An electromagnetic wave siphon self-defoaming bioreactor for natto fermentation

Abstract

An electromagnetic wave siphon self-defoaming bioreactor for natto fermentation relates to the field of biological production equipment. It includes a biological pre-reaction pressure vessel, a biological reaction pressure vessel and a gas-liquid mixer. The two sides of the biological pre-reaction pressure vessel are equipped with pulse magnetic field generators arranged oppositely. The biological reaction pressure vessel is fixedly equipped with an electromagnetic wave generator. One side of the upper part of the biological reaction pressure vessel is provided with a liquid inlet, and the other side of the upper part of the biological reaction pressure vessel is connected with a liquid outlet pipe. The bottom side of the biological reaction pressure vessel is provided with a reflux port. The liquid outlet pipe is connected to the inlet end of the gas-liquid mixer, and the outlet end of the gas-liquid mixer is connected to the reflux port of the biological reaction pressure vessel. The present invention can increase the biological conversion efficiency of the culture medium by more than 1 times, and the production capacity and the quality of the finished product can be improved. The siphon gas-liquid mixer can achieve zero addition of the defoaming process in the entire microbial growth process.

Description

Technical Field

[0001] The invention relates to the field of biological production equipment, in particular to an electromagnetic wave siphon self-defoaming bioreactor for natto fermentation. Background Art

[0002] Microbial fermentation refers to the process of using microorganisms to convert raw materials into products needed by humans through specific metabolic pathways under suitable conditions. The production level of microbial fermentation mainly depends on the genetic characteristics of the strain itself and the culture conditions. At present, natto fermentation uses Bacillus subtilis as a strain, and simply places the microbial raw materials in a fermentation tank. Conventional means such as heating and stirring are often used to accelerate the fermentation reaction. However, the bioconversion efficiency is low and the microbial cells cannot be changed. Summary of the invention

[0003] The purpose of the present invention is to provide an electromagnetic wave siphon self-defoaming bioreactor for natto fermentation, which can effectively solve the problems in the background technology.

[0004] The technical scheme for achieving the above object is: an electromagnetic wave siphon self-defoaming bioreactor for natto fermentation, characterized in that it includes a biopre-reaction pressure vessel, a bioreaction pressure vessel and a gas-liquid mixer,

[0005] An electromagnetic wave generator is fixedly installed in the bioreactor pressure vessel, a first exhaust port is arranged at the top of the bioreactor pressure vessel, the first exhaust port is connected to a second flow regulating valve, a liquid inlet is arranged on one side of the upper part of the bioreactor pressure vessel, a liquid outlet pipe is connected to the other side of the upper part of the bioreactor pressure vessel, a reflux port is arranged on one side of the bottom of the bioreactor pressure vessel, the liquid outlet pipe is connected to the inlet end of the gas-liquid mixer, and the outlet end of the gas-liquid mixer is connected to the reflux port of the bioreactor pressure vessel;

[0006] Pulse magnetic field generators arranged oppositely are installed on both sides of the biological pre-reaction pressure vessel, a second exhaust port is arranged at the top of the biological pre-reaction pressure vessel, the second exhaust port is connected to a third flow regulating valve, and the bottom end of the biological pre-reaction pressure vessel is connected to the liquid inlet of the biological reaction pressure vessel through the third control valve.

[0007] Furthermore, the electromagnetic wave generator is a TDP magic lamp, and two electromagnetic wave generators are provided and symmetrically installed on both sides of the top of the biological reaction pressure vessel.

[0008] Furthermore, the liquid outlet pipes are provided with a plurality of pipes arranged vertically, and the outlet ends are connected to each other and then connected to the gas-liquid mixer via a fourth control valve.

[0009] Furthermore, a plurality of vertically arranged siphon tubes are fixed on the inner wall of the bioreactor pressure vessel, an annular compressed air pipe is fixedly installed in the bioreactor pressure vessel above the siphon tube, the annular compressed air pipe is connected to an air inlet pipe, the air inlet pipe is connected to a compressed air source through a first flow regulating valve and a first control valve connected in series, and the annular compressed air pipe is also connected to jet pipes extending to the upper ports of the siphon tubes respectively.

[0010] Furthermore, a first stirring device is installed in the bioreactor pressure vessel.

[0011] Furthermore, a second stirring device is installed in the biological pre-reaction pressure vessel.

[0012] Furthermore, the biological pre-reaction pressure vessel is connected to a compressed air source via a second flow regulating valve and a second control valve which are connected in series.

[0013] Beneficial effects of the present invention:

[0014] When the present invention is working, the culture medium in the biological pre-reaction pressure vessel is fermented and expanded under the magnetic field environment of the pulse magnetic field generator. After the expansion is completed, the biological culture medium is inoculated into the biological reaction pressure vessel again. During the normal fermentation process, the electromagnetic wave generator in the biological reaction pressure vessel is turned on to improve the metabolic activity of the microorganisms; the compressed air is started, and the compressed air reaches the bottom of the reaction vessel through the siphon tube and then quickly siphons the foam generated on the surface of the culture medium to the gas-liquid mixer for mixing, and then transports it to the bottom of the liquid in the biological reaction pressure vessel, thereby achieving the purpose of replacing chemical defoaming with physical circulation defoaming, greatly saving costs and ensuring the fermentation effect.

[0015] The setting of magnetic field and TDP magic lamp can increase the bioconversion efficiency of culture medium by more than 1 times, and both production capacity and finished product quality can be improved. The siphon gas-liquid mixer can achieve zero addition in the defoaming process during the entire microbial growth process. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a structural schematic diagram of the present invention;

[0017] Figure 2 Schematic diagram of the distribution structure of siphons in the bioreactor pressure vessel. DETAILED DESCRIPTION

[0018] 1. Technical solution of the present invention

[0019] like Figure 1 , 2As shown, the present invention is mainly applied to natto fermentation with Bacillus subtilis as the strain, including a biological pre-reaction pressure vessel 1 with a volume of 30L, a biological reaction pressure vessel 2 with a volume of 10000L and a gas-liquid mixer 3. Both the biological pre-reaction pressure vessel 1 and the biological reaction pressure vessel 2 are closed pressure vessels.

[0020] Pulse magnetic field generators 1.1 are installed on both sides of the biological pre-reaction pressure vessel 1 and arranged oppositely. A second exhaust port 21 is provided at the top of the biological pre-reaction pressure vessel 1. The second exhaust port 21 is connected to a third flow regulating valve 22. The magnetic field strength is 0.5-1.5T and the number of pulses is 5-15.

[0021] An electromagnetic wave generator 4 is fixedly installed in the bioreactor pressure vessel 2. The electromagnetic wave generator 4 is a TDP magic lamp. Two electromagnetic wave generators 4 are provided and symmetrically installed on both sides of the top of the bioreactor pressure vessel 2. A first exhaust port 19 is provided at the top of the bioreactor pressure vessel 2. The first exhaust port 19 is connected to a second flow regulating valve 20. A liquid inlet 2.1 is provided on one side of the upper part of the bioreactor pressure vessel 2. A liquid outlet pipe 5 is connected to the other side of the upper part of the bioreactor pressure vessel 2. A reflux port 2.2 is provided on one side of the bottom of the bioreactor pressure vessel 2. The liquid inlet 2.1 of the bioreactor pressure vessel 2 is connected to the biopre-reaction pressure vessel 1 through a third control valve 13. A plurality of liquid outlet pipes 5 are provided in an upper and lower arrangement. The outlet ends of the liquid outlet pipes 5 are connected to each other and then connected to the inlet end of the gas-liquid mixer 3 through a fourth control valve 18. The outlet end of the gas-liquid mixer 3 is connected to the reflux port 2.2 of the bioreactor pressure vessel 2.

[0022] A plurality of vertically arranged siphon tubes 6 are fixed on the inner wall of the bioreactor pressure vessel 2, an annular compressed air pipe 7 is fixedly installed in the bioreactor pressure vessel 2 above the siphon tube 6, the annular compressed air pipe 7 is also connected to the jet tubes 9 extending to the upper ports of the siphon tube 6, the annular compressed air pipe 7 is connected to an air inlet pipe 8 extending outward from the bioreactor pressure vessel 2, the air inlet pipe 8 is connected to a compressed air source through a first flow regulating valve 14 and a first control valve 15 connected in series, and the compressed air source is also connected to the biopre-reaction pressure vessel 1 through a second flow regulating valve 16 and a second control valve 17 connected in series.

[0023] A stirring device is respectively arranged in the biological pre-reaction pressure vessel 1 and the biological reaction pressure vessel 2, and the stirring device comprises a stirring shaft 10 respectively arranged in the biological pre-reaction pressure vessel 1 and the biological reaction pressure vessel 2, and a stirring blade 11 is installed on the stirring shaft 10. A driving motor 12 for driving the stirring shaft 10 to rotate is installed at the top of the biological pre-reaction pressure vessel 1 and the biological reaction pressure vessel 2.

[0024] 2. Taking the natto Bacillus strain as the object, the effect of the pulse magnetic field generator 1.1 on microbial fermentation was tested. The test steps are as follows:

[0025] Test Example 1

[0026] 1) preparing a Bacillus natto strain;

[0027] 2) Prepare an ion culture medium for producing nattokinase: weigh 180 g of tryptone, 180 g of glucose, 18 g of yeast powder, 90 g of beef extract powder and 90 g of sodium chloride and place them in a biological pre-reaction pressure vessel 1, then add 14 kg of purified water and stir evenly to obtain an ion culture medium for producing nattokinase;

[0028] 3) The natto Bacillus natto strain is inoculated into the ion culture medium prepared in step 2), and placed in a shaker flask at 36-37° C., and cultured at 100-120 r / min for 8-12 hours to obtain fermentation seeds;

[0029] 4) The fermentation seeds are put into the biological pre-reaction pressure vessel 1 at an inoculation ratio of 3 to 10%, the second control valve 17 is opened, and the flow rate of the second flow control valve 16 is adjusted so that the pressure in the biological pre-reaction pressure vessel 1 is 0.1 MPa and the air input per kilogram of fermentation liquid is 0.1 m 3 / min, magnetic field strength of 0.5T, pulse number of 5, continued fermentation culture for 24h, and continuous stirring at a speed of 100r / min;

[0030] 5) During the continuous fermentation process, the fermentation liquid was sampled every two hours to measure the serine enzyme activity value;

[0031] 6) After the fermentation stops, the fermented seeds should be dried within 24 hours.

[0032] Test Example 2: The steps are the same as those of Test Example 1, except that:

[0033] In step (4), the fermentation process is carried out under an environment with a magnetic field strength of 1 T and a pulse number of 10;

[0034] Test Example 3: The steps are the same as those of Test Example 1, except that:

[0035] In step 4), the fermentation process is carried out under a magnetic field strength of 1.5 T and a pulse number of 15;

[0036] Test Example 4: The steps are the same as those of Test Example 1, except that:

[0037] In step 4), the fermentation process is carried out under a magnetic field strength of 3.3 T and a pulse number of 25;

[0038] Test Example 5: The steps are the same as those of Test Example 1, except that:

[0039] In step 4), the fermentation process is carried out under an environment with a magnetic field strength of 10 T and a pulse number of 48;

[0040] Activity values ​​of various implementation cases under different magnetic field strengths:

[0041]

[0042] Table 1

[0043] It can be seen from Table 1 that the magnetic field intensity and the number of pulses have a great influence on the growth effect of Bacillus subtilis. Bacillus subtilis is most sensitive to the pulsed magnetic field during the logarithmic growth period. With the increase of magnetic field intensity (>1.5T) and the number of pulses (>15), the metabolism of Bacillus subtilis decreases and the enzyme activity shows a downward trend; but when the magnetic field intensity (≤1.5T) and the number of pulses (≤15) are relatively low, the pulsed magnetic field promotes the growth of Bacillus subtilis and accelerates its metabolism.

[0044] 3. Using the natto Bacillus strain as the object, the effect of TDP magic lamp on microbial fermentation was tested. The test steps are as follows:

[0045] Use three test tubes, each containing 0.2 ml of Bacillus natto liquid, and irradiate them with TDP lamps at a distance of 30 cm and an irradiance of 54 mW / cm2. Irradiate for 15 minutes each time, at 10 a.m. and 4 p.m. After irradiation, place them in a 30°C incubator and observe them the next morning. The irradiation environment temperature is 26°C. If the test tube temperature is too high, use the rotating test tube method to achieve temperature balance.

[0046] OD (optical density absorbed by the test object) value determination After the irradiation treatment, the OD value of each group of bacterial liquid was measured using an ultraviolet spectrophotometer. The measurement results are shown in Table 2.

[0047] Table 2 OD values ​​of Bacillus natto culture after irradiation

[0048] Test tube number 1 2 3 OD value 0.650 0.813 0.519

[0049] Note: The initial OD of the Bacillus natto liquid is 0.054, OD = lg (1 / trans) where trans is the transmittance of the test object)

[0050] The results of this experimental study show that: under the same conditions, TDP magic lamp irradiation has a unique biological effect. An important premise is that the electromagnetic waves radiated by TDP must be absorbed by the body before they can play a role in the body. The growth of microorganisms requires nutrients such as carbon sources, nitrogen sources, energy, and inorganic salts. The TDP magic lamp can activate the activity of macromolecules in microorganisms, so that the molecules of microorganisms can be excited and in a higher vibration state, thus activating the activity of macromolecules such as proteins. Enzymes are water-soluble proteins with catalytic effects produced by living cells. They are composed of enzyme proteins and auxiliary factors. The characteristic is that one enzyme can only act on one type of substrate. Specific electromagnetic waves that are beneficial and harmless to microorganisms can promote changes in the enzyme system in microorganisms, increase enzyme metabolism, improve enzyme activity, and increase growth factors, thereby promoting the growth, development, reproduction and repair of body tissues.

[0051] 4. Using the natto Bacillus strain as the object, the effect of the technical solution described in this embodiment on microbial fermentation was tested. The test steps are as follows:

[0052] 1) preparing a Bacillus natto strain;

[0053] 2) According to the following mass components, 180 g of tryptone, 180 g of glucose, 18 g of yeast powder, 90 g of beef extract powder and 90 g of sodium chloride were weighed and placed in a biological pre-reaction pressure vessel 1, and 14 L of purified water was added and stirred to obtain an ion culture medium for producing nattokinase;

[0054] 3) According to the following mass ingredients, 250 kg of kidney bean powder, 100 kg of glucose, 25 kg of sodium chloride, 2.5 kg of sodium hydroxide, 0.15 kg of magnesium sulfate, 0.35 kg of calcium chloride, 2.5 kg of sodium hydroxide, 150 g of Tween (80), 1.5 kg of potassium dihydrogen phosphate, and 20 kg of disodium hydrogen phosphate were weighed and placed in a bioreactor pressure vessel 2, and 5000 L of purified water was added and stirred to obtain an ion culture medium.

[0055] 4) The natto Bacillus natto strain is inoculated into the ion culture medium prepared in step 2), and placed in a shaker flask at 36-37° C., and cultured at 100-120 r / min for 8-12 hours to obtain fermentation seeds;

[0056] 5) The fermentation seeds are added into the biological pre-reaction pressure vessel 1 in step 2) at an inoculation ratio of 3 to 10%, the second control valve 17 is opened, and the flow rate of the second flow control valve 16 is adjusted to adjust the pressure in the biological pre-reaction pressure vessel 1 to 0.06 MPa, and the air input per kilogram of fermentation liquid is 0.1 m 3 / min, adjust the pulse magnetic field generator 1.1, control the magnetic field strength to 1.4T, the pulse number to 14, and continue the fermentation culture at a speed of 400r / min for 12h;

[0057] 5) After the expansion is completed, the third control valve 13 is opened to inoculate the expanded bacterial solution into the bioreactor pressure vessel 2, and the first control valve 15 is opened to adjust the pressure in the bioreactor pressure vessel 2 to 0.05 MPa through the first flow regulating valve 14 and the second flow regulating valve 20 to ensure that the air input volume in the vessel is 10m 3 / min, exhaust volume 6.5m 3 / min, and the stirring speed of the bioreactor pressure vessel 2 is maintained at 80-120r / min.

[0058] 6) When the normal fermentation reaches 4 hours, turn on the TDP magic lamp, irradiate the surface of the fermentation liquid continuously for 30 minutes and then turn it off. When the fermentation reaches 8 hours, 12 hours, 16 hours, and 20 hours, turn on the TDP magic lamp respectively, and irradiate the surface of the fermentation liquid continuously for 30 minutes.

[0059] 7) During the fermentation process, if the foam height is too high, the fourth control valve 18 is opened, and the upper foam, under the action of compressed air, enters the gas-liquid mixer 7 through the liquid outlet pipe 5 for mixing and then enters the bioreactor pressure vessel 2 through the reflux port 2.2, thereby achieving physical circulation and defoaming.

[0060] 8) During the continuous fermentation process, the fermentation liquid was sampled every two hours, and the samples were taken four times in batches to determine the activity value of nattokinase;

[0061] 9) After fermentation stops, dry the mixture within 24 hours.

[0062] The final enzyme activities of the four batches are shown in Table 3:

[0063] batch Enzyme activity (U / g) 1 158200 2 165378 3 158653 4 169621 Conventional fermentation 78232

[0064] Table 3

[0065] Conclusion: The setting of electromagnetic waves can increase the bioconversion efficiency of the culture medium by more than 1 times, and both the production capacity and the quality of the finished product can be improved. The siphon gas-liquid mixer can achieve zero addition in the defoaming process during the entire microbial growth process.

Claims

1. An electromagnetic wave siphon self-defoaming bioreactor for natto fermentation, characterized in that: It comprises a biological pre-reaction pressure vessel, a biological reaction pressure vessel and a gas-liquid mixer, wherein an electromagnetic wave generator is fixedly installed in the biological reaction pressure vessel, a first exhaust port is arranged at the top of the biological reaction pressure vessel, the first exhaust port is connected to a second flow regulating valve, a liquid inlet is arranged on one side of the upper part of the biological reaction pressure vessel, a liquid outlet pipe is connected to the other side of the upper part of the biological reaction pressure vessel, a reflux port is arranged on one side of the bottom of the biological reaction pressure vessel, the liquid outlet pipe is connected to the inlet end of the gas-liquid mixer, and the outlet end of the gas-liquid mixer is connected to the reflux port of the biological reaction pressure vessel; The liquid outlet pipes are provided with a plurality of pipes arranged vertically, and the outlet ends are connected to each other and connected to the gas-liquid mixer through a fourth control valve; The two sides of the biological pre-reaction pressure vessel are installed with pulse magnetic field generators arranged opposite to each other. The pressure in the biological pre-reaction pressure vessel is 0.1 MPa, and the air input per kilogram of fermentation liquid is 0.1 m 3 / min, magnetic field strength of 1.5T, pulse number of 15, continued fermentation culture for 24h, and continuous stirring at a speed of 100r / min; The top of the biological pre-reaction pressure vessel is provided with a second exhaust port, the second exhaust port is connected to a third flow regulating valve, and the bottom of the biological pre-reaction pressure vessel is connected to the liquid inlet of the biological reaction pressure vessel through the third control valve; A plurality of vertically arranged siphon tubes are fixed on the inner wall of the bioreactor pressure vessel, an annular compressed air pipe is fixedly installed in the bioreactor pressure vessel above the siphon tube, the annular compressed air pipe is connected to an air inlet pipe, the air inlet pipe is connected to a compressed air source through a first flow regulating valve and a first control valve connected in series, and the annular compressed air pipe is also connected to jet pipes extending to upper ports of the siphon tubes respectively; The compressed air is started, and the compressed air reaches the bottom of the reaction vessel through the siphon tube and then quickly siphons the foam generated on the surface of the culture medium to the gas-liquid mixer for mixing, and then transported to the bottom of the liquid in the bioreactor pressure vessel.

2. The electromagnetic wave siphon self-defoaming bioreactor for natto fermentation according to claim 1, characterized in that: The electromagnetic wave generator is a TDP magic lamp, and two electromagnetic wave generators are provided and symmetrically installed on both sides of the top of the biological reaction pressure container.

3. The electromagnetic wave siphon self-defoaming bioreactor for natto fermentation according to claim 1, characterized in that: A first stirring device is installed in the bioreactor pressure vessel.

4. The electromagnetic wave siphon self-defoaming bioreactor for natto fermentation according to claim 1, characterized in that: A second stirring device is installed in the biological pre-reaction pressure vessel.

5. The electromagnetic wave siphon self-defoaming bioreactor for natto fermentation according to claim 4, characterized in that: The biological pre-reaction pressure container is connected to a compressed air source via a second flow regulating valve and a second control valve which are connected in series.

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