A fermentation method and device for producing sclareol

Abstract

The present invention relates to a fermentation device for producing sclareol, comprising a seed tank, a fermentation tank, and a feed tank. The fermentation tank is provided with a gas distributor, and the gas distributor is connected to a gas supply mechanism. The fermentation tank comprises a group of primary tank bodies and multiple groups of secondary tank bodies. The seed tank is connected to the primary tank body, the primary tank body is connected to each group of secondary tank bodies, and the feed tank is connected to the seed tank, the primary tank body, and each group of secondary tank bodies; the total volume of all secondary tank bodies is not less than 15 times of the primary tank body, and the volume of each group of secondary tank bodies is consistent. The present invention can improve the utilization rate of the fermentation tank and the yield of the fermented product; in addition, the present invention can improve the dissolved oxygen content of the fermentation liquid during high-density fermentation under low-speed stirring conditions, ensure the yield of the strain after fermentation, and thus improve the yield of sclareol.

Description

Technical Field

[0001] The invention relates to a fermentation method and a device for producing sclareol, belonging to the technical field of biosynthesis. Background Art

[0002] Sclareol is an important raw material for synthesizing high-grade ambergris flavoring agent ambroxan and tobacco flavoring agent sclareolide. The traditional method of extracting sclareol from sclareol is greatly limited by raw materials. The method that is currently being studied more is to use gene editing to recombinant Saccharomyces cerevisiae to prepare sclareol by fermentation, which can solve the problem of raw material limitation. In order to improve the yield of sclareol prepared by fermentation and realize industrial application, the current research direction is to achieve high-density fermentation by batch feeding. In the early stage of fermentation, the bacterial growth rate is slow. After a lag phase of 12 to 14 hours, the bacterial body enters the logarithmic growth phase. At this time, by feeding the culture medium in batches, the problem of high substrate concentration inhibiting bacterial growth can be solved, and high-density fermentation can be achieved. Since the culture medium is fed in batches during high-density fermentation, there is no effluent, and the filling coefficient of the fermentation tank in the early stage of fermentation is very low. In addition, a long lag phase is required in the early stage of fermentation. No feeding is done during the lag phase, and the fermentation tank has always maintained a low filling coefficient. After entering the logarithmic growth phase, batch feeding begins, and the filling coefficient gradually increases. Therefore, this method has the disadvantage of low equipment utilization, which limits the fermentation yield. In addition, high-density fermentation requires high dissolved oxygen. In process control, the dissolved oxygen content can be increased by increasing the speed of the stirring shaft and the pressure of the fermentation tank. However, the increase in speed, the increase in shear force of the fermentation liquid, and the increase in pressure will affect the growth of the bacteria. Summary of the invention

[0003] In view of the above problems, the present invention first provides a fermentation method and device for producing sclareol, and the specific scheme is as follows:

[0004] A fermentation device for producing sclareol comprises a seed tank, a fermentation tank and a feed tank. The fermentation tank is provided with a gas distributor, which is connected to a gas supply mechanism. The fermentation tank comprises a group of primary tank bodies and multiple groups of secondary tank bodies. The seed tank is connected to the primary tank body, which is connected to each group of secondary tank bodies, and the feed tank is connected to the seed tank, the primary tank body and each group of secondary tank bodies. The total volume of all the secondary tank bodies is not less than 15 times that of the primary tank body, and the volume of each group of secondary tank bodies is consistent.

[0005] Furthermore, the tank wall of the secondary tank body is provided with a plurality of annular partitions along the height direction, and the annular partitions include a bracket at the top, and an adsorption layer is fixed at the bottom of the bracket, and the adsorption layer is a polyester fiber layer.

[0006] Furthermore, the bottom surface of the annular spacer is inclined in the radial direction, and the inner edge is lower than the outer edge.

[0007] Furthermore, a hook is provided on the tank wall of the secondary tank body, a hanging ring is provided on the outer edge of the bracket, grooves are provided on the outer edge and inner edge of the lower part of the bracket, and the polyester fiber layer is embedded in the groove; a pin is provided on the upper part of the bracket, the pin is arranged in the guide cylinder, the lower part of the pin is embedded in the polyester fiber layer, and adjacent pins are cross-arranged.

[0008] Furthermore, the exhaust hole of the gas distributor of the secondary tank is located below the annular partition.

[0009] Furthermore, a dissolved oxygen tank is connected between the feed tank and the secondary tank body, and the dissolved oxygen tank is connected to the gas supply mechanism; the dissolved oxygen tank is connected to the secondary tank body through a feed pipe, and the opening of the feed pipe is located below the annular partition, a high-speed stirring shaft is provided in the dissolved oxygen tank, and a low-speed stirring shaft is provided in the secondary tank body.

[0010] According to the device of the present invention, the present invention also claims a fermentation method for producing sclareol, characterized in that it comprises the following steps:

[0011] 1) The engineered bacteria are connected to the seed tank, and the culture medium is added to obtain the seed solution. The seed solution first enters the primary tank, and the culture medium is added to obtain the culture solution after preliminary fermentation;

[0012] 2) The culture solution in the primary tank first enters the first group of secondary tanks and undergoes high-density fermentation through batch feeding;

[0013] 3) Repeat step 1), and the culture fluid after the initial fermentation in the tank enters the next group of secondary tanks in turn, and performs high-density fermentation through batch feeding;

[0014] Furthermore, the time for the initial fermentation is 18 to 20 hours, and the time for the high-density fermentation is 100 to 120 hours.

[0015] Furthermore, the culture medium in the feed tank first enters the dissolved oxygen tank, is mixed with the air introduced by the air supply mechanism, and then enters the secondary tank body; during fermentation, the speed of the high-speed stirring shaft is 2000r / min, and the speed of the low-speed stirring shaft is 200r / min.

[0016] Furthermore, in step 1), the filling coefficient of the primary tank body after the culture medium is added is 0.8, and in step 2), the initial filling coefficient of the secondary tank body is 0.3, and the filling coefficient is 0.8 when the fermentation is completed.

[0017] The present invention can improve the utilization rate of the fermentation tank and the yield of the fermented product; in addition, the present invention can increase the dissolved oxygen content of the fermentation liquid during high-density fermentation under low-speed stirring conditions, ensure the yield of the strain after fermentation, and thus increase the yield of sclareol. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1It is a schematic diagram of the structure of the fermentation device in the present invention;

[0019] Figure 2 It is a structural schematic diagram of the secondary tank body in the present invention;

[0020] Figure 3 It is a schematic diagram of the structure of the annular spacer in the present invention. DETAILED DESCRIPTION

[0021] The scheme of the present invention is described in detail below with reference to specific examples.

[0022] Example 1

[0023] like Figure 1-Figure 3 A fermentation device for producing sclareol includes a seed tank 1, a fermentation tank, and a feed tank 2. The fermentation tank is provided with a gas distributor 3, which is connected to a gas supply mechanism. The fermentation tank includes a group of primary tank bodies 4 and multiple groups of secondary tank bodies 5. The seed tank is connected to the primary tank body, the primary tank body is connected to each group of secondary tank bodies, and the feed tank is connected to the seed tank, the primary tank body, and each group of secondary tank bodies; the total volume of all secondary tank bodies is not less than 15 times that of the primary tank body, and the volume of each group of secondary tank bodies is consistent. Since the high-density fermentation stage is about 5 to 7 times longer than the initial fermentation stage, multiple groups of secondary tank bodies are set up to ensure that the primary tank body can supply culture fluid to each group of secondary tank bodies in turn, thereby improving the utilization rate of the primary tank body.

[0024] As shown in the figure, in this embodiment, a group of primary tanks are connected to 5 groups of secondary tanks. The primary tanks are used as containers for initial fermentation. The air supply mechanism uses an air compressor 6 to supply compressed air. The compressed air is first purified by a filter to remove bacteria to obtain clean air, and then used as an air source to supplement each fermentation tank through a gas distributor. The feed tank is used to configure the culture medium and add the culture medium to the seed tank and each fermentation tank.

[0025] The traditional batch feeding fermentation method is to add a small amount of seed liquid and culture medium to a single fermentation tank, and start batch feeding after entering the logarithmic growth phase until the fermentation is completed. During the initial fermentation period of about 20 hours, the fermentation tank always maintains a very low filling factor and the equipment utilization rate is very low.

[0026] This device uses a primary tank and a secondary tank to form a fermentation tank group. The initial fermentation is carried out in the primary tank, which can maintain a high filling factor. After the initial fermentation is completed, the culture solution is transferred to the secondary tank with a larger volume for high-density fermentation. When the secondary tank completes a cycle of high-density fermentation, the primary tank can carry out multiple cycles of initial fermentation. The culture solution obtained from each cycle of fermentation in the primary tank is transferred to a group of secondary tanks. By switching the secondary tanks, the high filling factor fermentation of the primary tank is achieved.

[0027] High-density fermentation requires maintaining a high dissolved oxygen content in the fermentation liquid. In the prior art, this is generally achieved by increasing the ventilation volume and the speed of the stirring shaft. Increasing the ventilation volume alone cannot increase the dissolved oxygen content. When the stirring shaft speed is increased, the turbulence effect of the fermentation liquid can be enhanced, thereby accelerating the dissolution of air. However, a high stirring shaft speed causes great damage to the bacteria and is not conducive to the reproduction of the bacteria.

[0028] In view of this problem, the present invention provides an improved structure of a fermentation tank body, in which a plurality of annular partitions 7 are arranged along the height direction of the tank wall of the secondary tank body, and the annular partitions include a bracket 8 at the top, and an adsorption layer is fixed at the lower part of the bracket, and the adsorption layer is a polyester fiber layer 9. The polyester fiber layer is loose and porous. Under the condition of low-speed stirring of the stirring shaft, the air that has no time to dissolve can be adsorbed by the polyester fiber layer, delaying the overflow speed of the air from the fermentation liquid, and the air adsorbed in the polyester fiber layer gradually dissolves into the fermentation liquid, thereby increasing the dissolved oxygen content of the fermentation liquid.

[0029] The bottom surface of the annular spacer is inclined radially, and the inner edge is lower than the outer edge. The bottom surface of the annular spacer is inverted cone shape. The rotation of the stirring shaft will drive the fermentation liquid to form a vortex. Under the action of centrifugal force, the bubbles entering the fermentation liquid will gather from the periphery to the center. The annular spacer is set in an inverted cone shape to block the flow of bubbles, which is conducive to the adsorption of air into the polyester fiber layer.

[0030] In order to facilitate the removal of the annular partition, a hook 10 is provided on the tank wall of the secondary tank body, and a hanging ring 11 is provided on the outer edge of the bracket. The bracket is hung on the hook through the hanging ring. The outer edge and inner edge of the lower part of the bracket are provided with grooves, and the polyester fiber layer is embedded in the grooves; the upper part of the bracket is provided with pins 12, which are arranged in the guide cylinder, and adjacent pins are arranged crosswise. When removing the polyester fiber layer, the pins are pulled out, and then the polyester fiber layer is taken out from the groove. The bracket can be made of a wire mesh frame, and the guide cylinder is fixed on the wire mesh frame.

[0031] The exhaust hole of the gas distributor of the secondary tank is located below the annular partition. The bubbles overflowing from the gas distributor can be blocked and adsorbed by the annular partition to prevent the bubbles from overflowing quickly.

[0032] In order to further increase the dissolved oxygen content, a dissolved oxygen tank 13 is connected between the feed tank and the secondary tank body, and the dissolved oxygen tank is connected to the gas supply mechanism; the dissolved oxygen tank is connected to the secondary tank body through a feed pipe 16, and the opening of the feed pipe is located below the annular partition. A high-speed stirring shaft 14 is provided in the dissolved oxygen tank, and a low-speed stirring shaft 15 is provided in the secondary tank body. The air is stirred and mixed with the liquid culture medium at a high speed in the dissolved oxygen tank, and can be quickly dissolved. The oxygen-rich culture medium enters the secondary tank body, and the dissolved oxygen content of the fermentation liquid is increased. The stirring speed of the stirring shaft in the secondary tank body can be reduced, which is conducive to the growth and reproduction of bacteria.

[0033] Example 2

[0034] According to the fermentation device of Example 1, the fermentation method of this embodiment comprises the following steps:

[0035] 1) The engineered bacteria are connected to the seed tank, and the culture medium is added to obtain the seed solution. The seed solution first enters the primary tank, and the culture medium is added to obtain the culture solution after preliminary fermentation;

[0036] 2) The culture solution in the primary tank first enters the first group of secondary tanks and undergoes high-density fermentation through batch feeding;

[0037] 3) Repeat step 1), and the culture fluid after the initial fermentation in the tank enters the next group of secondary tanks in turn, and performs high-density fermentation through batch feeding.

[0038] The time for initial fermentation is 18 to 20 hours, and the time for high-density fermentation is 100 to 120 hours. The culture medium in the feed tank first enters the dissolved oxygen tank, mixes with the air introduced by the air supply mechanism, and then enters the secondary tank body; during fermentation, the speed of the high-speed stirring shaft is 2000r / min, and the speed of the low-speed stirring shaft is 200r / min. In step 1), the filling coefficient of the primary tank body after the culture medium is added is 0.8, and the initial filling coefficient of the secondary tank body in step 2) is 0.3, and the filling coefficient is 0.8 when the fermentation is completed.

[0039] According to the traditional fermentation process, for a 160L fermentation tank, the initial fermentation time is 20 hours, the high-density fermentation time is 100 hours, and the utilization rate of the fermentation tank is about 50%. Taking a primary tank with a volume of 10L and a secondary tank with a single volume of 30L as an example, the total volume is 160L. Excluding the cleaning and sterilization time, the initial fermentation time is 20 hours, and the high-density fermentation time is 100 hours, the utilization rate of the fermentation tank can be increased to 55%.

Claims

1. A fermentation device for producing sclareol, comprising a seed tank, a fermentation tank, and a feed tank, wherein the fermentation tank is provided with a gas distributor, and the gas distributor is connected to a gas supply mechanism, characterized in that: The fermentation tank comprises a group of primary tank bodies and multiple groups of secondary tank bodies, the seed tank is connected to the primary tank body, the primary tank body is connected to each group of secondary tank bodies, and the feed tank is connected to the seed tank, the primary tank body and each group of secondary tank bodies; the total volume of all secondary tank bodies is not less than 15 times that of the primary tank body, and the volume of each group of secondary tank bodies is consistent; The tank wall of the secondary tank body is provided with a plurality of annular partitions along the height direction. The annular partitions include a bracket at the top. An adsorption layer is fixed at the bottom of the bracket. The adsorption layer is a polyester fiber layer.

2. The fermentation device for producing sclareol according to claim 1, characterized in that: The bottom surface of the annular spacer is inclined in the radial direction, and the inner side edge is lower than the outer side edge.

3. The fermentation device for producing sclareol according to claim 2, characterized in that: A hook is provided on the tank wall of the secondary tank body, a hanging ring is provided on the outer edge of the bracket, grooves are provided on the outer edge and inner edge of the lower part of the bracket, and the polyester fiber layer is embedded in the groove; a pin is provided on the upper part of the bracket, the pin is arranged in the guide cylinder, the lower part of the pin is embedded in the polyester fiber layer, and adjacent pins are cross-arranged.

4. The fermentation device for producing sclareol according to claim 3, characterized in that: The exhaust hole of the gas distributor of the secondary tank is located below the annular partition.

5. The fermentation device for producing sclareol according to claim 4, characterized in that: A dissolved oxygen tank is connected between the feed tank and the secondary tank body, and the dissolved oxygen tank is connected to the gas supply mechanism; the dissolved oxygen tank is connected to the secondary tank body through a feed pipe, and the opening of the feed pipe is located below the annular partition; a high-speed stirring shaft is provided in the dissolved oxygen tank, and a low-speed stirring shaft is provided in the secondary tank body.

6. A fermentation method for producing sclareol, characterized in that: The method comprises using the fermentation device for producing sclareol as claimed in claim 5, and comprising the following steps: 1) The engineered bacteria are connected to the seed tank, and the culture medium is added to obtain the seed solution. The seed solution first enters the primary tank, and the culture medium is added to obtain the culture solution after preliminary fermentation; 2) The culture solution in the primary tank first enters the first group of secondary tanks and undergoes high-density fermentation through batch feeding; 3) Repeat step 1), and the culture fluid after preliminary fermentation in the first tank enters the next group of second tanks in turn, and high-density fermentation is carried out through batch feeding.

7. The fermentation method for producing sclareol according to claim 6, characterized in that: The time for initial fermentation is 18~20h, and the time for high-density fermentation is 100~120h.

8. The fermentation method for producing sclareol according to claim 6, characterized in that: The culture medium in the feed tank first enters the dissolved oxygen tank, mixes with the air introduced by the air supply mechanism, and then enters the secondary tank body; during fermentation, the speed of the high-speed stirring shaft is 2000r / min, and the speed of the low-speed stirring shaft is 200r / min.

9. The fermentation method for producing sclareol according to claim 6, characterized in that: In step 1), the filling coefficient of the primary tank after the culture medium is added is 0.8, and in step 2), the initial filling coefficient of the secondary tank is 0.3, and the filling coefficient is 0.8 when the fermentation is completed.

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