CN111887102A - Online production method of bottle-cultivated pleurotus eryngii liquid strain - Google Patents
Online production method of bottle-cultivated pleurotus eryngii liquid strain Download PDFInfo
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- CN111887102A CN111887102A CN202010651673.9A CN202010651673A CN111887102A CN 111887102 A CN111887102 A CN 111887102A CN 202010651673 A CN202010651673 A CN 202010651673A CN 111887102 A CN111887102 A CN 111887102A
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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
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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
- A01G18/22—Apparatus for the preparation of culture media, e.g. bottling devices
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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
- A01G18/30—Accessories for use before inoculation of spawn, e.g. sterilisers
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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
- A01G18/50—Inoculation of spawn
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Abstract
The on-line production method of the bottle-cultivated pleurotus eryngii liquid strain comprises the following steps: (1) inspecting the inoculation chamber and equipment; (2) cleaning a fermentation tank and detecting leakage; (3) adding water to a constant volume and adjusting the pH value; (4) preparing a culture medium; (5) connecting a steam exhaust pipe: the fermentation tank is provided with a three-way pipe, the steam exhaust pipe is connected to one branch of the three-way pipe through an air inlet hose, and the other branch of the three-way pipe is respectively connected with the inoculation machine and the fermentation tank through an inoculation pipe; (6) sterilizing and cooling the culture material; (7) sterilizing and cooling by inoculating spray head and pipeline steam; (8) conveying and inoculating; (9) culturing and detecting; (10) and finishing inoculation. The invention can reduce the number of personnel in a clean workshop and the pollution risk, improve the inoculation efficiency, reduce the area of a seed production area and simplify the seed production and use process; effectively reduces the production cost of the strains, and realizes more centralized, reasonable, effective and accurate control on the production and use of the liquid strains.
Description
Technical Field
The invention relates to the technical field of production of bottle-cultured pleurotus eryngii liquid strains, and particularly relates to an online production method of bottle-cultured pleurotus eryngii liquid strains.
Background
The strain is the basis and key of the development of the edible fungus industry, the strain of the edible fungus mainly has two forms, namely a solid strain and a liquid strain, and the solid strain mainly comprises a branch strain and a wood chip strain under the condition of small scale and low mechanization degree in the past; with the great development of edible fungi, particularly pleurotus eryngii, the main defects of long culture period, multiple intermediate links, large amount of labor, high production cost, low inoculation efficiency and the like of solid strains are exposed more and more fully; the application and development of pleurotus eryngii liquid seeds in production are promoted due to the shortage of solid strains, but the liquid seed production is mature at present in an off-line production mode of the pleurotus eryngii liquid seeds in Japan and Korea; however, the liquid strain off-line production mode has the defects that a fermentation tank needs to occupy an autoclave independently, the fermentation tank occupies a large space, the batching, sterilization, inoculation, cleaning and the like cannot be finished in one place, and compared with the existing solid strain production, the production efficiency is improved and the production cost is reduced, but the production efficiency still needs to be further improved and the production cost also needs to be further reduced; the offline sterilization fermentation tank is limited by a sterilization pot and a sterilization mode, the volume of each fermentation tank can only reach 600L at most, the daily yield can only reach 6 ten thousand bottles at most, and the requirements of producing more liquid strains on the current production scale and unit area and the requirements of intensively producing cultivated species cannot be met.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects in the prior art and provide the on-line production method of the liquid strain of the bottle-cultivated pleurotus eryngii, which can realize intensive management, improve the yield, reduce the cost and improve the inoculation efficiency.
The technical scheme adopted by the invention for solving the technical problems is as follows: the on-line production method of the bottle-cultivated pleurotus eryngii liquid strain comprises the following steps: (1) inspecting inoculation chambers and equipment: starting a fan filter unit (FFU fan), and checking air pressure and temperature; checking and checking the seed sowing machine, and simultaneously opening an air pressure control valve to check whether the starting air pressure can be smoothly started; starting the inoculation machine, and adjusting the inoculation machine to a reset setting; (2) cleaning a fermentation tank and detecting leakage: opening a feed port and a drain pipe of the fermentation tank, repeatedly washing the tank wall by using a high-pressure water gun, washing strain residues completely, detaching the upper and lower perspective lenses, cleaning the lenses and the sealing rings and installing the lenses and the sealing rings; after the cleaning water in the tank is drained, closing the drain pipe and sealing the feed inlet; then opening an air inlet valve, closing the air inlet valve when the pressure in the tank is proper, standing, and then checking leakage of all valves and connectors on the fermentation tank by using soapy water; (3) adding water to fix the volume and adjusting the pH value: after the leakage detection is finished, opening an exhaust valve to exhaust and release pressure, opening a feed opening to start adding water to a constant volume when the pressure in the tank is reduced to 0, measuring the water level by using a water level positioning rod, and closing a water inlet valve when the water level is proper; measuring the pH value of water after the constant volume is finished, pouring phosphoric acid solution, opening an air inlet valve to roll the mixed solution, closing the air inlet valve, sampling and detecting the pH value, and finally regulating the mixed solution to reach the proper pH value; (4) preparing a culture medium: weighing white sugar, yeast extract solution, soybean meal, magnesium sulfate and potassium dihydrogen phosphate according to the formula of the culture medium, adding water into the soybean meal, grinding into soybean meal pulp, and mixing the white sugar with the soybean mealDissolving sugar in water to obtain white sugar solution, dissolving yeast extract in water to obtain yeast extract solution, sequentially adding yeast extract solution, magnesium sulfate, potassium dihydrogen phosphate and soybean meal slurry into white sugar solution, and stirring to obtain culture medium; (5) connecting a steam exhaust pipe: the fermentation tank is provided with a three-way pipe, the steam exhaust pipe is connected to one branch of the three-way pipe through an air inlet hose, the other branch of the three-way pipe is respectively connected with the inoculation machine and the fermentation tank through an inoculation pipe, the three-way pipe is provided with a three-way pipe main valve, and two branches of the three-way pipe are provided with branch valves; closing the three-way main valve, simultaneously opening the two branch valves, opening the steam exhaust pipe valve, turning on an inoculation valve on the inoculation machine control plate, and setting the inoculation valve to be in a normally open mode; (6) sterilizing and cooling the culture material: heating the fermentation tank, opening a feed opening, putting the culture medium which is uniformly stirred into the tank, pouring a bottle of defoaming agent, eliminating foam in the culture medium, closing a cover of the feed opening and screwing the cover in a diagonal manner, and adjusting an exhaust valve to be in a micro-discharge state; opening an air inlet valve for filtering clean air after sterilization is finished, opening an air exhaust valve, exhausting and decompressing, opening a cooling water valve for cooling, closing the air inlet valve and the air exhaust valve after the temperature of the culture medium in the tank is cooled, and preparing for inoculation; (7) inoculating a spray head, sterilizing the pipeline by steam, and cooling: when the inoculating nozzle sprays steam, timing disinfection is started, an inoculating valve on an inoculating machine control plate is closed and set to be in an automatic mode, and then a three-way main valve and a steam exhaust pipe valve are closed in sequence; after the steam disinfection is finished, closing two branches of the three-way valve and the steam main valve, taking down an air inlet hose on the three-way pipe, connecting a clean air hose to one branch of the three-way pipe, keeping the three-way main valve closed, opening a branch valve, allowing clean low-temperature air to enter a seed receiving pipe, cooling the seed receiving pipe, sequentially closing the branch valve and the clean air hose valve, and taking down the clean air hose; then adjusting an exhaust valve on the fermentation tank body to a micro-exhaust state, and opening a three-way main valve and a branch valve; during inoculation, the three-way pipe can directly start the inoculation pipeline to connect the fermentation tank with the inoculation machine, and during disinfection and cooling, the three-way assembly on the fermentation tankThe valve is closed, and the steam and the clean cold air can be directly disinfected and cooled without passing through the interior of the fermentation tank; (8) conveying and inoculating: starting an online manipulator and simultaneously starting an inoculation conveying line, and conveying the production bottles to an inoculation chamber; conveying the production bottles subjected to inoculation out of an inoculation chamber through a conveying line; after inoculation is started, whether the inoculation amount reaches the standard or not and the material surface coverage condition are confirmed, and relevant records are made; stacking qualified production bottles by using a manipulator, and then conveying the stacked production bottles to a culture room by using a forklift; (9) culturing and detecting: during strain culture, recording culture temperature and tank pressure every two hours within working time in daytime, and measuring CO every day after inoculating for one day2Concentration, observing the growth condition of the strain every day, smelling the smell of the strain, observing the color of the strain by eyes, measuring the size of a strain ball and the concentration of the strain; (10) end of inoculation: closing a seed metering valve of the fermentation tank, and then closing an inoculation valve on an inoculation machine control plate; connecting a steam exhaust pipe, closing a three-way main valve, simultaneously opening a fermentation tank valve and a steam main valve on a steam pipeline, and then opening an inoculation valve on an inoculation machine control panel and adjusting the inoculation valve to be in a normally open mode; when the inoculation nozzle sprays steam, timing disinfection is started, and the steam is pumped out of the inoculation chamber; after the disinfection is finished, firstly, an inoculation valve on an inoculation machine control panel is closed and is adjusted to be in an automatic mode, then a fermentation tank steam valve and a steam valve on a steam pipeline are closed in sequence, and finally, an inoculation machine power supply is closed.
Further, in the step (1), the wind pressure is 10-16pa, and the temperature is 15-18 ℃; in the step (2), the pressure in the tank is 0.1-0.2MPa, preferably 0.15MPa, and the standing time is 20-40min, preferably 30 min.
Further, in the step (3), the water level is 800L, the tumbling time of the mixed solution is 3-5min, and the pH value of the mixed solution is 6.1-6.3.
Further, in the step (4), the temperature for dissolving the white sugar and the yeast extract in the water is 40-60 ℃, preferably 50 ℃; in step (5), the steam pressure is adjusted to 0.3 to 0.5MPa, preferably 0.4 MPa.
Further, the step (6) comprises: 1) When the temperature of the fermentation tank reaches 110 ℃ and the steam pressure reaches 0.05MPa, opening a valve of the inoculation port and beginning to discharge cold air in the tank; 2) when the discharged gas is vertical pure white, closing the inoculation valve and finishing discharging cold air; 3) when the temperature of the fermentation tank reaches 122 ℃ and the steam pressure reaches 0.13MPa, starting heat preservation and pressure maintaining timing to sterilize the culture medium for 60 min; 4) when the culture medium is sterilized for 30min, connecting a steam pipe to the filter, and starting timing to sterilize the filter for 30 min; 5) when the sterilization of the filter and the culture medium is finished simultaneously, the steam inlet valve and the inoculation port valve are closed, a 75% alcohol cotton ball is plugged into the inoculation valve, and a PE bag is sleeved on the inoculation valve and then is fastened by a rubber band.
Further, in the step (6), the temperature is 60-80 ℃, and preferably 70 ℃; after the sterilization is finished, the air pressure is 0.01-0.03 MPa, preferably 0.02 MPa; the exhaust valve is opened 2/3; the cooling temperature of the culture medium is 20-30 ℃, and is preferably 22 ℃.
Further, in the step (7), the disinfection time is 0.8-1.2 hours, preferably 1 hour; the air pressure is adjusted to 0.2-0.3MPa, preferably 0.22MPa, and the inoculation tube is cooled for 5-6 min.
Further, the step (8) comprises: closing a refrigerating fan 15min before inoculation, wiping the periphery of a fermentation tank by using 75% alcohol cotton, wearing gloves and a mask by an inoculator, closing an air inlet valve and an exhaust valve of the inoculator, winding a circle of 95% alcohol cotton on an inoculation port, igniting to form a flame ring, holding the checked shake flask strain by a left hand, tightly holding tweezers by a right hand, clamping a copper pipe on a shake flask strain bottle by the tweezers, igniting back and forth, vertically burning the copper pipe on the alcohol flame above the inoculation port to form a pipe opening, pulling out the alcohol cotton ball of the inoculation port, slowly opening the inoculation valve under the condition of no air pressure, inserting the copper pipe into the inoculation port by the tweezers, quickly pouring the strain into the tank by lifting the left hand, immediately closing the inoculation valve after inoculation, extinguishing the flame ring, plugging in the 75% alcohol cotton ball, sleeving a PE bag, fastening by using a rubber band, sequentially opening the air inlet valve and the exhaust valve, culturing strains at constant tank temperature of 22 deg.C;
further, the step (9) comprises: culturing for 5 days later, samplingDetecting strain quality with microscope, and eliminating imperfect strain, such as strain ball size, strain concentration, color, odor, pH value, and CO2The concentration detection is qualified and can be used for production.
Further, in the step (8), the standard of the inoculation amount is 30 +/-1 g; in the step (10), the steam pressure is adjusted to 0.3-0.5MPa, preferably 0.4 MPa; the disinfection time is 30-50min, preferably 40 min.
By using the production method, the daily yield can reach 11 ten thousand bottles of liquid strains, and the cost of each bottle of strains is reduced to 3/4 of the original solid strains; the number of personnel in a cleaning workshop and the pollution risk can be reduced, the inoculation efficiency is improved, the production scale is improved, the area of a seed production area is reduced, and the seed production and use process is simplified; the space of an industrial seed production area is saved, the strain production cost is effectively saved, and more centralized, reasonable, effective and accurate control on the production and use of liquid strains is realized; the incoherence of the solid strains and the off-line liquid strains in the manufacturing and using processes is avoided, the continuity, the high efficiency, the accuracy and the simplification of the seed production are realized by the production method, and the potential pollution risk in the manufacturing and using processes of the solid strains and the off-line liquid strains is effectively solved; the volume limitation of the offline liquid fermentation tank to the maximum extent of 600L is avoided, the online sterilization adopts the mode that steam is directly introduced into the culture medium of the fermentation tank for sterilization, the volume of the tank body can be 1000L at least, the maximum volume can reach 3000L, the seed production quantity in unit area can be obviously improved, and the volume limitation of an offline sterilization pot on the fermentation tank is eliminated.
Detailed Description
The following examples are given to further illustrate the embodiments of the present invention:
example 1
The first embodiment includes the following steps: (1) inspecting inoculation chambers and equipment: starting a fan filter unit (FFU fan), and checking the air pressure and the temperature, wherein the air pressure is 10-16pa, and the temperature is 15-18 ℃; checking and checking the seed sowing machine, and simultaneously opening an air pressure control valve to check whether the starting air pressure can be smoothly started; starting the inoculation machine, and adjusting the inoculation machine to a reset setting; (2) cleaning ofAnd (3) fermenting the tank and detecting leakage: opening a feed port and a drain pipe of the fermentation tank, repeatedly washing the tank wall by using a high-pressure water gun, washing strain residues completely, detaching the upper and lower perspective lenses, cleaning the lenses and the sealing rings and installing the lenses and the sealing rings; after the cleaning water in the tank is drained, closing the drain pipe and sealing the feed inlet; then opening an air inlet valve, closing the air inlet valve when the pressure in the tank is 0.15MPa, standing for 30min, and then carrying out leak detection on all valves and connectors on the fermentation tank by using soapy water; (3) adding water to fix the volume and adjusting the pH value: after the leakage detection is finished, opening an exhaust valve to exhaust and release pressure, opening a feed opening to start adding water to a constant volume when the pressure in the tank is reduced to 0, measuring the water level by using a water level positioning rod, and closing a water inlet valve when the water level is 800L; measuring the pH value of water after the volume is fixed, pouring phosphoric acid solution, opening an air inlet valve to roll the mixed solution for 3-5min, closing the air inlet valve, sampling and detecting the pH value to enable the final mixed solution to be adjusted to reach the proper pH value of 6.1-6.3; (4) preparing a culture medium: weighing white sugar, yeast extract solution, bean pulp, magnesium sulfate and monopotassium phosphate according to a culture medium formula, adding water into the bean pulp, grinding the bean pulp into bean pulp slurry, dissolving the white sugar in water at 50 ℃ to prepare white sugar solution, dissolving the yeast extract in water at 50 ℃ to prepare yeast extract solution, sequentially adding the yeast extract solution, the magnesium sulfate, the monopotassium phosphate and the bean pulp slurry into the white sugar solution, and uniformly stirring to prepare a culture medium; (5) connecting a steam exhaust pipe: the fermentation tank is provided with a three-way pipe, the steam exhaust pipe is connected to one branch of the three-way pipe through an air inlet hose, the other branch of the three-way pipe is respectively connected with the inoculation machine and the fermentation tank through an inoculation pipe, the three-way pipe is provided with a three-way pipe main valve, and two branches of the three-way pipe are provided with branch valves; closing the three-way main valve, simultaneously opening the two branch valves, opening the steam exhaust pipe valve, adjusting the steam pressure to 0.4MPa, turning on an inoculation valve on a control plate of the inoculation machine, and setting the inoculation valve to be in a normally open mode; (6) sterilizing and cooling the culture material: heating the fermentation tank to 70 deg.C, opening the feed inlet, adding the culture medium, adding a bottle of defoaming agent, removing foam in the culture medium, closing the cover of the feed inlet, screwing in diagonal form, and regulating the exhaust valveSaving into a micro-row state; 1) when the temperature of the fermentation tank reaches 110 ℃ and the steam pressure reaches 0.05MPa, opening a valve of the inoculation port and beginning to discharge cold air in the tank; 2) when the discharged gas is vertical pure white, closing the inoculation valve and finishing discharging cold air; 3) when the temperature of the fermentation tank reaches 122 ℃ and the steam pressure reaches 0.13MPa, starting heat preservation and pressure maintaining timing to sterilize the culture medium for 60 min; 4) when the culture medium is sterilized for 30min, connecting a steam pipe to the filter, and starting timing to sterilize the filter for 30 min; 5) when the filter and the culture medium are sterilized at the same time, closing the steam inlet valve and the inoculation port valve, plugging a 75% alcohol cotton ball into the inoculation valve, sleeving a PE bag, and then fastening by a rubber band; opening an air inlet valve for filtering clean air after sterilization is finished, opening an exhaust valve, adjusting the air pressure to 0.02MPa, opening an exhaust valve 2/3, exhausting and relieving pressure, opening a cooling water valve for cooling, closing the air inlet valve and the exhaust valve after the temperature of the culture medium in the tank is cooled to 22 ℃, and preparing for inoculation; (7) inoculating a spray head, sterilizing the pipeline by steam, and cooling: when the inoculating spray head sprays steam, timing and disinfecting for 1h, closing an inoculating valve on an inoculating machine control plate, setting the inoculating valve to be in an automatic mode, and then closing a three-way main valve and a steam exhaust pipe valve in sequence; adjusting the air pressure to 0.22MPa, closing two branches of the tee joint and a main steam valve after steam sterilization is finished, taking down an air inlet hose on the tee joint, connecting a clean air hose to one branch of the tee joint, keeping the main three valve closed, opening a branch valve, allowing clean low-temperature air to enter a seed receiving pipe, cooling for 5-6min, sequentially closing the branch valve and the main clean air hose valve after the seed receiving pipe is cooled, and taking down the clean air hose; then adjusting an exhaust valve on the fermentation tank body to a micro-exhaust state, and opening a three-way main valve and a branch valve; (8) conveying and inoculating: starting an online manipulator and simultaneously starting an inoculation conveying line, and conveying the production bottles to an inoculation chamber; 15min before inoculation, the refrigerating fan is turned off, 75% alcohol cotton is used for wiping the periphery of the fermentation tank, the inoculation personnel wear gloves and masks, the air inlet valve and the exhaust valve of the inoculation machine are closed, a circle of 95% alcohol cotton is wound on the inoculation port, and a flame ring is formed by ignitionHolding the checked shake flask strain with the left hand, tightly holding the tweezers with the right hand, clamping the copper pipe on the shake flask strain bottle with the tweezers, firing back and forth, then vertically firing the copper pipe on alcohol flame above the inoculation port to remove the alcohol cotton ball of the inoculation port, slowly opening the inoculation valve under the condition of no air pressure, inserting the copper pipe into the inoculation port with the tweezers, lifting up the left hand to quickly pour the strain into the tank, immediately closing the inoculation valve after inoculation is completed, extinguishing a flame ring, plugging in the 75% alcohol cotton ball, sleeving a PE bag, then fastening the PE bag with a rubber band, sequentially opening an air inlet valve and an exhaust valve, and keeping the temperature of the tank constant at 22 ℃ to culture the strain; conveying the production bottles subjected to inoculation out of an inoculation chamber through a conveying line; after inoculation is started, whether the inoculation amount reaches 30g or not and the material surface coverage condition are confirmed, and relevant records are made; stacking qualified production bottles by using a manipulator, and then conveying the stacked production bottles to a culture room by using a forklift; (9) culturing and detecting: during strain culture, recording culture temperature and tank pressure every two hours within working time in daytime, and measuring CO every day after inoculating for one day2Concentration, observing the growth condition of the strain every day, smelling the smell of the strain, observing the color of the strain by eyes, measuring the size of a strain ball and the concentration of the strain; after the culture period is 5 days, sampling, detecting strain quality with microscope, eliminating imperfect strain, and determining strain ball size, strain concentration, color, odor, pH value, and CO2The qualified concentration detection can be used for production; (10) end of inoculation: closing a seed metering valve of the fermentation tank, and then closing an inoculation valve on an inoculation machine control plate; connecting a steam exhaust pipe, closing a three-way main valve, simultaneously opening a fermentation tank valve and a steam main valve on a steam pipeline, adjusting the steam pressure to 0.4MPa, and then opening an inoculation valve on an inoculation machine control panel and adjusting the inoculation valve to be in a normally open mode; when the inoculation nozzle sprays steam, timing and sterilizing are started for 40min, and the steam is pumped out of the inoculation chamber; after the disinfection is finished, firstly, an inoculation valve on an inoculation machine control panel is closed and is adjusted to be in an automatic mode, then a fermentation tank steam valve and a steam valve on a steam pipeline are closed in sequence, and finally, an inoculation machine power supply is closed.
Example 2
The second embodiment includes the following steps: (1) examination ofInoculation room and equipment: starting a fan filter unit (FFU fan), and checking the air pressure and the temperature, wherein the air pressure is 10-16pa, and the temperature is 15-18 ℃; checking and checking the seed sowing machine, and simultaneously opening an air pressure control valve to check whether the starting air pressure can be smoothly started; starting the inoculation machine, and adjusting the inoculation machine to a reset setting; (2) cleaning a fermentation tank and detecting leakage: opening a feed port and a drain pipe of the fermentation tank, repeatedly washing the tank wall by using a high-pressure water gun, washing strain residues completely, detaching the upper and lower perspective lenses, cleaning the lenses and the sealing rings and installing the lenses and the sealing rings; after the cleaning water in the tank is drained, closing the drain pipe and sealing the feed inlet; then opening an air inlet valve, closing the air inlet valve when the pressure in the tank is 0.2MPa, standing for 30min, and then carrying out leak detection on all valves and connectors on the fermentation tank by using soapy water; (3) adding water to fix the volume and adjusting the pH value: after the leakage detection is finished, opening an exhaust valve to exhaust and release pressure, opening a feed opening to start adding water to a constant volume when the pressure in the tank is reduced to 0, measuring the water level by using a water level positioning rod, and closing a water inlet valve when the water level is 800L; measuring the pH value of water after the volume is fixed, pouring phosphoric acid solution, opening an air inlet valve to roll the mixed solution for 3-5min, closing the air inlet valve, sampling and detecting the pH value to enable the final mixed solution to be adjusted to reach the proper pH value of 6.1-6.3; (4) preparing a culture medium: weighing white sugar, yeast extract solution, bean pulp, magnesium sulfate and monopotassium phosphate according to a culture medium formula, adding water into the bean pulp, grinding the bean pulp into bean pulp slurry, dissolving the white sugar in water at 60 ℃ to prepare white sugar solution, dissolving the yeast extract in water at 50 ℃ to prepare yeast extract solution, sequentially adding the yeast extract solution, the magnesium sulfate, the monopotassium phosphate and the bean pulp slurry into the white sugar solution, and uniformly stirring to prepare a culture medium; (5) connecting a steam exhaust pipe: the fermentation tank is provided with a three-way pipe, the steam exhaust pipe is connected to one branch of the three-way pipe through an air inlet hose, the other branch of the three-way pipe is respectively connected with the inoculation machine and the fermentation tank through an inoculation pipe, the three-way pipe is provided with a three-way pipe main valve, and two branches of the three-way pipe are provided with branch valves; closing the three-way main valve, simultaneously opening the two branch valves, opening the steam exhaust pipe valve, adjusting the steam pressure to 0.45MPa, and turning on the inoculation machine for controlThe inoculation valve on the plate is set to be in a normally open mode; (6) sterilizing and cooling the culture material: when the fermentation tank is heated to 70 ℃, opening a feed port, putting the culture medium which is uniformly stirred into the tank, pouring a bottle of antifoaming agent into the tank, eliminating foam in the culture medium, closing a cover of the feed port, screwing the cover in a diagonal manner, and adjusting an exhaust valve to be in a micro-discharge state; 1) when the temperature of the fermentation tank reaches 110 ℃ and the steam pressure reaches 0.05MPa, opening a valve of the inoculation port and beginning to discharge cold air in the tank; 2) when the discharged gas is vertical pure white, closing the inoculation valve and finishing discharging cold air; 3) when the temperature of the fermentation tank reaches 122 ℃ and the steam pressure reaches 0.13MPa, starting heat preservation and pressure maintaining timing to sterilize the culture medium for 60 min; 4) when the culture medium is sterilized for 30min, connecting a steam pipe to the filter, and starting timing to sterilize the filter for 30 min; 5) when the filter and the culture medium are sterilized at the same time, closing the steam inlet valve and the inoculation port valve, plugging a 75% alcohol cotton ball into the inoculation valve, sleeving a PE bag, and then fastening by a rubber band; opening an air inlet valve for filtering clean air after sterilization is finished, opening an exhaust valve, adjusting the air pressure to 0.02MPa, opening an exhaust valve 2/3, exhausting and relieving pressure, opening a cooling water valve for cooling, closing the air inlet valve and the exhaust valve after the temperature of the culture medium in the tank is cooled to 22 ℃, and preparing for inoculation; (7) inoculating a spray head, sterilizing the pipeline by steam, and cooling: when the inoculating nozzle sprays steam, timing disinfection is started for 1.2h, an inoculating valve on an inoculating machine control plate is closed, an automatic mode is set, and then a three-way main valve and a steam exhaust pipe valve are closed in sequence; adjusting the air pressure to 0.25MPa, closing two branches of the tee joint and a main steam valve after steam sterilization is finished, taking down an air inlet hose on the tee joint, connecting a clean air hose to one branch of the tee joint, keeping the main three valve closed, opening a branch valve, allowing clean low-temperature air to enter a seed connection pipe, cooling for 5-6min, sequentially closing the branch valve and the main clean air hose valve after the seed connection pipe is cooled, and taking down the clean air hose; then adjusting an exhaust valve on the fermentation tank body to a micro-exhaust state, and opening a three-way main valve and a branch valve; (8) conveying and inoculating: on the openingThe mechanical arm simultaneously starts the inoculation conveying line and conveys the production bottles to the inoculation chamber; closing a refrigerating fan 15min before inoculation, wiping the periphery of a fermentation tank by using 75% alcohol cotton, wearing gloves and a mask by an inoculator, closing an air inlet valve and an exhaust valve of the inoculator, winding a circle of 95% alcohol cotton on an inoculation port, igniting to form a flame ring, holding the checked shake flask strain by a left hand, tightly holding tweezers by a right hand, clamping a copper pipe on a shake flask strain bottle by the tweezers, igniting back and forth, vertically burning the copper pipe on the alcohol flame above the inoculation port to form a pipe opening, pulling out an inoculation port alcohol cotton ball, slowly opening the inoculation valve under the condition of no air pressure, inserting the copper pipe into the inoculation port by the tweezers, quickly pouring the strain into the tank by lifting the left hand, immediately closing the inoculation valve after inoculation, extinguishing the flame ring, filling the 75% alcohol cotton ball, sleeving a PE bag, fastening the PE bag by using a rubber band, and sequentially opening the air inlet valve and the exhaust valve, culturing strains at constant tank temperature of 22 deg.C; conveying the production bottles subjected to inoculation out of an inoculation chamber through a conveying line; after inoculation is started, whether the inoculation amount reaches 30g or not and the material surface coverage condition are confirmed, and relevant records are made; stacking qualified production bottles by using a manipulator, and then conveying the stacked production bottles to a culture room by using a forklift; (9) culturing and detecting: during strain culture, recording culture temperature and tank pressure every two hours within working time in daytime, and measuring CO every day after inoculating for one day2Concentration, observing the growth condition of the strain every day, smelling the smell of the strain, observing the color of the strain by eyes, measuring the size of a strain ball and the concentration of the strain; after the culture period is 5 days, sampling, detecting strain quality with microscope, eliminating imperfect strain, and determining strain ball size, strain concentration, color, odor, pH value, and CO2The qualified concentration detection can be used for production; (10) end of inoculation: closing a seed metering valve of the fermentation tank, and then closing an inoculation valve on an inoculation machine control plate; connecting a steam exhaust pipe, closing a three-way main valve, simultaneously opening a fermentation tank valve and a steam main valve on a steam pipeline, adjusting the steam pressure to 0.45MPa, and then opening an inoculation valve on an inoculation machine control panel and adjusting the inoculation valve to be in a normally open mode; when the inoculation nozzle sprays steam, timing and sterilizing are started for 50min, and the steam is pumped out of the inoculation chamber; end of sterilizationThen firstly closing the inoculation valve on the control panel of the inoculator, turning the inoculation valve into an automatic mode, then closing the steam valve on the fermentation tank and the steam valve on the steam pipeline in sequence, and finally closing the power supply of the inoculator.
By using the invention, the daily yield can reach 11 ten thousand bottles of liquid strains, and the cost of each bottle of strains is reduced to 3/4 of the original solid strains; the volume limitation of the offline liquid fermentation tank to 600L is avoided, the online sterilization adopts the mode of directly introducing steam into the culture medium of the fermentation tank for sterilization, the volume of the tank body can be 1000L at least, and the maximum volume can reach 3000L.
The specific experimental parameter comparison statistics are shown in table one.
Table one: comparison statistical condition table for key parameters of solid seed production, off-line liquid seed production and on-line liquid seed production
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as being within the protection scope of the present invention.
Those not described in detail in the specification are well within the skill of the art.
Claims (10)
1. The on-line production method of the bottle-cultivated pleurotus eryngii liquid strain is characterized by comprising the following steps: (1) inspecting inoculation chambers and equipment: starting a fan filter unit, and checking air pressure and temperature; checking and checking the seed sowing machine, and simultaneously opening an air pressure control valve to check whether the starting air pressure can be smoothly started; starting the inoculation machine, and adjusting the inoculation machine to a reset setting; (2) cleaning a fermentation tank and detecting leakage: opening a feed port and a drain pipe of the fermentation tank, repeatedly washing the tank wall by using a high-pressure water gun, washing strain residues completely, detaching the upper and lower perspective lenses, cleaning the lenses and the sealing rings and installing the lenses and the sealing rings; after the cleaning water in the tank is drained, the drainage is closedA tube sealing the feed inlet; then opening an air inlet valve, closing the air inlet valve when the pressure in the tank is proper, standing, and then checking leakage of all valves and connectors on the fermentation tank by using soapy water; (3) adding water to fix the volume and adjusting the pH value: after the leakage detection is finished, opening an exhaust valve to exhaust and release pressure, opening a feed opening to start adding water to a constant volume when the pressure in the tank is reduced to 0, measuring the water level by using a water level positioning rod, and closing a water inlet valve when the water level is proper; measuring the pH value of water after the constant volume is finished, pouring phosphoric acid solution, opening an air inlet valve to roll the mixed solution, closing the air inlet valve, sampling and detecting the pH value, and finally regulating the mixed solution to reach the proper pH value; (4) preparing a culture medium: weighing white sugar, yeast extract solution, bean pulp, magnesium sulfate and monopotassium phosphate according to a culture medium formula, adding water into the bean pulp, grinding the bean pulp into bean pulp slurry, dissolving the white sugar in the water to prepare white sugar solution, dissolving the yeast extract in the water to prepare yeast extract solution, sequentially adding the yeast extract solution, the magnesium sulfate, the monopotassium phosphate and the bean pulp slurry into the white sugar solution, and uniformly stirring to prepare a culture medium; (5) connecting a steam exhaust pipe: the fermentation tank is provided with a three-way pipe, the steam exhaust pipe is connected to one branch of the three-way pipe through an air inlet hose, the other branch of the three-way pipe is respectively connected with the inoculation machine and the fermentation tank through an inoculation pipe, the three-way pipe is provided with a three-way pipe main valve, and two branches of the three-way pipe are provided with branch valves; closing the three-way main valve, simultaneously opening the two branch valves, opening the steam exhaust pipe valve, turning on an inoculation valve on the inoculation machine control plate, and setting the inoculation valve to be in a normally open mode; (6) sterilizing and cooling the culture materials: heating the fermentation tank, opening a feed opening, putting the culture medium which is uniformly stirred into the tank, pouring a bottle of defoaming agent, eliminating foam in the culture medium, closing a cover of the feed opening and screwing the cover in a diagonal manner, and adjusting an exhaust valve to be in a micro-discharge state; opening an air inlet valve for filtering clean air after sterilization is finished, opening an air exhaust valve, exhausting and decompressing, opening a cooling water valve for cooling, closing the air inlet valve and the air exhaust valve after the temperature of the culture medium in the tank is cooled, and preparing for inoculation; (7) inoculating a spray head, sterilizing the pipeline by steam, and cooling: when the inoculating nozzle sprays steam, the timing is startedSterilizing, namely closing an inoculation valve on an inoculation machine control panel, setting the inoculation valve to be in an automatic mode, and then closing a three-way main valve and a steam exhaust pipe valve in sequence; after the steam disinfection is finished, closing two branches of the three-way valve and the steam main valve, taking down an air inlet hose on the three-way pipe, connecting a clean air hose to one branch of the three-way pipe, keeping the three-way main valve closed, opening a branch valve, allowing clean low-temperature air to enter a seed receiving pipe, cooling the seed receiving pipe, sequentially closing the branch valve and the clean air hose valve, and taking down the clean air hose; then adjusting an exhaust valve on the fermentation tank body to a micro-exhaust state, and opening a three-way main valve and a branch valve; (8) conveying and inoculating: starting an online manipulator and simultaneously starting an inoculation conveying line, and conveying the production bottles to an inoculation chamber; conveying the production bottles subjected to inoculation out of an inoculation chamber through a conveying line; after inoculation is started, whether the inoculation amount reaches the standard or not and the material surface coverage condition are confirmed, and relevant records are made; stacking qualified production bottles by using a manipulator, and then conveying the stacked production bottles to a culture room by using a forklift; (9) culturing and detecting: during strain culture, recording culture temperature and tank pressure every two hours within working time in daytime, and measuring CO every day after inoculating for one day2Concentration, observing the growth condition of the strain every day, smelling the smell of the strain, observing the color of the strain by eyes, measuring the size of a strain ball and the concentration of the strain; (10) end of inoculation: closing a seed metering valve of the fermentation tank, and then closing an inoculation valve on an inoculation machine control plate; connecting a steam exhaust pipe, closing a three-way main valve, simultaneously opening a fermentation tank valve and a steam main valve on a steam pipeline, and then opening an inoculation valve on an inoculation machine control panel and adjusting the inoculation valve to be in a normally open mode; when the inoculation nozzle sprays steam, timing disinfection is started, and the steam is pumped out of the inoculation chamber; after the disinfection is finished, firstly, an inoculation valve on an inoculation machine control panel is closed and is adjusted to be in an automatic mode, then a fermentation tank steam valve and a steam valve on a steam pipeline are closed in sequence, and finally, an inoculation machine power supply is closed.
2. The on-line production method of liquid spawn of pleurotus eryngii planted in bottles as claimed in claim 1, wherein the step (6) comprises: 1) when the temperature of the fermentation tank reaches 110 ℃ and the steam pressure reaches 0.05MPa, opening a valve of the inoculation port and beginning to discharge cold air in the tank; 2) when the discharged gas is vertical pure white, closing the inoculation valve and finishing discharging cold air; 3) when the temperature of the fermentation tank reaches 122 ℃ and the steam pressure reaches 0.13MPa, starting heat preservation and pressure maintaining timing to sterilize the culture medium for 60 min; 4) when the culture medium is sterilized for 30min, connecting a steam pipe to the filter, and starting timing to sterilize the filter for 30 min; 5) when the sterilization of the filter and the culture medium is finished simultaneously, the steam inlet valve and the inoculation port valve are closed, a 75% alcohol cotton ball is plugged into the inoculation valve, and a PE bag is sleeved on the inoculation valve and then is fastened by a rubber band.
3. The on-line production method of liquid spawn of pleurotus eryngii planted in bottles as claimed in claim 1 or 2, wherein the step (8) comprises: closing a refrigerating fan 15min before inoculation, wiping the periphery of a fermentation tank by using 75% alcohol cotton, wearing gloves and a mask by an inoculator, closing an air inlet valve and an exhaust valve of the inoculator, winding a circle of 95% alcohol cotton on an inoculation port, igniting to form a flame ring, holding the checked shake flask strain by a left hand, tightly holding tweezers by a right hand, clamping a copper pipe on a shake flask strain bottle by the tweezers, igniting back and forth, vertically burning the copper pipe on the alcohol flame above the inoculation port to form a pipe opening, pulling out the alcohol cotton ball of the inoculation port, slowly opening the inoculation valve under the condition of no air pressure, inserting the copper pipe into the inoculation port by the tweezers, quickly pouring the strain into the tank by lifting the left hand, immediately closing the inoculation valve after inoculation, extinguishing the flame ring, plugging in the 75% alcohol cotton ball, sleeving a PE bag, fastening by using a rubber band, sequentially opening the air inlet valve and the exhaust valve, the culture of the strain is carried out at a constant pot temperature of 22 ℃.
4. The on-line production method of liquid strains of bottle-cultivated pleurotus eryngii according to any one of claims 1 to 3, wherein in the step (1), the wind pressure is 10 to 16pa and the temperature is 15 to 18 ℃; in the step (2), the pressure in the tank is 0.1-0.2MPa, preferably 0.15MPa, and the standing time is 20-40min, preferably 30 min.
5. The on-line production method of liquid spawn of Pleurotus eryngii planted in bottle as claimed in any one of claims 1 to 4, wherein in step (3), the water level is 800L, the mixed solution is tumbled for 3-5min, and the pH of the mixed solution is 6.1-6.3.
6. The on-line production method of liquid spawn of pleurotus eryngii planted in bottles according to any one of claims 1 to 5, wherein in the step (4), the temperature of dissolving the white sugar and the yeast extract in the water is 40 to 60 ℃, preferably 50 ℃; in step (5), the steam pressure is adjusted to 0.3 to 0.5MPa, preferably 0.4 MPa.
7. The on-line production method of liquid strains of bottle-cultivated pleurotus eryngii according to any one of claims 1 to 6, wherein in step (6), the temperature is 60 to 80 ℃, preferably 70 ℃; after the sterilization is finished, the air pressure is 0.01-0.03 MPa, preferably 0.02 MPa; the exhaust valve is opened 2/3; the cooling temperature of the culture medium is 20-30 ℃, and is preferably 22 ℃.
8. The on-line production method of liquid spawn of pleurotus eryngii planted in bottles as claimed in one of claims 1 to 7, wherein in the step (7), the disinfection time is 0.8 to 1.2 hours, preferably 1 hour; the air pressure is adjusted to 0.2-0.3MPa, preferably 0.22MPa, and the inoculation tube is cooled for 5-6 min.
9. The on-line production method of liquid spawn of pleurotus eryngii planted in bottles as claimed in one of claims 1 to 8, wherein said step (9) comprises: after the culture period is 5 days, sampling, detecting strain quality with microscope, eliminating imperfect strain, and determining strain ball size, strain concentration, color, odor, pH value, and CO2The concentration detection is qualified and can be used for production.
10. The on-line production method of liquid spawn of pleurotus eryngii planted in bottles as claimed in one of claims 1 to 9, wherein in the step (8), the standard of the inoculum size is 30 ± 1 g; in the step (10), the steam pressure is adjusted to 0.3-0.5MPa, preferably 0.4 MPa; the disinfection time is 30-50min, preferably 40 min.
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