CN111763636B - Method for producing coenzyme Q10 by industrial fermentation - Google Patents

Method for producing coenzyme Q10 by industrial fermentation Download PDF

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CN111763636B
CN111763636B CN202010539816.7A CN202010539816A CN111763636B CN 111763636 B CN111763636 B CN 111763636B CN 202010539816 A CN202010539816 A CN 202010539816A CN 111763636 B CN111763636 B CN 111763636B
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韩祎君
巴国政
张玉东
弓吉龙
刘艳新
王瑞霞
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Shenzhou Biology & Technology Co ltd
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Abstract

The invention relates to a preparation method of coenzyme Q10, in particular to a method for producing coenzyme Q10 by industrial fermentation. The method adopts rhodobacter sphaeroides as a strain, and comprises the steps of flat plate culture of the strain, slant culture of the strain, 20L seed culture, primary seed tank culture, secondary seed culture, tertiary fermentation culture, seed pouring and secondary culture fermentation. The invention solves the problems of incomplete consumption of culture medium in fermentation broth, high impurity content, low effective unit, low extraction yield, difficult control of industrial salt quality, low yield and high pollution discharge caused by low unit hypha, and excessive COD discharge caused by unutilized organic matters in the discharge, thereby increasing environmental protection cost. The invention has simple process flow and low risk of bacteria infection, saves fermentation cost, improves fermentation yield, increases process reserve and increases production.

Description

Method for producing coenzyme Q10 by industrial fermentation
Technical Field
The invention relates to a preparation method of coenzyme Q10, in particular to a method for producing coenzyme Q10 by industrial fermentation.
Background
Coenzyme Q10 (CoenzymeQ, coQ 10), also known as ubiquinone, is a retinoid that is present in very low levels in cells of animals, plants, microorganisms, etc. Coenzyme Q10 is a natural antioxidant and cell metabolism activator synthesized by cells, and can protect proteins, DNA molecules and the like from free radical induced oxidation, so that the coenzyme Q10 is widely applied to the industries of medicines, cosmetics, foods and the like. Currently, coenzyme Q10 is a good clinical biochemical drug in medicine, and is commonly used for comprehensively treating liver diseases, cardiovascular diseases and cancers and improving the immunity of organisms. Can be used as a novel food nutrition additive in the food field, and has the effects of enhancing physical ability and improving immunity. Meanwhile, the coenzyme Q10 has irreplaceable effects on deferring senility and improving organism immunity, and is widely applied to the field of female cosmetics.
The production methods of coenzyme Q10 are generally classified into direct extraction, chemical synthesis, microbial fermentation, and the like. The direct extraction method is mainly used for separating and extracting from soybean, tobacco leaf or animal viscera. The extraction method has simple preparation process, but has high extraction cost, is limited by raw materials, seasons and the like, and is not suitable for modern industrial mass production. Chemical synthesis methods are mainly classified into full chemical synthesis methods not using solanesol as a raw material and semi chemical synthesis methods using solanesol as a raw material. The chemical synthesis method has complex reaction, multiple steps, low conversion efficiency and often has a plurality of byproducts, and all factors influence the industrialized development of the chemical synthesis method. Microbial fermentation is the main method for producing coenzyme Q10 at present. The method has the advantages of cheap and abundant raw materials, relatively simple product separation process, natural product, no chiral problem of the compound, good biological activity, easy absorption by human body, and realization of large-scale industrial production through a fermentation tank, thereby becoming the coenzyme Q10 production method with the most development potential.
The normal fermentation period of the coenzyme Q10 is longer, and nutrient substances such as carbon sources, nitrogen sources and the like need to be supplemented in order to meet the growth requirement of thalli in the fermentation process. However, with the increase of the feed supplement, the volume of the feed liquid in the fermentation tank is gradually increased, and the condition of feed liquid overflow exists. In order to improve the equipment utilization rate of the fermentation tank, three intermediate belt releases are required to be carried out regularly. The volume of the fermentation liquid with the unit of reduction is about one fourth of the total yield of a single batch. The bacterial body with the discharge liquid does not reach the aging period, and the culture liquid also contains partial non-metabolized complete nutrient substances, for example, the newly-fed liquid can be discharged along with the discharge, so that the waste of raw and auxiliary materials is caused. From the fermentation period, the first two times of band release are in the logarithmic growth phase of the thalli, and the third time of band release is in the Q10 accumulation peak period. So that the partial potency can be lost after 3 times of band release in the original process. Meanwhile, as the period of the fermentation liquor which is put in the original process for 3 times is shorter, the consumption of the culture medium in the fermentation liquor is incomplete, the impurity content is high, the effective unit is low, the refining yield is low, the quality of industrial salt is not easy to control, the low unit mycelium is high in yield, the pollution discharge is high, and the unused organic matters in the discharge are excessive in COD discharge, so that the environmental protection cost is increased. Therefore, the improvement of the utilization rate of the low-unit fermentation broth is significant for increasing the yield, improving the technical index and reducing the cost.
Disclosure of Invention
In order to solve the problems in the prior art, the present invention provides a method for producing coenzyme Q10 by industrial fermentation. Mainly collecting the fermentation liquor in a receiving tank with the aseptic feed liquor for secondary fermentation. The tape feed solution is required to provide a part of culture medium for growth and propagation of new hyphae and synthesis of metabolites. The feeding time and the feeding quantity are timely regulated and controlled through the detection of the fermentation process index, so that the mycelium maintains a good production environment, and the purpose of prolonging the growing period and improving the yield of fermentation liquor is achieved. As the fermentation liquor is in the vigorous growth period, the starting titer is high, and higher yield can be obtained in a shorter time. Meanwhile, the fermentation liquor with the fermentation liquor is continuously operated for a certain period in the culture tank, so that nutrient substances in the culture medium are further consumed and utilized, and meanwhile, the stable filtering yield of the mycelium quality can be obviously improved, and a certain effect is played on the stability of the product quality.
In order to achieve the above purpose, the invention provides a method for producing coenzyme Q10 by industrial fermentation, which comprises the following technical scheme:
The invention adopts rhodobacter sphaeroides (Rhodobacter sphaeroides) as the strain, and is preserved in the China general microbiological culture Collection center (CGMCC) of the China general microbiological culture Collection center (China general microbiological culture Collection center) with the accession number of CGMCC No.16625 in the 10 th month and 24 th day of 2018.
The method provided by the invention comprises the following steps:
S1: culturing strains on a flat plate: inoculating the strain to a flat plate, and culturing in an incubator with the temperature of 32.0+/-0.5 ℃ and the humidity of 35% -45% for 5-7 days;
S2: slant culture of strain; selecting mature strain from S1, culturing in incubator with temperature of 32.0+ -0.5deg.C and humidity of 35% -45%, and culturing for 5-7 days;
S3:20 Culturing L seeds; selecting a mature strain from the S2, placing the strain into a sterilized 20L seed tank, controlling the culture condition to be 60-80 rpm at 0-12 h rpm, 160-200 rpm at 13 h later, controlling the air flow to be 0.6+/-0.2 m 3/h at 32.0+/-1 ℃, and culturing for 22-30 h;
S4: primary seed tank culture: after the seed liquid in the S3 is cultivated and matured, the seed liquid is inoculated into a first-stage seed tank according to the inoculation amount of 0.1 to 0.5 percent by a differential pressure method, the cultivation condition of the first-stage seed tank is that the tank pressure is 0.03 to 0.05 MPa, the ventilation rate is 100+/-20 m 3/h, the tank temperature is 32 to 34 ℃, the rotating speed is 180 to 200 rpm, and the cultivation period is 24 to 32 h;
s5: secondary seed culture: inoculating the primary seed liquid which is cultivated and matured in the step S4 into a secondary seed tank according to the inoculation amount of 8-20% by using a differential pressure method, wherein the secondary seed tank is cultivated under the conditions of tank pressure of 0.03-0.05 MPa, ventilation rate of 400+/-200 m 3/h, tank temperature of 32-34 ℃, rotating speed of 120-160 rpm and cultivation period of 18-26 h;
S6: and (3) three-stage fermentation culture: inoculating the secondary seed liquid which is cultivated and matured in the step S5 into a tertiary fermentation tank according to the inoculum size of 20-30% by utilizing a differential pressure method, wherein the cultivation condition of the tertiary fermentation tank is that the tank pressure is 0.03-0.05 MPa, the ventilation rate of 40 h is controlled to be 800+/-200 m 3/h before fermentation, the ventilation rate is controlled to be 1000+/-200 m 3/h after the fermentation is 40 h, the rotating speed is controlled to be 80-100 rpm, the tank temperature is 32-34 ℃, and 3 times of feeding are needed in the fermentation process;
S7: pouring seeds, culturing and fermenting: and (3) selecting 1 batch or more than 1 batch of fermentation liquor which is cultured in the step (S6) until 50-65 h meets the pure culture standard, transferring the fermentation liquor to a receiving tank with the same sterilization completion for continuous culture, controlling the tank pressure of the receiving tank to be 0.03-0.05 MPa, controlling the ventilation rate to be 800+/-200 m 3/h before fermentation to be 40 h, controlling the ventilation rate to be 1000+/-200 m 3/h after fermentation to be 40 h, controlling the rotating speed to be 80-100 rpm, and controlling the tank temperature to be 32-34 ℃ and performing 3 times of feeding in the fermentation process.
Further, the formula of the culture medium used for the S1 plate culture and the step S2 slant culture is as follows: 1.0 to 10.0g/L of yeast powder, 0.1 to 2.5 g/L of monopotassium phosphate, 1.0 to 5.0g/L of magnesium sulfate, 0.1 to 1.0 g/L of ferrous sulfate, 0 to 5.0g/L of sodium chloride, 1.5 to 3.5 g/L of ammonium sulfate, 0.5 to 3.0g/L of sodium glutamate, 0.5 to 3.0g/L of corn steep liquor dry powder, 5 to 20g/L of glucose, 15 to 20g/L of agar and pH=6.5 to 7.3.
Further, the culture tank medium formula of the 20L seed culture in the step S3 is as follows: 30-60 g/L of glucose, 10-30 g/L of ammonium sulfate, 5-10 g/L of corn steep liquor dry powder, 1-10 g/L of sodium glutamate, 0.1-0.5 g/L of magnesium sulfate monohydrate, 5-20 g/L of yeast extract powder, 15-30 g/L of industrial salt, 5-10 g/L of dipotassium hydrogen phosphate, 5-10 g/L of potassium dihydrogen phosphate, 10-30 g/L of magnesium sulfate heptahydrate, 1-10 g/L of ferrous sulfate heptahydrate and pH=6.5-7.3.
Further, the formula of the medium of the first-level seed tank in the step S4 is as follows: glucose 0.1-0.4%, ammonium sulfate 0.2-0.5%, dipotassium phosphate 0.03-0.06%, potassium dihydrogen phosphate 0.03-0.06%, magnesium sulfate heptahydrate 0.1-0.3%, ferrous sulfate heptahydrate 0.01-0.03%, industrial salt 0.1-0.4%, calcium carbonate 0.5-1.0%, defoamer 0.01-0.05%, corn steep liquor dry powder 0.03-0.08%, sodium glutamate 0.03-0.08%, magnesium sulfate monohydrate 0.001-0.005%, cobalt chloride hexahydrate 0.0001-0.0005%, yeast extract 0.1-0.5%, and pH=6.5-7.3.
Further, the formula of the secondary seed tank culture medium in the step S5 is as follows: 1-2% of glucose, 0.1-0.5% of ammonium sulfate, 0.05-1.0% of dipotassium hydrogen phosphate, 0.05-1.0% of monopotassium phosphate, 0.1-0.5% of magnesium sulfate heptahydrate, 0.01-0.04% of ferrous sulfate heptahydrate, 0.1-0.5% of industrial salt, 0.5-1.0% of calcium carbonate, 0.01-0.05% of defoamer, 0.05-0.15% of corn steep liquor dry powder, 0.05-0.1% of sodium glutamate, 0.005-0.015% of magnesium sulfate monohydrate, 0.0001-0.0005% of cobalt chloride hexahydrate, 0.1-0.5% of yeast extract powder and pH=6.5-7.3.
Further, the formula of the medium of the third-stage fermentation tank in the third-stage fermentation culture in the step S6 is as follows: 0.2 to 0.6 percent of ammonium sulfate, 0.01 to 0.06 percent of monopotassium phosphate, 1.0 to 1.5 percent of magnesium sulfate heptahydrate, 0.1 to 0.5 percent of ferrous sulfate heptahydrate, 0.1 to 0.5 percent of industrial salt, 0.01 to 0.05 percent of defoamer, 0.005 to 0.01 percent of calcium chloride, 0.3 to 0.8 percent of corn steep liquor dry powder, 0.1 to 0.5 percent of sodium glutamate, 0.005 to 0.01 percent of magnesium sulfate monohydrate and pH=6.5 to 7.3.
Further, the fermentation end point judgment standard in S4 is: the OD of the fermentation liquid is more than or equal to 5.0, and the concentration of the reducing sugar is reduced to 0-0.5 g/L.
Further, the fermentation end point judgment standard in S5 is: the OD of the fermentation liquid is more than or equal to 10.0, and the concentration of the reducing sugar is reduced to 0-0.5 g/L.
Further, the bacteria shape is observed and the bacteria infection is monitored by sampling and measuring sugar, phosphorus and microscopic examination every 4h after inoculation in the S6 and the S7.
Further, the sugar concentration is controlled to be 0.8-1.2% by 40 h before the three-stage fermentation of S6 and the seed pouring and re-culturing fermentation of S7, the sugar concentration is controlled to be 0.5-0.8% by 40-80 hours in the middle of the fermentation, the sugar concentration is controlled to be about 0.2% after 80-h, the phosphorus supplementing speed is controlled to be 35-40L/h by 4-40 hours before the fermentation, the phosphorus supplementing speed is controlled to be 10-15L/h by 40-65 hours, and the phosphorus supplementing speed is controlled to be 25-30L/h by 65-80 hours.
Further, the three-stage fermentation of S6 and the seed pouring and re-culturing fermentation of S7 are carried out for 3 times, wherein the first time of feeding is 20-30 h, the second time of feeding is 40-50 h, and the third time of feeding is 90-105 h of culture period before seed pouring.
Further, the feed formula in the three-stage fermentation of S6 and the inverted seed and re-culture fermentation of S7 is as follows: 0.5 to 1.0 percent of ammonium sulfate, 0.1 to 0.5 percent of monopotassium phosphate, 1.5 to 3.5 percent of magnesium sulfate heptahydrate, 0.1 to 0.5 percent of ferrous sulfate heptahydrate, 0.1 to 0.3 percent of defoamer, 0.01 to 0.03 percent of calcium chloride, 1.0 to 3.0 percent of corn steep liquor dry powder, 0.5 to 1.0 percent of sodium glutamate and 0.01 to 0.03 percent of magnesium sulfate monohydrate.
The beneficial effects obtained by the invention are as follows:
1) The process flow is simplified, and the risk of bacteria infection is reduced: the prior art is about 60 hours and 70 hours before the third material supplementing (55 hours). And the 3 times of band release of the original process is simplified into 1 time of band release by adopting the band release and re-culture process, and meanwhile, the seed culture process is reduced. Not only simplifying the process flow, but also reducing the transfer times of the seed liquid and the fermentation liquid and reducing the probability of bacteria contamination.
2) Saving fermentation cost and improving fermentation yield: the three tape-laying of the original process is to directly put the tape into an extraction working section for extraction. The three times of belt discharge can lead to insufficient utilization of the culture medium, for example, newly-supplemented feed liquid can be discharged along with the belt, and waste of raw and auxiliary materials is caused. From the fermentation period, the first two times of band release are in the logarithmic growth phase of the thalli, and the third time of band release is in the Q10 accumulation peak period. So that the partial potency can be lost after 3 times of band release in the original process. By adopting the process of putting and re-culturing, the loss of the original putting process materials can be saved, and the metabolic growth period of the strain can be prolonged by culturing putting and re-culturing, so that the fermentation titer can be improved.
3) Is favorable for environmental protection: because the period of the fermentation liquor which is put in the original process for 3 times is shorter, the consumption of the culture medium in the fermentation liquor is incomplete, the impurity content is high, the effective unit is low, the refining yield is low, the quality of industrial salt is not easy to control, the low unit mycelium is high in yield, the pollution discharge is high, and the unused organic matters in the discharge are excessive in COD discharge, so that the environmental protection cost is increased. The above problems can be ameliorated by employing a ribbon-fed re-cultivation process.
4) Increase the process reserve, increase the production scheduling: the development of the secondary culture process is that a workshop is added with process selection, so that a proper process can be conveniently selected according to the actual fermentation condition. Meanwhile, the implementation of the process of re-culturing with the belt is beneficial to workshop production, and the production batch per month can be increased to a certain extent.
Drawings
FIG. 1 is a flow chart of the fermentation process of the present invention;
FIG. 2 is a graph showing the comparison of fermentation indexes of the seed pouring process with the seed pouring process and the original process in the embodiment 1;
FIG. 3 is a graph showing the comparison of fermentation indexes of the two-in-three seed pouring process and the original process in the embodiment 2.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.
The fermentation process flow chart of the invention is shown in figure 1.
The invention adopts rhodobacter sphaeroides (Rhodobacter sphaeroides) as the strain, and is preserved in the China general microbiological culture Collection center (CGMCC) of the China general microbiological culture Collection center (China general microbiological culture Collection center) with the accession number of CGMCC No.16625 in the 10 th month and 24 th day of 2018.
The medium formulation used in examples 1-2 was as follows:
Plate and slant medium (medium a): 10.0 g/L of yeast powder, 2.5 g/L of monopotassium phosphate, 2.0 g/L of magnesium sulfate, 0.02 g/L of ferrous sulfate, 0.03 g/L of manganese sulfate, 2.0 g/L of sodium chloride, 2.0 g/L of ammonium sulfate, 1.0g/L of sodium glutamate, 2.0 g/L of corn steep liquor dry powder, 10 g/L of glucose and 15-20 g/L of agar.
20 L seed tank medium (medium B): glucose 50 g/L, ammonium sulfate 20 g/L, corn steep liquor dry powder 5 g/L, sodium glutamate 5 g/L, magnesium sulfate monohydrate 0.3 g/L, yeast extract 10 g/L, industrial salt 20 g/L, dipotassium hydrogen phosphate 5.0 g/L, potassium dihydrogen phosphate 5.0 g/L, magnesium sulfate heptahydrate 20 g/L, ferrous sulfate heptahydrate 1.0 g/L.
First seed tank medium (medium C): glucose 0.4%, ammonium sulfate 0.25%, dipotassium hydrogen phosphate 0.05%, potassium dihydrogen phosphate 0.05%, magnesium sulfate heptahydrate 0.2%, ferrous sulfate heptahydrate 0.01%, industrial salt 0.2%, calcium carbonate 0.8%, defoamer 0.03%, corn steep liquor dry powder 0.05%, sodium glutamate 0.05%, magnesium sulfate monohydrate 0.003%, cobalt chloride hexahydrate 0.0001% and yeast extract powder 0.1%.
Secondary seed pot medium (medium D): 1.5% of glucose, 0.3% of ammonium sulfate, 0.08% of dipotassium hydrogen phosphate, 0.08% of monopotassium phosphate, 0.35% of magnesium sulfate heptahydrate, 0.02% of ferrous sulfate heptahydrate, 0.2% of industrial salt, 0.8% of calcium carbonate, 0.02% of defoamer, 0.1% of corn steep liquor dry powder, 0.08% of sodium glutamate, 0.01% of magnesium sulfate monohydrate, 0.00015% of cobalt chloride hexahydrate and 0.2% of yeast extract powder.
Three-stage fermenter Medium (Medium E): ammonium sulfate 0.415%, monopotassium phosphate 0.05%, magnesium sulfate heptahydrate 1.3%, ferrous sulfate heptahydrate 0.14%, industrial salt 0.25%, defoamer 0.015%, calcium chloride 0.008%, corn steep liquor dry powder 0.4%, sodium glutamate 0.3% and magnesium sulfate monohydrate 0.006%.
The formula of the feed supplement comprises the following components: ammonium sulfate 0.65%, monopotassium phosphate 0.2%, magnesium sulfate heptahydrate 2.3%, ferrous sulfate heptahydrate 0.21%, defoamer 0.1%, calcium chloride 0.01%, corn steep liquor dry powder 2.0%, sodium glutamate 0.6%, magnesium sulfate monohydrate 0.01%.
The sterilization conditions of the culture medium are 121 ℃ sterilization 30min.
Sugar supplementing and sterilizing conditions: 60% liquid sugar was sterilized at 115.5.+ -. 0.5 ℃ for 25 min.
Liquid phosphorus sterilization conditions: 0.08 g/L potassium dihydrogen phosphate is sterilized at 121+/-1 ℃ for 30 min.
Example 1 "one-two" seed pouring process
Plate culture of S1 strain: after preparing the culture medium according to the formula of the culture medium A and sterilizing, the plates are poured under aseptic conditions. Sucking 1 mL glycerin fungus suspension with a pipette, adding into a test tube filled with 9 mL sterile water, vibrating on a vortex oscillator, scattering, mixing uniformly, and diluting with the pipette in multiple ratio. Plates were inoculated with a dilution of 10 -5 of the bacterial suspension, and after 200-300. Mu.L of the bacterial suspension was placed in each plate, the plates were spread evenly with a sterilized spreader. Placing the evenly coated flat plate into an incubator with the temperature of 32.0+/-0.5 ℃ and the humidity of 35% -45% for culturing for 5-7 days.
S2, slant culture of strain: and (3) preparing a culture medium according to the formula of the culture medium A, sterilizing, and pouring the inclined plane under the aseptic condition. Selecting a plurality of single colonies with plump, convex and uniform size from a mother plate which is cultivated and matured by an inoculating shovel, transferring the single colonies into a test tube filled with 9 mL sterile water, vibrating on a vortex oscillator, scattering and uniformly mixing, and diluting to 10 -5 by using a pipetting gun ratio. The bacterial suspension was pipetted into a 0.5 mL pipette and spread evenly in a test tube. Culturing in an incubator with temperature of 32.0+ -0.5deg.C and humidity of 35% -45% for 5-7 days.
S3:20 L seed culture: taking 1-3 inclined planes, sterilizing the outer wall of a test tube by alcohol cotton, selecting a plurality of full, raised and uniform single colonies by an inoculating shovel, moving into a sterile water test tube filled with 50-60 mL for fully scattering, then moving the bacterial suspension into a sterilized 20L seed tank, controlling the rotation speed of the bacterial suspension to be 60-80 rpm under the culture condition of 0-12 h, controlling the rotation speed of the bacterial suspension to be 160-200 rpm after 13 hours, controlling the air flow to be 0.6+/-0.2 m 3/h, and culturing the bacterial suspension for 22-30 hours at the temperature of 32.0+/-1 ℃.
S4: primary seed tank culture: after 20L seed liquid is cultivated and matured, the seed liquid is inoculated into a first-stage seed tank according to the inoculation quantity of 0.1 to 0.5 percent by a differential pressure method, the cultivation condition of the first-stage seed tank is that the tank pressure is 0.03 to 0.05 MPa, the ventilation quantity is 100+/-20 m3/h, the tank temperature is 32 to 34 ℃, the rotating speed is 180 to 200 rpm, and the cultivation period is 24 to 32 h. Fermentation end point judgment standard: the OD of the fermentation liquid is more than or equal to 5.0, and the concentration of the reducing sugar is reduced to 0-0.5 g/L.
S5: secondary seed culture: the mature and sterile first-stage seed liquid is inoculated into a second-stage seed tank according to the inoculation amount of 8-20% by a differential pressure method, the culture condition of the second-stage seed tank is that the tank pressure is 0.03-0.05 MPa, the ventilation rate is 400+/-200 m 3/h, the tank temperature is 32-34 ℃, the rotating speed is 120-160 rpm, and the culture period is 18-26 h. Fermentation end point judgment standard: the OD of the fermentation liquid is more than or equal to 10.0, and the concentration of the reducing sugar is reduced to 0-0.5 g/L.
S6: the 'one-second' seed-reversing and re-culturing process comprises the following steps: and (3) inoculating the mature and sterile secondary seed liquid into a three-stage fermentation tank according to 20-30% of inoculum size by using a differential pressure method, selecting a fermentation tank which is fermented and cultivated for 45-48 hours and is detected to meet the pure culture standard as a fermentation liquid giving tank, and equally dividing the fermentation liquid into another receiving tank after sterilization and continuous cultivation. As can be seen from the figure 2, the pot release titer of the 'one-to-two' seed pouring process is basically the same as that of the original process. Because the fermentation process of the 'one-two' seed pouring process does not carry out 3 times of band discharge, the volume of the tank discharge is 30m 3 less than that of the original process, the corresponding tank discharge total is slightly lower than that of the original process, and the low content of coenzyme Q10 in the three times of band discharge liquid of the original process is indirectly proved. Meanwhile, compared with the original process, the process of pouring seeds in one-in-two mode can reduce the culture of one batch of seed tanks at all levels and the preparation of one batch of fermentation tank base materials under the condition that the sugar unit consumption is basically consistent, the phosphorus and ammonia supplementing amount of each batch is relatively reduced on average, and the cost is reduced while the workload is reduced. Meanwhile, the implementation of the process of re-culturing with the belt is beneficial to workshop production, the production batch per month can be increased to a certain extent, and the equipment utilization rate and the fermentation yield are greatly improved.
The specific process control is shown in the following table.
"One-two" seed pouring process control
Example 2 implementation of the "two-belt three" seed pouring Process
The fermentation process control of the earlier stage 'flat seed-inclined seed-20L seed-primary seed-secondary seed' is basically consistent. And (3) during the process of 'two-in-three' inverted seed re-culture, selecting a fermentation tank which is used for fermentation culture for 45-65 hours and is detected to meet the pure seed culture standard as a fermentation liquid feeding tank, and equally dividing the fermentation liquid into another receiving tank which is sterilized and is subjected to continuous culture. As can be seen from the figure 3, the tank release titer of the two-belt three-way seed release process is slightly higher than that of the original process, and mainly, after seed release of two belt seed release tanks, the fermentation liquid volume is reduced, so that the charging coefficient of the fermentation tank is effectively improved, the dissolved oxygen level of fermentation is improved, and the improvement of the tank release titer is promoted. And the fermentation process of the 'two-strip three' seed pouring process does not carry out 3-time strip discharge, so that the volume of the discharge tank is 27 m 3 less than that of the original process, the total volume of the corresponding discharge tank is slightly lower than that of the original process, and the fact that the coenzyme Q10 content of the material liquid discharged by the original process for three times is lower is indirectly proved. Compared with the original process, the process of pouring seeds in two bands and three ways can reduce the culture of seed tanks at all levels and the preparation of the base materials of a fermentation tank in every three batches under the condition of basically consistent sugar unit consumption, and simultaneously, the phosphorus and ammonia supplementing amount of each batch is relatively reduced, and the cost is reduced while the workload is reduced. Meanwhile, the implementation of the process of re-culturing with the belt is beneficial to workshop production, the production batch per month can be increased to a certain extent, and the equipment utilization rate and the fermentation yield are greatly improved.
The specific process control is shown in the following table.

Claims (2)

1. A method for producing coenzyme Q10 by industrial fermentation is characterized in that,
The rhodobacter sphaeroides (Rhodobacter sphaeroides) is adopted as a strain, and is preserved in the China general microbiological culture Collection center (CGMCC) with the accession number of CGMCC No.16625 in the 10 th month and 24 th day of 2018;
the method comprises the following steps:
s1: culturing strains on a flat plate: inoculating strains to a flat plate, and placing the flat plate into an incubator with the temperature of 32.0+/-0.5 ℃ and the humidity of 35% -45% for culturing for 5-7 days;
S2: slant culture of strain; selecting mature strain from the S1, and placing the strain into an incubator with the temperature of 32.0+/-0.5 ℃ and the humidity of 35% -45% for culturing for 5-7 days;
s3:20 Culturing L seeds; selecting a mature strain from the S2, placing the strain into a sterilized 20L seed tank, controlling the rotation speed to be 60-80 rpm after 0-12 h, controlling the rotation speed to be 160-200 rpm after 13 h, controlling the air flow to be 0.6+/-0.2 m 3/h, and culturing at the temperature of 32.0+/-1 ℃ for 22-30 h;
S4: primary seed tank culture: after the seed liquid in the step S3 is cultivated and matured, inoculating the seed liquid into a first-stage seed tank according to the inoculation amount of 0.1-0.5% by a differential pressure method, wherein the first-stage seed tank is cultivated under the conditions of tank pressure of 0.03-0.05 MPa, ventilation of 100+/-20 m < 3 >/h, the tank temperature of 32-34 ℃, the rotating speed of 180-200 rpm and the cultivation period of 24-32 h; and (4) judging the fermentation end point in the step (S4) as follows: the OD of the fermentation liquid is more than or equal to 5.0, and the concentration of the reducing sugar is reduced to 0-0.5 g/L;
S5: secondary seed culture: inoculating the primary seed liquid which is cultivated and matured in the step S4 into a secondary seed tank according to the inoculation amount of 8-20% by using a differential pressure method, wherein the secondary seed tank is cultivated under the conditions of tank pressure of 0.03-0.05 MPa, ventilation of 400+/-200 m < 3 >/h, tank temperature of 32-34 ℃, rotating speed of 120-160 rpm and cultivation period of 18-26 h; and (5) judging the fermentation end point standard in the step (S5) is as follows: the OD of the fermentation liquid is more than or equal to 10.0, and the concentration of reducing sugar is reduced to 0-0.5 g/L;
S6: and (3) three-stage fermentation culture: the second-level seed liquid which is cultivated and matured in the step S5 is inoculated into a third-level fermentation tank according to the inoculation amount of 20-30% by utilizing a differential pressure method, the cultivation condition of the third-level fermentation tank is that the tank pressure is 0.03-0.05 MPa, the ventilation rate of 40 h is controlled to be 800+/-200 m < 3 >/h before fermentation, the ventilation rate is controlled to be 1000+/-200 m < 3 >/h after 40 h and the rotating speed is controlled to be 80-100 rpm, the tank temperature is 32-34 ℃, and 3 times of material supplementing are needed in the fermentation process;
S7: pouring seeds, culturing and fermenting: selecting a fermentation tank which is used for fermentation culture for 45-65 h and is detected to meet the pure culture standard in the S6 as a fermentation liquid feeding tank, uniformly transferring the fermentation liquid in the fermentation tank to another sterilized receiving tank for continuous culture, wherein the tank pressure of the receiving tank is 0.03-0.05 MPa, the ventilation rate of 40 h is controlled to be 800+/-200 m < 3 >/h before fermentation, the ventilation rate is controlled to be 1000+/-200 m < 3 >/h after 40 h, the rotating speed is controlled to be 80-100 rpm, the tank temperature is 32-34 ℃, and 3 times of feeding are needed in the fermentation process;
Wherein, the formula of the culture medium used for the S1 flat-plate culture and the S2 slant culture is as follows: 1.0-10.0 g/L of yeast powder, 0.1-2.5 g/L of monopotassium phosphate, 1.0-5.0 g/L of magnesium sulfate, 0.1-1.0 g/L of ferrous sulfate, 0-5.0 g/L of sodium chloride, 1.5-3.5 g/L of ammonium sulfate, 0.5-3.0 g/L of sodium glutamate, 0.5-3.0 g/L of corn steep liquor dry powder, 5-20 g/L of glucose, 15-20 g/L of agar and pH=6.5-7.3;
The culture tank culture medium formula for 20L seed culture in the step S3 is as follows: 30-60 g/L of glucose, 10-30 g/L of ammonium sulfate, 5-10 g/L of corn steep liquor dry powder, 1-10 g/L of sodium glutamate, 0.1-0.5 g/L of magnesium sulfate monohydrate, 5-20 g/L of yeast extract powder, 15-30 g/L of industrial salt, 5-10 g/L of dipotassium hydrogen phosphate, 5-10 g/L of potassium dihydrogen phosphate, 10-30 g/L of magnesium sulfate heptahydrate, 1-10 g/L of ferrous sulfate heptahydrate and pH=6.5-7.3;
The formula of the medium of the first-level seed tank in the step S4 is as follows: 0.1-0.4% of glucose, 0.2-0.5% of ammonium sulfate, 0.03-0.06% of dipotassium hydrogen phosphate, 0.03-0.06% of monopotassium phosphate, 0.1-0.3% of magnesium sulfate heptahydrate, 0.01-0.03% of ferrous sulfate heptahydrate, 0.1-0.4% of industrial salt, 0.5-1.0% of calcium carbonate, 0.01-0.05% of defoamer, 0.03-0.08% of corn steep liquor dry powder, 0.03-0.08% of sodium glutamate, 0.001-0.005% of magnesium sulfate monohydrate, 0.0001-0.0005% of cobalt chloride hexahydrate, 0.1-0.5% of yeast extract powder and pH=6.5-7.3;
The formula of the secondary seed tank culture medium in the step S5 is as follows: 1-2% of glucose, 0.1-0.5% of ammonium sulfate, 0.05-1.0% of dipotassium hydrogen phosphate, 0.05-1.0% of monopotassium phosphate, 0.1-0.5% of magnesium sulfate heptahydrate, 0.01-0.04% of ferrous sulfate heptahydrate, 0.1-0.5% of industrial salt, 0.5-1.0% of calcium carbonate, 0.01-0.05% of defoamer, 0.05-0.15% of corn steep liquor dry powder, 0.05-0.1% of sodium glutamate, 0.005-0.015% of magnesium sulfate monohydrate, 0.0001-0.0005% of cobalt chloride hexahydrate, 0.1-0.5% of yeast extract powder and pH=6.5-7.3;
The formula of the medium of the three-stage fermentation tank in the three-stage fermentation culture in the step S6 is as follows: 0.2-0.6% of ammonium sulfate, 0.01-0.06% of monopotassium phosphate, 1.0-1.5% of magnesium sulfate heptahydrate, 0.1-0.5% of ferrous sulfate heptahydrate, 0.1-0.5% of industrial salt, 0.01-0.05% of defoamer, 0.005-0.01% of calcium chloride, 0.3-0.8% of corn steep liquor dry powder, 0.1-0.5% of sodium glutamate, 0.005-0.01% of magnesium sulfate monohydrate and pH=6.5-7.3;
The three-stage fermentation of the S6 and the seed pouring and re-culturing fermentation of the S7 are carried out for 3 times, wherein the first time of feeding is 20-30 hours, the second time of feeding is 40-50 hours, and the third time of feeding is 90-105 hours before seed pouring;
the sugar concentration is controlled to be 0.8-1.2% by 40 h before the three-stage fermentation of S6 and the seed pouring and re-culturing fermentation of S7, the sugar concentration is controlled to be 0.5-0.8% by 40-80 hours in the middle of the fermentation, the sugar concentration is controlled to be about 0.2% after 80-h, the phosphorus supplementing speed is controlled to be 35-40L/h by 4-40 hours before the fermentation, the phosphorus supplementing speed is controlled to be 10-15L/h by 40-65 hours, and the phosphorus supplementing speed is controlled to be 25-30L/h by 65-80 hours;
The feed formula in the three-stage fermentation of S6 and the seed pouring and re-culture fermentation of S7 is as follows: 0.5-1.0% of ammonium sulfate, 0.1-0.5% of monopotassium phosphate, 1.5-3.5% of magnesium sulfate heptahydrate, 0.1-0.5% of ferrous sulfate heptahydrate, 0.1-0.3% of defoamer, 0.01-0.03% of calcium chloride, 1.0-3.0% of corn steep liquor dry powder, 0.5-1.0% of sodium glutamate and 0.01-0.03% of magnesium sulfate monohydrate.
2. The method for producing coenzyme Q10 by industrial fermentation according to claim 1, wherein: and in the steps S6 and S7, sampling and measuring sugar and phosphorus every 4h, observing the fungus shape by microscopic examination, and monitoring the bacteria infection.
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