CN103782801B - White mushroom liquid spawn and preparation method thereof - Google Patents
White mushroom liquid spawn and preparation method thereof Download PDFInfo
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- CN103782801B CN103782801B CN201310755235.7A CN201310755235A CN103782801B CN 103782801 B CN103782801 B CN 103782801B CN 201310755235 A CN201310755235 A CN 201310755235A CN 103782801 B CN103782801 B CN 103782801B
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Abstract
The present invention discloses a kind of White mushroom liquid spawn and preparation method thereof, White mushroom liquid spawn is expanded numerous obtained by inclined-plane, Shake flask medium and fermentation tank culture medium through three grades, inclined-plane and Shake flask medium component: potato 100 ~ 200g, cow dung 50 ~ 150g, glucose 10 ~ 30g, potassium dihydrogen phosphate 1 ~ 2.5g, magnesium sulfate 0.5 ~ 2g, peptone 1 ~ 4g; Each component in every 50 premium on currency of fermentation tank culture medium: 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.The invention solves the problem easily occurring aging regression in mycelial growth, liquid spawn is coccoid mycelium, cell age neat and consistent, and mycelia is energetic, and sprout fast, be inoculated in solid culture medium, form multiple bacterium center, fruiting is neat; Breeding cost is 1/10 of solid spawn; In 7 ~ 9 days fermentation tank growth cycles, bacterial classification in enormous quantities can be cultivated in a short time.
Description
Technical field
The invention belongs to edible fungi liquid strain technical field, particularly relate to the preparation method of White mushroom liquid spawn in edible mushroom and this liquid spawn thereof.
Background technology
Bacterial classification used in the production process of White mushroom has solid spawn and liquid spawn.Current edible mushroom White mushroom adopts traditional solid spawn cultivation mode usually, cultivating White mushroom bacterial classification is raw material mainly with wheat, one-level PDA test tube slant mother is planted to receive in wheat medium make secondary solid spawn, three grades of solid spawns are made again by numerous for the expansion of secondary kind, in actual production, there is 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 covers with bottle from being inoculated into mycelia, generally needs 35-40 days, and the mycelia of solid spawn often at the bottom of bottle 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, and send out the advantages such as bacterium is fast, biological transformation ratio is high, bacterial classification purity is high, being conducive to the advantages such as mechanized operation, is one of important directions of large-scale production development.But at present White mushroom liquid spawn culture medium still also exists and is prepared into that power is low, mycelial growth is not suitable with, easily there is the problems such as the aging regression of mycelia in the phase after incubation, and operation professional technique requires high, in large-scale production process, cultivation quality and the output of White mushroom directly can be affected.
Summary of the invention
The present invention seeks to the many defects overcoming the existence of above-mentioned prior art, the preparation method of a kind of White mushroom liquid spawn and this liquid spawn thereof is provided.
Technical problem solved by the invention proposes following technical scheme to realize: a kind of White mushroom liquid spawn, be made up of each solid constituent and liquid components, it is characterized in that: a kind of White mushroom liquid spawn, be made up of each solid constituent and liquid components, it is characterized in that: this White mushroom liquid spawn expands numerous cultivation by slant medium, Shake flask medium and fermentation tank culture medium through three grades to obtain, and in the numerous cultivation of described expansion, the weight composition of medium at different levels is:
1) composition of slant medium: in every 1000 ml waters add each component weight be:
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 Shake flask medium: in every 1000 ml waters add each component weight be:
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: in every 50 premium on currency add each component weight portion be: 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: in every 1000 ml waters add each component weight be:
Potato 200g, cow dung 150g, glucose 20g, potassium dihydrogen phosphate 2g, magnesium sulfate 1g, peptone 2g, agar 20g, pH nature;
2) composition of Shake flask medium: in every 1000 ml waters add each component weight be:
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: in every 50 premium on currency add each component weight portion be: 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 White mushroom liquid spawn comprises the steps:
1) activated spawn: former for White mushroom mother is planted in access slant medium, cultivates 5 ~ 7 days in 23 ~ 28 DEG C, obtained inclined-plane mother plants bacterial classification sheet;
2) shaking flask strain cultivation: the Shake flask medium loading bottle capacity 3/5, in 120 ± 2 DEG C of sterilizings 30 minutes, bacterial classification sheet 5 ~ 6 is planted inoculating hood access 0.4 ~ 0.5 centimetre of inclined-plane mother after cooling, bacterial classification sheet is made to swim on liquid level, in 23 ~ 28 DEG C of quiescent culture 60 ~ 72 hours in incubator, then in 23 ~ 28 DEG C, 200r/min shakes cultivation 5 ~ 8 days, obtain shaking flask liquid spawn, its standard is: have mycelia wall built-up after shaking flask shake, and detect that culture fluid mycelium pellet unit intensity touches the mark more than 80% can use;
3) fermentor liquid Spawn incubation: a) sterilizing of medium: the fermentation tank culture medium liquid of inserting tankage size 4/5 in fermentation tank, then heat up, air bleeding valve is opened when manometer rises to 0.05MPa, close after being vented to zero, 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 DEG C, sterilizing 30 ~ 50 minutes under pressure 0.12 ~ 0.14MPa, crack discharge gate after sterilizing, discharge 1.5 ~ 2 liters is sterilized to valve, b) cool: adopt the interlayer type of cooling, cooling makes the discharge pressure of fermentation tank be down to 0.05 ~ 0.08MPa, and open air pump and vent valve, regulating tank is depressed into 0.02MPa, cools the temperature to 26 ~ 28 DEG C, c) inoculated and cultured: when fermentation jar temperature is down to 26 ~ 28 DEG C, open tank body inoculation mouth and protected with flame ring, by flame ring by shaking flask liquid spawn according to 5 ~ 8% inoculum concentration drop in tank, cover inoculation lid immediately, open air bleeding valve and remove flame ring, tank pressure is made to remain on 0.01MPa, control cultivation temperature 25 ~ 28 DEG C, inoculated and cultured detected strain quality after 48 hours, detect and qualifiedly at 25 ~ 28 DEG C of temperature, continue cultivation 5 ~ 7 days, detect that culture fluid mycelium pellet unit intensity touches the mark more than 80%, i.e. obtained White mushroom three grades of liquid spawns.
The invention solves the problem easily occurring aging regression in mycelial growth, because liquid spawn is coccoid mycelium, also there is many bacterium liquid fragments in bacterium liquid, dispersity is large, good fluidity, and vitality is vigorous, sprout fast, be inoculated in solid culture medium, with the deep layer position being seeped into solid culture medium under culture fluid, many Fa Jun centers can be formed; And liquid spawn breeds under stereoscopic culture state, cell age neat and consistent, and be mostly in vigorous period, after inoculation, mycelia restoration ecosystem is fast; Inoculate convenient and swift, after inoculation, energy restoration ecosystem rapidly, adopts special inoculating gun, bacterium liquid is injected in bacterium bag, removes inoculation cave mouth from and seals operation, raising effect 4 ~ 5 times; Mycelia is energetic, so fruiting is neat; Breeding cost is low, and liquid spawn breeding cost is only about 1/10 of solid spawn; Adopt liquid spawn to have with short production cycle, in only 7 ~ 9 days fermentation tank growth cycle, large batch of bacterial classification can be cultivated in a short time, 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: White mushroom mother in Shouguang, Shandong 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) Shake 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: sieve after the process of cow dung dried and crushed, tan by the sun under sunlight dry for subsequent use.
3, activated spawn: by White mushroom master clock access slant medium, cultivate 6 days at 25 ~ 26 DEG C, obtained inclined-plane mother plants bacterial classification sheet;
4, the cultivation of shaking flask liquid spawn: with 1000 milliliters of triangular flasks, load Shake flask medium 600 milliliters, in 120 ± 2 DEG C of sterilizings 30 minutes, bacterial classification sheet 5 ~ 6 is planted inoculating hood access about 0.5 centimetre of inclined-plane mother after cooling, bacterial classification sheet is made to swim on liquid level, in 26 DEG C of quiescent culture 72 hours in incubator, again in 25 ~ 26 DEG C, 6-7 days is cultivated in 200r/min concussion, obtain shaking flask liquid spawn, the standard of this liquid spawn is: have mycelia wall built-up after shake shaking flask, detecting culture fluid mycelium pellet unit intensity (the mycelium pellet number in often liter of zymotic fluid) is 238 ~ 252/600m1.
5, fermentor liquid Spawn incubation:
A) sterilizing of medium: adopt 50 liters of automatic fermenters, fermentation tank culture medium liquid 40 liters is inserted in fermentation tank, then sterilizing intensification is carried out, air bleeding valve is opened when manometer rises to 0.05MPa, close after being vented to zero, again rise to 0.05MPa, then open the micro-row of air bleeding valve, close after 1 ~ 2 minute; Temperature rises to 121 ~ 125 DEG C, and pressure, when 0.12 ~ 0.14MPa, keeps sterilizing 50 minutes, after sterilizing terminates, and crack discharge gate, discharge 1.5 ~ 2 liters carries out valve sterilization, and valve-off cools;
B) cool: adopt the interlayer type of cooling, running water pipe is received cooling water inlet, cools when the 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 when cooling the temperature to 26 ~ 28 DEG C;
C) inoculated and cultured: when fermentation jar temperature is down to 26 ~ 28 DEG C, open tank body inoculation mouth, and inoculate mouth with flame ring protection, that 95% alcohol-pickled sliver is wrapped on the inoculation mouth of fermentation tank, inoculation mouth is opened while lighting sliver, by flame ring, shaking flask liquid spawn is dropped in tank, inoculum concentration (weight) is 5%, then inoculation mouth is covered immediately, open air bleeding valve and remove flame ring, tank pressure is made to remain on 0.01MPa, control cultivation temperature 25 ~ 26 DEG C, inoculated and cultured was inspected by random samples bacterial classification after 48 hours, detect and qualifiedly at 25 ~ 26 DEG C of temperature, continue cultivation 5 ~ 6 days, i.e. obtained White mushroom three grades of liquid spawns, detecting culture fluid mycelium pellet unit intensity (the mycelium pellet number in often liter of zymotic fluid) is 384 ~ 402.
White mushroom liquid fungus seed result of the test shows: in fermentation process, content of reducing sugar is relatively stable, and pH value is little change in about 2 days, but pH then obviously declines subsequently; When fermentation reaches more than 80% of standard to the biomass of button mushroom pompon when 6 ~ 7 days, reach maximum to the mycelium pellet biomass in fermentation tank when 8th ~ 9 days, the biomass of mycelium pellet keeps relative stability and starts progressively to decline subsequently.By to index analysis such as pH value in fermentation process and mycelium pellet biomasss, the 9th day can be defined as fermentation termination, and aborning, fermentation 8th ~ 9 days can be selected as liquid fungus seed terminal, mycelium pellet biomass now and activity the highest.
Below give the detection data (accessing fermentation tank by shaking flask kind) that different cultivation temperature and inoculum concentration affect mycelium pellet biomass, wherein mycelium pellet Board Lot and density (being the mycelium pellet number in often liter of zymotic fluid).
Table 1 cultivation temperature is on the impact of fermentation tank White mushroom hypha biomass
| Cultivation temperature/DEG C | 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 |
Table 2 different vaccination amount is on the impact of fermentor liquid strain bio amount
The data of table 1 show: during cultivation temperature 25 DEG C, and the index such as biomass, mycelium pellet unit intensity of mycelium pellet is best, and the White mushroom liquid spawn of the present invention cultivation temperature expanded when cultivating is selected 25 ~ 26 DEG C and is advisable.
The data of table 2 show: the mycelium pellet biomass that (accesses 3 grades of fermentation tanks by 2 grades of shaking flask kinds) when liquid-spawn inoculation amount is 5% comparatively inoculum concentration 2.5% time be significantly improved, but inoculum concentration more than 5% after each process between the biomass of mycelium pellet, diameter and mycelium pellet Board Lot difference little; Therefore when White mushroom liquid spawn expansion of the present invention is cultivated, 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) Shake 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 White mushroom liquid spawn 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) Shake 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 White mushroom liquid spawn is with embodiment 1.
Claims (2)
1. the preparation method of a White mushroom liquid spawn, it is characterized in that: described White mushroom liquid spawn expands numerous cultivation by slant medium, Shake flask medium and fermentation tank culture medium through three grades to obtain, and in the numerous cultivation of described expansion, the weight composition of medium at different levels is:
1) composition of slant medium: in every 1000 ml waters add each component weight be:
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 Shake flask medium: in every 1000 ml waters add each component weight be:
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, VB11 0 ~ 30 milligram, pH nature;
3) composition of fermentation tank culture medium: in every 50 premium on currency add each component weight portion be: 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, cobalamin 0 ~ 50 milligram, defoamer 40 ~ 50g, pH nature;
The method of above-mentioned each medium preparing White mushroom liquid spawn is utilized to comprise the steps:
1) activated spawn: former for White mushroom mother is planted in access slant medium, cultivates 5 ~ 7 days in 23 ~ 28 DEG C, obtained inclined-plane mother plants bacterial classification sheet;
2) shaking flask strain cultivation: the Shake flask medium loading bottle capacity 3/5, in 120 ± 2 DEG C of sterilizings 30 minutes, bacterial classification sheet 5 ~ 6 is planted inoculating hood access 0.4 ~ 0.5 centimetre of inclined-plane mother after cooling, bacterial classification sheet is made to swim on liquid level, in 23 ~ 28 DEG C of quiescent culture 60 ~ 72 hours in incubator, then in 23 ~ 28 DEG C, 200r/min shakes cultivation 5 ~ 8 days, obtain shaking flask liquid spawn, its standard is: have mycelia wall built-up after shaking flask shake, and detect that culture fluid mycelium pellet unit intensity touches the mark more than 80% can use;
3) fermentor liquid Spawn incubation: a) sterilizing of medium: the fermentation tank culture medium liquid of inserting tankage size 4/5 in fermentation tank, then heat up, air bleeding valve is opened when manometer rises to 0.05MPa, close after being vented to zero, 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 DEG C, sterilizing 30 ~ 50 minutes under pressure 0.12 ~ 0.14MPa, crack discharge gate after sterilizing, discharge 1.5 ~ 2 liters is sterilized to valve, b) cool: adopt the interlayer type of cooling, cooling makes the discharge pressure of fermentation tank be down to 0.05 ~ 0.08MPa, and open air pump and vent valve, regulating tank is depressed into 0.02MPa, cools the temperature to 26 ~ 28 DEG C, c) inoculated and cultured: when fermentation jar temperature is down to 26 ~ 28 DEG C, open tank body inoculation mouth and protected with flame ring, by flame ring by shaking flask liquid spawn according to 5 ~ 8% inoculum concentration drop in tank, cover inoculation lid immediately, open air bleeding valve and remove flame ring, tank pressure is made to remain on 0.01MPa, control cultivation temperature 25 ~ 28 DEG C, inoculated and cultured detected strain quality after 48 hours, detect and qualifiedly at 25 ~ 28 DEG C of temperature, continue cultivation 5 ~ 7 days, detect that culture fluid mycelium pellet unit intensity touches the mark more than 80%, i.e. obtained White mushroom three grades of liquid spawns.
2. the preparation method of White mushroom liquid spawn according to claim 1, is characterized in that: the weight composition of preferred medium at different levels is:
1) composition of slant medium: in every 1000 ml waters add each component weight be:
Potato 200g, cow dung 150g, glucose 20g, potassium dihydrogen phosphate 2g, magnesium sulfate 1g, peptone 2g, agar 20g, pH nature;
2) composition of Shake flask medium: in every 1000 ml waters add each component weight be:
Potato 200g, cow dung 150g, corn flour 10g, wheat bran 10g, glucose 20g, potassium dihydrogen phosphate 2g, magnesium sulfate 1g, peptone 2g, cobalamin 0 milligram, pH nature;
3) composition of fermentation tank culture medium: in every 50 premium on currency add each component weight portion be: potato 2.5kg, cow dung 1kg, wheat bran 0.5kg, corn flour 0.25kg, sucrose 0.5kg, potassium dihydrogen phosphate 50g, magnesium sulfate 25g, orotic acid 0 milligram, defoamer 50g, pH nature.
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| CN102550293B (en) * | 2012-02-03 | 2014-05-21 | 连云港市农业科学院 | Method for liquid fermentation cultivation of Agaricus bisporus strain |
| CN102964170A (en) * | 2012-08-30 | 2013-03-13 | 上海雪榕生物科技股份有限公司 | Formula and preparation method of agaricus bisporus liquid spawn |
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