WO2019157785A1

WO2019157785A1 - Production process and equipment for continuously supplying bacterial liquid in exponential growth phase

Info

Publication number: WO2019157785A1
Application number: PCT/CN2018/086428
Authority: WO
Inventors: 刘正初; 苏维利; 李云峰
Original assignee: 大庆天之草生物新材料科技有限公司
Priority date: 2018-02-13
Filing date: 2018-05-11
Publication date: 2019-08-22
Also published as: CN108192817A

Abstract

The invention discloses a production process and equipment for continuously supplying a bacterial liquid in the exponential growth phase. The process comprises the following steps: (1) cultivating a typical bacterial colony; (2) selecting a bacterial liquid; (3) activating the bacterial liquid; (4) amplifying the bacterial liquid in a seed tank; (5) amplifying the bacterial liquid in a fermenter; (6) buffering and storing the bacterial liquid; and (7) mixing and inoculating the bacterial liquid. The technical problem solved by the invention is to overcome the problem in which existing methods of microbial culture or fermentation cannot continuously supply a bacterial liquid or cannot ensure that the function of the bacterial species is obtained. The present invention provides a production process and equipment for continuously supplying a bacterial liquid in the exponential growth phase.

Description

Production process and equipment for continuously supplying logarithmic growth phase bacterial liquid

Technical field

The invention relates to the field of hemp fiber production. Specifically, it is a continuous production process and equipment for supplying logarithmic growth phase.

Background technique

In industrial (medicine, food, beverage, energy, chemical, etc.) and agricultural (molecular operation breeding, biological nitrogen fixation, biological pesticides, feed) production and environmental protection, it is usually necessary to cultivate or ferment microorganisms by appropriate methods. According to the method of operation, microbial culture or fermentation is divided into two modes: “batch” and “continuous”. The former is a production mode in which the culture medium is once loaded into a culture (fermentation) tank, inoculated under suitable conditions, and cultured (fermented) for a certain period of time, and all the culture (fermentation) products are taken out. The latter is a method in which the culture medium is continuously supplied to the culture (fermentation) tank on the one hand, the microbial growth process is stabilized, the culture conditions are not changed with time, and the culture (fermentation) product is continuously taken out at the same time. Production mode.

According to the growth rate constant of microorganisms, that is, the division algebra per hour, the typical curve of microbial growth is generally divided into four periods: lag phase, log phase, stationary phase and decay phase. When the microorganisms are cultured in a closed system, when the microorganisms grow for a certain period of time, the specific growth rate of the microorganisms reaches a maximum, and at this time, the logarithmic growth phase is entered. In the logarithmic growth phase, if there is no factor that inhibits or limits the growth of microorganisms, the microorganisms will maintain a constant maximum growth rate, and the number of cells will increase exponentially. However, the logarithmic growth period is limited and generally lasts from a few hours to a dozen hours. For the microbial fermentation production process that needs to continuously supply the bacterial liquid in the “logarithmic growth phase”, there are some key technical obstacles in the above two production modes. The “batch” mode obviously cannot meet the requirements of “continuousization”. The “continuous” mode is difficult to avoid because the bacterial liquid is in the culture (fermentation) tank for a long time, and may have passed the logarithmic growth phase of microorganisms when supplied. The phenomenon that the function of the strain is degraded leads to unstable production.

Summary of the invention

The technical problem solved by the invention is to overcome the problem that the existing microbial culture or fermentation mode can not continuously supply the bacterial liquid or can not guarantee the function of the bacterial species, and provide a continuous production process for supplying the logarithmic growth phase.

The continuous production process of the logarithmic growth phase of the present invention comprises the following steps:

(1) cultivating a typical colony, that is, diluting and separating the purified and reconstituted original species, and statically cultivating the diluted and isolated cells by using a selective solid medium to obtain a colony that meets the requirements;

(2) selecting the bacterial liquid, inoculation of the typical colony obtained in the step (1) into a test tube containing the selective liquid medium, and dynamically cultured at a set temperature and an oscillation frequency to a set time to obtain logarithmic growth. Selective culture of bacterial liquid;

(3) activating the bacterial liquid, that is, transferring the selective culture liquid in the logarithmic growth phase obtained in the step (2) to the cone type bottle containing the liquid activation medium and setting the liquid amount, temperature and Dynamically culturing to a set time under an oscillating frequency condition to obtain an activated culture broth in a logarithmic growth phase;

(4) The seed tank amplifies the bacterial liquid, that is, transfers the activated culture liquid in the logarithmic growth phase to the seed tank containing the liquid expansion medium and sets the aeration amount, the tank pressure, the temperature, the stirring rate. Performing a deep liquid culture at a set time to obtain a seed tank amplifying bacterial solution in a logarithmic growth phase;

(5) The fermenter amplifies the bacterial liquid, and transfers the seed tank amplifying liquid in the logarithmic growth phase to the fermenting tank containing the liquid fermentation medium and sets the aeration amount, the tank pressure, the temperature, and the stirring. Performing a deep liquid fermentation at a set rate at a rate to obtain a fermenter-enriched bacterial solution in a logarithmic growth phase and having a stable function;

(6) Buffer storage bacteria liquid, that is, the fermentation tank liquid in the fermenter in the logarithmic growth phase is transferred to the buffer storage tank close to the emptying, and the bacterial liquid in the buffer storage tank is at the set ventilation volume and the tank pressure. Deep liquid fermentation is continued at a temperature and a stirring rate, and the fermentation broth of the fermenter is continuously delivered to the mixed inoculating tank;

(7) compounding the inoculating bacterial solution, that is, diluting the buffered bacterial liquid with a set inorganic salt solution, and combining the vortex rotation and the blocking impact to prepare a bacterial suspension uniformly distributed with the bacterial body, The bacterial suspension was continuously discharged at a set flow rate to supply a subsequent inoculation procedure.

Preferably, the step of preparing the expansion medium performed before the step (4) is further included, and the preparation of the expansion medium is completed in the expansion medium preparation tank, and the completed amplification medium is delivered to the seed tank. Step (4) is carried out. Also included is a step of preparing a fermentation medium performed before the step (5), and the preparation of the fermentation medium is completed in a dedicated fermentation medium preparation tank, and the completed fermentation medium is sent to the fermentation tank for the step ( 5).

Preferably, the method further comprises a step of sterilizing to ensure that there is no source of contaminated bacteria from the culture medium and equipment during the pure culture of the target strain, the step comprising first evacuating the heat-insulating water in the jacket for holding the inner liner, secondly Amplification medium preparation tanks containing medium components and/or expansion medium preparation tanks and/or seed tanks and/or fermentors or to empty seed tanks and/or fermentors and/or buffer tanks Steam is set in the inner tank for sterilization of set time and/or temperature. After the set time, the sterile compressed air is injected into the inner tank to drive the steam to be drained, and cold water is injected into the jacket for cooling the inner tank. And maintain the internal pressure of the bladder ≥ 0.01MPa.

Preferably, in the sterilization step, the gas pressure in the inner tank of the expansion medium preparation tank and/or the fermentation medium preparation tank and/or the seed tank and/or the ferment tank and/or the buffer storage tank is detected, and according to the gas pressure To control the intake and output of the liner.

Preferably, when the temperature of the inner bladder drops to a set culture temperature, the inner bladder is kept warm to maintain the temperature of the inner bladder.

Preferably, the level of the liquid in the inner tank of the expansion medium preparation tank and/or the fermentation medium preparation tank and/or the seed tank and/or the ferment tank and/or the buffer storage tank is detected and controlled according to the liquid level. The opening and closing of the feeding and discharging parts of the bladder.

The invention also provides a production device for continuously supplying a logarithmic growth phase bacterial liquid, comprising:

a seed tank containing a liquid expansion medium for receiving and cultivating the activated culture liquid cultured in the bacterial activation bottle to obtain a seed tank amplification liquid in a logarithmic growth phase;

a fermenter containing a liquid fermentation medium for receiving and cultivating the seed tank cultured seed tank to amplify the bacterial liquid to obtain a fermenting tank amplifying liquid in a logarithmic growth phase;

a buffer storage tank for receiving the fermentation tank of the fermentation tank cultured in the fermenter to be subjected to deep liquid fermentation, and outputting the fermenter to amplify the bacterial liquid;

The mixed inoculating tank is configured to receive the amplified bacterial liquid delivered from the buffer storage tank, dilute it, and prepare a bacterial suspension uniformly distributed with the bacterial cells, and output the bacterial suspension.

Preferably, the production apparatus further comprises an expansion medium preparation tank for preparing an expansion medium required for multi-batch production of the seed tank amplification bacterial solution, the expansion medium preparation tank and the seed A tank connection is used to deliver the expansion medium to the seed tank. The production apparatus further includes a fermentation medium preparation tank for preparing a fermentation medium required for a multi-batch production fermenter to amplify the bacterial liquid, the fermentation medium preparation tank being connected to the fermentor for use in the The fermentation tank conveys the fermentation medium.

Advantageously, said expansion medium preparation tank and/or fermentation medium preparation tank and/or seed tank and/or fermentor and/or buffer storage tank comprises a liner enclosed by a casing and enclosing said liner a cover body, a jacket surrounding the inner tank for accommodating the heat insulating liquid, a stirring member on the cover body, and a motor for driving the stirring member to rotate, the inner tank having the feeding member and the discharging a component, the feed component may be filled or inhaled, and the cover body is provided with an air outlet component communicating with the inner tank, the jacket having a first liquid circulation component that can serve as a hot water inlet or a cold water outlet, A second liquid flow-through member and a venting member that can serve as a hot water outlet or a cold water inlet.

Preferably, a control device is also included, the control device being coupled to a motor for agitating the liquid in the bladder to control the opening and closing of the motor. The inner tank is internally provided with a liquid level detecting member connected to the control device, and the control device is respectively connected with the feeding and discharging members of the inner tank to control the feeding member according to the detected liquid level and Opening and closing of the discharge unit. The cover body is provided with the control device for detecting a gas pressure detecting member in the inner tank, and the control device is connected with the air outlet member to control the air intake amount and the air outlet member of the feeding member according to the detected air pressure. The amount of gas. a temperature detecting component connected to the control device is disposed inside the inner tank, and the control device is further connected to the liquid inlet component and the liquid outlet component of the jacket to control the liquid inlet component according to the data of the temperature detecting component. And opening and closing of the liquid discharge parts.

Preferably, a baffle plate is provided on the inner wall of the inner casing for generating resistance to the flow of the bacterial liquid. A defoaming paddle is also included in the inner casing, the defoaming paddle is fixed to the same rotating shaft as the agitating paddle of the agitating member, and the position of the defoaming paddle is higher than the agitating paddle.

Preferably, the compounding inoculating tank comprises a mixing tank body and a buffer tank body which is connected with the mixed tank body and receives the bacterial suspension formed by mixing the mixed tank body, wherein the compounding tank body is provided a bacterial liquid inflowing member, a diluent inflowing member, and a ventilation member, wherein the mixing tank is provided with a mixing member for uniformly mixing the bacterial liquid and the diluent, and the buffer tank is provided with a liquid level detecting for detecting the liquid level Parts and discharge parts. The bacterial liquid inflow member, the diluent inflow member, the ventilation member, the mixing member, and the discharge member and the liquid level detecting member of the mixing tank are connected to the control device.

Compared with the prior art, the production process and equipment for continuously supplying the logarithmic growth phase of the present invention have the following beneficial effects:

1. The production process and equipment of the invention increase the buffer storage bacteria liquid and the mixed inoculation liquid solution, and the buffered storage liquid liquid can ensure the bacterial liquid in the logarithmic growth phase, and when the bacterial liquid is needed, the present invention is now available. By using the mixed inoculum, it is possible to continuously supply the bacterial liquid in the logarithmic growth phase.

2. The expansion medium and the fermentation medium are respectively completed in the separately prepared expansion medium preparation tank H and the fermentation medium preparation tank, and the seed tank and the ferment tank are no longer occupied, so that the expansion medium and the fermentation medium are expanded. The preparation and expansion of the bacterial liquid can be carried out simultaneously, thereby improving the efficiency of the production of the bacterial liquid.

3. Amplification medium preparation tank and/or fermentation medium preparation tank and/or seed tank and/or ferment tank and/or buffer storage tank are provided with a gas pressure detecting device capable of detecting the pressure in the inner tank, the air pressure detecting device and the control The device is connected, and the control device can control the intake and exhaust of the intake and exhaust parts of the inner tank according to the received air pressure, thereby realizing automatic control of the inner air pressure.

4. Amplification medium preparation tank and/or fermentation medium preparation tank and/or seed tank and/or ferment tank and/or buffer storage tank are provided with a liquid level detecting device capable of detecting the liquid level in the inner tank, and the liquid level detecting The device is connected to the control device, and the control device can control the feeding part and the discharging part of the inner tank according to the liquid level.

5. Amplification medium preparation tank and/or fermentation medium preparation tank and/or seed tank and/or fermentor and/or buffer storage tank are provided with temperature detecting means capable of detecting the inner temperature of the inner tank, temperature detecting means and control The device is connected, and the control device can control the liquid inlet part and the liquid discharge part of the jacket according to the temperature of the inner tank.

6. The bacterial liquid inflow component, the diluent inflow component, the ventilation component, and the mixing component of the mixing tank and the discharge component of the buffer tank are all connected with the control device to realize mixing, exhausting and discharging. Automated control. And the discharging component can continuously supply the mixed bacterial liquid at a set flow rate under the control of the control device.

DRAWINGS

Fig. 1 is a schematic structural view showing a production apparatus for continuously supplying a logarithmic growth phase bacterial liquid according to a first embodiment of the present invention.

2 is a schematic view showing the structure of an expansion medium preparation tank or a fermentation medium preparation tank or a seed tank or a ferment tank or a buffer storage tank for continuously supplying a logarithmic growth phase bacterial liquid production apparatus according to a first embodiment of the present invention.

Fig. 3 is a schematic view showing the structure of a compounding inoculating tank for continuously supplying a logarithmic growth phase bacterial liquid production apparatus according to a first embodiment of the present invention.

Reference numeral

A colony culture device, B bacteria liquid selection test tube, C bacteria liquid activation bottle, D seed tank, E fermentation tank, F buffer storage tank, G mixed seeding tank, H amplification medium preparation tank, I fermentation medium preparation tank , J control device;

11 housing, 111 motor, 112 stirring paddle, 113 defoaming paddle, 114 spoiler, 115 level detection unit, 116 hand holes, 117 observation window, 118 cover, 12 liner, 121 feeding parts, 122 out Material component, 123 gas outlet component, 124 temperature detecting component, 13 jacket, 131 first liquid-passing component, 132 ventilation component, 133 second liquid-passing component, 14 supporting component;

21 mixed tank, 211 spoiler, 212 dilution inflow part, 213 bacterial liquid inflow part, 214 ventilation part, 22 buffer tank, 221 liquid level detecting part, 222 discharging part, 23 supporting part.

Detailed ways

The continuous supply of the logarithmic growth phase bacterial liquid production process of the present invention is used in the industrial and agricultural fields to culture or ferment microorganisms, including the following steps:

(1) The typical colonies are cultured, that is, the purified and reconstituted original species are diluted and separated, and the diluted and isolated cells are statically cultured by using a selective solid medium to obtain a colony that meets the requirements. During the static culture, the cells remain stationary.

(2) selecting the bacterial liquid, that is, the typical colony obtained in the step (1) is transferred to a test tube containing the selective liquid medium and dynamically cultured at a set temperature and an oscillation frequency to a set time to obtain logarithmic growth. Selective culture of bacterial cells.

(3) activating the bacterial liquid, that is, transferring the selective culture bacterial liquid in the logarithmic growth phase obtained in the step (2) to a bottle containing the liquid activation medium, in this embodiment, a conical flask, and The culture medium was in the logarithmic growth phase by dynamically culturing to a set time under the conditions of the liquid content and the oscillation frequency.

(4) The seed tank amplifies the bacterial liquid, and transfers the activated culture liquid in the logarithmic growth phase to the seed tank D containing the liquid expansion medium and sets the aeration amount, the tank pressure, the temperature and the stirring. A deep liquid culture at a set time was performed at a rate to obtain a seed tank amplifying liquid in a logarithmic growth phase. Among them, deep liquid culture is a culture method in which a strain is inoculated into a tank, and the cells are suspended in a liquid medium and grown or fermented. Deep liquid culture generally requires air supply and stirring, and is a traditional microbial fermentation culture method. The seed tank augmentation medium formulation is a chemically pure reagent of known major component, which is close to the liquid selective medium used in step (2).

(5) The fermenter amplifies the bacterial liquid, and the seed tank amplifying liquid in the logarithmic growth phase is transported to the fermenter E containing the liquid fermentation medium and set at aeration, tank pressure, temperature and stirring. A deep liquid fermentation at a set time is performed at a rate to obtain a fermenter-enriched bacterial solution which is stable in a logarithmic growth phase and is functionally stable. The fermenter expansion medium is formulated as an unseparated natural organic substance (such as soybean meal), which is close to the fermented material (non-cellulose in the phloem).

(6) buffering the bacterial liquid, that is, transferring the fermentation liquid of the fermenter in the logarithmic growth phase to the buffer storage tank F which is emptyed or close to the emptying, and the bacterial liquid in the buffer storage tank F is set. The deep liquid fermentation is continued under aeration, tank pressure, temperature and agitation rate, and the fermentation tank amplification liquid is continuously delivered to the compound inoculation tank G.

(7) compounding the inoculating bacteria solution, that is, diluting the buffer storage liquid solution with a set inorganic salt solution, and combining the vortex rotation and the blocking impact to prepare a bacterial suspension uniformly distributed with the bacteria body, and The bacterial suspension was continuously discharged at a set flow rate to supply a subsequent inoculation procedure.

Through the seven steps of the production process of the present invention, especially by adding the buffer storage liquid solution and the step of compounding the inoculating bacterial liquid, the buffered storage liquid liquid can ensure that the bacterial liquid in the logarithmic growth phase is needed. When the bacterial liquid is used, it can be used now. By mixing the inoculum, the bacterial liquid can be continuously supplied in the logarithmic growth phase.

As a preferred embodiment, the production process of the present invention further comprises the step of preparing an amplification medium before the step (4), and the preparation of the expansion medium is completed in the expansion medium preparation tank H, and the preparation is completed. The increasing medium is transferred to the seed tank D for the step (4). Also included is a step of preparing a fermentation medium performed before the step (5), and the preparation of the fermentation medium is completed in a dedicated fermentation medium preparation tank I, and the completed fermentation medium is transferred to the fermentation tank E. Step (5).

In the prior art, the expansion medium is prepared in a seed tank, and the fermentation medium is prepared in a fermenter. In the present invention, the expansion medium and the fermentation medium are separately prepared in an additional expansion medium. The tank H and the fermentation medium are completed in the preparation tank I, and the seed tank and the ferment tank are no longer occupied, so that the preparation of the expansion medium and the fermentation medium can be simultaneously performed with the expansion and culture of the bacterial liquid, thereby improving the production of the bacterial liquid. s efficiency.

As a preferred method, the production process of the present invention further comprises a sterilization step to ensure that there is no source of contaminated bacteria from the culture medium and equipment during the pure culture of the target species, the step comprising first emptying the clamp for holding the inner liner The insulating water in the jacket, followed by the preparation of the tank H and/or the fermentation medium preparation tank I and/or the seed tank D and/or the fermenter E of the expansion medium containing the medium components or the emptying Setting time for the expansion medium preparation tank H and/or the fermentation medium preparation tank I and/or the seed tank D and/or the fermenter E and/or the tank 12 of the buffer storage tank F are passed through the sterilization steam. And / or temperature sterilization. That is, the amplification medium preparation tank H and/or the fermentation medium preparation tank I and/or the seed tank D and/or the ferment tank E may be sterilized in an emptied state or in a state in which a corresponding liquid is contained. For sterilization, the buffer tank F can only be sterilized in an empty state.

In the present embodiment, the temperature of the sterilization steam is 101-121 ° C, and the set period is 15-30 min. After the heat is kept for a set time, the compressed air is injected into the inner tank to drive the steam to be drained, and cold water is injected into the jacket for cooling the inner tank. When the temperature of the inner liner drops to the set culture temperature, the inner tank is subjected to cooling. Keep warm to maintain the temperature of the bladder. The air pressure of the bladder 12 is maintained ≥ 0.01 MPa to maintain the sterile environment of the bladder 12.

In the sterilization step, the amplification medium preparation tank H and/or the fermentation medium preparation tank I and/or the seed tank D and/or the ferment tank E and/or the buffer storage tank F and/or the mixed inoculation tank G are detected. The air pressure in the inner casing 12 controls the intake air amount of the feed member 121 of the inner casing 12 and the air output amount of the air outlet member 123 in accordance with the air pressure. In the present embodiment, the feeding member 121 and the air outlet member 123 are each provided with a valve controllable by the control device. Preferably, the valve is further provided with a flow detecting member (not shown) for detecting the intake air flow rate and the air output flow rate. With this preferred embodiment, control of the pressure of the bladder 12 can be achieved.

The temperature of the inner liner 12 is lowered after sterilization, and can be naturally reduced or artificially reduced. In the present embodiment, cold water is introduced into the jacket surrounding the inner casing 12 to lower the temperature. When the temperature of the liner 12 drops to a set culture temperature, the tank H and/or the fermentation medium preparation tank I and/or the seed tank D and/or the ferment tank E and/or the buffer tank are prepared for the expansion medium. The inner bulb 12 of F is kept warm to maintain the set culture temperature. Depending on the liquid in the tank, different culture temperatures can be set for different tanks to best suit the growth of microorganisms in the tank.

Detecting the level of liquid in the inner chamber 12 of the expansion medium preparation tank H and/or the fermentation medium preparation tank I and/or the seed tank D and/or the ferment tank E and/or the buffer storage tank F, and according to the liquid level The opening and closing of the feeding member 121 and the discharging member 122 of the inner liner 12 are controlled. Preferably, the feed member 121 is closed when the liquid level reaches 65-75% of the internal height of the bladder 12. Preferably, the feeding member 121 and the discharging member 122 each have a valve (not shown) controllable by a control device, and the valve may be provided with a flow detecting component for detecting the feed flow rate and the discharge flow rate (in the figure) Not shown).

The invention also provides a production device for continuously supplying a logarithmic growth phase bacterial liquid, which can realize the above production process, comprising:

The seed tank contains a liquid expansion medium for receiving and cultivating the activated culture liquid of the culture liquid activation bottle C culture to obtain the seed tank amplification liquid in the logarithmic growth phase.

The fermenter E contains a liquid fermentation medium for receiving and cultivating the seed tank cultured seed tank to amplify the bacterial liquid to obtain a fermenting tank amplifying liquid in a logarithmic growth phase.

The buffer storage tank F is configured to receive the fermentation tank of the fermenter E culture in the state of being emptied and to continue the deep liquid fermentation, and simultaneously output the fermenter to amplify the bacterial liquid.

The inoculation tank G is mixed and used to receive the amplified bacterial liquid delivered from the buffer storage tank F, and is diluted into a bacterial suspension uniformly distributed with the bacterial cells, and the bacterial suspension is output.

In addition to the above structure, in the present embodiment, the production apparatus may further comprise a colony culture apparatus A for diluting and separating the purely reconstituted stock and performing static culture on the solid plate medium to obtain an intrinsic colony morphology characteristic description. Colonies. The bacterial solution is selected from a test tube B containing a liquid selective medium for receiving and cultivating the colonies cultured in the colony culture apparatus A to obtain a selective culture bacterial liquid in a logarithmic growth phase. The bacterial liquid activation bottle C contains a liquid activation medium for receiving and cultivating the selective culture liquid of the bacterial liquid selection test tube B to obtain the activated culture bacterial liquid in the logarithmic growth phase, in this embodiment, The bacterial solution activation bottle C is a conical flask.

Preferably, the production apparatus further comprises an expansion medium preparation tank H for preparing a culture expansion medium for multi-batch production of the seed tank amplification bacterial liquid, the expansion medium preparation tank H and The seed tank connection is used to deliver an expansion medium to the seed tank. The volume of the expansion medium preparation tank H is at least twice the volume of the seed tank, and an adequate supply of the expansion medium to the seed tank can be ensured.

The production apparatus further includes a fermentation medium preparation tank I for preparing a fermentation medium for multi-batch production of a fermenter to amplify the bacterial liquid, and the fermentation medium preparation tank I is connected to the fermenter E The fermentation medium is delivered to the fermentor E. The volume of the fermentation medium preparation tank I is at least twice the volume of the fermenter E, and an adequate supply of the fermentation medium to the fermenter E can be ensured.

As a preferred embodiment, the amplification medium preparation tank H and/or the fermentation medium preparation tank I and/or the seed tank and/or the ferment tank E and/or the buffer storage tank F comprise an inner casing 11 a bladder 12 and a lid 118 enclosing the bladder 12, a jacket 13 surrounding the liner 12 from the bottom for containing the heat insulating liquid, a stirring member on the lid 118, and a rotation for driving the stirring member Motor 111. The housing 11, the inner casing 12 and the jacket 13 may be integrally formed or may be separate. In the present embodiment, the three are integrally formed and supported by the support member 14 to stand on the ground.

The outer wall of the cover body 118 is provided with a hand hole 116, and a finger can be inserted into the hand hole 116 when the can body is moved to facilitate the application of force. The cover 118 is also provided with a transparent viewing window 117 for observing the interior of the bladder. An air outlet member 123 is also provided on the cover body for adjusting the air pressure in the inner casing 12. The inner casing 12 has a feeding member 121 and a discharging member 122, and the feeding member 121 can be filled or inhaled.

The jacket 13 has a first liquid circulation member 131 that can serve as a hot water inlet or a cold water outlet, a second liquid circulation member 133 that can serve as a hot water outlet or a cold water inlet, and a ventilation member 132. The first liquid circulation member 131 is provided at the highest point of the jacket 13, and the second liquid circulation member 133 is disposed at the bottom of the jacket 13. When it is necessary to cool the inner tank, cold water is injected into the jacket from the second liquid circulation member 133, and the cold water flows out from the first liquid circulation member 131 to form a circulation. When it is necessary to heat the inner liner, hot water is injected into the jacket from the first liquid circulation member 131, and the hot water flows out from the second liquid circulation member 133 to form a circulation. When hot water or cold water is injected, the gas in the jacket is discharged from the venting member 132, and when the water is emptied, the air enters the jacket through the venting member 132. After long-term use, the dirt in the water is inevitably left in the jacket 13, and the dirt can be removed by the second liquid circulation member 133.

As a preferred solution, the production apparatus of the present invention further includes a control device J that is coupled to the motor 111 for agitating the member to control the opening and closing of the motor 111.

A liquid level detecting member 115 connected to the control device J is disposed inside the inner casing 12, and the control device J is connected to the feeding member 121 and the discharging member 122 of the inner casing 12, respectively, according to the detection. The level controls the opening and closing of the feed member 121 and the discharge member 122. Preferably, the feed member 121 is closed when the liquid level reaches 65-75% of the internal height of the bladder 12. As a preferred solution, the feed member 121 and the discharge member 122 each have a valve (not shown) controllable by the control device J, and the valve may be provided with a flow detecting member for detecting the feed flow rate and the discharge flow rate (Fig. Not shown).

The inner casing 12 is provided with a gas pressure detecting member connected to the control device J, the control device J is connected to the feeding member 121 of the inner casing 12, and the feeding member 121 can enter a liquid and a gas, and the control device J is also connected to the air outlet member 123 to control the air intake and the air outlet member 123 of the feed member 121 to be ventilated according to the detected air pressure.

The inside of the inner casing 12 is provided with a temperature detecting member 124 connected to the control device J, and the control device J is also connected to the first liquid-passing member 131 and the second liquid-passing member 133 of the jacket 13 to be in accordance with the temperature. The data of the detecting member 124 controls the opening and closing of the first liquid passing member 131 and the second liquid passing member 133. The control device J is also coupled to the venting member 132 of the jacket to control opening and closing of the venting member 132.

The inner casing 12 is provided with a baffle 114 for generating resistance to the flow of the bacterial liquid. In the embodiment, the baffle 114 is fixed to the inner wall of the inner casing 12 to generate resistance to the rotating bacterial liquid, thereby Mix the bacteria solution with the diluent better.

The inner tank 12 is further provided with a defoaming paddle 113. The defoaming paddle 113 and the agitating paddle 112 of the agitating member are fixed on the same rotating shaft, and the position of the defoaming paddle 113 is higher than the agitation. Paddle 112. The defoaming paddle 113 agitates the foam floating above the liquid level to break it, thereby eliminating or reducing the foam.

The compounding inoculating tank G includes a mixing tank 21 and a buffer tank 22 that communicates with the mixing tank 21 to receive the mixed liquid formed by mixing the mixing tank 21, and mixes the tank 21 and the buffer. The can body 22 is integral in this embodiment, supported on the ground by the support member 23, and the buffer can 22 is located below the compound can body 21. The mixed tank body 21 is provided with a bacterial liquid inflow member 213, a diluent inflow member 212, and a ventilation member 214, and a mixed member for uniformly mixing the bacterial liquid and the diluted liquid is provided inside the mixed tank body 21. The bacterial liquid inflow member 213, the diluent inflow member 212, the ventilation member 214, and the compounding member of the mixed tank 21 are connected to the control device J.

In the present embodiment, the mixing tank 21 is provided with a spoiler 211 that generates resistance to the moving bacteria liquid and the diluent.

The buffer tank 22 is further provided with a liquid level detecting component 221 for detecting the liquid level of the bacterial liquid in the buffer tank 22. The bottom of the buffer tank 22 is provided with a discharging member 222, and the discharging member 222 is under control. Under the control of the device J, the continuous supply of the mixed bacterial liquid is completed at the set flow rate.

The above embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the invention, and the scope of the invention is defined by the claims. It is also within the scope of the present invention to make various modifications or equivalents to the present invention within the spirit and scope of the invention.

Claims

A process for continuously supplying a logarithmic growth phase bacterial liquid, characterized in that it comprises the following steps:

(1) cultivating a typical colony, that is, diluting and separating the purified and reconstituted original species, and statically cultivating the diluted and isolated cells by using a selective solid medium to obtain a colony that meets the requirements;

(2) selecting the bacterial liquid, inoculation of the typical colony obtained in the step (1) into a test tube containing the selective liquid medium, and dynamically cultured at a set temperature and an oscillation frequency to a set time to obtain logarithmic growth. Selective culture of bacterial liquid;

(3) activating the bacterial liquid, that is, transferring the selective culture liquid in the logarithmic growth phase obtained in the step (2) to the cone type bottle containing the liquid activation medium and setting the liquid amount, temperature and Dynamically culturing to a set time under an oscillating frequency condition to obtain an activated culture broth in a logarithmic growth phase;

(4) The seed tank amplifies the bacterial liquid, that is, transfers the activated culture liquid in the logarithmic growth phase to the seed tank containing the liquid expansion medium and sets the aeration amount, the tank pressure, the temperature, the stirring rate. Performing a deep liquid culture at a set time to obtain a seed tank amplifying bacterial solution in a logarithmic growth phase;

(5) The fermenter amplifies the bacterial liquid, and transfers the seed tank amplifying liquid in the logarithmic growth phase to the fermenting tank containing the liquid fermentation medium and sets the aeration amount, the tank pressure, the temperature, and the stirring. Performing a deep liquid fermentation at a set rate at a rate to obtain a fermenter-enriched bacterial solution in a logarithmic growth phase and having a stable function;

(6) Buffer storage bacteria liquid, that is, the fermentation tank liquid in the fermenter in the logarithmic growth phase is transferred to the buffer storage tank close to the emptying, and the bacterial liquid in the buffer storage tank is at the set ventilation volume and the tank pressure. Deep liquid fermentation is continued at a temperature and a stirring rate, and the fermentation broth of the fermenter is continuously delivered to the mixed inoculating tank;

(7) compounding the inoculating bacterial solution, that is, diluting the buffered bacterial liquid with a set inorganic salt solution, and combining the vortex rotation and the blocking impact to prepare a bacterial suspension uniformly distributed with the bacterial body, The bacterial suspension was continuously discharged at a set flow rate to supply a subsequent inoculation procedure.
The process for continuously producing a logarithmic growth phase bacterial liquid according to claim 1, further comprising the step of preparing an expansion medium performed before the step (4), and the preparation of the expansion medium is The amplification medium preparation tank is completed, and the prepared amplification medium is delivered to the seed tank for the step (4).
The process for producing a continuous supply logarithmic growth phase bacterial liquid according to claim 2, further comprising the step of preparing a fermentation medium before the step (5), and preparing the fermentation medium in a special design The fermentation medium is prepared in a tank, and the prepared fermentation medium is transferred to the fermentor for the step (5).
The process for continuously producing a logarithmic growth phase bacterial liquid according to claim 1, further comprising a sterilization step of ensuring that there is no source of contamination of the bacteria from the culture medium and the device during the pure culture of the target species, The steps include first emptying the insulating water in the jacket for holding the inner liner, and secondly preparing the can and/or the expansion medium preparation tank and/or the seed tank and/or the expansion medium containing the medium components. Or fermenting the tank or steaming into the drain of the seed tank and/or fermentor and/or buffer storage tank for set time and/or temperature, and then injecting into the inner tank after holding for a set time. Sterile compressed air drives the steam to drain, while injecting cold water into the jacket for cooling the liner and maintaining the internal pressure of the bladder ≥0.01MPa.
The process for producing a continuous supply logarithmic growth phase bacterial liquid according to claim 4, wherein the sterilization steam has a temperature of 101 to 121 °C.
The process for producing a continuous supply logarithmic growth phase bacterial liquid according to claim 4, wherein the set period of time is 15-30 min.
The process for continuously producing a logarithmic growth phase bacterial liquid according to claim 4, wherein in the sterilization step, the amplification medium preparation tank and/or the fermentation medium preparation tank and/or the seed tank are detected. And / or the pressure inside the tank of the fermenter and / or buffer storage tank, and according to the air pressure to control the amount of air intake and outlet of the tank.
The process for producing a continuous supply logarithmic growth phase bacterial liquid according to claim 4, wherein the inner liner is kept warm to maintain the temperature of the inner liner when the temperature of the inner liner drops to a set culture temperature.
The process for producing a continuous supply logarithmic growth phase bacterial liquid according to claim 4, characterized in that the amplification medium preparation tank and/or the fermentation medium preparation tank and/or the seed tank and/or the ferment tank are detected and / or buffer the level of liquid in the inner tank of the storage tank, and according to the liquid level to control the opening and closing of the feeding and discharging parts of the inner tank.
The process for continuously producing a logarithmic growth phase bacterial liquid according to claim 9, wherein the liquid level reaches an expansion medium preparation tank and/or a fermentation medium preparation tank and/or a seed tank and/or The liquid inlet component is closed when the inner height of the inner tank of the fermentor and/or the buffer storage tank is 65-75%.
A production facility for continuously supplying a logarithmic growth phase bacterial liquid, characterized in that it comprises:

a seed tank containing a liquid expansion medium for receiving and cultivating the activated culture liquid cultured in the bacterial activation bottle to obtain a seed tank amplification liquid in a logarithmic growth phase;

a fermenter containing a liquid fermentation medium for receiving and cultivating the seed tank cultured seed tank to amplify the bacterial liquid to obtain a fermenting tank amplifying liquid in a logarithmic growth phase;

a buffer storage tank for receiving the fermentation tank of the fermentation tank cultured in the fermenter to be subjected to deep liquid fermentation, and outputting the fermenter to amplify the bacterial liquid;

The mixed inoculating tank is configured to receive the amplified bacterial liquid delivered from the buffer storage tank, dilute it, and prepare a bacterial suspension uniformly distributed with the bacterial cells, and output the bacterial suspension.
The apparatus for continuously supplying a logarithmic growth phase bacterial liquid according to claim 11, wherein the production apparatus further comprises an expansion culture for preparing a multi-batch production seed tank to amplify the bacterial liquid. A base expansion medium preparation tank is coupled to the seed tank for delivering the expansion medium to the seed tank.
The apparatus for continuously supplying a logarithmic growth phase bacterial liquid according to claim 12, wherein the volume of the expansion medium preparation tank is at least twice the volume of the seed tank.
The apparatus for continuously supplying a logarithmic growth phase bacterial liquid according to claim 12, wherein the production apparatus further comprises a fermentation medium for preparing a multi-batch production fermenter to amplify the bacterial liquid. The fermentation medium preparation tank is connected to the fermenter for delivering the fermentation medium to the fermentor.
The apparatus for continuously supplying a logarithmic growth phase bacterial liquid according to claim 14, wherein the fermentation medium preparation tank has a volume of at least twice the volume of the fermenter.
The apparatus for continuously supplying a logarithmic growth phase bacterial liquid according to claim 11, wherein the amplification medium preparation tank and/or the fermentation medium preparation tank and/or the seed tank and/or the ferment tank And/or the buffer storage tank includes a liner enclosed by the casing and a cover body enclosing the inner casing, a jacket surrounding the inner liner from the bottom for accommodating the heat insulating liquid, and a stirring member on the cover body And a motor for driving the agitating member to rotate, the inner casing having a feeding member and a discharging member, the feeding member being capable of introducing or inhaling, the cover being provided with an outlet member communicating with the inner casing The jacket has a first liquid flow-through member that can serve as a hot water inlet or a cold water outlet, a second liquid flow-through member and a venting member that can serve as a hot water outlet or a cold water inlet.
A production apparatus for continuously supplying a logarithmic growth phase bacterial liquid according to claim 16, further comprising control means for connecting a motor for agitating the liquid in said inner casing to control the motor Turn it on and off.
The apparatus for continuously supplying a logarithmic growth phase bacterial liquid according to claim 16, wherein the inner tank is provided with a liquid level detecting member connected to the control device, the control device and the The feed and discharge components of the liner are separately connected to control the opening and closing of the feed and discharge components based on the detected level.
The apparatus for continuously supplying a logarithmic growth phase bacterial liquid according to claim 16, wherein the cover body is provided with a pressure detecting member connected to the control device for detecting a pressure inside the inner casing, the control A device is coupled to the air outlet member to control the amount of intake air of the feed member and the amount of air output from the air outlet member based on the detected air pressure.
The apparatus for continuously supplying a logarithmic growth phase bacterial liquid according to claim 16, wherein the inner casing is provided with a temperature detecting member connected to the control device, and the control device is further provided with a jacket The liquid inlet part and the liquid discharge part are respectively connected to control opening and closing of the liquid inlet part and the liquid discharge part according to data of the temperature detecting part.
The apparatus for continuously supplying a logarithmic growth phase bacterial liquid according to claim 16, further comprising a baffle plate provided on an inner wall of said inner liner for generating resistance to the flow of the bacterial liquid.
The apparatus for continuously supplying a logarithmic growth phase bacterial liquid according to claim 16, further comprising a defoaming paddle located in said inner tank, said defoaming paddle and a stirring paddle of said stirring member Fixed on the same rotating shaft, and the position of the defoaming paddle is higher than the stirring paddle.
The apparatus for continuously supplying a logarithmic growth phase bacterial liquid according to claim 11, wherein the compounding inoculating tank comprises a compounding tank body and communicating with the compounding tank body to receive the compounding tank a buffer tank for the bacterial suspension formed by the body mixing, wherein the mixed tank body is provided with a bacterial liquid inflow member, a diluent inflow member, and a ventilation member, and the mixed tank is provided with a liquid and a diluent A uniformly mixed compounding member provided with a liquid level detecting member and a discharging member for detecting a liquid level.
The apparatus for continuously supplying a logarithmic growth phase bacterial liquid according to claim 23, wherein the bacterial liquid inflow member, the diluent inflow member, the ventilation member, the compounding member, and the buffer tank of the tank body are mixed. Both the discharge member and the liquid level detecting member are connected to the control device.
The apparatus for continuously supplying a logarithmic growth phase bacterial liquid according to claim 23, wherein the compounding tank body is provided with a baffle plate for generating resistance to the moving bacterial liquid and the diluted liquid.