CN113801774B - Thallus culture equipment and method - Google Patents
Thallus culture equipment and method Download PDFInfo
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- CN113801774B CN113801774B CN202111114562.5A CN202111114562A CN113801774B CN 113801774 B CN113801774 B CN 113801774B CN 202111114562 A CN202111114562 A CN 202111114562A CN 113801774 B CN113801774 B CN 113801774B
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- 238000000034 method Methods 0.000 title claims description 13
- 238000000855 fermentation Methods 0.000 claims abstract description 95
- 230000004151 fermentation Effects 0.000 claims abstract description 95
- 239000000463 material Substances 0.000 claims abstract description 46
- 239000007788 liquid Substances 0.000 claims abstract description 42
- 241001052560 Thallis Species 0.000 claims abstract description 36
- 238000004062 sedimentation Methods 0.000 claims abstract description 29
- 238000001514 detection method Methods 0.000 claims abstract description 17
- 238000000926 separation method Methods 0.000 claims abstract description 15
- 238000007599 discharging Methods 0.000 claims abstract description 10
- 238000003756 stirring Methods 0.000 claims description 99
- 230000001580 bacterial effect Effects 0.000 claims description 12
- 238000012258 culturing Methods 0.000 claims description 9
- 239000001963 growth medium Substances 0.000 claims description 9
- 238000004113 cell culture Methods 0.000 claims description 8
- 230000005540 biological transmission Effects 0.000 claims description 3
- 230000001502 supplementing effect Effects 0.000 claims description 3
- 241000233866 Fungi Species 0.000 claims 2
- 230000000694 effects Effects 0.000 abstract description 13
- 238000012136 culture method Methods 0.000 abstract description 6
- 239000012530 fluid Substances 0.000 abstract 1
- 238000011897 real-time detection Methods 0.000 abstract 1
- 240000006024 Lactobacillus plantarum Species 0.000 description 7
- 235000013965 Lactobacillus plantarum Nutrition 0.000 description 7
- 230000003698 anagen phase Effects 0.000 description 7
- 229940072205 lactobacillus plantarum Drugs 0.000 description 7
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- 230000012010 growth Effects 0.000 description 5
- 238000011081 inoculation Methods 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 241000235342 Saccharomycetes Species 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 4
- 241000235033 Zygosaccharomyces rouxii Species 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 230000035764 nutrition Effects 0.000 description 3
- 230000002035 prolonged effect Effects 0.000 description 3
- 230000001276 controlling effect Effects 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
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- 241000186660 Lactobacillus Species 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
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- 238000010276 construction Methods 0.000 description 1
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- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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Abstract
The utility model belongs to the technical field of thallus culture, and particularly discloses thallus culture equipment and a thallus culture method, aiming at solving the problems that the existing thallus culture equipment is complex in structure and is unfavorable for timely separation of thalli. According to the thallus culture equipment, the first chamber, the second chamber and the third chamber which are sequentially connected from top to bottom are formed in the fermentation tank, the thallus obtained by fermentation in the first chamber can be enriched by utilizing the second chamber, and the thallus enriched in the second chamber can be fully accumulated and settled by utilizing the third chamber, so that the thallus can be concentrated without adding devices such as a cyclone sedimentation tank, a circulating pump and a circulating pipe, and the equipment structure is simplified; simultaneously, through setting up turbidity detection device in the third cavity, do benefit to the turbidity of real-time detection liquid in the third cavity to be connected centrifugal device's inlet and the leakage fluid dram of third cavity lower extreme through vertical pipeline, do benefit to in time discharging the material to centrifugal device after satisfying turbidity requirement, in order to obtain thallus activity mud with the quick centrifugal separation of material.
Description
Technical Field
The utility model belongs to the technical field of thallus culture, and particularly relates to thallus culture equipment and a thallus culture method.
Background
The thallus culture equipment is facility equipment for providing proper environment for thallus growth according to the principle of biological fermentation. At present, more methods of high-density culture are adopted, namely, culture conditions are optimized, nutrients and oxygen are supplemented, or inhibition factors are removed, so that the amount of thalli in single fermentation is increased, and higher substrate utilization efficiency or product yield is obtained.
The thallus is timely separated from the fermentation tank in the fermentation process, so that the excessive consumption of nutrients and the generation of wastes can be reduced, the growth time of the thallus is prolonged, and the efficient fermentation effect is obtained. For example: the Chinese patent with the publication number of CN213624107U discloses a yeast fermentation separation device, which comprises a fermentation tank and a cyclone sedimentation tank, wherein the fermentation tank is connected with the cyclone sedimentation tank through a circulating pipe, a centrifugal pump and a cyclone sedimentation feed valve are respectively arranged on the circulating pipe, an air inlet pipe and a fermentation return pipe are connected on the circulating pipe between the centrifugal pump and the cyclone sedimentation feed valve, the other end of the fermentation return pipe is connected with the upper part of the fermentation tank, a centrifugal clear liquid pipe and a cyclone sedimentation return pipe are connected on the fermentation return pipe between the fermentation return valve and the fermentation tank, the other end of the centrifugal clear liquid pipe is connected with a solid-liquid centrifuge, and the other end of the cyclone sedimentation return pipe is connected with the cyclone sedimentation tank; the bottom of the rotary sedimentation tank is connected with a solid-liquid centrifuge through a sedimentation material valve, and a concentrated yeast valve is arranged at the bottom of the solid-liquid centrifuge.
Although the yeast fermentation separation device can fully utilize the operation of the centrifugal pump to drive the fermentation liquor to be pre-separated in the cyclone sedimentation tank, so that the operation time of the centrifugal machine is greatly reduced, the operation cost is saved, the production cost of yeast is reduced, and the fermentation effect is improved; however, when the cyclone sedimentation tank, the centrifugal pump and the circulating pipe are added, and the thalli are separated, the fermentation liquor is pumped into the cyclone sedimentation tank, the sediment is discharged into the centrifugal machine after the fermentation liquor is precipitated in the cyclone sedimentation tank, and meanwhile, the supernatant flows back into the fermentation tank, so that the device has a complex structure, the thalli are cultured and separated, the on-line separation of the thalli cannot be realized, the thalli are not separated timely, and the activity of the thalli is not maintained.
Disclosure of Invention
The utility model provides a thallus culture device, which aims to solve the problems that the existing thallus culture device is complex in structure and is unfavorable for timely separation of thalli.
The technical scheme adopted for solving the technical problems is as follows: the thallus culture equipment comprises a fermentation tank, a stirring device, a centrifugal device and a turbidity detection device; the fermentation tank is provided with a first chamber, a second chamber and a third chamber which are sequentially connected from top to bottom, and the lower end of the third chamber is provided with a liquid outlet; the stirring device is arranged on the fermentation tank, and stirring paddles of the stirring device are positioned in the first chamber and the second chamber; the turbidity detection device is arranged in the third chamber, and a liquid inlet of the centrifugal device is connected with the liquid outlet through a vertical pipeline.
Further, the stirring device comprises a stirring shaft which is vertically arranged on the fermentation tank and extends into the first chamber and the second chamber, stirring paddles which are arranged on the stirring shaft, and a stirring motor which is arranged on the fermentation tank and is in transmission connection with the stirring shaft; the stirring shaft and the stirring blade jointly form a stirring paddle of the stirring device.
Further, a valve is arranged on the vertical pipeline.
Further, the centrifugal device is a disc centrifuge.
Further, the volume of the first chamber is 65-75% of the total volume of the fermentation tank, the volume of the second chamber is 20-30% of the total volume of the fermentation tank, and the volume of the third chamber is 2-5% of the total volume of the fermentation tank.
Further, the first chamber comprises a cylindrical main body and an inverted conical bottom arranged at the lower end of the cylindrical main body, the second chamber and the third chamber are of inverted conical structures, and the first chamber, the second chamber and the third chamber are coaxially arranged.
Further, the diameter of the joint of the first chamber and the second chamber is 40-50% of the diameter of the cylindrical main body of the first chamber, and the diameter of the joint of the second chamber and the third chamber is 30-40% of the diameter of the upper end of the third chamber.
Further, the taper of the third chamber is 0.7:1-1.2:1.
The utility model also provides a thallus culture method capable of improving the substrate utilization rate and the product yield, and the thallus culture device is adopted to produce thallus.
Further, the method comprises a fermentation step, a sedimentation step and a separation step;
fermentation: firstly, adding a sterilized culture medium into a fermentation tank through a first chamber, then adding a thallus seed liquid into the fermentation tank through the first chamber, and simultaneously starting a stirring device to perform stirring fermentation; sedimentation step: stopping the stirring device after the fermentation of the thallus seeds reaches the logarithmic growth phase; after 3-8 min, starting the stirring device to stir for 0.5-2 min at a rotating speed of 2-10 rpm, so that part of thalli is settled into the third chamber; then, stirring and fermenting are continuously carried out according to the stirring mode of the fermentation step;
separating: circularly operating according to a sedimentation step, and discharging materials to a centrifugal device through a vertical pipeline after the turbidity detection device detects that the turbidity of the liquid in the third chamber exceeds a set value, wherein the volume of the discharged materials is 80-150% of the volume of the third chamber; after the materials are discharged, adding culture solution into the first chamber, wherein the volume of the added culture solution is equal to the volume of the discharged materials; and (3) discharging the materials into a centrifugal device, and performing centrifugal separation on the materials in the centrifugal device at the rotating speed of 10000-20000 rpm to obtain the bacterial active mud.
The beneficial effects of the utility model are as follows: according to the thallus culture equipment, the first chamber, the second chamber and the third chamber which are sequentially connected from top to bottom are formed in the fermentation tank, the thallus obtained by fermentation in the first chamber can be enriched by utilizing the second chamber, and the thallus enriched in the second chamber can be fully accumulated and settled by utilizing the third chamber, so that the thallus can be concentrated without adding devices such as a cyclone sedimentation tank, a circulating pump and a circulating pipe, the equipment structure is simplified, and the convenience and the production efficiency of equipment control are improved; meanwhile, the stirring paddles of the stirring device are arranged in the first chamber and the second chamber, so that on one hand, the culture medium and the thallus seed liquid added into the first chamber can be stirred, and the thallus seeds can fully absorb nutrition to achieve a good fermentation culture effect, and on the other hand, the thallus enriched in the second chamber can be stirred to maintain the activity of the thallus; in addition, the turbidity detection device is arranged in the third chamber, so that the turbidity of liquid in the third chamber is detected in real time, the liquid inlet of the centrifugal device is connected with the liquid outlet at the lower end of the third chamber through the vertical pipeline, and the material is discharged into the centrifugal device in time after the turbidity requirement is met, so that the material is rapidly centrifugally separated to obtain the bacterial active mud. According to the thallus culture method, the thallus is produced by adopting the thallus culture equipment, and the same volume of culture solution is added to continue culture after materials are discharged, so that the thallus culture and separation can be performed simultaneously, the fermentation time is prolonged, the substrate utilization rate is further improved, the product yield is improved, the on-line separation of the thallus is realized, and the thallus can be separated in time and keeps good activity.
Drawings
FIG. 1 is a schematic diagram showing the construction of a cell culture apparatus according to the present utility model;
marked in the figure as: the device comprises a first chamber 1, a second chamber 2, a third chamber 3, a stirring device 4, a turbidity detection device 5, a liquid outlet 6, a vertical pipeline 7, a valve 8 and a centrifugal device 9.
Detailed Description
The utility model is further described below with reference to the drawings and examples.
As shown in FIG. 1, the thallus culture equipment comprises a fermentation tank, a stirring device 4, a centrifugal device 9 and a turbidity detection device 5; the fermentation tank is provided with a first chamber 1, a second chamber 2 and a third chamber 3 which are sequentially connected from top to bottom, and the lower end of the third chamber 3 is provided with a liquid outlet 6; the stirring device 4 is arranged on the fermentation tank, and stirring paddles of the stirring device are arranged in the first chamber 1 and the second chamber 2; the turbidity detection device 5 is arranged in the third chamber 3, and a liquid inlet of the centrifugal device 9 is connected with the liquid outlet 6 through a vertical pipeline 7.
According to the thallus culture equipment, the first chamber 1, the second chamber 2 and the third chamber 3 which are sequentially connected from top to bottom are arranged in the fermentation tank, the thallus can be fermented and cultured by utilizing the first chamber 1, the thallus can be enriched by utilizing the second chamber 2, the thallus can be fully accumulated and settled by utilizing the third chamber 3, and the volume of culture solution brought out when materials are discharged is reduced, so that the thallus can be concentrated without adding a cyclone sedimentation tank, a circulating pump, a circulating pipe and other devices, the equipment structure is simplified, and the convenience and the production efficiency of equipment control are improved; meanwhile, the stirring paddles of the stirring device 4 are arranged in the first chamber 1 and the second chamber 2, so that on one hand, the culture medium and the thallus seed liquid added into the first chamber 1 can be stirred, and the thallus seeds can fully absorb nutrition to achieve a good fermentation culture effect, and on the other hand, the thallus enriched in the second chamber 2 can be stirred to maintain the activity of the thallus; in addition, the turbidity detection device 5 is arranged in the third chamber 3, so that the turbidity of liquid in the third chamber 3 can be detected in real time, the liquid inlet of the centrifugal device 9 is connected with the liquid outlet 6 at the lower end of the third chamber 3 through the vertical pipeline 7, the material can be discharged into the centrifugal device 9 in time after the turbidity requirement is met, the material can be rapidly centrifuged to obtain the bacterial active mud, and the bacterial activity can be maintained.
Wherein the fermentation tank is a main component of the thallus culture equipment, the first chamber 1 is a main body part of the thallus culture of the fermentation tank, the first chamber 1 generally needs to ensure a large enough volume, and the volume of the first chamber 1 preferably accounts for 65-75% of the total volume of the fermentation tank; the second chamber 2 of the fermentation tank is mainly used as a sedimentation chamber of the thalli, the second chamber 2 needs to be smaller than the first chamber 1 to achieve the purpose of enriching the thalli, and meanwhile, the volume of the second chamber 2 needs to be capable of accommodating a stirring paddle extending into the second chamber so as to facilitate stirring to keep the activity of the thalli, and the volume of the second chamber 2 preferably accounts for 20-30% of the total volume of the fermentation tank; the third chamber 3 of the fermenter is mainly used for fully gathering and settling the thalli, the volume of the third chamber 3 should be small enough to reduce the volume of the culture solution carried out when the thalli are discharged, and the volume of the third chamber 3 is preferably 2-5% of the total volume of the fermenter; the liquid outlet 6 arranged at the lower end of the third chamber 3 is mainly used for the materials formed after the outer bacterial cells are fully gathered and settled, and a control switch for opening and closing the liquid outlet 6 is usually arranged at the liquid outlet 6.
In order to facilitate the fermentation culture, enrichment and sedimentation of the thalli, as shown in fig. 1, the first chamber 1 preferably comprises a cylindrical main body and an inverted conical bottom arranged at the lower end of the cylindrical main body, the second chamber 2 and the third chamber 3 are both of inverted conical structures, and the first chamber 1, the second chamber 2 and the third chamber 3 are coaxially arranged. The cylindrical main body of the first chamber 1 is favorable for stirring, and can provide a proper environment for the growth of thalli; the inverted conical bottom of the first chamber 1 facilitates the grown thalli to flow towards the second chamber 2; the second chamber 2 with the inverted conical structure is beneficial to enrichment of thalli and beneficial to flowing of the enriched thalli towards the third chamber 3; the third chamber 3 with the inverted conical structure is beneficial to the sedimentation of enriched thalli, and ensures that only a small amount of culture solution is carried out during discharging materials.
On the basis of the above, in order to further optimize the structure of the fermenter, the diameter of the connecting part of the first chamber 1 and the second chamber 2 is 40-50% of the diameter of the cylindrical main body of the first chamber 1, so as to ensure that the thalli in the second chamber 2 cannot easily return to the first chamber 1, and the air in the first chamber 1 is facilitated to enter the second chamber 2; the diameter of the joint of the second chamber 2 and the third chamber 3 is 30-40% of the diameter of the upper end of the third chamber 3, so that the third chamber 3 can fully enrich and settle thalli, and excessive culture solution is further prevented from being discharged when materials are discharged; the taper of the third chamber 3 is 0.7:1-1.2:1 so as to ensure that thalli fully settle at the bottom of the third chamber 3, the third chamber 3 has enough height, the funnel flow is lightened, the turbidity detection device 5 is convenient to install, and the cleaning is convenient.
The stirring device 4 is mainly used for stirring the culture medium and the thallus seed liquid added into the first chamber 1 so as to enable the thallus seeds to fully absorb nutrition and achieve good fermentation culture effect; the stirring device 4 can also stir the thalli enriched in the second chamber 2 so as to maintain the activity of the thalli; the stirring device 4 generally comprises a stirring shaft vertically arranged on the fermentation tank and extending into the first chamber 1 and the second chamber 2, stirring paddles arranged on the stirring shaft, and a stirring motor arranged on the fermentation tank and in transmission connection with the stirring shaft; the stirring shaft and the stirring blade jointly form a stirring paddle of the stirring device 4. The stirring motor is used for driving the stirring shaft to rotate, and is preferably a servo motor.
The turbidity detecting device 5 is mainly used for detecting the turbidity of the liquid in the third chamber 3 so as to enable a worker to judge the concentration of the thalli in the liquid; the turbidity detecting device 5 may be various, for example: turbidity detectors, turbidity detection sensors, etc.
The vertical pipeline 7 is a vertically arranged conveying pipeline and is mainly used for conveying materials discharged by the fermentation tank; for the purpose of transport control, a valve 8 is usually provided on the vertical pipe 7, and the valve 8 may be various, for example: manual valves, solenoid valves, etc.
The centrifugal device 9 is mainly used for separating thalli from materials, and is usually a solid-liquid centrifuge, preferably a disc centrifuge.
The utility model also provides a thallus culture method capable of improving the substrate utilization rate and the product yield, and the thallus culture device is adopted to produce thallus.
Specifically, the method comprises a fermentation step, a sedimentation step and a separation step;
fermentation: firstly, adding a sterilized culture medium into a fermentation tank through a first chamber 1, then adding a thallus seed liquid into the fermentation tank through the first chamber 1, and simultaneously starting a stirring device 4 for stirring fermentation; in this step, the stirring rotation speed of the stirring device 4 is generally such that the bacterial seeds can be suspended in the culture medium; the inoculation density of the bacterial seeds in the bacterial seed liquid is usually 1×10 5 ～5×10 5 CFU/mL；
Sedimentation step: circularly operating according to the sedimentation step, and stopping the stirring device 4 when the thallus seed fermentation reaches the logarithmic growth phase; after 3-8 min, starting the stirring device 4 again to stir for 0.5-2 min at the rotating speed of 2-10 rpm, so that part of thalli is settled into the third chamber 3; then, stirring and fermenting are continuously carried out according to the stirring mode of the fermentation step; the logarithmic growth phase is the phase of the maximum specific growth speed after a certain time of growth when the microorganism fermentation system is proliferated;
separating: when the turbidity detection device 5 detects that the turbidity of the liquid in the third chamber 3 exceeds a set value, discharging materials to the centrifugal device 9 through the vertical pipeline 7, wherein the volume of the discharged materials is 80-150% of the volume of the third chamber 3; after the materials are discharged, adding culture solution into the first chamber 1, wherein the volume of the added culture solution is equal to the volume of the discharged materials; the materials discharged into the centrifugal device 9 are centrifugally separated at the rotating speed of 10000-20000 rpm of the centrifugal device 9 to obtain the bacterial active mud; the number of times of discharging the material in this step is more than the number of times of replenishing the culture solution, i.e., the culture solution is not replenished after the last time of discharging the material, and the material is usually discharged 2 to 5 times. The value set in the turbidity detecting device 5 is generally set according to the type of the cultured cells, and is usually 7000 to 10000NTU.
According to the thallus culture method, the thallus is produced by adopting the thallus culture equipment, the stirring rotation speed and the sedimentation time are effectively controlled, so that the thallus is cultured and effectively sedimented into the third chamber 3, the thallus amount in fermentation is effectively reduced, and the substrate utilization rate is improved; in addition, the culture solution with the same volume is added after the materials are discharged for continuous culture, so that the culture and separation of the thalli are ensured, the fermentation time is prolonged, the utilization rate of the substrate is further improved, the product yield is improved, the on-line separation of the thalli is realized, and the thalli can be separated in time and keep good activity.
Example 1
By utilizing the thallus culture equipment and the thallus culture equipment method provided by the utility model, saccharomycetes are continuously cultured;
in this example, the total volume of the fermenter used was about 650L, the height of the cylindrical body of the first chamber 1 was 75cm and the diameter was 80cm, the height of the inverted conical bottom of the first chamber 1 was 20cm and the diameter of the lower end was 35cm; the height of the inverted conical second chamber 2 is 100cm, and the diameters of the upper end and the lower end are 80cm and 10cm respectively; the height of the inverted conical third chamber 3 is 53cm, and the diameter of the upper end is 42cm; the volumes of the first chamber 1, the second chamber 2 and the third chamber 3 respectively account for about 66%, 30% and 4% of the total volume of the fermentation tank.
During cultivation, 500L of yeast wort culture medium is pumped into a fermentation tank, and sterilized, and then the seed liquid of the Lu's combined yeast (Zygosaccharomyces rouxii) is inoculated into the fermentation tank, and the inoculation density is 10 5 CFU/mL, culturing in 30 ℃ after inoculation, and simultaneously starting a stirring device 4 to perform stirring fermentation at a rotating speed of 200 rpm;
after culturing for 24 hours, the fermentation broth reaches the logarithmic growth phase, stirring is stopped, and the fermentation broth is allowed to stand for 5min; then, starting the stirring device 4 to stir for 1min at a rotating speed of 5rpm, so that part of thalli are settled into the third chamber 3; then, regulating and controlling the stirring device 4 to 200rpm, and continuing stirring and fermenting; continuously culturing for 5h, then reaching logarithmic growth phase again, stopping stirring, and standing the fermentation broth for 5min; then starting the stirring device 4 to stir for 1min at a rotating speed of 5rpm, so that part of thalli are settled into the third chamber 3, and performing reciprocating circulation operation;
when the turbidity detection device 5 detects that the turbidity of the liquid in the third chamber 3 reaches 10000NTU, the valve 8 is opened to discharge the material to the centrifugal device 9 through the vertical pipeline 7, and the volume of the discharged material is about 30L; after discharging materials, supplementing glucose solution with the mass concentration of 3% into the first chamber 1 through a feed inlet of the fermentation tank, wherein the volume of the supplemented glucose solution is equal to the volume of the discharged materials; and the materials discharged into the centrifugal device 9 are centrifugally separated at the rotating speed of 10000rpm of the centrifugal device 9, so as to obtain the saccharomycete active bacterial sludge.
And continuing the culture operation to perform continuous culture until all materials are discharged after the glucose solution is added for 2 times, and ending the production.
Proved by verification, 2-2.5 kg of saccharomycete mud can be obtained per 100L of culture solution of the zygosaccharomyces rouxii cultivated by the embodiment, compared with the zygosaccharomyces rouxii cultivated by a traditional method (1.8-2 kg of saccharomycete mud can be obtained per 100L of culture solution), the yield is obviously improved, and the active freeze-dried powder prepared by adding the protective agent has no obvious difference in viable count compared with the traditional method.
Example 2
By utilizing the thallus culture equipment and the thallus culture equipment method provided by the utility model, lactobacillus plantarum is continuously cultured;
in this example, the total volume of the fermenter used was about 2500L, the height of the cylindrical body of the first chamber 1 was 1m, the diameter was 1.36cm, the height of the inverted conical bottom of the first chamber 1 was 30cm, and the diameter of the lower end was 43cm; the height of the inverted conical second chamber 2 is 107cm, and the diameters of the upper end and the lower end are 136cm and 36.5cm respectively; the height of the inverted conical third chamber 3 is 57cm, and the diameter of the upper end is 57cm; the volumes of the first chamber 1, the second chamber 2 and the third chamber 3 respectively account for about 68%, 30% and 2% of the total volume of the fermenter.
During culture, 2000LMRS culture medium is pumped into the fermentation tank, sterilized, and lactobacillus plantarum (Lactobacillus plantarum) seed liquid is inoculated into the fermentation tank with inoculation density of 10 5 CFU/mL, culturing in 37 ℃ environment after inoculation, and simultaneously starting a stirring device 4 to stir and ferment at a rotating speed of 100 rpm;
after culturing for 24 hours, the fermentation broth reaches the logarithmic growth phase, stirring is stopped, and the fermentation broth is allowed to stand for 8min; then, the stirring device 4 is started again to stir for 30 seconds at the rotating speed of 2rpm, so that part of thalli are settled into the third chamber 3; then, regulating and controlling the stirring device 4 to 100rpm, and continuing stirring and fermenting; continuously culturing for 5h, then reaching logarithmic growth phase again, stopping stirring, and standing the fermentation broth for 8min; then starting the stirring device 4 to stir for 30 seconds at a rotating speed of 2rpm, so that part of thalli are settled into the third chamber 3, and performing reciprocating circulation operation;
when the turbidity detection device 5 detects that the turbidity of the liquid in the third chamber 3 reaches 7000NTU, the valve 8 is opened to discharge the material to the centrifugal device 9 through the vertical pipeline 7, and the volume of the discharged material is about 40L; after discharging materials, supplementing glucose solution with the mass concentration of 5% into the first chamber 1 through a feed inlet of the fermentation tank, wherein the volume of the supplemented glucose solution is equal to the volume of the discharged materials; the material discharged into the centrifugal device 9 was subjected to centrifugal separation at a rotation speed of 15000rpm in the centrifugal device 9 to obtain lactobacillus active bacterial sludge.
Continuously culturing the above culture operation until all materials are discharged after the glucose solution is added for 3 times, and ending the production.
Through verification, 1.2-1.8 kg of lactobacillus plantarum mud can be obtained per 100L of culture solution of lactobacillus plantarum cultivated by the embodiment, compared with lactobacillus plantarum cultivated by a traditional method (1-1.5 kg of lactobacillus plantarum mud can be obtained per 100L of culture solution), the yield is obviously improved, and the active freeze-dried powder is prepared by adding a protective agent, so that the number of viable bacteria is not obviously different from that of the traditional method.
Claims (9)
1. Thallus culture equipment, including fermentation cylinder, agitating unit (4) and centrifugal device (9), its characterized in that: also comprises a turbidity detection device (5); the fermentation tank is provided with a first chamber (1), a second chamber (2) and a third chamber (3) which are sequentially connected from top to bottom, and the lower end of the third chamber (3) is provided with a liquid outlet (6); the stirring device (4) is arranged on the fermentation tank, and stirring paddles of the stirring device are arranged in the first chamber (1) and the second chamber (2); the turbidity detection device (5) is arranged in the third chamber (3), and a liquid inlet of the centrifugal device (9) is connected with the liquid outlet (6) through a vertical pipeline (7); the first chamber (1) comprises a cylindrical main body and an inverted conical bottom arranged at the lower end of the cylindrical main body, the second chamber (2) and the third chamber (3) are of inverted conical structures, and the first chamber (1), the second chamber (2) and the third chamber (3) are coaxially arranged.
2. The cell culture apparatus according to claim 1, wherein: the stirring device (4) comprises a stirring shaft which is vertically arranged on the fermentation tank and extends into the first chamber (1) and the second chamber (2), stirring paddles which are arranged on the stirring shaft, and a stirring motor which is arranged on the fermentation tank and is in transmission connection with the stirring shaft; the stirring shaft and the stirring blade jointly form a stirring paddle of the stirring device (4).
3. The cell culture apparatus according to claim 1, wherein: and a valve (8) is arranged on the vertical pipeline (7).
4. The cell culture apparatus according to claim 1, wherein: the centrifugal device (9) is a disc type centrifugal machine.
5. The cell culture apparatus according to any one of claims 1 to 4, wherein: the volume of the first chamber (1) is 65-75% of the total volume of the fermentation tank, the volume of the second chamber (2) is 20-30% of the total volume of the fermentation tank, and the volume of the third chamber (3) is 2-5% of the total volume of the fermentation tank.
6. The cell culture apparatus according to claim 5, wherein: the diameter of the joint of the first chamber (1) and the second chamber (2) is 40-50% of the diameter of the cylindrical main body of the first chamber (1), and the diameter of the joint of the second chamber (2) and the third chamber (3) is 30-40% of the diameter of the upper end of the third chamber (3).
7. The cell culture apparatus according to claim 6, wherein: the taper of the third chamber (3) is 0.7:1-1.2:1.
8. A method for culturing a fungus body, characterized by comprising the steps of: producing a cell using the cell culture apparatus according to any one of claims 1 to 7.
9. The method for culturing a fungus according to claim 8, wherein: comprises a fermentation step, a sedimentation step and a separation step;
fermentation: firstly, adding a sterilized culture medium into a fermentation tank through a first chamber (1), then adding a thallus seed liquid into the fermentation tank through the first chamber (1), and simultaneously starting a stirring device (4) to perform stirring fermentation;
sedimentation step: stopping the stirring device (4) after the fermentation of the thallus seeds reaches the logarithmic phase; after 3-8 min, starting the stirring device (4) to stir for 0.5-2 min at the rotating speed of 2-10 rpm, so that part of thalli is settled into the third chamber (3); then, stirring and fermenting are continuously carried out according to the stirring mode of the fermentation step;
separating: circularly operating according to a sedimentation step, and discharging materials to a centrifugal device (9) through a vertical pipeline (7) after the turbidity detection device (5) detects that the turbidity of the liquid in the third chamber (3) exceeds a set value, wherein the volume of the discharged materials is 80-150% of the volume of the third chamber (3); after the materials are discharged, supplementing culture solution into the first chamber (1), wherein the volume of the supplemented culture solution is equal to that of the discharged materials; and (3) centrifugally separating the material discharged into the centrifugal device (9) at the rotating speed of 10000-20000 rpm to obtain the bacterial active mud.
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