A process for preparing long-chain dibasic acid by bioconversion

Abstract

The invention relates to the technical field of preparation of oxygen-containing organic compounds, in particular to a process for preparing long-chain dibasic acid by bioconversion; the process comprises the following steps: firstly, adding a non-polar macroporous carrier into an impregnation liquid containing a conditioning agent and an alkane to obtain an impregnation mixture; then, inoculating activated long-chain dibasic acid fermentation bacteria (Candida) into an expansion culture medium for cultivation; then, adding the impregnation mixture to activate the strain to obtain a seed liquid; finally, using a constant temperature shaker to ferment the seed liquid to obtain the desired long-chain dibasic acid; the invention realizes dropwise addition of the expansion culture medium in the constant temperature shaker through the above process, and compared with adding all the expansion culture medium at one time, the gradual addition can not only reduce the accumulation of metabolites caused by excess nutrition, but also is conducive to the stability of the cell growth environment, and improves the efficiency of the expansion culture of Candida.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of oxygen-containing organic compounds, in particular to a process for preparing long-chain dibasic acids by biological transformation. Background Art

[0002] Long chain dicarboxylic acids refer to aliphatic dicarboxylic acids (DCn for short) with more than 10 carbon atoms in the carbon chain, including saturated and unsaturated dicarboxylic acids. They are a kind of fine chemical products with important and extensive industrial applications. They are also important raw materials for synthesizing high-grade fragrances, high-performance nylon engineering plastics, high-grade nylon hot melt adhesives, high-temperature dielectrics, high-grade paints and coatings, high-grade lubricants, cold-resistant plasticizers, resins, medicines and pesticides in the chemical industry.

[0003] There are also some technical solutions for preparing long-chain dibasic acids by bioconversion in the prior art. For example, a Chinese patent with publication number CN116497071A discloses a method for preparing long-chain dibasic acids by bioconversion, which comprises the following contents: (1) adding a non-polar macroporous carrier to an impregnation solution containing a conditioning agent and an alkane, and performing an impregnation treatment to obtain an impregnation mixture; (2) expanding the culture of the activated long-chain dibasic acid fermentation bacteria, adding the impregnation mixture prepared in step (1) in the middle and late stages of the culture, and activating the strain by pH control. , further obtaining a seed liquid of the fermentation strain; (3) adding the seed liquid obtained in step (2) to the fermentation medium, and preparing the long-chain dibasic acid by aerobic fermentation mode; the present invention aims at the microbial metabolic characteristics of the long-chain dibasic acid fermentation strain expansion stage, and uses a specific carrier adsorption mode to achieve the concentration enrichment of the induction substrate, which is conducive to the formation of a strain / substrate contact unit, can improve the induction effect, and improve the strain activity; however, this technical solution cannot avoid the slow reproduction speed of the long-chain dibasic acid fermentation bacteria, resulting in a reduction in the preparation rate of the entire long-chain dibasic acid;

[0004] In view of this, in order to overcome the above technical problems, the present invention proposes a process for preparing long-chain dibasic acids by bioconversion, which solves the above technical problems. Summary of the invention

[0005] In order to make up for the shortcomings of the prior art, the present invention proposes a process for preparing long-chain dibasic acids by bioconversion. The present invention adds the expansion culture medium drop by drop in a constant temperature shaker in a gradual addition manner, so that the culture bottle does not need to be taken out, thereby avoiding instantaneous changes in the ambient temperature of the culture bottle, preventing the temperature from affecting the growth and expansion efficiency of Candida, and at the same time, avoiding the risk of introducing exogenous pollutants caused by taking out the culture bottle; and compared with adding all the expansion culture medium at one time, the gradual addition can not only reduce the accumulation of metabolites caused by excess nutrition; it is also beneficial to the stability of the cell growth environment; and improve the efficiency of the expansion culture of Candida.

[0006] The technical solution adopted by the present invention to solve the technical problem is: a process for preparing long-chain dibasic acid by bioconversion according to the present invention comprises the following steps:

[0007] S1: adding a non-polar macroporous carrier to an impregnation solution containing a conditioning agent and an alkane, wherein the ratio of the conditioning agent to the alkane is 1:2 to 1:3; and the mass ratio of the non-polar macroporous carrier to the impregnation solution is 1:3 to 1:4, the impregnation temperature is 30 to 35° C., and the impregnation time is 5 to 10 hours, to finally obtain an impregnation mixture;

[0008] S2: The activated long-chain dibasic acid fermentation bacteria (Candida) are inoculated into the expansion medium. Before inoculation, an appropriate amount of expansion medium is dripped into the culture bottle for inoculation of the activated long-chain dibasic acid fermentation bacteria. The culture bottle is then placed in a constant temperature shaker. The remaining expansion medium is continuously dripped into the culture bottle in the constant temperature shaker in a drop-by-drop manner. The culture temperature is 25-30°C.

[0009] S3: When the pH in the culture bottle drops to 4.5, the prepared impregnation mixture is added to control the pH value in the range of 3 to 5 to activate the strains and further obtain the seed liquid of the fermentation strains;

[0010] S4: Add the fermentation medium dropwise into the culture bottle of the seed solution, and use a constant temperature shaker to ferment the seed solution in the culture bottle. The fermentation temperature is 25-35°C; the oxygen ventilation volume is 0.2-0.35 vvm; the fermentation time is 130-155 hours; and the desired long-chain dibasic acid is obtained.

[0011] Preferably, in S2 and S4, the droplets of the expansion medium and the fermentation medium added dropwise are both filled with oxygen.

[0012] Preferably, the fermentation seed liquid accounts for 10% to 20% of the fermentation medium.

[0013] Preferably, the temperature of the oxygen introduced into S2 and S4 is 25°C.

[0014] Preferably, the constant temperature shaking incubator comprises:

[0015] A box body, wherein a box cover is provided at the upper end of the box body; a box door is provided at one side of the box body; a heating resistor is embedded in the side wall of the box body; a tray is provided inside the box body; the tray is fixedly connected to the bottom of the box body through a supporting spring; a driving motor is fixedly installed at the bottom of the box body; an eccentric wheel is fixedly connected to the output end of the driving motor; the eccentric wheel is rotatably connected to the bottom of the tray;

[0016] A dripping plate, wherein a support plate is installed on the inner wall of the box body; the dripping plate is placed on the support plate; the dripping plate is located above the tray; a handle is fixedly connected to the upper end of the dripping plate; a liquid storage cavity is provided at the upper end of the dripping plate; a dripping hole connected to the liquid storage cavity is provided at the lower end of the dripping plate; a cavity is provided inside the dripping plate; the liquid storage cavity and the dripping hole are connected by a hose; a pushing unit is installed in the cavity; the pushing unit is used to push the solution in the hose to be discharged.

[0017] Preferably, the pushing unit includes a pushing wheel; the pushing wheel is rotatably connected in the cavity; the hose is wrapped around the pushing wheel and the inner wall of the cavity; a servo motor is installed on one side of the box; the servo motor is used to drive the pushing wheel to rotate.

[0018] Preferably, there are at least two liquid storage chambers; a connecting tube is fixedly connected to the bottom of the liquid storage chamber; a liquid inlet is provided on the surface of the connecting tube; a solenoid valve is fixedly installed inside the connecting tube; and both connecting tubes are connected to a hose.

[0019] Preferably, a rotating rod is rotatably connected in the cavity; there are two rotating rods; the two rotating rods are connected by a belt drive; a rectangular groove is provided at the output end of the servo motor; a connecting rod is slidably connected in the rectangular groove; the connecting rod is connected to the bottom of the rectangular groove by a fixed spring; an electromagnetic sheet is inlaid at the bottom of the rectangular groove; a slot matching the connecting rod is provided at one end of the rotating rod close to the servo motor.

[0020] Preferably, a gas pipe is fixedly installed on the side wall of the box; a needle groove is provided on the inner wall of the drip hole; a gas injection needle is slidably and sealingly connected in the needle groove; the gas injection pipe is fixedly connected to the end of the gas injection needle away from the drip hole; a pull rod is slidably connected to the handle and the lower end; the pull rod and the plug are connected by a steel wire rope; a slot is provided on the side wall of the drip plate; a plug is fixedly connected to the end of the gas injection pipe away from the gas injection needle; the plug is slidably and sealingly connected in the slot; the plug and the bottom of the slot are connected by a reset spring.

[0021] Preferably, a push groove is provided on one side of the cavity; a push rod is connected to the push groove in a sliding seal; the push rod and the bottom of the push groove are fixedly connected by a push spring; a slide groove is provided at the bottom of the needle groove; a sealing plate is connected to the slide groove in a sliding seal; the push groove and the needle groove are connected through an airway.

[0022] The beneficial effects of the present invention are as follows:

[0023] 1. The present invention adds the expansion culture medium dropwise in a constant temperature shaker in a gradual addition manner, so that the culture bottle does not need to be taken out, thereby avoiding instantaneous changes in the ambient temperature of the culture bottle, preventing the temperature from affecting the growth and expansion efficiency of Candida, and at the same time, avoiding the risk of introducing exogenous pollutants caused by taking out the culture bottle; and compared with adding all the expansion culture medium at one time, the gradual addition can not only reduce the accumulation of metabolites caused by excess nutrition, but also be beneficial to the stability of the cell growth environment, and improve the efficiency of the expansion culture of Candida.

[0024] 2. The present invention fills oxygen into the droplets of expansion culture medium added drop by drop, and fills the culture bottle with oxygen instead of air, thereby avoiding the entry of external carbon dioxide and water vapor, so as to ensure the stability of the pH value, humidity and concentration of the culture medium in the culture bottle, thereby ensuring that the Candida in the culture bottle can grow and reproduce rapidly and stably, accelerating the metabolic activity of the Candida and the synthesis of products; thereby improving the expansion culture rate of the Candida, so that the yield and quality of the dibasic acid in the fermentation process are improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention is further described below in conjunction with the accompanying drawings and implementation modes.

[0026] Figure 1 It is a process flow chart of the present invention;

[0027] Figure 2 is a stereogram of a constant temperature shaking table used in the present invention;

[0028] Figure 3 It is a structural schematic diagram of the constant temperature shaking table used in the present invention;

[0029] Figure 4 yes Figure 3 The enlarged view of point A in the middle;

[0030] Figure 5 yes Figure 3 The enlarged view of point B in the middle;

[0031] Figure 6 Schematic diagram of the structure of the drip plate used in the present invention;

[0032] Figure 7yes Figure 6 Enlarged view of point C in the middle;

[0033] Figure 8 It is a schematic diagram of the structure of the rotating rod used in the present invention;

[0034] In the figure: 1, box body; 11, box cover; 12, heating resistor; 13, tray; 131, support spring; 132, drive motor; 133, eccentric wheel; 14, support plate; 15, air pipe; 16, pull rod; 17, box door; 2, drip plate; 21, handle; 22, liquid storage chamber; 221, drip hole; 23, cavity; 231, driving wheel; 232, hose; 233, servo motor; 234, connecting pipe; 23 5. Solenoid valve; 236. Rotating rod; 24. Rectangular groove; 241. Connecting rod; 242. Fixed spring; 243. Electromagnetic sheet; 25. Card slot; 26. Needle slot; 261. Gas injection needle; 262. Gas injection tube; 27. Slot; 271. Plug; 272. Return spring; 273. Wire rope; 28. Pushing groove; 281. Pushing rod; 282. Pushing spring; 29. ​​Sliding groove; 291. Sealing plate; 292. Airway. DETAILED DESCRIPTION

[0035] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0036] like Figures 1 to 8 As shown, the process for preparing long-chain dibasic acid by bioconversion according to the present invention comprises the following steps:

[0037] S1: adding a non-polar macroporous carrier to an impregnation solution containing a conditioning agent and an alkane, wherein the ratio of the conditioning agent to the alkane is 1:2 to 1:3; and the mass ratio of the non-polar macroporous carrier to the impregnation solution is 1:3 to 1:4, the impregnation temperature is 30 to 35° C., and the impregnation time is 5 to 10 hours, to finally obtain an impregnation mixture;

[0038] S2: The activated long-chain dibasic acid fermentation bacteria (Candida) are inoculated into the expansion medium. Before inoculation, an appropriate amount of expansion medium is dripped into the culture bottle for inoculation of the activated long-chain dibasic acid fermentation bacteria. The culture bottle is then placed in a constant temperature shaker. The remaining expansion medium is continuously dripped into the culture bottle in the constant temperature shaker in a drop-by-drop manner. The culture temperature is 25-30°C.

[0039] S3: When the pH in the culture bottle drops to 4.5, the prepared impregnation mixture is added to control the pH value in the range of 3 to 5 to activate the strains and further obtain the seed liquid of the fermentation strains;

[0040] S4: Add the fermentation medium dropwise into the culture bottle of the seed solution, and use a constant temperature shaker to ferment the seed solution in the culture bottle. The fermentation temperature is 25-35°C; the oxygen ventilation volume is 0.2-0.35 vvm; the fermentation time is 130-155 hours; and the desired long-chain dibasic acid is obtained.

[0041] As an embodiment of the present invention, in S2 and S4, the droplets of the expansion medium and the fermentation medium added dropwise are both filled with oxygen.

[0042] As an embodiment of the present invention, the fermentation seed liquid accounts for 10% to 20% of the fermentation medium.

[0043] As an embodiment of the present invention, the temperature of the oxygen introduced into S2 and S4 is 25°C;

[0044] During operation, the expansion medium is 10g / L sucrose, 2g / L corn steep liquor, 2.0g / L yeast extract, 1.2g / L sodium chloride, 3.0g / L potassium dihydrogen phosphate, and 1.8g / L magnesium sulfate; the fermentation medium is 25g / L sucrose, 1.0g / L corn steep liquor, 2.0g / L yeast extract, 0.8g / L sodium chloride, 3.0g / L potassium dihydrogen phosphate, 1.2g / L magnesium sulfate, 4.0g / L urea, 2.0g / L ammonium sulfate, 1.5g / L sodium acetate, and 160g / L alkane;

[0045] In the prior art, when Candida is inoculated into an expansion medium, the expansion medium required for expansion is usually added directly into a culture bottle, and then the Candida is inoculated, and then the culture bottle inoculated with Candida is directly placed in a constant temperature shaker for expansion culture. Compared with adding successively, there is no need to repeatedly take out the culture bottle from the constant temperature shaker, so as to reduce the instantaneous changes of conditions such as culture temperature, thereby reducing the impact on the growth and expansion efficiency of Candida. At the same time, it also reduces the risk of introducing exogenous contaminants caused by taking out the culture bottle. However, adding all the expansion medium to the culture bottle at one time is likely to cause excess nutrients in the culture medium, which will have an impact on the growth of the strain and the quality of the product. Negative effects, such as excessive carbon source will promote excessive cell growth and acid production, leading to the accumulation of metabolites, thus causing cell growth stagnation; in this regard, the present invention adds the expansion culture medium drop by drop in a constant temperature shaker by gradual addition, so that there is no need to take out the culture bottle, avoiding instantaneous changes in the ambient temperature of the culture bottle, preventing the temperature from affecting the growth and expansion efficiency of Candida, and at the same time, avoiding the risk of introducing exogenous pollutants caused by taking out the culture bottle; and compared with adding all the expansion culture medium at one time, the gradual addition can not only reduce the accumulation of metabolites caused by excess nutrition, but also be beneficial to the stability of the cell growth environment; and improve the efficiency of the expansion culture of Candida;

[0046] Moreover, before inoculation, if all the expansion medium is added to the culture bottle at once and then inoculated, the Candida inoculated into the culture bottle will be affected by the sudden change of environmental parameters such as temperature and pH. This sudden change of environment will cause excessive shock and stress to the Candida. Moreover, the cells need a certain amount of time to adapt to the new environmental conditions. If all the expansion medium is added at once, the cells will be unable to adjust their metabolic activities in time due to excess nutrients, resulting in metabolic imbalance and cell stress, and even inoculation failure, affecting the efficiency of the entire biotransformation to prepare long-chain dibasic acids. For this, an appropriate amount of expansion medium is dripped into the culture bottle, and the culture bottle with the expansion medium needs to be heated to ensure that the temperature of the expansion medium is consistent with the temperature of the inoculated Candida. The effect of lowering temperature on Candida inoculation is consistent, and the appropriate amount of expansion medium is conducive to the gradual adaptation of Candida cells to new environmental conditions, so that the metabolic activities of Candida are gradually adjusted, the production of growth inhibitors is reduced, and it is beneficial to cell growth and expansion. In addition, the carbon source (such as sucrose) in the expansion medium will be used by Candida to produce acidic metabolites, thereby affecting the pH value of the medium, so that the culture speed of Candida will be reduced. The gradual addition of expansion medium gradually increases the nutrients and carbon sources in the medium, thereby slowing down the drastic fluctuations in the pH value, and helping to maintain the stability of the acid-base balance in the medium, so as to maintain the appropriate pH range for the growth and reproduction of Candida, thereby improving the expansion efficiency of Candida.

[0047] Since Candida releases heat during cell metabolism, if a large amount of culture medium is added at one time, the concentration of nutrients will suddenly increase, which will increase cell metabolism and release more heat. Therefore, the temperature in the culture bottle will change greatly, and the temperature change will affect the growth and reproduction of Candida. Adding culture medium drop by drop will make the increase of nutrients and carbon sources in the culture medium more gradual. Compared with adding a large amount of nutrients at one time, cell metabolism is stable and heat release is relatively small. Moreover, the gradually added culture medium will slow down the temperature fluctuation in the culture bottle, ensuring the stability of the expansion culture of Candida and improving the efficiency of the expansion culture of Candida.

[0048] Although Candida is a facultative anaerobe, that is, it can grow and reproduce in both aerobic and anaerobic environments, in an aerobic environment, there is sufficient oxygen, and Candida obtains more energy through oxidative respiration, thereby promoting the growth and reproduction of Candida. Industrial oxygen filling is to supply oxygen to Candida by introducing air, but the oxygen content in the air is low, which limits the oxidative respiration process of Candida, affects the cell energy supply, and leads to a slow expansion culture rate and fermentation culture rate of Candida. In addition, the air also contains other gases, which will also have a certain impact on the fermentation process of Candida to produce dibasic acid.

[0049] For example, although nitrogen in the air will not directly participate in chemical reactions during expansion culture and fermentation culture, excessive nitrogen will occupy the internal space of the constant temperature shaker, affecting the mixing uniformity of oxygen and other gases, thereby affecting the efficiency of Candida albicans to utilize oxygen; the carbon dioxide content in the air is relatively high, and the addition of carbon dioxide will cause the acid-base balance in the culture bottle to be disturbed, affecting the metabolic activity of Candida albicans and the synthesis of products; thus affecting the yield and quality of dibasic acids in the fermentation process; in addition, the air also contains water vapor, which directly affects the humidity in the fermentation tank, the concentration of the culture medium and other parameters, thereby indirectly affecting the growth of Candida albicans and product synthesis;

[0050] In this regard, the present invention fills oxygen into the droplets of expansion culture medium added drop by drop, and fills the culture bottle with oxygen instead of air, thereby avoiding the entry of external carbon dioxide and water vapor, so as to ensure the stability of pH value, humidity and concentration of culture medium in the culture bottle, thereby ensuring that Candida in the culture bottle can grow and reproduce rapidly and stably, accelerating the metabolic activity of Candida and the synthesis of products; thereby improving the expansion culture rate of Candida, so that the yield and quality of dibasic acid in the fermentation process are improved;

[0051] Moreover, the production cost of oxygen is relatively high. If oxygen is directly delivered to the culture medium through a transmission pipeline, the oxygen concentration in the constant temperature shaker will be too high. On the one hand, it will cause the oxygen to be not fully utilized and cause oxygen waste. On the other hand, when Candida is in a high-oxygen environment for a long time, it will increase the lipid peroxidation of the cell membrane, DNA damage, etc., affecting the normal physiological function of the cells; and then directly affecting the growth and reproduction rate of Candida; and the droplets of the expansion culture medium and the fermentation culture medium added drop by drop are filled with oxygen. In the process of ensuring that oxygen can be filled into the culture medium, on the one hand, it can reduce the oxygen filling rate and reduce the oxygen waste caused by excessive oxygen filling, thereby reducing the preparation cost of long-chain dibasic acids. On the other hand, it avoids problems such as lipid peroxidation and DNA damage of the cell membrane of Candida, so as to ensure the normal growth and reproduction of Candida, and improve the growth and reproduction rate of Candida; by setting the temperature of oxygen to 25°C, in the process of dropwise addition of droplets of expansion medium and fermentation medium, the 25°C oxygen filled into the droplets of expansion medium and fermentation medium can cool down the aerobic respiration and heat release of Candida, so as to ensure that the environmental temperature for the growth and reproduction of Candida can be stabilized at 25-30°C, thereby improving the growth and reproduction rate of Candida in the expansion medium, and further improving the yield and rate of Candida oxidizing alkanes to obtain dibasic acids in the fermentation medium.

[0052] As an embodiment of the present invention, the constant temperature shaking table comprises:

[0053] A box body 1, wherein a box cover 11 is provided at the upper end of the box body 1; a box door 17 is provided at one side of the box body 1; a heating resistor 12 is inlaid on the side wall of the box body 1; a tray 13 is provided inside the box body 1; the tray 13 is fixedly connected to the bottom of the box body 1 through a supporting spring 131; a driving motor 132 is fixedly installed at the bottom of the box body 1; an eccentric wheel 133 is fixedly connected to the output end of the driving motor 132; the eccentric wheel 133 is rotatably connected to the bottom of the tray 13;

[0054] A dripping plate 2, wherein a support plate 14 is installed on the inner wall of the box body 1; the dripping plate 2 is placed on the support plate 14; the dripping plate 2 is located above the tray 13; a handle 21 is fixedly connected to the upper end of the dripping plate 2; a liquid storage cavity 22 is provided at the upper end of the dripping plate 2; a dripping hole 221 connected to the liquid storage cavity 22 is provided at the lower end of the dripping plate 2; a cavity 23 is provided inside the dripping plate 2; the liquid storage cavity 22 and the dripping hole 221 are connected by a hose 232; a pushing unit is installed in the cavity 23; the pushing unit is used to push the solution in the hose 232 to be discharged.

[0055] As an embodiment of the present invention, the pushing unit includes a pushing wheel 231; the pushing wheel 231 is rotatably connected in the cavity 23; the hose 232 is wrapped around the pushing wheel 231 and the inner wall of the cavity 23; a servo motor 233 is installed on one side of the box body 1; the servo motor 233 is used to drive the pushing wheel 231 to rotate.

[0056] As an embodiment of the present invention, the number of the liquid storage chambers 22 is set to at least two; a connecting pipe 234 is fixedly connected to the bottom of the liquid storage chamber 22; a liquid inlet is opened on the surface of the connecting pipe 234; a solenoid valve 235 is fixedly installed inside the connecting pipe 234; and the two connecting pipes 234 are connected to the hose 232.

[0057] As an embodiment of the present invention, a rotating rod 236 is rotatably connected in the cavity 23; the number of the rotating rods 236 is set to two; the two rotating rods 236 are connected by a belt transmission; a rectangular groove 24 is provided at the output end of the servo motor 233; a connecting rod 241 is slidably connected in the rectangular groove 24; the connecting rod 241 is connected to the bottom of the rectangular groove 24 by a fixing spring 242; an electromagnetic sheet 243 is inlaid at the bottom of the rectangular groove 24; a card slot 25 matching the connecting rod 241 is provided at one end of the rotating rod 236 close to the servo motor 233;

[0058] During operation, a temperature sensor is installed inside the box 1, and a bracket is installed on the upper end of the tray 13; before inoculating Candida, the user first drips an appropriate amount of expansion culture medium into the culture bottle, and then inoculates Candida into the culture bottle, then opens the box cover 11 and places the culture bottle in the bracket on the upper end of the tray 13, and the two liquid storage chambers 22 on the upper end of the drip plate 2 are filled with expansion culture medium and fermentation culture medium respectively, and then the user first controls the electromagnetic sheet 243 to energize, so that the electromagnetic sheet 243 can absorb the connecting rod 241 and squeeze the fixed spring 242 into the rectangular groove 24, thereby preventing the connecting rod 241 from blocking the drip plate 2 from entering the box 1; then the drip plate 2 is placed in the box by holding the handle 21 The support plate 14 in the box 1 is placed on the support plate 14; at this time, the drip hole 221 under the drip plate 2 is directly opposite to the culture bottle; the box cover 11 is closed, and the electromagnetic sheet 243 is controlled to be powered off. The connecting rod 241 extends out of the rectangular groove 24 under the push of the restoring force of the fixed spring 242 and connects with the rotating rod 236; at the same time, the heating resistor 12 in the box 1 is controlled to operate so that the heating resistor 12 heats the temperature in the box 1. When the temperature sensor detects that the internal temperature of the knife reaches 30°C, the heating resistor 12 is controlled to stop. At the same time, the driving motor 132 drives the tray 13 to swing through the eccentric wheel 133, so that the tray 13 can drive the culture bottle in the bracket to shake, so that the Candida in the culture bottle and the inside of the culture bottle The expansion culture medium is fully mixed, and the precipitation of cells or other substances in the culture medium is reduced, and the uniformity and stability of the culture solution are maintained; it is beneficial to the growth and metabolic activities of cells, thereby accelerating the expansion culture efficiency of Candida; since the culture bottle is shaken with the tray 13 with a small amplitude, excessive shaking amplitude will cause the culture solution in the culture bottle to splash out, so during the small shaking of the culture bottle, the drip hole 221 under the drip plate 2 is always aligned with the bottle mouth of the culture bottle; it is ensured that the solution dripped from the drip hole 221 can flow into the culture bottle; since the connecting rod 241 is in contact with the rotating rod 236, the connecting rod 241 is rotated to the position aligned with the card slot during the process of the servo motor 233 driving the connecting rod 241 to rotate 25 matching state, so that the connecting rod 241 can be inserted into the card slot 25, so that the servo motor 233 drives the rotating rod 236 to rotate through the connecting rod 241, so that the rotating rod 236 drives the driving wheel 231 in the cavity 23 to rotate. There are three driving wheels 231 in the same cavity 23, and the three driving wheels 231 are all connected to the rotating rod 236. When the driving wheel 231 is driven by the rotating rod 236 to rotate, the driving wheel 231 can squeeze the hose 232, so that the gas at the squeezed part of the hose 232 is discharged through the drip hole 221 under the push of the driving wheel 231. At this time, the air pressure in the hose 232 is reduced, and the liquid in the connecting pipe 234 connected with the hose 232 enters the hose 232;Because the two connecting tubes 234 are connected to the hose 232, in order to prevent the fermentation medium from entering the hose 232 through the connecting tube 234 when the Candida is expanded, an electromagnetic valve 235 is installed in the connecting tube 234, so that the electromagnetic valve 235 is in a closed state in the initial state. When the Candida is expanded and cultured, only the electromagnetic valve 235 in the connecting tube 234 inserted with the expansion medium is opened, so that the expansion medium in the liquid storage chamber 22 can flow into the hose 232 through the connecting tube 234, and the expansion medium flowing into the hose 232 is pushed by the driving wheel 231 to the culture bottle through the drip hole 221. By controlling the rotation speed of the servo motor 233, the time for the interval dripping of the expansion medium can be adjusted, the rate of dripping of the expansion medium can be controlled, and then the stability of the expansion culture of Candida is controlled, the efficiency of the expansion culture of Candida is improved, and the overall time for preparing long-chain dibasic acids can be shortened. The preparation efficiency of long-chain dibasic acid is improved; when the expansion is completed, it is only necessary to open the box door 17 and add the impregnation mixture into the culture bottle, so that the culture bottle does not need to be taken out, which not only avoids the instantaneous change of the ambient temperature of the culture bottle, but also reduces the risk of exogenous pollutants entering; after the addition is completed, the box door 17 is closed, and the electromagnetic valve 235 in the connecting pipe 234 inserted into the expansion culture medium is controlled to be closed, and the electromagnetic valve 235 in the connecting pipe 234 inserted into the fermentation culture medium is opened, so that the driving wheel 231 drives the fermentation culture liquid to drip into the culture bottle through the hose 232, thereby realizing the addition of the fermentation culture medium in a drop-by-drop manner, so that the Candida can grow and reproduce rapidly and stably during the fermentation culture process, and accelerate the metabolic activity of the Candida and the synthesis of the product; thereby improving the fermentation culture rate of the Candida, so that the yield and quality of the dibasic acid in the fermentation process are also correspondingly improved. ;

[0059] As an embodiment of the present invention, a gas supply pipe 15 is fixedly installed on the side wall of the box body 1; a needle groove 26 is provided on the inner wall of the drip hole 221; a gas injection needle 261 is slidably and sealedly connected in the needle groove 26; the end of the gas injection needle 261 away from the drip hole 221 is fixedly connected to the gas injection pipe 262; the handle 21 is slidably connected to the lower end with a pull rod 16; the pull rod 16 and the plug 271 are connected by a steel wire rope 273; a slot 27 is provided on the side wall of the drip plate 2; the end of the gas injection pipe 262 away from the gas injection needle 261 is fixedly connected to the plug 271; the plug 271 is slidably and sealedly connected in the slot 27; the plug 271 and the bottom of the slot 27 are connected by a reset spring 272.

[0060] As an embodiment of the present invention, a push groove 28 is provided on one side of the cavity 23; a push rod 281 is slidably and sealably connected in the push groove 28; the push rod 281 is fixedly connected to the bottom of the push groove 28 via a push spring 282; a slide groove 29 is provided at the bottom of the needle groove 26; a sealing plate 291 is slidably and sealably connected in the slide groove 29; the push groove 28 and the needle groove 26 are connected via an airway 292;

[0061] During operation, because the expansion medium is sucrose 10g / L, corn syrup 2g / L, yeast extract 2.0g / L, sodium chloride 1.2g / L, potassium dihydrogen phosphate 3.0g / L, magnesium sulfate 1.8g / L; among which the concentrations of sucrose, corn syrup and yeast extract are relatively high; so the mixed solution is slightly viscous, while the fermentation medium is sucrose 25g / L, corn syrup 1.0g / L, yeast extract 2.0g / L, sodium chloride 0.8g / L, potassium dihydrogen phosphate 3.0g / L, magnesium sulfate 1.2g / L, urea 4.0g / L, ammonium sulfate 2.0g / L, sodium acetate 1.5g / L, alkane 160g / L; the concentrations of sucrose, corn syrup, yeast extract, sodium chloride, potassium dihydrogen phosphate, magnesium sulfate, urea, ammonium sulfate and sodium acetate are relatively high, and the concentration of alkane is even higher; so the mixed solution is viscous;

[0062] When oxygen is injected into a viscous solution, the oxygen bubbles will be suspended in the solution, and will not float up quickly like in water; this is because when the bubbles are injected into the viscous solution, they will be subject to the viscous resistance of the surrounding solution, and due to the effect of viscosity, the bubbles will also be suspended in the solution for a relatively long time, and will not disperse quickly; therefore, when the viscous solution drips from the droplet mouth, the oxygen bubbles wrapped in the solution will drip into the culture bottle together with the droplets, thereby achieving oxygen filling in the culture bottle; when in use, the user, while holding the handle 21, Grip the pull rod 16, pull the plug 271 through the pull rod 16 to squeeze the return spring 272 into the slot 27, after the drip plate 2 is placed in the box 1, the slot 27 of the drip plate 2 is directly opposite to the gas pipe 15, when the user releases the grip 21, the plug 271 extends out of the slot 27 and enters the gas pipe 15 under the push of the restoring force of the return spring 272; so that the gas pipe 15 can be transported to the slot 27 through the plug 271, and enter the gas injection pipe 262 through the slot 27, so that the oxygen in the gas injection pipe 262 can be transported to the drip hole 221 through the gas injection needle 261; to ensure the drip The liquid droplets dripping from the liquid hole 221 can effectively wrap the oxygen injected by the gas injection needle 261. The present invention sets a push rod 281. When the rotating rod 236 drives the push wheel 231 to rotate, the push wheel 231 can contact the push rod 281, so that the push rod 281 squeezes the push spring 282 into the push groove 28 under the push of the push wheel 231, so that the hydraulic oil in the push groove 28 is squeezed into the slide groove 29 through the air channel 292, so that the hydraulic oil pushes the sealing plate 291 in the slide groove 29 to move toward the direction close to the liquid drop hole 221, so that the sealing plate 291 pushes the gas injection. The needle 261 extends out of the needle slot 26 and is inserted into the solution in the drip hole 221, so that the gas injection needle 261 inserted into the drip hole 221 can be injected into the center of the solution in the drip hole 221, so that the solution dripping from the drip hole 221 can bend and wrap the oxygen bubbles, thereby preventing the oxygen bubbles from escaping, ensuring that the droplets dripped into the culture bottle can stably bring oxygen into the culture bottle; ensuring that the Candida in the culture bottle can be fermented quickly and stably, thereby improving the fermentation culture rate of the Candida, and the yield and quality of the dibasic acid in the fermentation process are also correspondingly improved.

[0063] In the description of the present invention, it should be noted that the terms "center", "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate directions or positional relationships based on the attached Figure 1The orientation or positional relationship shown is only for the convenience of describing the present invention and simplifying the description, and does not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation. Therefore, it cannot be understood as limiting the scope of protection of the present invention. In addition, the terms "first", "second", "third", etc. are only used to distinguish the description and cannot be understood as indicating or implying relative importance.

[0064] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A process for preparing long-chain dibasic acids by bioconversion, characterized in that: The following steps are involved: S1: adding a non-polar macroporous carrier to an impregnation solution containing a conditioning agent and an alkane, wherein the ratio of the conditioning agent to the alkane is 1:2 to 1:3; and the mass ratio of the non-polar macroporous carrier to the impregnation solution is 1:3 to 1:4, the impregnation temperature is 30 to 35° C., and the impregnation time is 5 to 10 hours, to finally obtain an impregnation mixture; S2: The activated long-chain dibasic acid fermentation bacteria are inoculated into the expansion medium. The long-chain dibasic acid fermentation bacteria are Candida. Before inoculation, an appropriate amount of expansion medium is dripped into the culture bottle for inoculation of the activated long-chain dibasic acid fermentation bacteria. The culture bottle is then placed in a constant temperature shaker. The remaining expansion medium is continuously dripped into the culture bottle in the constant temperature shaker in a drop-by-drop manner. The culture temperature is 25-30°C. S3: When the pH in the culture bottle drops to 4.5, the prepared impregnation mixture is added to control the pH value in the range of 3 to 5 to activate the strains and further obtain the seed liquid of the fermentation strains; S4: adding the fermentation medium dropwise into the culture bottle of the seed solution, fermenting the seed solution in the culture bottle using a constant temperature shaker, the fermentation temperature is 25-35°C; the oxygen ventilation volume is 0.2-0.35 vvm; the fermentation time is 130-155 hours; and the desired long-chain dibasic acid is obtained; The constant temperature shaking table comprises: A box body (1), wherein a box cover (11) is provided at the upper end of the box body (1); a box door (17) is provided at one side of the box body (1); a heating resistor (12) is embedded in the side wall of the box body (1); a tray (13) is provided inside the box body (1); the tray (13) is fixedly connected to the bottom of the box body (1) via a supporting spring (131); a driving motor (132) is fixedly installed at the bottom of the box body (1); an eccentric wheel (133) is fixedly connected to the output end of the driving motor (132); the eccentric wheel (133) is rotatably connected to the bottom of the tray (13); A dripping plate (2), wherein a support plate (14) is installed on the inner wall of the box body (1); the dripping plate (2) is placed on the support plate (14); the dripping plate (2) is located above the tray (13); a handle (21) is fixedly connected to the upper end of the dripping plate (2); a liquid storage cavity (22) is provided at the upper end of the dripping plate (2); a dripping hole (221) connected to the liquid storage cavity (22) is provided at the lower end of the dripping plate (2); a cavity (23) is provided inside the dripping plate (2); the liquid storage cavity (22) and the dripping hole (221) are connected via a hose (232); a pushing unit is installed in the cavity (23); the pushing unit is used to push the solution in the hose (232) to be discharged; The pushing unit comprises a pushing wheel (231); the pushing wheel (231) is rotatably connected in the cavity (23); the hose (232) is wrapped around the pushing wheel (231) and the inner wall of the cavity (23); a servo motor (233) is installed on one side of the box body (1); the servo motor (233) is used to drive the pushing wheel (231) to rotate; The number of the liquid storage chambers (22) is at least two; a connecting pipe (234) is fixedly connected to the bottom of the liquid storage chamber (22); a liquid inlet is provided on the surface of the connecting pipe (234); a solenoid valve (235) is fixedly installed inside the connecting pipe (234); the two connecting pipes (234) are both connected to the hose (232); A rotating rod (236) is rotatably connected in the cavity (23); there are two rotating rods (236); the two rotating rods (236) are connected via a belt transmission; a rectangular groove (24) is provided at the output end of the servo motor (233); a connecting rod (241) is slidably connected in the rectangular groove (24); the connecting rod (241) is connected to the bottom of the rectangular groove (24) via a fixing spring (242); an electromagnetic sheet (243) is embedded in the bottom of the rectangular groove (24); and a clamping groove (25) matching with the connecting rod (241) is provided at one end of the rotating rod (236) close to the servo motor (233).

2. The process for preparing long-chain dibasic acid by bioconversion according to claim 1, characterized in that: In S2 and S4, the droplets of the expansion medium and fermentation medium added dropwise are filled with oxygen.

3. The process for preparing long-chain dibasic acid by bioconversion according to claim 1, characterized in that: The fermentation seed liquid accounts for 10% to 20% of the fermentation medium.

4. The process for preparing long-chain dibasic acid by bioconversion according to claim 1, characterized in that: The temperature of the oxygen introduced into S2 and S4 is 25°C.

5. The process for preparing long-chain dibasic acid by bioconversion according to claim 1, characterized in that: An air supply pipe (15) is fixedly installed on the side wall of the box body (1); a needle groove (26) is provided on the inner wall of the drip hole (221); an air injection needle (261) is slidably and sealingly connected in the needle groove (26); an air injection pipe (262) is fixedly connected to the end of the air injection needle (261) away from the drip hole (221); a pull rod (16) is slidably connected to the lower end of the handle (21); a steel wire rope (273) is connected between the pull rod (16) and the plug (271); a slot (27) is provided on the side wall of the drip plate (2); a plug (271) is fixedly connected to the end of the air injection pipe (262) away from the air injection needle (261); the plug (271) is slidably and sealingly connected in the slot (27); the plug (271) and the bottom of the slot (27) are connected via a return spring (272).

6. The process for preparing long-chain dibasic acid by bioconversion according to claim 5, characterized in that: A push groove (28) is provided on one side of the cavity (23); a push rod (281) is slidably and sealably connected inside the push groove (28); the push rod (281) is fixedly connected to the bottom of the push groove (28) via a push spring (282); a slide groove (29) is provided at the bottom of the needle groove (26); a sealing plate (291) is slidably and sealably connected inside the slide groove (29); the push groove (28) and the needle groove (26) are connected via an airway (292).

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