Directed micro-ecology expansion apparatus, expansion method and computer-readable storage medium

Abstract

A directed micro-ecology expansion apparatus. The apparatus may comprise: a housing; an expansion container disposed in the housing, used to provide a place for expanding a micro-ecology microbial agent; a liquid injection port disposed on the housing and in communication with the expansion container, used to inject a first liquid into the expansion container; a microbial agent adding unit disposed on the shell and in communication with the expansion container, used to add the micro-ecology microbial agent into the expansion container; a temperature control unit disposed in the shell, used to control the temperature of the first liquid in the expansion container; a stirrer disposed in the expansion container, used to stir the first liquid in the expansion container to promote expansion; and a discharge port disposed on the shell and in communication with the expansion container, used to discharge material in the expansion container after expansion. Also provided are an expansion method for the directed micro-ecology expansion apparatus, a computer-readable storage medium and an electronic device.

Description

Directed microecological expansion culture device, expansion culture method and computer-readable storage medium

[0001] Priority Declaration

[0002] This application claims priority to the Chinese invention patent application number 202310943775.1 filed on July 31, 2023, entitled “Directed microecological expansion device, expansion method, medium and electronic equipment”. All contents of the above patent are incorporated herein in their entirety. Technical Field

[0003] The present invention relates to the technical field of microbial cultivation, in particular to a microecological quantitative expansion cultivation system, and more specifically to a directional microecological expansion cultivation device, an expansion cultivation method and a computer-readable storage medium. Background Art

[0004] Microecological expansion technology mainly involves the screening and cultivation of microecological strains and the large-scale expansion of microecological species. The expansion of microecological species requires consideration of various growth conditions of microecological species, including temperature, pH value, oxygen content, seed age, inoculum size, etc.

[0005] During the process of implementing the present invention, the inventors discovered that the related art suffers from at least the following problems: Microbial fermentation tank systems are large in scale, have high investment and operating costs, complex process flows, and require specialized personnel for operation. Furthermore, after centralized production, the expanded culture products require long-distance transportation to reach end users. Furthermore, upon arrival, the microbial survival rate is low, failing to meet end-user needs.

[0006] Therefore, technical personnel in this field urgently need to develop a microecological expansion and cultivation equipment to solve the problems of large scale of microbial inoculant products, high investment and operating costs, complex process flow, and the need for professional personnel to operate. In addition, after centralized production, inoculant products need to be transported over long distances to reach end users. Moreover, after the products reach end users, the survival rate of microorganisms is not high and cannot meet the needs of end users.

[0007] Summary of the Invention

[0008] In view of this, the technical problem to be solved by the present invention is to provide a directional microecological expansion culture device, expansion culture method and computer-readable storage medium, which solves the problems in the related technology that the microbial inoculant products are large in scale, high in investment cost and operating cost, complex in process flow, and require professional personnel to operate. In addition, after the inoculant products are centrally produced, they need to be transported over long distances to reach the end users. Moreover, after the products reach the end users, the survival rate of the microorganisms is not high and cannot meet the needs of the end users.

[0009] In order to solve the above technical problems, a specific embodiment of the present invention provides a directional microecological expansion and cultivation device, comprising: a shell; an expansion and cultivation container, arranged in the shell, for providing a place for the expansion and cultivation of a microecological microbial agent; a liquid injection port arranged on the shell, connected to the expansion and cultivation container, for injecting a first liquid into the expansion and cultivation container; a microbial agent adding unit arranged on the shell, connected to the expansion and cultivation container, for adding the microecological microbial agent into the expansion and cultivation container; a temperature control unit, arranged in the shell, for controlling the temperature of the first liquid in the expansion and cultivation container; a stirrer, arranged in the expansion and cultivation container, for stirring the first liquid in the expansion and cultivation container to promote expansion and cultivation; and a discharge port arranged on the shell, connected to the expansion and cultivation container, for discharging the material in the expansion and cultivation container after expansion and cultivation.

[0010] A specific embodiment of the present invention also provides an expansion method of a directional microecological expansion device, comprising: controlling an electronic valve to inject a predetermined amount of a first liquid into an expansion container; sterilizing the injected first liquid using a non-complete sterilization method; adding a microecological agent into the expansion container according to the liquid level of the first liquid after non-complete sterilization; and expanding the first liquid to which the microecological agent is added using set conditions according to the type of the microecological agent.

[0011] Another aspect of an embodiment of the present invention provides a computer program, which includes computer-executable instructions. When the instructions are executed, they are used to implement the method of the embodiment of the present invention.

[0012] Another aspect of an embodiment of the present invention provides a computer-readable storage medium storing computer-executable instructions, which are used to implement the method of the embodiment of the present invention when executed by a processor.

[0013] Another aspect of an embodiment of the present invention provides an electronic device, including one or more processors and a storage device, wherein the storage device is used to store executable instructions, and when the executable instructions are executed by the processor, the method of the embodiment of the present invention is implemented.

[0014] According to the above-mentioned embodiment of the present invention, the use of an integrated directional microecological expansion and cultivation device can at least partially solve the problems in the related art that the microbial inoculant products are large in scale, high in investment cost and operating cost, complex in process flow, and require professional personnel to operate. In addition, after the expansion and cultivation products are centrally produced, they need to be transported over long distances to reach the end users. Moreover, after the products reach the end users, the microbial survival rate is not high and cannot meet the needs of the end users. Therefore, the technical effects of low cost, one-click standardized production, no need for transportation, immediate use, and high microbial survival rate can be achieved.

[0015] It should be understood that the foregoing general description and the following detailed description are merely exemplary and illustrative and are not intended to limit the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings, which are part of the specification of the present invention, illustrate exemplary embodiments of the present invention and, together with the description, serve to explain the principles of the present invention.

[0017] FIG1 is a first structural schematic diagram of a directional microecological expansion culture device provided by a specific embodiment of the present invention.

[0018] FIG2A is a schematic structural diagram of a stirrer blade provided in a specific embodiment of the present invention.

[0019] FIG2B is a schematic diagram of the flow direction of the first liquid when a stirrer provided by a specific embodiment of the present invention is stirring in a culture vessel.

[0020] FIG3 is a second structural schematic diagram of a directional microecological expansion culture device provided in a specific embodiment of the present invention.

[0021] FIG4 is a third structural schematic diagram of a directional microecological expansion culture device provided by a specific embodiment of the present invention.

[0022] FIG5 is a fourth structural schematic diagram of a directional microecological expansion culture device provided by a specific embodiment of the present invention.

[0023] FIG6 is a fifth structural schematic diagram of a directional microecological expansion culture device provided by a specific embodiment of the present invention.

[0024] FIG7 is a schematic structural diagram of an electronic switch provided in a specific embodiment of the present invention.

[0025] FIG8 is a sixth structural schematic diagram of a directional microecological expansion culture device provided by a specific embodiment of the present invention.

[0026] FIG9 is a first schematic flow chart of a method for expanding culture of a directional microecological expansion culture device provided by a specific embodiment of the present invention.

[0027] FIG10 is a second schematic flow chart of a method for expanding culture of a directional microecological expansion culture device provided by a specific embodiment of the present invention.

[0028] FIG11 is a third schematic flow chart of a method for expanding culture of a directional microecological expansion culture device provided in a specific embodiment of the present invention.

[0029] FIG12 is a fourth schematic flow chart of a method for expanding culture of a directional microecological expansion culture device provided by a specific embodiment of the present invention.

[0030] FIG13 is a schematic flow chart of a method for sterilizing the injected first liquid using a non-complete sterilization method according to a specific embodiment of the present invention.

[0031] FIG14 is a schematic flow chart of a method for aerobic expansion of a first liquid to which a microbial agent is added using set conditions according to a specific embodiment of the present invention.

[0032] FIG15 is a schematic flow chart of an embodiment of the present invention providing a method for anaerobic culture of a first liquid to which a microbial agent is added using set conditions.

[0033] Explanation of Reference Numerals: 1 housing 2 expansion container 3 liquid injection port 4 microbial agent addition unit 5 temperature control unit 6 agitator 7 discharge port 8 liquid storage tank 9 nozzle 10 first drive unit 11 first electronic valve 12 second electronic valve 13 third electronic valve 14 fourth electronic valve 15 drain port 16 fifth electronic valve 17 aeration unit 18 air filter 19 liquid level monitor 20 controller 21 temperature monitor 4-1 microbial agent box 4-2 microbial agent transmission pipeline 4-3 electronic switch 4-3-1 rotating shaft 4-3-2 blade 4-3-3 third drive unit 5-1 liquid jacket 5-2 heater 5-3 cooler 5-4 second drive unit DETAILED DESCRIPTION

[0034] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention more clearly understood, the spirit of the contents disclosed in the present invention will be clearly illustrated with the accompanying drawings and detailed descriptions below. After understanding the embodiments of the contents of the present invention, any technician in the relevant technical field can change and modify the contents of the present invention based on the techniques taught by the contents of the present invention without departing from the spirit and scope of the contents of the present invention.

[0035] The exemplary embodiments of the present invention and their description are used to explain the present invention, but are not intended to limit the present invention. In addition, elements / components with the same or similar reference numerals used in the drawings and embodiments are used to represent the same or similar parts.

[0036] The terms “first,” “second,” etc. used herein do not particularly refer to an order or sequence, nor are they intended to limit the present invention. They are merely used to distinguish elements or operations described with the same technical terms.

[0037] The directional terms used herein, such as up, down, left, right, front, or back, are only used to refer to the directions in the accompanying drawings. Therefore, the directional terms used are used to illustrate and not to limit the present invention.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] As used herein, "and / or" includes any and all combinations of the items mentioned.

[0040] Regarding "plurality" in this document, "plurality" includes "two" and "more than two"; regarding "plurality groups" in this document, "plurality groups" includes "two groups" and "more than two groups".

[0041] As used herein, the terms "substantially" and "approximately" are used to modify any quantity or error that may vary slightly, but such variation or error does not alter the essence of the quantity. Generally speaking, the range of such variation or error modified by such terms may be 20% in some embodiments, 10% in some embodiments, 5% in some embodiments, or other values. Those skilled in the art will appreciate that the aforementioned values ​​may be adjusted based on actual needs and are not intended to be limiting.

[0042] All terms used herein (including technical and scientific terms) have the meanings commonly understood by those skilled in the art unless otherwise defined. It should be noted that the terms used herein should be interpreted as having a meaning consistent with the context of this specification and should not be interpreted in an idealized or overly rigid manner.

[0043] When expressions such as “at least one of A, B, and C, etc.” are used, they should generally be interpreted in accordance with the meaning commonly understood by those skilled in the art (e.g., “a system having at least one of A, B, and C” should include, but is not limited to, systems having A alone, B alone, C alone, A and B, A and C, B and C, and / or A, B, and C, etc.). When expressions such as “at least one of A, B, or C, etc.” are used, they should generally be interpreted in accordance with the meaning commonly understood by those skilled in the art (e.g., “a system having at least one of A, B, or C” should include, but is not limited to, systems having A alone, B alone, C alone, A and B, A and C, B and C, and / or A, B, and C, etc.). Those skilled in the art should also understand that any transitional conjunctions and / or phrases that essentially represent two or more optional items, whether in the specification or the drawings, should be understood to provide the possibility of including one, either, or both of these items. For example, the phrase "A or B" should be understood to include the possibilities of "A" or "B", or "A and B".

[0044] Conventional liquid fermentation equipment requires significant investment and high technical requirements, requiring only specialized personnel to operate. Furthermore, to ensure product stability upon reaching the customer, the fermentation broth typically needs to be processed into specialized preparations. By the time microbial preparations reach the customer, not only are the microbial activity significantly reduced through multiple distribution channels, but the terminal price is also very high, making them unacceptable to users or requiring insufficient dosage within an acceptable cost range. Consequently, the desired results cannot be achieved, leading to a loss of confidence in the use of microbial agents.

[0045] By optimizing equipment, processes, and culture media, this invention achieves automated and standardized control of microbial inoculum expansion, simplifying equipment and reducing production costs. No specialized operating skills are required; simply by activating the equipment's automated control button, fresh, high-yield, low-cost microbial culture can be obtained within a specified timeframe, making the microbial culture a readily available, low-cost, and affordable product for most users.

[0046] FIG1 is a first structural schematic diagram of a directional microecological expansion culture device provided by a specific embodiment of the present invention.

[0047] As shown in FIG1 , the directional microecological expansion and cultivation device may include a housing 1 , an expansion and cultivation container 2 , a liquid injection port 3 , a bacterial agent adding unit 4 , a temperature control unit 5 , an agitator 6 and a discharge port 7 .

[0048] Specifically, the expansion container 2 is arranged in the shell 1, and the expansion container 2 is used to provide a place for the expansion of the microecological microbial agent. The liquid injection port 3 is arranged on the shell 1, and the liquid injection port 3 is connected to the expansion container 2. The liquid injection port 3 is used to inject the first liquid into the expansion container 2. The microbial agent adding unit 4 is arranged on the shell 1, and the microbial agent adding unit 4 is connected to the expansion container 2. The microbial agent adding unit 4 is used to add the microecological microbial agent into the expansion container 2. The temperature control unit 5 is arranged in the shell 1, and the temperature control unit 5 is used to control the temperature of the first liquid in the expansion container 2. The stirrer 6 is arranged in the expansion container 2, and the stirrer 6 is used to stir the first liquid in the expansion container 2 to promote expansion. The discharge port 7 is arranged on the shell 1, and the discharge port 7 is connected to the expansion container 2. The discharge port 7 is used to discharge the material in the expansion container 2 that has completed expansion.

[0049] In an embodiment of the present invention, the liquid injection port 3 can be connected with the faucet by a hose, and the faucet injects the first liquid into the expansion culture container 2 by a hose. Required raw materials can be added to clean water or tap water to form the first liquid, and the first liquid can be a culture medium. According to the bacterial strain to be cultivated, the pH value of the first liquid can be adjusted to meet the needs of cultivating bacterial strains. Clean water or tap water can also be placed in a bucket, and then the raw materials are dropped into the bucket to form the first liquid. The hose is placed in the bucket, and the first liquid is drawn into the expansion culture container 2. The workplace of the directional microecological expansion culture device is unrestricted. Clean water or tap water can also be directly injected into the expansion culture container 2, and raw materials are dropped from the top of the expansion culture container 2. The liquid injection port 3 can also be provided with a manual switch, as shown in the figure, and the manual switch is turned on so that the first liquid can be injected into the expansion culture container 2. The manual switch is set to prevent user misoperation.

[0050] In embodiments of the present invention, one-touch automation and standardized control of microecological expansion and cultivation are realized, the equipment is simple, does not need to be transported, is produced and used, microecological survival rate is high, production cost is low, and professional operating skills are not required, only the automation control button of the equipment needs to be started, and fresh, high bacterial count, low-cost microecological bacterial liquid can be obtained within a specific time. The directional microecological expansion and cultivation device is small in size, light in weight, and has a small floor space. The entire expansion and cultivation process can be completed in less than 24 hours, and the entire device has only the size of a washing machine, and the user can utilize a general vehicle for transportation at will.

[0051] Figure 2A is a schematic diagram of the structure of a stirrer blade provided by a specific embodiment of the present invention. Figure 2B is a schematic diagram of the flow direction of a first liquid when a stirrer provided by a specific embodiment of the present invention is stirring in a culture vessel.

[0052] As shown in Figures 2A and 2B, blades 6-1 of stirrer 6 are arranged at an angle. As blades 6-1 rotate in the direction indicated by the arrow in the figure, a front side b of blade 6-1 tilts upward, while a rear side a of blade 6-1 droops downward. That is, the distance between front side b and the bottom of culture container 2 is greater than the distance between rear side a and the bottom of culture container 2. As blades 6-1 of stirrer 6 rotate, the first liquid in culture container 2 flows in the direction indicated in Figure 2B, and stirrer 6 can fully stir the first liquid in culture container 2.

[0053] FIG3 is a second structural schematic diagram of a directional microecological expansion culture device provided in a specific embodiment of the present invention.

[0054] As shown in FIG3 , the directional microecological expansion and cultivation device may further include a liquid storage tank 8 , a nozzle 9 and a first driving unit 10 .

[0055] Specifically, a liquid storage tank 8 is disposed within the housing 1 and is in communication with the liquid inlet 3 and the expansion container 2. The liquid storage tank 8 is used to store a first liquid used for expansion and / or a liquid for cleaning residues on the inner wall of the expansion container 2. A nozzle 9 is disposed within the expansion container 2 and is in communication with the liquid inlet 3 and the liquid storage tank 8. The nozzle 9 is used to clean residues on the inner wall of the expansion container 2. A first drive unit 10 is disposed between the nozzle 9 and the liquid storage tank 8. The first drive unit 10 is used to drive the liquid in the liquid storage tank 8 to clean residues on the inner wall of the expansion container 2. The first drive unit 10 is also used to drive the material in the expansion container 2 that has been expanded to be discharged from the discharge port 7.

[0056] In an embodiment of the present invention, the liquid in the liquid storage tank 8 for cleaning the residue on the inner wall of the expansion container 2 can be a first liquid, or it can be clean water or tap water. The liquid storage tank 8 can only store the liquid for cleaning the residue on the inner wall of the expansion container 2. The liquid in the liquid storage tank 8 is used to clean the residue on the inner wall of the expansion container 2. The water supply is sufficient, and the residue on the inner wall of the expansion container 2 can be quickly rinsed and cleaned thoroughly. The liquid storage tank 8 can separately store the first liquid used for expansion and the liquid for cleaning the residue on the inner wall of the expansion container 2. For example, the liquid storage tank 8 contains two cavities, one cavity for storing the first liquid used for expansion, and the other cavity for cleaning the liquid on the residue on the inner wall of the expansion container 2. The first drive unit 10 drives the expanded material in the expansion container 2 to be discharged from the discharge port 7. The material that cannot be discharged remains on the inner wall of the expansion container 2. Therefore, the residue on the inner wall of the expansion container 2 is mainly residual material.

[0057] In an embodiment of the present invention, the liquid storage tank 8 may be a water tank, the spray head 9 may be a shower head, and the first driving unit 10 may be a water pump.

[0058] In an embodiment of the present invention, the liquid storage tank 8 stores liquid, and the liquid stored in the liquid storage tank 8 is used to clean the liquid remaining on the inner wall of the expansion container 2. The liquid storage tank 8 supplies sufficient liquid, which can quickly rinse the residue on the inner wall of the expansion container 2 and clean it thoroughly.

[0059] In an embodiment of the present invention, the directional microecological expansion and cultivation device may further include: a first electronic valve 11 , a second electronic valve 12 , a third electronic valve 13 and a fourth electronic valve 14 .

[0060] Specifically, the first electronic valve 11 is arranged between the nozzle 9 and the liquid injection port 3. The second electronic valve 12 is arranged between the liquid storage tank 8 and the liquid injection port 3. The first electronic valve 11 and the second electronic valve 12 jointly control the first liquid in the liquid storage tank 8 to clean the residue on the inner wall of the expansion container 2 through the nozzle 9. The third electronic valve 13 is arranged between the discharge port 7 and the liquid injection port 3. The fourth electronic valve 14 is arranged between the bottom opening 2-1 of the expansion container 2 and the liquid injection port 3. The third electronic valve 13 and the fourth electronic valve 14 jointly control the discharge of the expanded material in the expansion container 2 through the discharge port 7, or jointly control the discharge of the cleaned residue through the discharge port 7.

[0061] In this embodiment of the present invention, the second electronic valve 12 is opened, and the first liquid and / or liquid for cleaning residue on the inner wall of the expansion container 2 is injected into the liquid storage tank 8 through the liquid injection port 3. The first and second electronic valves 11, 12 are opened, and the first drive unit 10 is activated, so that the liquid in the liquid storage tank 8 can be used to clean the residue on the inner wall of the expansion container 2. The third and fourth electronic valves 13, 14 are opened, and the first drive unit 10 is activated, so that the material after expansion in the expansion container 2 is discharged through the discharge port 7, and the residue cleaned in the expansion container 2 can also be discharged through the discharge port 7.

[0062] In an embodiment of the present invention, a first liquid is injected into the expansion container 2, and the material expanded in the expansion container 2 is discharged, and a portion of the pipeline is shared, thereby saving raw materials for producing the directional microecological expansion device and reducing the floor space of the directional microecological expansion device. The residue on the inner wall of the expansion container 2 is cleaned, the material expanded in the expansion container 2 is discharged, and the residue cleaned in the expansion container 2 is discharged, and a portion of the pipeline is shared, further saving raw materials for producing the directional microecological expansion device and further reducing the floor space of the directional microecological expansion device.

[0063] FIG4 is a third structural schematic diagram of a directional microecological expansion culture device provided by a specific embodiment of the present invention.

[0064] As shown in FIG4 , the directional microecological expansion and cultivation device may further include a sewage outlet 15 and a fifth electronic valve 16 .

[0065] Specifically, a drain port 15 is connected to the bottom opening 2-1 via a pipe. The drain port 15 is provided on the housing 1 and is used to discharge cleaning residues. A fifth electronic valve 16 is provided between the bottom opening 2-1 and the drain port 15 and is used to control the discharge of cleaning residues through the drain port 15.

[0066] In an embodiment of the present invention, the fourth electronic valve 14 is closed, the fifth electronic valve 16 is opened, and the cleaned residue is discharged through the drain port 15. After discharging the material that has been expanded in the expansion container 2, the residue in the expansion container 2 is directly cleaned and discharged through the drain port 15 to prevent the residue in the expansion container 2 from affecting the next expansion, making it convenient for continued use next time. It is a one-button operation that does not require professional operating skills and is safe and reliable. The drain port 15 can also be provided with a manual switch, as shown in the figure. The residue can only be discharged through the drain port 15 by turning on the manual switch. Setting a manual switch can prevent users from operating it incorrectly.

[0067] FIG5 is a fourth structural schematic diagram of a directional microecological expansion culture device provided by a specific embodiment of the present invention.

[0068] As shown in FIG5 , the directional microecological expansion and cultivation device may further include an aeration unit 17 and an air filter 18 .

[0069] Specifically, an aeration unit 17 is disposed within the housing 1 and communicates with the culture container 2. The aeration unit 17 is configured to introduce air into the culture container 2 to facilitate aerobic culture of the microbial agent. An air filter 18 is disposed between the aeration unit 17 and the culture container 2 to filter impurities and moisture from the air.

[0070] In an embodiment of the present invention, the aeration unit 17 may include a blower, etc. The air filter 18 may include a filter membrane, etc. The aeration unit 17 directly introduces air into the expansion culture container 2. The air source is wide, and the working place of the directional microecological expansion culture device is not restricted. The air filter 18 filters out impurities and water vapor in the air to prevent impurities and water vapor in the air from affecting the expansion culture effect. The directional microecological expansion culture device is provided with the aeration unit 17, which can not only expand aerobic strains, but also expand anaerobic strains, and can also enrich microbial metabolites, further expanding the scope of use of the directional microecological expansion culture device.

[0071] FIG6 is a fifth structural schematic diagram of a directional microecological expansion culture device provided by a specific embodiment of the present invention.

[0072] As shown in FIG6 , the temperature control unit 5 may include a liquid jacket 5 - 1 , a heater 5 - 2 , a cooler 5 - 3 and a second driving unit 5 - 4 .

[0073] A liquid jacket 5-1 is disposed within the outer shell of the incubation vessel 2. The liquid jacket 5-1 is configured to heat or cool the first liquid within the incubation vessel 2 using a second liquid therein through heat transfer. The second liquid within the liquid jacket 5-1 is isolated from the first liquid within the incubation vessel 2. A heater 5-2 is configured to heat the second liquid within the liquid jacket 5-1. The liquid outlet of the heater 5-2 is connected to the liquid inlet of the liquid jacket 5-1, and the liquid inlet of the heater 5-2 is connected to the liquid outlet of the liquid jacket 5-1. A cooler 5-3 is connected in parallel to the heater 5-2 and is configured to cool the second liquid within the liquid jacket 5-1. A second drive unit 5-4 is connected in series with the heater 5-2 and is configured to drive the second liquid within the liquid jacket 5-1 to circulate.

[0074] In an embodiment of the present invention, the second liquid may include at least one of oil, water, and liquid gas. The heater 5-2 may include an electromagnetic heating system, etc. The cooler 5-3 may include a tubular cooler, a plate cooler, and an air-cooled cooler, etc. If the second liquid is oil, the heater 5-2 may heat the oil to more than 100 degrees to pasteurize the first liquid (culture medium) in the expansion culture container 2. Then, the cooler 5-3 cools the first liquid to below 40 degrees so that a microbial agent can be added for expansion culture.

[0075] In an embodiment of the present invention, the microbial agent adding unit 4 may include a microbial agent box 4 - 1 , a microbial agent delivery pipeline 4 - 2 and an electronic switch 4 - 3 .

[0076] Specifically, a microbial inoculum box 4-1 is disposed within the housing 1 and is used to store the microbial inoculum. A microbial inoculum delivery line 4-2 is disposed between the microbial inoculum box 4-1 and the expansion container 2 and is used to deliver the microbial inoculum stored in the microbial inoculum box 4-1 to the expansion container 2. The microbial inoculum delivery line 4-2 is made of a heat-insulating material. An electronic switch 4-3 is disposed between the microbial inoculum box 4-1 and the microbial inoculum delivery line 4-2 and is used to, under signal control, deliver a predetermined amount of the microbial inoculum through the microbial inoculum delivery line 4-2 to the expansion container 2.

[0077] In the embodiment of the present invention, the inoculum delivery line 4-2 is made of a thermally insulating material. When the inoculum container 2 is heated by the liquid jacket 5-1, the heat from the inoculum container 2 is not transferred to the inoculum cartridge 4-1. This ensures that the inoculum cartridge 4-1 remains at a low temperature and does not affect the activity of the microbial inoculum within the inoculum cartridge 4-1. The inoculum delivery line 4-2 can be made of wood or a thermally insulating polymer material, but the present invention is not limited thereto.

[0078] FIG7 is a schematic structural diagram of an electronic switch provided in a specific embodiment of the present invention.

[0079] As shown in FIG7 , the inoculant box 4 - 1 is conical, and the electronic switch 4 - 3 may include a rotating shaft 4 - 3 - 1 , a plurality of blades 4 - 3 - 2 and a third driving unit 4 - 3 - 3 .

[0080] Specifically, a rotating shaft 4-3-1 is disposed at the bottom of the inoculum box 4-1. Multiple blades 4-3-2 are evenly arranged around the rotating shaft 4-3-1. The rotating shaft 4-3-1 and the blades 4-3-2 prevent the microbial inoculum in the inoculum box 4-1 from entering the inoculum delivery line 4-2. A third drive unit 4-3-3 is connected to the rotating shaft 4-3-1 and is configured to rotate the rotating shaft 4-3-1, thereby rotating the blades 4-3-2 to draw the microbial inoculum in the inoculum box 4-1 into the inoculum delivery line 4-2.

[0081] In embodiments of the present invention, the rotation angle of shaft 4-3-1 is proportional to the amount of probiotic agent introduced into microbial agent delivery line 4-2. Therefore, the amount of probiotic agent introduced into microbial agent delivery line 4-2 can be controlled by controlling the rotation angle of shaft 4-3-1. The present invention allows precise control of the amount of microbiotic agent added by controlling the rotation angle of shaft 4-3-1.

[0082] FIG8 is a sixth structural schematic diagram of a directional microecological expansion culture device provided by a specific embodiment of the present invention.

[0083] As shown in FIG8 , the directional microecological expansion and cultivation device may further include a liquid level monitor 19 , a controller 20 and a temperature monitor 21 .

[0084] Specifically, a liquid level monitor 19 is disposed within the incubation container 2 and is configured to monitor the liquid level of the first liquid within the incubation container 2. A controller 20 is electrically connected to the liquid level monitor 19 and the third drive unit 4-3-3 and is configured to control the rotation angle of the rotating shaft 4-3-1 based on the liquid level of the first liquid. A temperature monitor 21 is disposed within the incubation container 2 and is electrically connected to the controller 20, the heater 5-2, the cooler 5-3, and the second drive unit 5-4, respectively. The temperature monitor 21 is configured to monitor the temperature of the first liquid within the incubation container 2. The controller 20 controls the operation of the heater 5-2, the cooler 5-3, and the second drive unit 5-4 based on the temperature of the first liquid within the incubation container 2.

[0085] In an embodiment of the present invention, the controller 20 controls the rotation angle of the rotating shaft 4-3-1 according to the liquid level of the first liquid, thereby controlling the amount of the microecological microbial agent inserted into the microbial agent transmission pipeline 4-2, and accurately controlling the amount of microecological microbial agent added. When the first liquid in the expansion container 2 needs to be pasteurized, the heater 5-2 and the second drive unit 5-4 work, and the heater 5-2 heats the second liquid in the liquid jacket 5-1, and the second liquid heats the first liquid in the expansion container 2. When the first liquid in the expansion container 2 needs to be cooled, the cooler 5-3 and the second drive unit 5-4 work, and the cooler 5-3 cools the second liquid in the liquid jacket 5-1, and the second liquid cools the first liquid in the expansion container 2.

[0086] In an embodiment of the present invention, the directional microecological expansion device may further include a display screen and a control key (or a touch screen). The user can set the expansion amount (half tank expansion or full tank expansion) through the control key, and can choose aerobic strain expansion or anaerobic strain expansion, or even choose a specific expansion strain. It can also be selected whether to store the material in the expansion container 2 after the expansion is completed or to discharge it through the discharge port 7. After the material is discharged through the discharge port 7, it can be set to automatically clean the expansion container 2, which is controlled by one button and does not require professional operating skills.

[0087] The present invention achieves automated and standardized control of microbial inoculum expansion, with simple equipment and low production costs. No specialized operating skills are required; simply by activating the equipment's automated control button, fresh, high-yield, low-cost microbial inoculum can be obtained within a specified timeframe.

[0088] The most widely used microecological strains at present include Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Lactobacillus plantarum, Lactobacillus casei, Rhizobium, etc., which can basically be divided into aerobic strains and anaerobic strains. Aerobic strains include Bacillus phlei, Bacillus licheniformis and Bacillus amyloliquefaciens, etc. Anaerobic strains include Lactobacillus plantarum, Lactobacillus casei and Rhizobium, etc. Below, in combination with aerobic strains and anaerobic strains, an aerobic microecological strain expansion culture embodiment, an anaerobic microecological strain expansion culture embodiment, and a polyglutamic acid, rhamnolipid microecological strain expansion culture enrichment embodiment are respectively provided.

[0089] FIG9 is a first schematic flow chart of a method for expanding culture of a directional microecological expansion culture device provided by a specific embodiment of the present invention.

[0090] As shown in FIG9 , the expansion method of the directional microecological expansion device may include the following operations S101 to S104:

[0091] In operation S101 , an electronic valve is controlled to inject a predetermined amount of first liquid into the expansion container.

[0092] In the embodiment of the present invention, the first liquid can be a culture medium, which can be formed by adding the culture medium into clean water or tap water. The liquid level of the first liquid in the expansion culture container 2 can be monitored by a liquid level monitor 19 .

[0093] Then, in operation S102 : the injected first liquid is sterilized by adopting a non-complete sterilization method.

[0094] In an embodiment of the present invention, after the first liquid is sterilized by a non-complete sterilization method, theoretically, target bacteria and miscellaneous bacteria exist in the first liquid at the same time. The target bacteria can only begin to proliferate rapidly after a certain delay period. Sterilizing the first liquid by a non-complete sterilization method can effectively suppress the rapid proliferation of miscellaneous bacteria and effectively reduce the base number of miscellaneous bacteria in the culture medium, thereby providing favorable conditions for the rapid proliferation of target bacteria.

[0095] Next, in operation S103 : adding a microbial agent into the expansion container according to the liquid level of the first liquid after incomplete sterilization.

[0096] In an embodiment of the present invention, the user can arbitrarily set the amount of the first liquid injected into the expansion container 2 as needed, and the amount of the first liquid injected into the expansion container 2 is reflected by the liquid level of the first liquid. The larger the amount of the first liquid, the more microbial agent is added, and the user can set the expansion amount as needed.

[0097] Then, in operation S104 : according to the type of the microbial agent, the first liquid to which the microbial agent is added is cultured using set conditions.

[0098] In an embodiment of the present invention, the microbial agent may include anaerobic agents and aerobic agents. Aerobic agents may include Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens. Anaerobic agents may include Lactobacillus plantarum, Lactobacillus casei, and Rhizobium. The set conditions may include the culture temperature, stirring speed, ventilation volume, and culture time.

[0099] In embodiments of the present invention, one-touch automation and standardized control of microecological agent expansion and cultivation are realized, the equipment is simple, does not need to be transported, is ready for use, microecological survival rate is high, production cost, and professional operating skills are not required. Only the automation control button of the equipment needs to be started, and fresh, high bacterial count, low-cost microecological bacterial liquid can be obtained within a specific time. The whole expansion and cultivation process can be completed in less than 24 hours, and the expansion and cultivation efficiency is high.

[0100] FIG10 is a second schematic flow chart of a method for expanding culture of a directional microecological expansion culture device provided by a specific embodiment of the present invention.

[0101] As shown in FIG10 , after the first liquid to which the microecological agent is added is expanded using set conditions according to the type of the microecological agent in operation S104, the expansion method of the directional microecological expansion device may further include the following operation S105:

[0102] In operation S105 , the liquid pre-stored in the directional microecological expansion device is used to clean the residue on the inner wall of the expansion container.

[0103] In an embodiment of the present invention, the liquid used for cleaning the residue can be stored in a liquid storage tank 8 built into the directional microecological agent expansion device. The liquid used for cleaning the residue can include water or a special liquid. The liquid used for cleaning the residue is stored in the liquid storage tank 8, and a large amount of liquid can be used for flushing and thorough cleaning.

[0104] FIG11 is a third schematic flow chart of a method for expanding culture of a directional microecological expansion culture device provided in a specific embodiment of the present invention.

[0105] As shown in FIG11 , before adding the microecological agent into the expansion container according to the liquid level of the first liquid after incomplete sterilization in operation S103, the expansion method of the directional microecological expansion device may further include the following operation S106:

[0106] In operation S106 : the pH value of the first liquid is adjusted according to the type of the microbial agent to be added.

[0107] In the embodiments of the present invention, the initial pH of the first liquid directly affects the pH of the first liquid after partial sterilization, which in turn affects the growth efficiency. Experiments have shown that for aerobic inoculants, the initial pH of the first liquid can be adjusted to 7.6-7.8 to achieve maximum growth efficiency. For anaerobic inoculants, the initial pH of the first liquid can be adjusted to 6.8-7.0.

[0108] FIG12 is a fourth schematic flow chart of a method for expanding culture of a directional microecological expansion culture device provided by a specific embodiment of the present invention.

[0109] As shown in FIG12 , after the first liquid to which the microecological agent is added is expanded using set conditions according to the type of the microecological agent in operation S104, the expansion method of the directional microecological expansion device may further include the following operation S107:

[0110] In operation S107 , the end point of the culturing is determined according to a curve showing the OD value of the first liquid changing with time.

[0111] In the embodiments of the present invention, the OD value can reflect the content of the target bacteria in the first liquid. The larger the OD value, the higher the content of the target bacteria in the first liquid. As the expansion time increases, the OD value continues to increase. After a certain period of time (for example, 12 to 18 hours), the OD value reaches a peak value, and then the OD value begins to decrease. The peak value of the OD value is the end point of the expansion.

[0112] FIG13 is a schematic flow chart of a method for sterilizing the injected first liquid using a non-complete sterilization method according to a specific embodiment of the present invention.

[0113] As shown in FIG13 , operation S102 of sterilizing the injected first liquid using a non-complete sterilization method may include the following operations S1021 to S1024:

[0114] The heater is controlled to heat a second liquid in a liquid jacket, wherein the liquid jacket is disposed in an outer shell of the expansion container, and the second liquid is isolated from the first liquid.

[0115] In embodiments of the present invention, the second liquid may include at least one of oil, water, and liquid gas. If the second liquid is oil, the boiling point of the oil may exceed 100°C, which can heat the first liquid to 100°C, thereby eliminating more bacteria in the first liquid and facilitating the efficient growth of the microbial agent in the first liquid.

[0116] The first liquid in the expansion container is heated by utilizing the second liquid.

[0117] In an embodiment of the present invention, the second liquid is isolated from the first liquid, and the second liquid heats the first liquid in the expansion container by heat transfer.

[0118] The second liquid in the liquid jacket is cooled by a cooler.

[0119] In the embodiment of the present invention, the cooler 5 - 3 may not be started, and the second liquid in the liquid jacket may be allowed to automatically cool to room temperature, but the cooling process is relatively long.

[0120] The first liquid in the expansion container is cooled by utilizing the second liquid.

[0121] In an embodiment of the present invention, the second liquid and the first liquid are isolated from each other, and the first liquid transfers heat to the second liquid by heat transfer.

[0122] In the embodiment of the present invention, the heater 5 - 2 and the cooler 5 - 3 cooperate to sterilize the second liquid and promote efficient proliferation of the microbial agent in the first liquid, thereby achieving efficient utilization of the temperature control unit 5 .

[0123] FIG14 is a schematic flow chart of a method for aerobic expansion of a first liquid to which a microbial agent is added using set conditions according to a specific embodiment of the present invention.

[0124] As shown in FIG14 , if the probiotic agent is an aerobic agent, operation S104 may include the following operations S1041 to S1044 according to the type of the probiotic agent and the set conditions for expanding the first liquid to which the probiotic agent is added:

[0125] In operation S1041 : the temperature of the first liquid to which the microbial agent is added is controlled by a temperature control unit.

[0126] In the embodiments of the present invention, experiments have shown that the temperature control unit can be used to maintain the first liquid at a constant temperature (for example, 30 degrees to 40 degrees) to achieve the stability of aerobic bacterial culture. The culture is not easily affected by the environment and can be carried out in fields, woods, waterside and other places, with a wide range of applications.

[0127] In operation S1042 , impurities and moisture in the air are filtered out using an air filter.

[0128] In an embodiment of the present invention, the air filter may be a filter membrane that filters out impurities and moisture in the air, thereby preventing bacteria in the air from entering the first liquid and allowing the target bacteria to be stably cultured.

[0129] In operation S1043 , air is supplied to the first liquid to which the microbial agent is added by using an aeration unit at a predetermined ventilation volume.

[0130] In the embodiments of the present invention, experiments have shown that a ventilation volume of 2-3 vvm is sufficient for the expansion of aerobic bacteria.

[0131] In operation S1044 , the first liquid to which the microbial agent is added is stirred by a stirrer at a predetermined rotation speed.

[0132] In the embodiment of the present invention, experiments have shown that stirring the first liquid to which the microbial agent is added at a rotation speed of 200-300 rpm can meet the requirements for the expansion and cultivation of aerobic bacteria.

[0133] In an embodiment of the present invention, operation S1043, using an aeration unit to supply air to the first liquid to which the probiotic agent is added at a predetermined ventilation volume, may include the following operations: using the aeration unit to supply air to the first liquid to which the probiotic agent is added at a ventilation volume of 2-3 vvm. Operation S1044, using an agitator to stir the first liquid to which the probiotic agent is added at a predetermined rotational speed, may include the following operations: using an agitator to stir the first liquid to which the probiotic agent is added at a rotational speed of 200-300 rpm.

[0134] FIG15 is a schematic flow chart of an embodiment of the present invention providing a method for anaerobic culture of a first liquid to which a microbial agent is added using set conditions.

[0135] As shown in FIG15 , if the probiotic agent is an anaerobic agent, operation S104 may include the following operations S1041 ′ to S1042 ′ based on the type of the probiotic agent and the set conditions for incubating the first liquid to which the probiotic agent is added:

[0136] In operation S1041 ′: the temperature of the first liquid to which the microbial agent is added is controlled by a temperature control unit.

[0137] In the embodiments of the present invention, experiments have shown that a temperature control unit can be used to maintain the first liquid at a constant temperature (for example, 33 degrees to 37 degrees) to achieve stability in the expansion of anaerobic bacteria. The expansion is not easily affected by the environment and can be carried out in fields, woods, waterside and other places, with a wide range of applications.

[0138] In operation S1042 ′: the first liquid to which the microbial agent is added is periodically stirred by a stirrer.

[0139] In the embodiment of the present invention, experiments have shown that stirring the first liquid for one minute every three hours can achieve efficient expansion of the microecological agent in the first liquid.

[0140] In an embodiment of the present invention, operation S1042′ of periodically stirring the first liquid to which the probiotic agent is added using a stirrer may include the following operation: stirring the first liquid for one minute every three hours using the stirrer.

[0141] In an embodiment of the present invention, users can choose to expand aerobic or anaerobic bacterial agents according to their needs, and can also enrich microbial metabolites. According to the type of microecological bacterial agent, expansion parameters such as expansion temperature, stirring speed and ventilation volume are automatically set. It can be completed with one click, and the operation is simple and clear. No professional guidance is required. Users can operate it by themselves, and it can be used as soon as it is produced, without the need for transportation, with a high microbial survival rate and low production cost. It is suitable for wide application in agriculture, forestry, animal husbandry and fishery and other fields.

[0142] Example of aerobic microecological strain expansion culture

[0143] 1. Determine the optimal pH value of the culture medium

[0144] Open the manual switch and the fourth electronic valve 14 of the liquid injection port 3 of the directional microecological expansion and cultivation device, inject clean water or tap water into the expansion and cultivation container 2, or directly inject the first liquid into the expansion and cultivation container 2. If clean water or tap water is injected into the expansion and cultivation container 2, culture medium can be added through the top opening of the expansion and cultivation container 2 to form the first liquid (culture medium). Test the first liquid pH value, and the aerobic microecological pH value is alkaline. The initial pH value of the first liquid directly affects the pH value of the culture medium after pasteurization, and the pH value has a direct impact on the expansion and cultivation of the bacterial strain. Based on existing more mature expansion and cultivation culture medium, using bacillus subtilis as the representative strain of aerobic expansion and cultivation, the most suitable initial pH value screening is obtained by experiment as shown in Table 1 below, and Table 1 is the impact of the initial pH value of aerobic microecological bacterial strain expansion on the amount of expansion and cultivation:

[0145] Table 1

[0146] Conclusion: Taking Bacillus subtilis as an example, the most suitable initial pH value for aerobic strain expansion is 7.6-7.8.

[0147] 2. Determine the optimal sterilization temperature

[0148] By heater 5-2, heat the second liquid, the second liquid heats the first liquid (culture medium) in the expansion culture container 2, pasteurizes the culture medium (heating power 5kw~7kw), and under stirring, the culture medium is progressively warmed to 90 ℃~100 ℃ from room temperature, and the heating time is about 2h~3h; Then cool the second liquid by cooler 5-3, the second liquid cools the first liquid in the expansion culture container 2, and progressively lowers the temperature to 30 ℃~40 ℃, and the cooling time is about 1.5h~3h (specific cooling time is relevant with room temperature). Microecological microbial agent (i.e. Bacillus subtilis species) is added to the expansion culture container 2 after cooling by microbial agent adding unit 4.

[0149] Pasteurization is a non-complete sterilization system. Theoretically, both target bacteria and contaminants can coexist in the culture medium. However, after the target bacteria enter the expansion culture container 2, it takes a certain lag period before they begin to rapidly proliferate. Therefore, it is necessary to effectively suppress the rapid growth of contaminants in the early stages of the culture. Pasteurization can effectively reduce the base number of contaminants in the culture medium, is simple to operate, and is economical, thus providing favorable conditions for the rapid growth of the target bacteria.

[0150] Referring to the tracking results of the changes in the amount of aerobic culture bacteria and OD value with the culture time (see Table 7), after the target bacteria were inoculated, the OD value of the culture solution began to increase rapidly after 4 hours, and the strain proliferation entered the logarithmic phase at 8 hours. Therefore, in order to ensure that the target bacteria are the dominant bacteria before the end of the culture, it is necessary to control the rapid proliferation of miscellaneous bacteria within 8 hours of blank culture.

[0151] To simulate pasteurization conditions, the same culture medium (LB medium) was treated at different temperatures for a certain period of time, and then cultured at 37°C with sterile air (no microbial agents were added). The changes in the OD values ​​(absorbance values) of the various treated bacteria were tracked and detected. The results are shown in Table 2 below. Table 2 shows the effects of pasteurization conditions on the growth of various bacteria during the expansion of aerobic microbial strains.

[0152] Table 2

[0153] Conclusion: After pasteurization, the OD values ​​of the 90°C treatments varied significantly less than those of the other temperature treatments within 8 hours of incubation. This suggests that heating the culture medium to 90°C to 100°C minimized the number of contaminants, effectively suppressing their rapid proliferation. Further verification confirmed the pasteurization process as follows: heating the culture medium to 90°C to 100°C and then directly cooling it.

[0154] 3. Screening of culture temperature

[0155] In this embodiment, the inoculation volume is 100 million / mL to 500 million / mL (the target bacterial amount is not less than 1 billion / mL to 5 billion / mL to achieve a hundredfold amplification), the bacterial strain is inoculated into the expansion container 2, the expansion temperature is controlled at 30 ° C to 40 ° C, the stirring speed is 200 rpm to 300 rpm, and the ventilation volume is 2 vvm to 3 vvm. The expansion culture is carried out under the conditions of approximately 14h to 18h.

[0156] Different strains have different optimal propagation temperatures. Using Bacillus subtilis as a representative strain for aerobic propagation, the changes in the OD value of the propagation solution at different temperatures over time were investigated. The results are shown in Table 3 below. Table 3 shows the changes in the OD value of the propagation solution of aerobic microecological strains as a function of culture temperature:

[0157] Table 3

[0158] Conclusion: Bacillus subtilis can grow normally under the conditions of 30℃~40℃.

[0159] 4. Screening of expansion temperature

[0160] The culture medium (first liquid) is pasteurized and cooled by air cooling, which takes a long time and is basically unable to avoid the introduction of external bacteria. The longer the cooling time, the higher the probability of bacterial contamination.

[0161] Referring to Table 3 above, Bacillus subtilis can metabolize normally at 30°C to 40°C. Therefore, it is advisable to consider inoculating above the optimal growth temperature range to shorten the cooling time and reduce the chance of bacterial contamination. The experimental data for exploring the optimal inoculation temperature are shown in Table 4 below. Table 4 shows the effect of the initial inoculation temperature on the growth of aerobic microecological strains.

[0162] Table 4

[0163] Conclusion: When the inoculation temperature of the culture medium (first liquid) exceeds 42°C, the lag phase of Bacillus subtilis is significantly prolonged, resulting in an inability to increase the culture yield or a prolonged culture cycle. Therefore, considering all factors, Bacillus subtilis inoculation can be performed at a culture medium temperature not exceeding 42°C.

[0164] 5. Selection of aeration device

[0165] The smaller the bubbles in the expansion vessel, the more complete the oxygen supply interface, the greater the oxygen exchange rate, and the more vigorous the microbial growth. In this equipment, due to equipment investment and space limitations, stirring cannot further reduce the bubble size. Therefore, the size of the bubbles entering the equipment directly affects the oxygen supply in the expansion vessel.

[0166] The results of the evaluation using commercially available aeration equipment are shown in Table 5 below. Table 5 shows the effect of the aeration unit aperture on the proliferation of aerobic microecological strains:

[0167] Table 5

[0168] Conclusion: The smaller the aperture of the aeration device, the better the aeration effect. In order to facilitate procurement and replacement, microporous aeration rods with an aperture of 5μm to 10μm are directly selected.

[0169] 6. Ventilation volume screening

[0170] Conventional aerobic expansion culture generally adopts a combination of high-speed stirring and ventilation to provide sufficient oxygen to the strains. Among them, the ventilation volume is generally 0.8vvm to 1.2vvm to meet the needs of aerobic expansion culture strains.

[0171] Compared with conventional fermentation systems, the present invention significantly reduces the cost of equipment. Conventional magnetic stirring combined with bottom ventilation is used for air supply. To ensure the supply of oxygen, the stirring speed and ventilation volume are screened. The specific data are shown in Table 6 below. Table 6 Effect of ventilation volume on the amount of cultured bacteria in aerobic microecological strain expansion:

[0172] Table 6

[0173] Conclusion: The rotation speed is selected as 200rpm~300rpm and the ventilation of 2vvm~3vvm is sufficient for the expansion of aerobic bacteria (Bacillus subtilis).

[0174] 7. Expansion training completed

[0175] After 14-16 hours of incubation, the pH of the incubation solution is approximately 6.0 and the viable bacterial content is not less than 5 billion / mL. At this time, the incubation can be stopped. The determination of the incubation endpoint can be determined by measuring the viable bacterial growth curve. The specific data is shown in Table 7 below. Table 7 shows the bacterial count of aerobic microecological strains at different incubation times:

[0176] Table 7

[0177] Conclusion: The endpoint of aerobic expansion culture can be selected between 14h and 16h of culture.

[0178] 8. Discharging and cleaning

[0179] After the expansion is complete, the expansion liquid can be pumped directly into the inoculum application system or into the temporary storage expansion container 2. After discharge, the first and second electronic valves 11, 12 are opened, and the first drive unit 10 is activated. The liquid in the liquid storage tank 8 can be used to clean the residue on the inner wall of the expansion container 2, preparing to enter the next expansion cycle.

[0180] Example of anaerobic microecological strain expansion

[0181] The directional microecological expansion device provided in the embodiment of the present invention can implement both aerobic microecological strain expansion and anaerobic microecological expansion. Compared with aerobic microecological strain expansion, anaerobic microecological expansion is only carried out without ventilation during the cultivation process and using intermittent stirring.

[0182] 1. Determine the optimal pH value of the culture medium

[0183] First, add a suitable culture medium to the expansion container 2 and determine that the initial pH value of the first liquid is approximately 6.8 to 7.0. The initial pH value of the culture medium directly affects the pH value of the culture medium after pasteurization, thereby having a direct impact on the expansion of the strain. Based on the existing more mature expansion culture medium, using Lactobacillus plantarum as a representative strain for anaerobic expansion, the most suitable initial pH value is screened as shown in Table 8 below. Table 8 shows the effect of the initial pH value of anaerobic strain expansion on the amount of expanded bacteria:

[0184] Table 8

[0185] Conclusion: The most suitable initial pH value for anaerobic culture (Lactobacillus plantarum) is 6.8-7.0.

[0186] 2. Determine the optimal sterilization temperature

[0187] The second liquid is heated by the heater 5-2, and the second liquid heats the first liquid (culture medium) in the expansion container 2 to pasteurize the culture medium (heating power 5kw~7kw). Under stirring conditions, the temperature is gradually raised from room temperature to 90℃~100℃, and the heating time is about 2h~3h; then the temperature is gradually lowered to below 30℃~40℃ by air cooling, and the cooling time is about 1.5h~3h (related to the room temperature).

[0188] The screening of pasteurization conditions is the same as that of aerobic expansion.

[0189] 3. Screening of culture temperature

[0190] The bacteria were introduced into the expansion culture system at an inoculum size of 100 million / mL to 500 million / mL. The expansion culture temperature was controlled at 30°C to 40°C. The expansion culture was carried out under intermittent stirring and non-ventilated conditions. The culture cycle was about 14h to 18h.

[0191] Different strains have different optimal culture temperatures. Lactobacillus plantarum was used as a representative strain for anaerobic culture. The changes in the OD values ​​of the culture solutions at different temperatures were investigated. The results are shown in Table 9 below. Table 9 shows the changes in the OD values ​​of the culture solutions of anaerobic strains as a function of culture temperature.

[0192] Table 9

[0193] Conclusion: Lactobacillus plantarum can grow normally under the conditions of 30℃~40℃.

[0194] 4. Screening of stirring methods

[0195] Proper stirring helps to mix nutrients during anaerobic culture, but excessive stirring may introduce too much oxygen, thereby inhibiting the proliferation of anaerobic bacteria. Therefore, it is necessary to screen suitable stirring culture conditions to obtain a higher bacterial yield in anaerobic culture.

[0196] The treatments of no stirring, continuous stirring, and stirring for 1 minute every 3 hours (stirring for 1 minute every 3 hours) were set respectively. The results are shown in Table 10 below. Table 10 shows the effect of stirring mode on the proliferation of anaerobic strain expansion:

[0197] Table 10

[0198] Conclusion: The method of stirring for 1 minute every 3 hours is more conducive to the expansion of anaerobic bacteria.

[0199] 5. Expansion training completed

[0200] At the end of the expansion culture, the pH value of the expansion medium is about 4.0, and the viable bacterial content is not less than 5 billion / mL. At this time, the expansion culture can be terminated.

[0201] The determination of the end point of the culture can be determined by measuring the viable bacterial growth curve. The specific data are shown in Table 11 below. Table 11 shows the effect of the culture time of anaerobic strains on the amount of cultured bacteria:

[0202] Table 11

[0203] Conclusion: Anaerobic expansion is completed after 12h to 18h.

[0204] 6. Discharging and cleaning

[0205] After the expansion is complete, the expansion liquid can be pumped directly into the inoculum application system or into the temporary storage expansion container 2. After discharge, the first and second electronic valves 11, 12 are opened, and the first drive unit 10 is activated. The liquid in the liquid storage tank 8 can be used to clean the residue on the inner wall of the expansion container 2, preparing to enter the next expansion cycle.

[0206] Example of expansion and enrichment of polyglutamic acid and rhamnolipid microecological strains

[0207] The directional microecological expansion culture device provided in the embodiment of the present invention can not only implement aerobic and anaerobic microecological expansion culture, but also support the function of enriching microbial metabolites.

[0208] 1. Selection of strains and culture medium

[0209] A high-yield polyglutamic acid strain, Bacillus subtilis, was isolated from soybean meal residue, cultured on an expanded scale, and then spray-dried to obtain 100 billion bacterial powder. The polyglutamic acid enrichment medium was selected as follows: 40 g / L glucose, 6 g / L yeast powder, 50 g / L sodium glutamate, 4 g / L ammonium chloride, 7 g / L dipotassium hydrogen phosphate, 2 g / L magnesium sulfate heptahydrate, and 0.5 g / L manganese sulfate monohydrate. The pH value of the polyglutamic acid enrichment medium was adjusted to 7.0.

[0210] The high-rhamnolipid-producing strain Pseudomonas aeruginosa was selected from Northwestern University. The rhamnolipid enrichment medium was selected to contain 6 g / L sodium nitrate, 0.2 g / L potassium chloride, 0.5 g / L magnesium sulfate, 5.13 g / L disodium hydrogen phosphate, 0.76 g / L sodium dihydrogen phosphate, 2 mL / L glycerol, 30 mL / L olive oil, and 0.2 mL / L trace elements. The pH of the rhamnolipid enrichment medium was adjusted to 7.0 using sodium hydroxide (NaOH). The trace element formula (1 L) was as follows: 39.9 mg manganese sulfate, 42.8 mg zinc sulfate, and 34.7 mg ammonium molybdate.

[0211] 2. Selection of culture conditions

[0212] The two strains involved in this embodiment are both aerobic bacteria, so their culture conditions are mainly based on the aerobic microecological fermentation embodiment.

[0213] For Bacillus subtilis, the culture conditions were modified as follows: After heating the culture medium to 90°C–100°C, the temperature was immediately lowered to maintain the culture temperature at 37°C, with agitation at 200–300 rpm and aeration at 2.5 v / m. The culture cycle lasted approximately 20 hours. The initial inoculum size was 50 million CFU / mL (CFU stands for Colony-Forming Units). After the culture was complete, a 500 mL sample was collected.

[0214] For Pseudomonas aeruginosa, the culture conditions were modified as follows: After heating the culture medium to 90-100°C, the temperature was immediately lowered to 30°C, with a stirring speed of 200-300 rpm and aeration of 2.5 v / m. The culture was continued for approximately 40 hours. The initial inoculum size was 20 million CFU / mL. After the incubation period, a 500 mL sample was retained.

[0215] 3. Discharging and cleaning

[0216] After the expansion is complete, the expansion liquid can be pumped directly into the inoculant application system or temporarily stored in the expansion container 2. After discharge, the first and second electronic valves 11, 12 are opened, and the first drive unit 10 is activated. The liquid in the liquid storage tank 8 can be used to clean the residue on the inner wall of the expansion container 2, preparing to begin the next expansion cycle.

[0217] 4. Preliminary purification and determination of polyglutamic acid in fermentation broth

[0218] Polyglutamic acid (γ-PGA) solution can react with sodium hydroxide solution of cetyltrimethylammonium bromide (CTAB) to form a suspension. The turbidity of this suspension can be reflected by measuring the absorbance. By preparing γ-PGA standard solutions of different concentrations and measuring the absorbance values ​​to draw standard curves at different wavelengths, a linear relationship between concentration and absorbance values ​​is established, and the γ-PGA content in the fermentation culture medium can be quickly and accurately determined.

[0219] 1. Initial Purification

[0220] The polyglutamic acid fermentation broth was centrifuged at 4800 r / min for 25 min to remove the bacteria, and the supernatant was taken. 3 times the volume of anhydrous ethanol was added and allowed to stand at 4°C overnight. The precipitate was taken by centrifugation at 8000 r / min for 15 min and the precipitate was dissolved in distilled water of the same volume as the fermentation broth to prepare the sample solution, which was diluted 4 times before determination.

[0221] 2. Preparation of CTAB solution

[0222] Prepare 2% sodium hydroxide (NaOH) solution and use it as solvent. Add CTAB and after CTAB is dissolved (the temperature can be increased appropriately to accelerate the dissolution), prepare a 25g / L CTAB test solution.

[0223] 3. CTAB turbidimetry

[0224] Take 2mL of standard solution or sample solution into a test tube, accurately add 2mL of CTAB test solution, start timing from the time of addition, shake thoroughly, try to avoid foaming of the reaction solution, let it stand for nearly 3 minutes, then pour the reaction solution into a cuvette, and measure the absorbance at a wavelength of 250nm after 3 minutes.

[0225] 4. Preparation of γ-PGA standard solution and standard curve

[0226] Accurately weigh 3 g, 2.5 g, 2 g, 1.5 g, 1 g, and 0.5 g of γ-polyglutamic acid standard respectively and place them in a clean beaker. Add appropriate amount of distilled water to dissolve, then dilute to 100 mL volumetric flask and shake well to obtain 30 g / L, 25 g / L, 20 g / L, 15 g / L, 10 g / L, and 5 g / L of γ-PGA standard respectively.

[0227] After calculation (see table below), the standard curve is y=0.0357x(R 2 =0.9931), the linear fit is good and can be used for quantitative analysis. The absorbance value of the fermentation sample diluted four times was 0.63, and the final calculated γ-PGA concentration of the sample was 70g / L, as shown in Table 12 below.

[0228] Table 12

[0229] 5. Determination of rhamnolipids in fermentation broth

[0230] The surfactant glycolipid molecular structure contains pentose, which is dehydrated to form aldehyde compounds under the action of hot concentrated sulfuric acid. The aldehyde compounds react with phenolic compounds (3,5-dihydroxytoluene, orcinol) and condense to form colored substances with a maximum absorption peak at a visible wavelength (620nm). The corresponding glycolipid concentration is determined on the standard curve to perform quantitative analysis of the biosurfactant.

[0231] 1. Preparation of standard solution

[0232] Weigh 8.6 mg of the dry constant weight rhamnose reference substance, dissolve it in water, and dilute it to volume in a 100 mL volumetric flask to prepare a 0.086 mg / mL standard solution.

[0233] 2. Preparation of Anthrone Reagent

[0234] Take 0.20 g of anthrone and dissolve it in 100 mL of 85% sulfuric acid.

[0235] 3. Drawing of the standard curve

[0236] Accurately pipette 1mL, 2mL, 3mL, 4mL, 5mL, 6mL, 7mL, 8mL, and 9mL of standard solution into a 10mL volumetric flask, add distilled water to make up to volume and shake well, then pipette 1mL of rhamnose solution of each concentration respectively, add 4mL of anthrone test solution, add 4mL of 0.2% sulfuric acid-anthrone reagent in an ice water bath, mix well after adding, and immerse in a boiling water bath. Boil again in the water bath for 15 minutes, take out, cool with tap water, and place at room temperature for 20 minutes. Replace the standard solution with distilled water as a control, and measure the absorbance at a wavelength of 620nm.

[0237] With rhamnose concentration as the horizontal axis and absorbance as the vertical axis, regression processing was performed to obtain the regression equation shown in Table 13 below:

[0238] Table 13

[0239] 4. Determination of Rhamnolipids in Fermentation Broth

[0240] Accurately place 1 mL of the 100-fold diluted fermentation broth in a test tube. Add 4 mL of 0.2% sulfuric acid-anthrone reagent in an ice-water bath. Mix thoroughly and immerse in a boiling water bath. Remove after 15 minutes, cool with tap water, and let stand at room temperature for 20 minutes. Use uninoculated fermentation broth as a blank control and measure the absorbance at 620 nm. Record the absorbance value, A, as 0.416. Rhamnolipid yield (g / L) = (A - 0.2661) / 3.8157 × 3 × n (n is the dilution factor). Therefore, the final rhamnolipid content in the fermentation broth is calculated to be 11.8 g / L.

[0241] According to an embodiment of the present invention, the method flow according to an embodiment of the present invention can be implemented as a computer software program. For example, an embodiment of the present invention includes a computer program product, which includes a computer program carried on a computer-readable storage medium, and the computer program includes program code for executing the method shown in the flowchart. According to an embodiment of the present invention, the electronic devices, devices, apparatuses, modules, units, etc. described above can be implemented by computer program modules.

[0242] The present invention also provides a computer-readable storage medium, which may be included in the device / apparatus / system described in the above embodiments, or may exist independently and not incorporated into the device / apparatus / system. The computer-readable storage medium carries one or more programs, which, when executed, implement the method according to the embodiments of the present invention.

[0243] According to an embodiment of the present invention, a computer-readable storage medium may be a non-volatile computer-readable storage medium, such as, but not limited to, a portable computer disk, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or flash memory), a portable compact disk read-only memory (CD-ROM), an optical storage device, a magnetic storage device, or any suitable combination thereof. In the present invention, a computer-readable storage medium may be any tangible medium containing or storing a program that can be used by or in conjunction with an instruction execution system, apparatus, or device.

[0244] The flowcharts and block diagrams in the accompanying drawings illustrate the possible implementation architecture, functions and operations of the systems, methods and computer program products according to various embodiments of the present invention. In this regard, each box in the flowchart or block diagram can represent a module, program segment, or a part of code, and the above-mentioned module, program segment, or a part of code contains one or more executable instructions for implementing the specified logical function. It should also be noted that in some alternative implementations, the functions marked in the box can also occur in an order different from that marked in the accompanying drawings. For example, two boxes represented in succession can actually be executed substantially in parallel, and they can sometimes be executed in the opposite order, depending on the functions involved. It should also be noted that each box in the block diagram or flowchart, and the combination of boxes in the block diagram or flowchart, can be implemented with a dedicated hardware-based system that performs the specified function or operation, or can be implemented with a combination of dedicated hardware and computer instructions.

[0245] Those skilled in the art will appreciate that the features described in the various embodiments of the present invention may be combined and / or coupled in various ways, even if such combinations and / or couplings are not explicitly described in the present invention. In particular, the features described in the various embodiments of the present invention may be combined and / or coupled in various ways without departing from the spirit and teachings of the present invention. All such combinations and / or couplings fall within the scope of the present invention.

[0246] The above describes embodiments of the present invention. However, these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Although each embodiment has been described separately above, this does not mean that the measures in each embodiment cannot be advantageously used in combination. Without departing from the scope of the present invention, those skilled in the art may make various substitutions and modifications, which should all fall within the scope of the present invention.

Claims

1. A directional microecological expansion culture device, characterized in that: The device includes: Housing (1); A culture expansion container (2), arranged in the housing (1), for providing a culture expansion site for the microecological bacterial agent; A liquid injection port (3) provided on the housing (1), connected to the expansion culture container (2), and used for injecting a first liquid into the expansion culture container (2); A temperature control unit (5) is arranged in the housing (1) and is used to heat the first liquid in the culture container (2) to perform non-complete sterilization on the first liquid; A bacterial agent adding unit (4) disposed on the housing (1), connected to the expansion culture container (2), and used for adding the microecological bacterial agent into the expansion culture container (2); a stirrer (6), arranged in the expansion and cultivation container (2), for stirring the first liquid in the expansion and cultivation container (2) to promote expansion and cultivation, wherein the temperature control unit (5) is used to control the temperature of the first liquid in the expansion and cultivation container (2); and A discharge port (7) provided on the shell (1) is communicated with the expansion container (2) and is used to discharge the expanded material in the expansion container (2).

2. The directional microecological expansion culture device according to claim 1, characterized in that: The expansion device also includes: A liquid storage tank (8) disposed in the housing (1) is connected to the liquid injection port (3) and the culture container (2) and is used to store a first liquid used for culture and / or a liquid for cleaning residues on the inner wall of the culture container (2).

3. The directional microecological expansion culture device according to claim 2, characterized in that: The expansion device also includes: A nozzle (9) disposed inside the expansion and cultivation container (2), connected to the liquid injection port (3) and the liquid storage tank (8), and used for cleaning the residue on the inner wall of the expansion and cultivation container (2); and The first driving unit (10) is arranged between the nozzle (9) and the liquid storage tank (8) and is used to drive the liquid in the liquid storage tank (8) to clean the residue on the inner wall of the expansion culture container (2).

4. The directional microecological expansion culture device according to claim 1, characterized in that: The expansion device also includes: An aeration unit (17) disposed in the housing (1) is communicated with the culture expansion container (2) and is used to introduce air into the culture expansion container (2) so as to allow the microecological bacterial agent to be aerobically cultured.

5. The directional microecological expansion culture device according to claim 4, characterized in that: The expansion device also includes: An air filter (18) is provided between the aeration unit (17) and the culture expansion container (2) for filtering Filter impurities and moisture in the air.

6. The directional microecological expansion culture device according to claim 1, characterized in that: The temperature control unit (5) comprises: a liquid jacket (5-1) disposed in the outer shell of the expansion culture container (2) and used for heating or cooling the first liquid in the expansion culture container (2) by utilizing the second liquid therein through heat transfer, wherein the second liquid in the liquid jacket (5-1) is isolated from the first liquid in the expansion culture container (2); a heater (5-2) for heating the second liquid in the liquid jacket (5-1) to perform incomplete sterilization on the first liquid, wherein the liquid outlet of the heater (5-2) is connected to the liquid inlet of the liquid jacket (5-1), and the liquid inlet of the heater (5-2) is connected to the liquid outlet of the liquid jacket (5-1); a cooler (5-3), connected in parallel with the heater (5-2), and used for cooling the second liquid in the liquid jacket (5-1); and The second driving unit (5-4) is connected in series with the heater (5-2) and is used to drive the second liquid in the liquid jacket (5-1) to circulate.

7. The directional microecological expansion and cultivation device according to claim 6, characterized in that: The microbial agent adding unit (4) comprises: A microbial agent box (4-1), arranged in the housing (1) and used for storing the microecological microbial agent; a microbial agent delivery pipeline (4-2), arranged between the microbial agent box (4-1) and the expansion culture container (2), and used for delivering the microecological microbial agent stored in the microbial agent box (4-1) to the expansion culture container (2), wherein the microbial agent delivery pipeline (4-2) is made of a heat-insulating material; and The electronic switch (4-3) is arranged between the microbial agent box (4-1) and the microbial agent delivery pipeline (4-2), and is used to deliver a predetermined amount of the microecological microbial agent to the expansion culture container (2) through the microbial agent delivery pipeline (4-2) under the control of a signal.

8. The directional microecological expansion culture device according to claim 1, characterized in that: The blade (6-1) of the stirrer (6) is arranged obliquely. When the blade (6-1) of the stirrer (6) rotates counterclockwise, the front side (b) of the blade (6-1) is tilted, and the rear side (a) of the blade (6-1) is drooped. The distance between the front side (b) and the bottom of the expansion and cultivation container (2) is greater than the distance between the rear side (a) and the bottom of the expansion and cultivation container (2).

9. The method for expanding the culture of the directional microecological expansion device according to any one of claims 1 to 8, characterized in that: The expansion method includes: Controlling the electronic valve to inject a predetermined amount of the first liquid into the expansion container; sterilizing the injected first liquid by a non-complete sterilization method; Adding a microecological agent into the expansion container according to the liquid level of the first liquid after incomplete sterilization; and According to the type of the microbial agent, the first liquid to which the microbial agent is added is expanded using set conditions.

10. The method for expanding the culture of the directional microecological expansion device according to claim 9, characterized in that: According to the type of the microecological agent, after the step of expanding the first liquid to which the microecological agent is added using set conditions, the expansion method further comprises: The liquid pre-stored in the directional microecological bacterial agent expansion device is used to clean the residue on the inner wall of the expansion container.

11. The method for expanding the culture of the directional microecological expansion device according to claim 9, characterized in that: Before the step of adding a microecological agent into the expansion culture container according to the liquid level of the first liquid after incomplete sterilization, the expansion culture method further comprises: The pH value of the first liquid is adjusted according to the type of microbial agent to be added.

12. The method for expanding the culture of the directional microecological expansion device according to claim 9, characterized in that: According to the type of the microecological agent, after the step of expanding the first liquid to which the microecological agent is added using set conditions, the expansion method further comprises: The end point of the culture is determined according to a curve showing the OD value of the first liquid changing with time.

13. The method for expanding the culture of the directional microecological expansion device according to claim 9, characterized in that: The step of sterilizing the injected first liquid in a non-complete sterilization manner comprises: Controlling the heater to heat the second liquid in the liquid jacket, wherein the liquid jacket is disposed in the shell of the expansion culture container, and the second liquid is isolated from the first liquid; Using the second liquid to heat the first liquid in the expansion container; cooling the second liquid in the liquid jacket using a cooler; and The first liquid in the expansion container is cooled by the second liquid.

14. The method for expanding the culture of the directional microecological expansion device according to claim 13, characterized in that: The second liquid includes at least one of oil, water and liquid gas.

15. The method for expanding the culture of the directional microecological expansion device according to claim 9, characterized in that: If the microbial agent is an aerobic agent, the step of expanding the first liquid to which the microbial agent is added under set conditions according to the type of the microbial agent includes: Using a temperature control unit to control the temperature of the first liquid to which the microecological agent is added; Using an aeration unit to supply air to the first liquid to which the microbial agent is added at a predetermined ventilation volume; and The first liquid to which the microecological agent is added is stirred by a stirrer at a predetermined rotation speed.

16. The method for expanding the culture of the directional microecological expansion device according to claim 15, characterized in that: Before the step of supplying air to the first liquid to which the microbial agent is added with a predetermined ventilation volume by using an aeration unit, the step of expanding the first liquid to which the microbial agent is added with set conditions according to the type of the microbial agent also includes: Use an air filter to filter out impurities and moisture in the air.

17. The method for expanding the culture of the directional microecological expansion device according to claim 15, characterized in that: The step of supplying air to the first liquid to which the microbial agent is added with an aeration unit at a predetermined ventilation volume comprises: The aeration unit is used to supply air to the first liquid to which the microbial agent is added at a ventilation rate of 2vvm to 3vvm. The step of stirring the first liquid to which the microecological agent is added by a stirrer at a predetermined speed comprises: The first liquid to which the microecological agent is added is stirred by a stirrer at a rotation speed of 200 rpm to 300 rpm.

18. The method for expanding the culture of the directional microecological expansion device according to claim 9, characterized in that: If the microbial agent is an anaerobic agent, the step of expanding the first liquid to which the microbial agent is added under set conditions according to the type of the microbial agent includes: Using a temperature control unit to control the temperature of the first liquid to which the microecological agent is added; and The first liquid to which the microecological agent is added is periodically stirred by a stirrer.

19. The method for expanding the culture of the directional microecological expansion device according to claim 18, characterized in that: The step of periodically stirring the first liquid to which the microecological agent is added by a stirrer comprises: The first liquid was stirred for one minute using a stirrer at intervals of three hours.

20. A computer-readable storage medium having executable instructions stored thereon, wherein the instructions are executed by a processor to implement the method according to any one of claims 9 to 19.

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