CN103782801A - Agaricus Bisporus liquid spawn and preparation method thereof - Google Patents
Agaricus Bisporus liquid spawn and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an Agaricus Bisporus liquid spawn and a preparation method thereof. The Agaricus Bisporus liquid spawn is produced by means of three-level propagation through slant and shake-flask media and a fermenter medium. The slant and shake-flask media comprise components including 100-200g of potato, 50-150g of cow dung, 10-30g of glucose, 1-2.5g of mono potassium phosphate, 0.5-2g of magnesium sulfate and 1-4g of peptone; the fermenter medium of every 50 liters of water is composed of 2-4kg of potato, 1-3kg of cow dung, 0.5-1kg of bran, 0.2-0.4 kg of corn powder, 0.5-1kg of sucrose, 30-60g of mono potassium phosphate, 20-30g of magnesium sulfate, 120-150 mg of vitamin B and 40-50g of defoamer. By application of the Agaricus Bisporus liquid spawn and the preparation method thereof, the problem that mycelia are prone to aging and degradation during growth is solved; the liquid spawn is of globular mycelia with uniform age, high activity and fast running; a high-running spawn center is formed after the liquid spawn is inoculated to solid media, with uniform fruiting achieved; the preparation cost is only one-tenth of that of solid spawn; the growth cycle of fermenter spawn is 7-9 days, and thus spawns on a large scale can be cultured within a short time.
Description
Technical field
The invention belongs to edible fungi liquid strain technical field, particularly relate to two spore mushroom liquid strains in edible mushroom and the preparation method of this liquid spawn thereof.
Background technology
In the production process of two spore mushrooms, bacterial classification used has solid spawn and liquid spawn.The two spore mushrooms of edible mushroom adopt traditional solid spawn cultivation mode conventionally at present, cultivating button mushroom kind is raw material mainly with wheat, be by female one-level PDA test tube slant plant to receive in wheat medium, make secondary solid spawn, again secondary kind is expanded to numerous three grades of solid spawns of making, in actual production, exist the production cycle long, cost of manufacture is high; The shortcomings such as pollution rate is high, and cell age is long, and fruiting is irregular, inconsistent.Solid spawn is covered with bottle from being inoculated into mycelia, generally needs 35-40 days, and the solid spawn often mycelia at the bottle end just sprouts, and its top layer mycelia is just aging, and therefore fruiting is uneven.Adopt liquid spawn to have with short production cycle, good fluidity, vitality is vigorous, sends out the advantages such as bacterium is fast, biological transformation ratio is high, bacterial classification purity height, is conducive to the advantages such as mechanized operation, is one of important directions of large-scale production development.But at present two spore mushroom liquid strain medium still exist and are prepared into that power is low, mycelial growth inadaptable, easily there is the problems such as the aging regression of mycelia in late stage of culture, and operation professional technique requires high, in large-scale production process, directly cultivation quality and the output of the two spore mushrooms of impact.
Summary of the invention
The present invention seeks to overcome many defects that above-mentioned prior art exists, the preparation method of a kind of pair of spore mushroom liquid strain and this liquid spawn thereof is provided.
Technical problem solved by the invention proposes following technical scheme and realizes: a kind of two spore mushroom liquid strains, formed by each solid constituent and liquid components, it is characterized in that: a kind of two spore mushroom liquid strains, formed by each solid constituent and liquid components, it is characterized in that: this pair of spore mushroom liquid strain is to expand numerous cultivation by slant medium, shaking flask medium and fermentation tank culture medium through three grades to make, and in the numerous cultivation of described expansion, the weight of medium at different levels composition is:
1) composition of slant medium: the weight of adding each component in every 1000 ml waters is:
Potato 100～200g, cow dung 50～150g, glucose 10～30g, potassium dihydrogen phosphate 1～2.5g, magnesium sulfate 0.5～2g, peptone 1～4g, agar 15～25g, pH nature;
2) composition of shaking flask medium: the weight of adding each component in every 1000 ml waters is:
Potato 100～200g, cow dung 50～150g, corn flour 5～20g, wheat bran 5～20g, glucose 10～30g, potassium dihydrogen phosphate 1～2.5g, magnesium sulfate 0.5～2g, peptone 1～4g, vitamin
b110～30 milligrams, pH nature;
3) composition of fermentation tank culture medium: the weight portion that adds each component in every 50 premium on currency is: potato 2～4kg, cow dung 1～3kg, wheat bran 0.5～1kg, corn flour 0.2～0.4kg, sucrose 0.5～1kg, potassium dihydrogen phosphate 30～60g, magnesium sulfate 20～30g, vitamin
b120～50 milligrams, defoamer 40～50g, pH nature.
The weight composition of the preferred medium at different levels of the present invention is:
1) composition of slant medium: the weight of adding each component in every 1000 ml waters is:
Potato 200g, cow dung 150g, glucose 20g, potassium dihydrogen phosphate 2g, magnesium sulfate 1g, peptone 2g, agar 20g, pH nature;
2) composition of shaking flask medium: the weight of adding each component in every 1000 ml waters is:
Potato 200g, cow dung 150g, corn flour 10g, wheat bran 10g, glucose 20g, potassium dihydrogen phosphate 2g, magnesium sulfate 1g, peptone 2g, vitamin
b120 milligrams, pH nature;
3) composition of fermentation tank culture medium: the weight portion that adds each component in every 50 premium on currency is: potato 2.5kg, cow dung 1kg, wheat bran 0.5kg, corn flour 0.25kg, sucrose 0.5kg, potassium dihydrogen phosphate 50g, magnesium sulfate 25g, vitamin
b130 milligrams, defoamer 50g, pH nature.
The method that the present invention prepares two spore mushroom liquid strains comprises the steps:
1) activated spawn: by former two spore mushrooms female kind in access slant medium, in 23～28 ℃ of cultivations 5～7 days, make the female bacterial classification sheet of planting in inclined-plane;
2) shaking flask strain cultivation: the shaking flask medium that packs bottle capacity 3/5 into, in 120 ± 2 ℃ of sterilizings 30 minutes, after cooling, plant 5～6 of bacterial classification sheets 0.4～0.5 centimetre of inclined-plane mother of inoculating hood access, bacterial classification sheet is swum on liquid level, leave standstill and cultivate 60～72 hours in incubator in 23～28 ℃, then in 23～28 ℃, 200r/min concussion is cultivated 5～8 days, obtain shaking flask liquid spawn, its standard is: after shaking flask shake, have mycelia wall built-up, what detection culture fluid mycelium pellet unit intensity touched the mark more than 80% can use;
3) fermentation tank strain cultivation: a) sterilizing of medium: the fermentation tank culture medium liquid of inserting tankage size 4/5 in fermentation tank, then heat up, in the time that rising to 0.05MPa, opens by manometer air bleeding valve, after being vented to zero, close, again rise to 0.05MPa, then open the micro-row of air bleeding valve, after 1～2 minute, close air bleeding valve, in 121～125 ℃, sterilizing 30～50 minutes under pressure 0.12～0.14MPa, crack discharge gate after sterilizing, 1.5～2 liters of discharges are sterilized to valve, b) cooling: adopt the interlayer type of cooling, the cooling discharge pressure that makes fermentation tank is down to 0.05～0.08MPa, opens air pump and vent valve, and regulating tank is depressed into 0.02MPa, cools the temperature to 26～28 ℃, c) inoculated and cultured: in the time that fermentation jar temperature is down to 26～28 ℃, open tank body inoculation mouthful and protected with flame ring, by flame ring, shaking flask liquid spawn is dropped in tank according to 5～8% inoculum concentration, cover immediately inoculation lid, open air bleeding valve and remove flame ring, make tank pressure remain on 0.01MPa, control 25～28 ℃ of cultivation temperature, inoculated and cultured detected strain quality after 48 hours, detecting qualified continuation at 25～28 ℃ of temperature cultivates 5～7 days, detect that culture fluid mycelium pellet unit intensity touches the mark more than 80%, make two three grades of liquid spawns of spore mushroom.
The invention solves the problem that is prone to aging regression in mycelial growth, because liquid spawn is coccoid mycelium, in bacterium liquid, also there are many bacterium liquid fragments, dispersity is large, good fluidity, and vitality is vigorous, sprout fast, be inoculated in solid culture medium, can, with the deep layer position that is seeped into solid culture medium under culture fluid, form many Fa Jun center; And liquid spawn breeds under stereoscopic culture state, cell age neat and consistent, and mostly in vigorous period, after inoculation mycelia to recover growth fast; Inoculate convenient and swiftly, after inoculation, can recover rapidly growth, adopt special inoculating gun, bacterium liquid is injected in bacterium bag, remove inoculation cave mouth from and seal operation, improve 4～5 times of effects; Mycelia is energetic, so fruiting is neat; Breeding cost is low, and liquid spawn breeding cost is only 1/10 left and right of solid spawn; Adopt liquid spawn to have with short production cycle, only 7～9 days fermentation tank growth cycle, can cultivate in a short time large batch of bacterial classification, meet the needs of mushroom industry large-scale production.
Embodiment
The invention will be further described by the following examples.
Embodiment 1:
1, strains tested: the two spore mushrooms in Shouguang, Shandong are female plants 2796;
2, culture medium prescription
A) slant medium: potato 200g, cow dung 150g, glucose 20g, potassium dihydrogen phosphate 2g, magnesium sulfate 1g, peptone 2g, agar 20g, PH nature, 1000 milliliters, water.
B) shaking flask medium: potato 200g, cow dung 150g, corn flour 10g, wheat bran 10g, glucose 20g, potassium dihydrogen phosphate 2g, magnesium sulfate 1g, peptone 2g, Cobastab
120 milligrams, PH nature, 1000 milliliters, water.
C) fermentation tank culture medium (by 50 liters of preparations): potato 2.5kg, cow dung 1kg, wheat bran 0.5kg, corn flour 0.25kg, sucrose 0.5kg, potassium dihydrogen phosphate 50g, magnesium sulfate 25g, vitamin
b130 milligrams, defoamer 50g, pH nature, 50 liters, water.
Cow dung process: cow dung dried and crushed process after sieve, under sunlight, tan by the sun dry for subsequent use.
3, activated spawn: by two spore mushroom master clock access slant mediums, cultivate at 25～26 ℃ 6 days, make the female bacterial classification sheet of planting in inclined-plane;
4, the cultivation of shaking flask liquid spawn: with 1000 milliliters of triangular flasks, pack 600 milliliters of shaking flask medium into, in 120 ± 2 ℃ of sterilizings 30 minutes, after cooling, access female 5～6 of the bacterial classification sheets of planting in approximately 0.5 centimetre of inclined-plane at inoculating hood, bacterial classification sheet is swum on liquid level, leave standstill and cultivate 72 hours in incubator in 26 ℃, again in 25～26 ℃, 6-7 days is cultivated in 200r/min concussion, obtain shaking flask liquid spawn, the standard of this liquid spawn is: after shake shaking flask, have mycelia wall built-up, detecting culture fluid mycelium pellet unit intensity (the mycelium pellet number in every liter of zymotic fluid) is 238～252/600m1.
5, fermentation tank strain cultivation:
A) sterilizing of medium: adopt 50 liters of automatic fermenters, in fermentation tank, insert 40 liters of fermentation tank culture medium liquid, then carry out sterilizing intensification, in the time that rising to 0.05MPa, opens by manometer air bleeding valve, after being vented to zero, close, again rise to 0.05MPa, then open the micro-row of air bleeding valve, after 1～2 minute, close; Temperature rise to 121～125 ℃, pressure, in the time of 0.12～0.14MPa, keeps sterilizing 50 minutes, after sterilizing finishes, crack discharge gate, 1.5～2 liters of discharges carry out valve sterilization, and valve-off is cooling;
B) cooling: to adopt the interlayer type of cooling, running water pipe is received to cooling water inlet, cooling when discharge pressure in fermentation tank is down to 0.05～0.08MPa, open air pump and vent valve, regulating tank is depressed into 0.02MPa, prepares inoculation while cooling the temperature to 26～28 ℃;
C) inoculated and cultured: in the time that fermentation jar temperature is down to 26～28 ℃, open tank body inoculation mouthful, and with flame ring protection inoculation mouthful, that 95% alcohol-pickled sliver is wrapped on the inoculation mouth of fermentation tank body, when lighting sliver, open inoculation mouthful, by flame ring, shaking flask liquid spawn is dropped in tank, inoculum concentration (weight) is 5%, then cover immediately inoculation mouthful, open air bleeding valve and remove flame ring, make tank pressure remain on 0.01MPa, control 25～26 ℃ of cultivation temperature, inoculated and cultured was inspected by random samples bacterial classification after 48 hours, detecting qualified continuation at 25～26 ℃ of temperature cultivates 5～6 days, make two three grades of liquid spawns of spore mushroom, detecting culture fluid mycelium pellet unit intensity (the mycelium pellet number in every liter of zymotic fluid) is 384～402.
Two spore mushroom liquid strain fermentation test results show: in fermentation process, content of reducing sugar is relatively stable, and pH value changes not quite about 2 days, but pH obviously declines subsequently; In the time fermenting to 6～7 days, the biomass of button mushroom pompon reaches the more than 80% of standard, and the mycelium pellet biomass during to 8th～9 days in fermentation tank reaches maximum, and the biomass of mycelium pellet keeps relative stability and starts and progressively declines subsequently.By to the index analysis such as pH value and mycelium pellet biomass in fermentation process, can be defined as fermentation termination by the 9th day, and aborning, can select to ferment 8th～9 days as liquid fungus seed terminal, mycelium pellet biomass and activity are now the highest.
Below give different cultivation temperature and the inoculum concentration detection data (by shaking flask kind access fermentation tank) on the impact of mycelium pellet biomass, wherein mycelium pellet Board Lot is density (for the mycelium pellet number in every liter of zymotic fluid).
The impact of table 1 cultivation temperature on fermentation tank button mushroom silk biomass
| Cultivation temperature/℃ | Mycelium pellet biomass | Mycelium pellet Board Lot | Mycelium pellet diameter |
| 21 | 8.0～9.1 | 270～390 | 0.8～1.1 |
| 25 | 8.2～9.4 | 350～437 | 0.8～1.2 |
| 29 | 3.5～4.7 | 210～252 | 0.9～1.24 |
The impact of table 2 different vaccination amount on fermentation tank liquid spawn biomass
The data of table 1 show: when 25 ℃ of cultivation temperature, and index the best such as biomass, mycelium pellet unit intensity of mycelium pellet, the two spore mushroom liquid strains of the present invention expand cultivation temperature while cultivation and select 25～26 ℃ and be advisable.
The data of table 2 show: when when liquid-spawn inoculation amount is 5%, (by 3 grades of fermentation tanks of 2 grades of shaking flask kind accesses) mycelium pellet biomass is compared with inoculum concentration 2.5%, be significantly improved, but inoculum concentration exceedes the biomass of mycelium pellet between the each processing after 5%, diameter and mycelium pellet Board Lot difference are little; Therefore when the two spore mushroom liquid strains of the present invention expand cultivation, inoculum concentration selection 5% is comparatively suitable.
Embodiment 2:
1, culture medium prescription
A) slant medium: potato 150g, cow dung 100g, glucose 15g, potassium dihydrogen phosphate 1.5g, magnesium sulfate 0.8g, peptone 1.5g, agar 15g, PH nature, 1000 milliliters, water.
B) shaking flask medium: potato 150g, cow dung 100g, corn flour 8g, wheat bran 8g, glucose 15g, potassium dihydrogen phosphate 1.5g, magnesium sulfate 0.8g, peptone 1.5, Cobastab
115 milligrams, PH nature, 1000 milliliters, water.
C) fermentation tank culture medium (by 50 liters of preparations): potato 2.0kg, cow dung 1kg, wheat bran 0.5kg, corn flour 0.2kg, sucrose 0.5kg, potassium dihydrogen phosphate 40g, magnesium sulfate 20g, vitamin
b120 milligrams, defoamer 40g, pH nature, 50 liters, water.
2, the preparation method of two spore mushroom liquid strains is with embodiment 1.
Embodiment 3:
1, culture medium prescription
A) slant medium: potato 200g, cow dung 120g, glucose 18g, potassium dihydrogen phosphate 1.8g, magnesium sulfate 0.8g, peptone 2g, agar 25g, PH nature, 1000 milliliters, water.
B) shaking flask medium: potato 200g, cow dung 120g, corn flour 15g, wheat bran 15g, glucose 20g, potassium dihydrogen phosphate 1.8g, magnesium sulfate 0.8g, peptone 2, Cobastab
120 milligrams, PH nature, 1000 milliliters, water.
C) fermentation tank culture medium (by 50 liters of preparations): potato 2.5kg, cow dung 2.5kg, wheat bran 1.0kg, corn flour 0.3kg, sucrose 0.5kg, potassium dihydrogen phosphate 55g, magnesium sulfate 25g, vitamin
b130 milligrams, defoamer 50g, pH nature, 50 liters, water.
2, the preparation method of two spore mushroom liquid strains is with embodiment 1.
Claims (3)
1. a two spore mushroom liquid strain, formed by each solid constituent and liquid components, it is characterized in that: this pair of spore mushroom liquid strain is to expand numerous cultivation by slant medium, shaking flask medium and fermentation tank culture medium through three grades to make, and in the numerous cultivation of described expansion, the weight of medium at different levels composition is:
1) composition of slant medium: the weight of adding each component in every 1000 ml waters is:
Potato 100～200g, cow dung 50～150g, glucose 10～30g, potassium dihydrogen phosphate 1～2.5g, magnesium sulfate 0.5～2g, peptone 1～4g, agar 15～25g, pH nature;
2) composition of shaking flask medium: the weight of adding each component in every 1000 ml waters is:
Potato 100～200g, cow dung 50～150g, corn flour 5～20g, wheat bran 5～20g, glucose 10～30g, potassium dihydrogen phosphate 1～2.5g, magnesium sulfate 0.5～2g, peptone 1～4g, vitamin
b110～30 milligrams, pH nature;
3) composition of fermentation tank culture medium: the weight portion that adds each component in every 50 premium on currency is: potato 2～4kg, cow dung 1～3kg, wheat bran 0.5～1kg, corn flour 0.2～0.4kg, sucrose 0.5～1kg, potassium dihydrogen phosphate 30～60g, magnesium sulfate 20～30g, vitamin
b120～50 milligrams, defoamer 40～50g, pH nature.
2. according to claim 1 pair of spore mushroom liquid strain, is characterized in that: the weight composition of preferred medium at different levels is:
1) composition of slant medium: the weight of adding each component in every 1000 ml waters is:
Potato 200g, cow dung 150g, glucose 20g, potassium dihydrogen phosphate 2g, magnesium sulfate 1g, peptone 2g, agar 20g, pH nature;
2) composition of shaking flask medium: the weight of adding each component in every 1000 ml waters is:
Potato 200g, cow dung 150g, corn flour 10g, wheat bran 10g, glucose 20g, potassium dihydrogen phosphate 2g, magnesium sulfate 1g, peptone 2g, vitamin
b120 milligrams, pH nature;
3) composition of fermentation tank culture medium: the weight portion that adds each component in every 50 premium on currency is: potato 2.5kg, cow dung 1kg, wheat bran 0.5kg, corn flour 0.25kg, sucrose 0.5kg, potassium dihydrogen phosphate 50g, magnesium sulfate 25g, vitamin
b130 milligrams, defoamer 50g, pH nature.
3. the preparation method of according to claim 1 pair of spore mushroom liquid strain, is characterized in that: utilize the method for the two spore mushroom liquid strains of this medium preparation to comprise the steps:
1) activated spawn: by former two spore mushrooms female kind in access slant medium, in 23～28 ℃ of cultivations 5～7 days, make the female bacterial classification sheet of planting in inclined-plane;
2) shaking flask strain cultivation: the shaking flask medium that packs bottle capacity 3/5 into, in 120 ± 2 ℃ of sterilizings 30 minutes, after cooling, plant 5～6 of bacterial classification sheets 0.4～0.5 centimetre of inclined-plane mother of inoculating hood access, bacterial classification sheet is swum on liquid level, leave standstill and cultivate 60～72 hours in incubator in 23～28 ℃, then in 23～28 ℃, 200r/min concussion is cultivated 5～8 days, obtain shaking flask liquid spawn, its standard is; After shaking flask shake, have mycelia wall built-up, what detection culture fluid mycelium pellet unit intensity touched the mark more than 80% can use;
3) fermentation tank strain cultivation: a) sterilizing of medium: the fermentation tank culture medium liquid of inserting tankage size 4/5 in fermentation tank, then heat up, in the time that rising to 0.05MPa, opens by manometer air bleeding valve, after being vented to zero, close, again rise to 0.05MPa, then open the micro-row of air bleeding valve, after 1～2 minute, close air bleeding valve, in 121～125 ℃, sterilizing 30～50 minutes under pressure 0.12～0.14MPa, crack discharge gate after sterilizing, 1.5～2 liters of discharges are sterilized to valve, b) cooling: adopt the interlayer type of cooling, the cooling discharge pressure that makes fermentation tank is down to 0.05～0.08MPa, opens air pump and vent valve, and regulating tank is depressed into 0.02MPa, cools the temperature to 26～28 ℃, c) inoculated and cultured: in the time that fermentation jar temperature is down to 26～28 ℃, open tank body inoculation mouthful and protected with flame ring, by flame ring, shaking flask liquid spawn is dropped in tank according to 5～8% inoculum concentration, cover immediately inoculation lid, open air bleeding valve and remove flame ring, make tank pressure remain on 0.01MPa, control 25～28 ℃ of cultivation temperature, inoculated and cultured detected strain quality after 48 hours, detecting qualified continuation at 25～28 ℃ of temperature cultivates 5～7 days, detect that culture fluid mycelium pellet unit intensity touches the mark more than 80%, make two three grades of liquid spawns of spore mushroom.
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