cost; the culture period of the hyphae is long, the difference of the hyphae age in the culture medium is large, when the front end hyphae are in a budding state, the hyphae on the surface of the substrate are nearly aged, the fruiting of the cultivated species is irregular, the scale production of the mushrooms cannot be achieved, and the production requirements of people are difficult to meet. In recent years, due to the rapid development of industrial cultivation technology, in order to shorten the strain preparation period and improve the strain quality and production efficiency, manufacturers at home and abroad research and develop liquid strains to replace solid strains commonly used in the production of the shiitake mushrooms at present and start to apply the liquid strains in actual production.
In the prior art, the culture process of the mother strains of the pleurotus eryngii liquid strains is lagged, and the spawn running efficiency and the disease-resistant effect of the prepared liquid strains are not ideal and still need to be further improved.
Disclosure of Invention
Aiming at the problems, the invention provides a method for culturing a mother strain of a pleurotus eryngii liquid strain.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a mother culture method of pleurotus eryngii liquid strains comprises the following steps:
s1: activating the PSA slant strain of the pleurotus eryngii, inoculating the activated strain into an active culture medium, and culturing the activated strain into a flat plate mother strain of the pleurotus eryngii;
s2: carrying out aeration treatment on tap water at 10-15 ℃ by using an aeration instrument, and stopping aeration when the oxygen content in the tap water is 15-20 mg/L;
s3: mixing the tap water subjected to oxygen exposure treatment with an active culture medium according to the mass ratio of 1: 2-3, placing the mixture into a shake flask and mixing the mixture into slurry, and then placing the shake flask containing the slurry into a sterilization pot for sterilization treatment;
s4: adding the flat mother seeds of the pleurotus eryngii cultured in the S1 into a homogenizer after sterilization treatment for homogenizing treatment;
s5: and inoculating the serous fluid in the S4 into a shake flask of S3, standing for 24h, transferring to a constant-temperature shaking table at 25 ℃ and culturing for 36-48h to obtain the mother strain of the pleurotus eryngii liquid strain.
Further, the preparation method of the active culture medium comprises the following steps:
a) the method comprises the following steps Inoculating the mixed microbial inoculum in a sterilized culture medium according to an inoculation amount of 5%, loading a sterile air system, and culturing at a constant temperature of 33-35 ℃ for 2 days, wherein the ventilation amount is 8-10L/min;
b) the method comprises the following steps Filtering the culture medium after culturing for 1-3 days with 500 mesh filter screen, treating the filtrate at 150 deg.C for 60-90s, and taking out;
c) mixing the filtrate obtained in the step b) according to a mass ratio of 1: 15-20 are added into the liquid culture medium and mixed evenly to prepare the active culture medium.
Further, the mixed microbial inoculum is prepared by mixing bacillus subtilis, bacillus megaterium, bacillus amyloliquefaciens and azotobacter chroococcum according to the mass ratio of 5: 3: 2: 6, and the effective viable count in the mixed microbial inoculum is not less than 2.0 × 109cfu/g。
Further, the pH value of the culture medium in the step a) is 6.2-7.0, and the culture medium comprises the following components in percentage by weight: 10-12% of corn flour; 7-9% of sucrose; 3-8% of light calcium carbonate; 2-5% of sodium chloride; 0.06-0.08% of defoaming agent; peptone 1-5%, calcium superphosphate 0.2-0.8%, and water in balance.
Further, the pH of the liquid culture medium in the step b) is 6.5-7.0, and the components of the liquid culture medium in percentage by weight comprise: 5-8% of soybean meal, 10-12% of soybean meal, 6-8% of white sugar, 5-6% of glucose, 0.5-0.8% of monopotassium phosphate, 0.05-0.08% of defoaming agent, VB 11 tablets/15L and the balance of water.
Preferably, the sterilization temperature in S3 is 110-120 ℃, and the sterilization time is 45-60 min.
Preferably, the rotation speed of the homogenizing machine in S4 is 120-150r/min, and the homogenizing time is 30-60 min.
Preferably, the volume ratio of the slurry of S4 in S5 to the slurry in the S3 shake flask is 1: 9-10.
Compared with the prior art, the invention has the following beneficial effects:
the liquid strain mother culture of the pleurotus eryngii cultured by the method has the advantages of high growth rate and strong anti-bacteria capability, greatly improves the yield and fruiting efficiency of the pleurotus eryngii, and has excellent application prospect. According to the invention, a large amount of oxygen is needed in the growth stage of the pleurotus eryngii hyphae, and the oxygen is injected into the culture solution in the mother culture, so that the oxygen content in the culture solution is improved, and the growth of the hyphae in the processes of shake flask culture and subsequent fermentation is promoted. In addition, the promoting factors produced in the process of culturing the mixed microbial inoculum in the active culture medium can not only accelerate the spawn running rate of the mother seeds, but also improve the ability of the mother seeds to resist the infection of the mixed bacteria, greatly reduce the occurrence probability of the yellow rot and the blight, and are suitable for further popularization and application.
Drawings
FIG. 1 is a graph showing the detection of the diameter of a bacterial pellet at different time points.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely in the following with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
the method for culturing the mother strain of the pleurotus eryngii B012 liquid strain comprises the following steps:
s1: activating the PSA slant strain of the pleurotus eryngii, inoculating the activated strain into an active culture medium, and culturing the activated strain into a flat plate mother strain of the pleurotus eryngii;
s2: carrying out aeration treatment on tap water at 10-15 ℃ by using an aeration instrument, and stopping aeration when the oxygen content in the tap water is 15-20 mg/L;
s3: mixing the tap water subjected to oxygen exposure treatment with an active culture medium according to the mass ratio of 1: 2, placing the mixture into a shake flask and mixing the mixture into slurry, and then placing the shake flask filled with the slurry into a sterilization pot for sterilization treatment, wherein the sterilization temperature is 110 ℃, and the sterilization time is 60 min;
s4: adding the mother strains of the oyster mushroom plates cultured in the S1 into a homogenizer after sterilization treatment, and homogenizing for 60min at the rotating speed of 120 r/min;
s5: mixing the slurry in the S4 in a volume ratio of 1: and 9, standing the mixture in a shake flask of S3 for 24 hours, and then transferring the mixture to a constant-temperature shaking table at 25 ℃ for culturing for 36 hours to obtain the mother strain of the pleurotus eryngii liquid strain.
Further, the preparation method of the active culture medium comprises the following steps:
a) the method comprises the following steps Inoculating the mixed microbial inoculum in a sterilized culture medium according to an inoculation amount of 5%, loading a sterile air system, and culturing at a constant temperature of 33-35 ℃ for 2 days, wherein the ventilation amount is 8-10L/min;
the pH value of the culture medium in the step a) is 6.2-7.0, and the culture medium comprises the following components in percentage by weight: 12% of corn flour; 9% of sucrose; 8 percent of light calcium carbonate; 5% of sodium chloride; 0.08 percent of defoaming agent; 5% of peptone, 0.8% of calcium superphosphate and the balance of water.
b) The method comprises the following steps Filtering the culture medium after culturing for 1 day with 500 mesh filter screen, treating the filtrate at 150 deg.C for 80s, and taking out;
the pH value of the liquid culture medium in the step b) is 6.5-7.0, and the liquid culture medium comprises the following components in percentage by weight: the seed tank and the shake flask in S1 are composed of culture media: 8% of soybean meal, 12% of soybean meal, 8% of white sugar, 6% of glucose, 0.8% of monopotassium phosphate, 0.08% of defoaming agent, VB 11 tablets/15L and the balance of water.
c) Mixing the filtrate obtained in the step b) according to a mass ratio of 1: 15 is added into the liquid culture medium and mixed evenly to prepare the active culture medium.
The mixed microbial inoculum is prepared by mixing bacillus subtilis, bacillus megatherium, bacillus amyloliquefaciens and azotobacter chroococcum according to the mass ratio of 5: 3: 2: 6, and the effective viable count in the mixed microbial inoculum is 2.0-2.3 × 109cfu/g。
Example 2:
substantially the same as in embodiment 1, except that, in step S5: transferring to a constant temperature shaker at 25 ℃ for culturing for 40h to obtain the mother strain of the pleurotus eryngii liquid strain.
Example 3:
substantially the same as in embodiment 1, except that, in step S5: transferring to a constant temperature shaker at 25 ℃ for culturing for 44h to obtain the mother strain of the pleurotus eryngii liquid strain.
Example 4:
substantially the same as in embodiment 1, except that, in step S5: transferring to a constant temperature shaker at 25 ℃ for culturing for 48h to obtain the mother strain of the pleurotus eryngii liquid strain.
Comparative example 1:
substantially the same as in embodiment 1, except that, in step S5: transferring to a constant temperature shaker at 25 ℃ for culturing for 56h to obtain the mother strain of the pleurotus eryngii liquid strain.
Comparative example 2:
substantially the same as in embodiment 1, except that, in step S5: transferring to a constant temperature shaker at 25 ℃ for culturing for 64h to obtain the mother strain of the pleurotus eryngii liquid strain.
Comparative example 3:
substantially the same as in embodiment 1, except that, in step S5: transferring to a constant temperature shaker at 25 ℃ for culturing for 72h to obtain the mother strain of the pleurotus eryngii liquid strain.
And (3) strain testing:
1) 1ml of mother seed slurry of the pleurotus eryngii B102 liquid strain prepared in examples 1-4 and comparative examples 1-3 of the present invention was put into a culture dish, 20 pellets were randomly arranged in a row in the culture dish, the total length was measured, the diameter of a single pellet was calculated, the average value was taken at least 5 times, and the curve was plotted as shown in fig. 1.
From examples 1 to 4 and comparative examples 1 to 3 of the present invention, it is seen that the activity of the liquid strain of Pleurotus eryngii can be significantly improved and the reaction time can be reduced by controlling the culture time and the culture solution. It can be understood that in the initial stage of the reaction, as the culture solution of the fermentation tank is more suitable for the rapid division and proliferation of the strains, the diameter is reduced and the activity is improved in the proliferation process; in the middle reaction period, due to the consumption of nutrient substances and the generation of waste, the competition among strains is intensified, the proliferation speed is reduced, the strains grow up, and the activity is reduced. Therefore, it can be seen from the figure that the optimum culture time of the culture solution of the present invention can be shortened from about 6 days to about 3 days.
2) 10mL of the mother seed slurry of the pleurotus eryngii B102 liquid strain prepared in the examples 1-4 and the comparative examples 1-3 of the invention and the mother seed slurry of the commercial pleurotus eryngii B102 liquid strain are taken and poured into a shake flask and cultured by constant temperature oscillation at 24 ℃, after 6 days of shake flask culture, hyphae in the shake flask are filtered out, washed for 3 times, then put into a drying oven for drying at 100 ℃ for 90min, and weighed after being cooled to room temperature, the biomass of the pleurotus eryngii hyphae is measured, and the test results are shown in the following table: (the control group is the mother strain of commercial Pleurotus eryngii B102 liquid strain)
| Group of | Mycelium Biomass (g) |
| Example 1 | 0.67 |
| Example 2 | 0.72 |
| Example 3 | 0.74 |
| Example 4 | 0.75 |
| Comparative example 1 | 0.60 |
| Comparative example 2 | 0.45 |
| Comparative example 3 | 0.38 |
| Control group | 0.33 |
As can be seen from the above table, the mycelium biomass of the liquid strain stock seed of Pleurotus eryngii prepared by the present invention is larger than that of the commercial Pleurotus eryngii strain stock seed. Compared with the commercial pleurotus eryngii strain mother culture, the pleurotus eryngii liquid strain mother culture prepared by the invention has the advantages of short growth cycle and high spawn running rate. In addition, it can be seen from the table that the mother strain of the liquid strain of Pleurotus eryngii in example 4 has the best activity.
3) The mother strains of the pleurotus eryngii B102 liquid strains prepared in the embodiments 1-4 of the invention and the mother strains of the commercial pleurotus eryngii B102 liquid strains are inoculated in a fermentation tank filled with sterilized fermentation culture solution by 10 percent of inoculum size, the ventilation volume is 10L/min, and the pleurotus eryngii B102 liquid strains are prepared by constant temperature culture at 25 ℃ for 36 hours. Are described as example 1, example 2, example 3, example 4 and control, respectively.
Wherein the fermentation culture solution comprises the following components in percentage by weight: 10-12% of corn flour, 7-9% of white sugar, 3-5% of glucose, 2-3% of soybean meal, 0.5-0.8% of monopotassium phosphate, 0.2-0.5% of magnesium sulfate, 0.06-0.09% of defoaming agent, 0.03-0.05% of active agent, VB 11 tablets/10L and the balance of water.
Respectively inoculating the liquid strains prepared by fermenting the mother strains of the liquid strains of the embodiments 1 to 4 and the commercial pleurotus eryngii B102 onto the bacterial sticks, and pricking 5 holes on each bacterial stick; then placing the fungus sticks in a culture room for shading culture, controlling the temperature to be 24-26 ℃ and the relative humidity of air to be 66-68%, and pricking holes for increasing oxygen for 2 times; pricking holes after hypha grows over the fungus sticks to increase oxygen, irradiating with light, controlling fruiting temperature at 12-25 deg.C, day and night temperature difference greater than 10 deg.C, air relative humidity at 86-88%, fruiting, and harvesting.
The infectious microbe infection condition, the fungus growing time and the biological conversion rate in the cultivation process of each group of test strains are observed and counted, and the statistical results are shown in the following table: (wherein the control group is liquid strain cultured by commercial Pleurotus eryngii liquid strain mother strain.)
As can be seen from the above table, compared with the commercial pleurotus eryngii liquid strain mother strains, the pleurotus eryngii liquid strain mother strains prepared by the invention have higher activity, stronger anti-mixed bacteria capability, high biological conversion rate and excellent application prospect.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (8)
1. A mother culture method of pleurotus eryngii liquid strains is characterized by comprising the following steps:
s1: activating the PSA slant strain of the pleurotus eryngii, inoculating the activated strain into an active culture medium, and culturing the activated strain into a flat plate mother strain of the pleurotus eryngii;
s2: carrying out aeration treatment on tap water at 10-15 ℃ by using an aeration instrument, and stopping aeration when the oxygen content in the tap water is 15-20 mg/L;
s3: mixing the tap water subjected to oxygen exposure treatment with an active culture medium according to the mass ratio of 1: 2, placing the mixture into a shake flask and mixing the mixture into slurry, and then placing the shake flask filled with the slurry into a sterilization pot for sterilization treatment;
s4: adding the flat mother seeds of the pleurotus eryngii cultured in the S1 into a homogenizer after sterilization treatment for homogenizing treatment;
s5: and inoculating the serous fluid in the S4 into a shake flask of S3, standing for 24h, transferring to a constant-temperature shaking table at 25 ℃ and culturing for 36-48h to obtain the mother strain of the pleurotus eryngii liquid strain.
2. The fermentation method of high-activity pleurotus eryngii liquid spawn according to claim 1, wherein the preparation method of the active culture medium is as follows:
a) the method comprises the following steps Inoculating the mixed microbial inoculum in a sterilized culture medium according to an inoculation amount of 5%, loading a sterile air system, and culturing at a constant temperature of 33-35 ℃ for 2 days, wherein the ventilation amount is 8-10L/min;
b) the method comprises the following steps Filtering the culture medium after culturing for 1-3 days with 500 mesh filter screen, treating the filtrate at 150 deg.C for 80s, and taking out;
c) mixing the filtrate obtained in the step b) according to a mass ratio of 1: 15 is added into the liquid culture medium and mixed evenly to prepare the active culture medium.
3. The mother culture method of liquid spawn of pleurotus eryngii according to claim 2, wherein the mixed microbial inoculum is prepared by mixing bacillus subtilis, bacillus megaterium, bacillus amyloliquefaciens and azotobacter chroococcum according to a mass ratio of 5: 3: 2: 6, and the effective viable count in the mixed microbial inoculum is not less than 2.0 × 109cfu/g。
4. The method for culturing the mother culture of the liquid spawn of pleurotus eryngii according to claim 2, wherein the pH of the culture medium in the step a) is 6.2-7.0, and the components of the culture medium in percentage by weight comprise: 12% of corn flour; 9% of sucrose; 8 percent of light calcium carbonate; 5% of sodium chloride; 0.08 percent of defoaming agent; 5% of peptone, 0.8% of calcium superphosphate and the balance of water.
5. The method for culturing mother strains of liquid strains of pleurotus eryngii according to claim 2, wherein the pH of the liquid culture medium in step b) is 6.5-7.0, and the liquid culture medium comprises the following components in percentage by weight: 8% of soybean meal, 12% of soybean meal, 8% of white sugar, 6% of glucose, 0.8% of monopotassium phosphate, 0.08% of defoaming agent, VB 11 tablets/15L and the balance of water.
6. The method for culturing mother strains of liquid strains of pleurotus eryngii according to claim 1, wherein the sterilization temperature in S3 is 110 ℃ and the sterilization time is 60 min.
7. The method for culturing the mother strain of the liquid spawn of pleurotus eryngii according to claim 1, wherein the rotation speed of a homogenizer in S4 is 120r/min, and the homogenizing time is 30-60 min.
8. The method for culturing mother strains of liquid spawn of Pleurotus eryngii according to claim 1, wherein the volume ratio of the S4 serous fluid in S5 to the S3 shake flask is 1: 9.
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Cited By (1)
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| CN115152532A (en) * | 2022-06-08 | 2022-10-11 | 湖南果秀食品有限公司 | Quantitative and visual pleurotus eryngii liquid shake flask stock seed production method |
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| CN115152532A (en) * | 2022-06-08 | 2022-10-11 | 湖南果秀食品有限公司 | Quantitative and visual pleurotus eryngii liquid shake flask stock seed production method |
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