CN111378699A - Method for producing DHA by schizochytrium limacinum fermentation - Google Patents
Method for producing DHA by schizochytrium limacinum fermentation Download PDFInfo
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Abstract
The invention provides a method for producing DHA by schizochytrium limacinum fermentation, which comprises the following steps: (1) activating, expanding culture and fermenting schizochytrium to obtain fermentation liquor; (2) discharging the fermentation liquor part into an enzymolysis tank for enzymolysis, transferring the obtained enzymolysis liquid to separation equipment, extracting DHA crude oil, and recovering the bacterial meal for later use; (3) supplementing part of culture medium components and the recycled bacterial dregs to the residual fermentation liquor, continuing to ferment, and repeating the step (2) until the fermentation period is finished. The fermentation method provided by the invention recycles the mushroom dregs as nutrient substances, can provide nutrition for microorganisms, keeps the production activity of the microorganisms, can improve the production efficiency of the traditional semi-continuous fermentation, simultaneously reduces the risk of bacteria contamination, reduces the cost of producing DHA by schizochytrium, accords with the concept of green production, and is beneficial to environmental protection.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, particularly relates to a method for producing DHA through microbial fermentation, and particularly relates to a method for producing DHA through schizochytrium limacinum fermentation.
Background
Docosahexaenoic acid (DHA) is an Omega-3 polyunsaturated fatty acid, has important physiological functions of promoting brain cell development, protecting eyesight, improving intelligence, improving memory and the like, is an important nutrient which can not be synthesized by a human body and is indispensable, and is widely applied to the food and medicine industry of infants. DHA obtained by extraction and purification of deep sea fish oil has low yield, is influenced by seasons, has limited supply and is far from meeting market demands.
Since the marine microorganisms Thraustochytrium (Thraustochytrium), Schizochytrium (Schizochytrium), crypthecodinium cohnii (crypthecodinium cohnii) and the like can synthesize DHA in vivo, and the microorganisms have the characteristics of high growth and propagation speed, high grease content, easiness in large-scale culture and the like, the DHA obtained by microbial fermentation becomes a preferred mode for industrial large-scale production of DHA.
At present, the main source for obtaining DHA by a microbiological method is the fermentation production of schizochytrium limacinum, and the DHA-rich grease is obtained by carrying out mechanical wall breaking or enzymolysis wall breaking treatment on the schizochytrium limacinum thalli through mass accumulation of the schizochytrium limacinum thalli. But the market price of DHA is high due to its high production cost. Therefore, the method has great significance for reducing the production cost and expanding the promotion level in common consumers by improving the DHA fermentation production process and continuously increasing the biomass and DHA content of industrial fermentation production.
CN102888348A discloses Schizochytrium limacinum and a method for producing DHA grease by high-density fermentation of Schizochytrium limacinum, wherein the fermentation conditions of strains are optimized from the perspective of element supply and fermentation control, and high-density fermentation is realized by combining sugar supplement operation, the final dry cell weight reaches 70.43g/L, and the grease content reaches 50.1 g/L. The whole process is convenient to operate, obtains higher biomass and DHA content, reduces the fermentation cost, and is suitable for industrial fermentation production. The method adopts the concept of batch fermentation, the consumption of manpower and material resources of the batch fermentation is large, the operations of loading, sterilizing, inoculating, discharging, cleaning and the like are required for each batch of fermentation, the working procedures are complicated, the fermentation period is long, and the production efficiency is low.
CN104263770A discloses a schizochytrium semi-continuous staged fermentation, which adopts the traditional continuous fermentation concept, the main fermentation tank adopts continuous fermentation, and the after-fermentation adopts an intermittent fermentation mode, but the method has the problems that large-area contamination can occur as long as mixed bacteria enter one of the fermentation tanks, and abnormal fermentation is finally caused. At present, continuous and semi-continuous fermentation adopt single-line or double-line production, each line relates to a plurality of fermentation tanks, and the production line is complex.
Therefore, how to improve the production efficiency, ensure the production activity of the schizochytrium limacinum, and simultaneously save the cost, save the energy and reduce the emission is the problem to be further solved in the existing production system for producing the DHA by fermenting the schizochytrium limacinum.
Disclosure of Invention
In view of the problems in the prior art, the invention provides a method for producing DHA by schizochytrium limacinum fermentation. The production method provided by the invention recycles the fungus meal as nutrient substances in the whole production process, can provide nutrition for microorganisms, keeps the production activity of the microorganisms, improves the yield, improves the material utilization rate by adopting a semi-continuous circulating fermentation mode, and reduces the production cost; meanwhile, a fresh culture medium is not introduced in the fermentation period, and the fungus meal is not discharged outwards, so that relatively closed continuous production is realized, foreign bacteria are prevented from entering a fermentation system along with external substances, the risk of bacterial contamination is reduced, the unicity of microbial colonies is ensured, and the product quality is improved; meanwhile, the method also conforms to the concept of green production and is beneficial to protecting the environment. In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a method for producing DHA by schizochytrium limacinum fermentation, which comprises the following steps:
(1) activating, expanding culture and fermenting schizochytrium to obtain fermentation liquor;
(2) discharging the fermentation liquor part into an enzymolysis tank for enzymolysis, transferring the obtained enzymolysis liquid to separation equipment, extracting DHA crude oil, and recovering the bacterial meal for later use;
(3) and (3) supplementing part of culture medium components and the recovered bacterial meal for standby use to the residual fermentation liquid, continuing fermentation, and repeating the step (2) until the fermentation period is finished.
Compared with the traditional continuous or semi-continuous fermentation mode, the method for producing DHA by fermenting schizochytrium does not need a plurality of complicated fermentation tank production lines, only a proper amount of carbon sources and nitrogen sources are needed to be supplemented in the culture medium feeding, and the bacterial meal in the separated waste is used as nutrient substances, so that the growth and metabolism of the fermentation bacteria are stimulated, the activity of the bacteria is maintained, the degeneration of the bacteria is delayed, the oil content of the schizochytrium is improved, the traditional fermentation period is prolonged, the long-time fermentation production is realized, the production efficiency in the cycle is higher, and the production yield is increased. Meanwhile, the fungus meal is recycled as a nutrient substance in the whole production process, so that the pollution of mixed bacteria is reduced, the stability of the activity of microorganism thalli is ensured, the control on the product quality is realized, the green production is realized, and the environment is protected.
As a preferred technical solution of the present invention, the partial discharging in the step (2) is discharging 20-70% of the fermentation liquid in the fermentation tank volume to the enzymolysis tank, for example, 20%, 25%, 30%, 40%, 50%, 60% or 70%, preferably 40-50%.
If the volume of the discharged fermentation liquor is more than 70 percent, the fermentation liquor is replaced too much, the concentration of the bacterial colony is difficult to increase in the next cycle, and if the volume of the discharged fermentation liquor is less than 20 percent, the discharging and replacing amount is too small, the product yield is not high, and the accumulation amount of harmful metabolites in the fermentation liquor is large, so that the fermentation system is abnormal, the fermentation cannot be continued, the continuous production cannot be realized, and the cycle is ended in advance.
In a preferred embodiment of the present invention, the part of the medium components comprises a carbon source.
Preferably, the carbon source comprises any one of glucose, glycerol, fructose, xylose, sucrose, maltose, a saccharification liquid of starch or a saccharification liquid of lignocellulose or a combination of at least two of the two, preferably glucose.
Preferably, the concentration of the carbon source in the fermentation broth after the supplementation reaches 20-50% of the mass concentration of the carbon source in the fermentation medium, for example, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or the like. The detection method of the carbon source is determined according to the determination method of reducing sugar in GB5009.7-2016 food.
Preferably, in the invention, the carbon source can be supplemented according to the consumption of the carbon source after fermenting for 25-30h each time, so as to ensure the sufficiency of the carbon source.
Meanwhile, the content of the product cannot be directly used for evaluating whether the fermentation liquor is infected with bacteria or not in the fermentation process. Biomass, total oil or sugar consumption rates in the fermentation broth are typically used to assess whether a production system is contaminated. If the fermentation is infected with bacteria, the specific expression is that the sugar consumption rate in the fermentation process is low, the accumulated sugar supplement amount in the total fermentation process is low, and the biomass and total oil indexes in the corresponding fermentation system are relatively reduced. Therefore, whether the fermentation system is infected with bacteria or not can be reflected by the amount of the carbon source supplemented in the invention.
Preferably, the continuous fermentation time in step (3) is 60-72 h.
Preferably, the fermentation period is 490-745 h (without seed activation and culture time), for example, 490h, 492h, 528h, 576h, 600h, 624h, 648h, 672h, 720h, 744h or 745h, etc.
Preferably, 8-10 times of discharging are carried out in the fermentation period, for example, 8 times, 9 times or 10 times of discharging can be carried out.
The method can fully utilize the schizochytrium limacinum and the bacterial meal, in order to maximize the utilization of raw materials and ensure the production activity of the schizochytrium limacinum in the system, the continuous fermentation time is selected to be 60-72 hours, and the cycle period and the emptying frequency are respectively set to be 490-745 hours and 8-10 times.
As a preferred embodiment of the present invention, the medium component in step (3) includes a nitrogen source.
Preferably, the nitrogen source is selected from any one or a combination of at least two of peanut cake powder, soybean cake powder, cotton seed cake powder, corn steep liquor, yeast extract, yeast powder, fish meal, silkworm chrysalis meal, peptone, bran or sodium glutamate, and preferably sodium glutamate and/or yeast extract.
Preferably, the mass concentration of nitrogen in the fermentation broth after the supplementation reaches 60-80% of the mass concentration of nitrogen in the fermentation medium, for example, 60%, 62%, 65%, 70%, 75%, 78%, or 80%.
The mass concentration of nitrogen is measured by Kjeldahl method based on amino nitrogen. In the invention, the nitrogen content in the mushroom dregs needs to be measured to determine the amount of the nitrogen source which needs to be supplemented.
Preferably, the nitrogen source is supplemented into the remaining fermentation broth together with the meal.
In a preferred embodiment of the present invention, the temperature of the expanding culture in step (1) is 25 to 30 ℃, and may be, for example, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃ or 30 ℃.
Preferably, the time for the propagation in the step (1) is 48-96 h, for example, 48h, 60h, 72h, 84h or 96h, etc.
The amount of inoculation in the fermentation in step (1) is preferably 10% to 30% of the fermentation medium, and may be, for example, 10%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, or the like, and is preferably 16 to 30%.
Preferably, the fermentation time in step (1) is 72-96h, such as 72h, 78h, 80 h, 84h, 85h, 86h, 90h or 96 h.
Preferably, the fermentation temperature in step (1) is 25 to 30 ℃, and may be, for example, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃ or 30 ℃.
As a preferable technical scheme of the invention, the enzyme used for enzymolysis in the step (2) is any one or a combination of at least two of protease, cellulase, helicase and chitinase, and is preferably a combination of protease and cellulase. In a preferred embodiment, the protease to cellulase mass ratio is 7: 3.
Preferably, the pH of the enzymolysis solution in the step (2) is 6-9, such as 6, 7, 7.5, 8.0, 8.6 or 9.
Preferably, the enzymatic hydrolysis temperature is 40-70 ℃, for example, 40, 45, 50, 55, 60, 65 or 70 ℃ and the like.
Preferably, the volume concentration of the enzyme in step (2) is 0.08-0.20%, for example, 0.08%, 0.09%, 0.10%, 0.12%, 0.15%, 0.18%, or 0.20%.
Preferably, the enzymolysis time in the step (2) is 5-10 h, for example, 5h, 5.5h, 6h, 6.5h, 7h, 7.5h, 8h, 9h, 9.5h or 10h, etc.
In a preferred embodiment of the present invention, the medium used for the activation in step (1) is an activation medium.
Preferably, the culture medium used in the expanding culture in the step (1) is a seed culture medium.
Preferably, the medium used for the fermentation in step (1) is a fermentation medium.
In a preferred embodiment of the present invention, the activating medium comprises 10-20g/L (e.g., 10g/L, 15g/L, 20g/L, etc.) carbon source, 20-40 g/L (e.g., 20g/L, 25g/L, 30g/L, 35g/L, 40g/L, etc.) nitrogen source, and 10-25 g/L (e.g., 10g/L, 12g/L, 15g/L, 18g/L, 20g/L, 25g/L, etc.) inorganic salt;
preferably, the seed culture medium comprises 20-40 g/L (for example, 20g/L, 30g/L, 35g/L, 40g/L and the like) of carbon source, 20-40 g/L (for example, 20g/L, 25g/L, 30g/L, 35g/L, 40g/L and the like) of nitrogen source and 5-20 g/L (for example, 5g/L, 10g/L, 12g/L, 15g/L, 18g/L, 20g/L and the like) of inorganic salt.
Preferably, the fermentation medium comprises 20-40 g/L (for example, 20g/L, 30g/L, 35g/L, 40g/L and the like) of carbon source, 15-35 g/L (for example, 15g/L, 20g/L, 25g/L, 30g/L, 35g/L and the like) of nitrogen source and 5-20 g/L (for example, 5g/L, 10g/L, 12g/L, 15g/L, 18g/L, 20g/L and the like) of inorganic salt.
The schizochytrium limacinum has wide tolerance and requirement range of inorganic salt, and the bacterial dregs contain a large amount of inorganic salt, so the inorganic salt can not be supplemented in the invention. If inorganic salt is needed to be supplemented, the inorganic salt can be added into the fermentation tank together with clear water. The inorganic salt is any one or combination of at least two of sodium chloride, monopotassium phosphate, magnesium sulfate heptahydrate, calcium chloride, sodium bicarbonate, sodium sulfate, ammonium sulfate or potassium chloride.
As a preferred technical scheme of the invention, the method uses a fermentation system which comprises the following steps: the device comprises a seed tank, a fermentation tank, an enzymolysis tank, a separation device, a waste material tank, a material supplementing tank and a collection tank, wherein the material supplementing tank comprises a nitrogen source material supplementing tank and a carbon source material supplementing tank; the seed tank, the fermentation tank, the enzymolysis tank and the separation equipment are sequentially connected, the collection tank is connected with the separation equipment, the waste tank and the fermentation tank are sequentially connected, the nitrogen source supplement tank is connected with the fermentation tank, and the carbon source supplement tank is connected with the fermentation tank. The seed tank can be provided with a plurality of levels according to the fermentation scale.
Preferably, the seed tank and the fermentation tank in the fermentation system are connected through a seed transferring station.
Preferably, the fermentation tank, the enzymolysis tank, the separation equipment, the waste material tank, the feed supplement tank and the collection tank are connected through sterile pipelines.
Preferably, the separation device is a centrifuge.
The method has the advantages that the schizochytrium limacinum is used for preparing the DHA in the system, the process system is simple, the phenomenon that mixed bacteria enter a fermentation system along with external substances can be avoided, the risk of bacteria infection is reduced, the stable production activity of the schizochytrium limacinum is ensured, the strain aging is delayed, and the stable control on the yield of the DHA product is realized; meanwhile, the schizochytrium dregs are used as nutrient components for repeated utilization, so that the production cost is reduced.
As a preferable technical scheme of the invention, the fermentation method comprises the following steps:
(1) activating schizochytrium limacinum, transferring the activated schizochytrium limacinum to a seed pot, and culturing to obtain a seed solution, wherein the propagation time is 48-96 hours, and the propagation temperature is 25-30 ℃; transferring the seed liquid into a fermentation tank according to the inoculation amount of 10-30% for fermentation to obtain fermentation liquid, wherein the fermentation time is 72-96h, and the fermentation temperature is 25-30 ℃;
(2) discharging 20-70% of the fermentation liquor into an enzymolysis tank, carrying out enzymolysis for 5-10 h by using protease and cellulase, transferring the obtained enzymolysis liquid into separation equipment, extracting DHA crude oil, and discharging the bacterial meal into a waste tank for recycling for later use;
(3) and (3) supplementing a carbon source and a nitrogen source to the residual fermentation liquor through a supplementing tank, supplementing bacterial dregs through a waste tank, continuously fermenting for 60-72 hours, and repeating the step (2) until the fermentation period is finished, wherein the concentration of the carbon source in the fermentation liquor reaches 20-50% of the mass concentration of the carbon source in the fermentation medium after the carbon source is supplemented, the concentration of nitrogen in the culture medium reaches 60-80% of the concentration of nitrogen in the fermentation medium after the nitrogen source and the bacterial dregs are supplemented, the fermentation period is 490-745 hours, and discharging is carried out for 8-10 times in the fermentation period.
The recitation of numerical ranges herein includes not only the above-recited numerical values, but also any numerical values between any of the above-recited numerical ranges not recited, and for the sake of brevity and clarity, the present invention is not intended to be exhaustive of the specific numerical values encompassed within the range.
Compared with the prior art, the invention has at least the following beneficial effects:
(1) compared with the traditional fermentation method, the method for producing DHA through fermentation has the advantages that the technological process is simple, the method provided by the invention utilizes the fungus dregs of schizochytrium limacinum as nutrient components for repeated utilization, the raw materials are saved, the long-time fermentation production is realized, and the production efficiency of fermentation is improved; meanwhile, the fungus meal can also keep the production activity of schizochytrium limacinum, delay the strain degeneration, ensure the DHA yield and reduce the production cost;
(2) the fermentation method provided by the invention does not use an organic solvent, has high efficiency, ensures the quality and the yield of DHA, conforms to the concept of green production and is beneficial to environmental protection;
(3) the method for producing DHA through fermentation provided by the invention is relatively closed continuous production in the whole production process, reduces the risk of microbial contamination, and ensures the unicity of microbial colonies.
Drawings
FIG. 1 is a schematic diagram of a fermentation system for producing DHA by fermentation of Schizochytrium in examples 1-4.
Detailed Description
The technical solutions of the present invention are further described in the following embodiments with reference to the drawings, but the following examples are only simple examples of the present invention and do not represent or limit the scope of the present invention, which is defined by the claims.
The fermentor used in the examples below was a 50L pilot plant fermentor.
The formulation of the activation medium in the following examples is: 10g/L of glucose, 25g/L of sodium glutamate, 10g/L of yeast extract, 20g/L of sodium chloride and 0.5g/L of magnesium sulfate.
The formulation of the seed culture medium in the following examples was: 40g/L glucose, 25g/L sodium glutamate, 10g/L yeast extract, 10g/L sodium chloride, 1g/L potassium dihydrogen phosphate, 5g/L magnesium sulfate heptahydrate and 0.5g/L calcium chloride.
The formula of the fermentation medium in the following examples was: 40g/L glucose, 30g/L sodium glutamate, 2g/L yeast extract, 5g/L sodium chloride, 1g/L monopotassium phosphate, 5g/L magnesium sulfate heptahydrate, 0.5g/L calcium chloride, 0.5g/L sodium bicarbonate, 8g/L sodium sulfate, 6g/L ammonium sulfate and 0.5g/L potassium chloride.
The fermentation system for producing DHA by fermentation of Schizochytrium in the following examples is shown in FIG. 1. Because the fermentation scale is less, the seed tank only needs one level can, specifically include: a seed tank, a fermentation tank, an enzymolysis tank, a separation device, a waste material tank, a nitrogen source supplement tank, a carbon source supplement tank and a collection tank. Wherein the seeding tank is connected with the fermentation tank through a seed transferring station; the fermentation tank, the enzymolysis tank, the separation equipment, the waste material tank, the feed supplement tank and the collection tank are connected through a sterile pipeline.
And (3) transferring the activated culture solution formed by the activated schizochytrium into a seed tank through a seed transferring station, performing propagation, and transferring the seed solution into the fermentation tank for fermentation by using an inoculation station after propagation.
After fermentation for a period of time, transferring part of fermentation liquor in the fermentation tank into an enzymolysis tank through a sterile pipeline, carrying out enzymolysis reaction after inactivation in the enzymolysis tank, and inactivating microorganisms and releasing a product; and transferring the enzymolysis liquid to a separation device through a sterile tank guide, transferring DHA crude oil in the enzymolysis liquid to a collection tank through a sterile pipeline in the separation device, and transferring the fungus meal to a waste tank.
The fungus meal in the waste material tank provides nitrogen source and trace elements for the schizochytrium in the residual fermentation tank, and other nitrogen sources can be supplemented in advance in the nitrogen source supplement tank to be supplemented into the residual fermentation liquid together with the fungus meal. After the system starts working, transferring part of fermentation liquor to an enzymolysis tank after first fermentation for 72-96h, and controlling the time interval and times of discharging. Before secondary fermentation, the concentrations of carbon, nitrogen and nitrogen in the residual fermentation liquor are measured, glucose is supplemented to 20-50% of the concentration of the glucose in the fermentation medium, the nitrogen source and the bacterial meal supplement the concentration of the nitrogen in the fermentation liquor to 60-80% of the mass concentration of the nitrogen in the fermentation medium (calculated by amino nitrogen), and finally clear water is used for supplementing the fermentation volume, the tolerance and demand range of the schizochytrium for inorganic salt is wide, in the embodiment of the invention, the inorganic salt is not supplemented, and the repeated circulating fermentation time is 60-72 hours.
After continuous fermentation, the activity of schizochytrium limacinum bacteria is gradually reduced, the nutrient content in the bacterial meal is gradually reduced, the fermentation period is finished for about 490-745 h, the materials are discharged for 8-10 times during the fermentation period, products are collected, and the next circular fermentation is started after the fermentation system is cleaned.
Before the system starts to work, nutrients such as a sterile seed culture medium, a fermentation medium and a carbon source need to be filled in. After the system starts to work, the system becomes a relatively closed production system, the fungus dregs generated during the fermentation of the schizochytrium limacinum are not removed outwards, and other nutrient substances except clean water are not required to be provided for the interior of the system, so that the material exchange frequency between the interior and the exterior of the system is reduced, and the risk of bacterial contamination is reduced.
Example 1
The embodiment provides a method for producing DHA by schizochytrium limacinum, which comprises the following steps:
(1) activation, propagation and fermentation of schizochytrium: culturing the preserved Schizochytrium CABIO-A-2-II (Schizochytrium sp. CABIO-A-2-II) (with the preservation number of CCTCC NO: M2019990; the preservation address of ChinA Center for Type Culture Collection (CCTCC), ChinA, Wuhan university, zip code: 430070; the preservation date of 2019, 12 months and 2 days), transferring the culture medium into A seed tank for expanding culture, wherein the expanding culture temperature is 30 ℃, and continuously culturing for 60 hours to obtain A seed solution; inoculating the seed solution into a fermentation tank, wherein the fermentation tank contains 36L of fermentation liquor after inoculation is finished, and continuously fermenting for 96 hours at 30 ℃;
(2) discharging 18L of fermentation liquor into an enzymolysis tank, inactivating, cooling to 55 ℃, adding sodium hydroxide to adjust the pH to 7.5, and carrying out enzymolysis for 5 hours by using protease (50 ten thousand U/g) with the final concentration of 0.7% and cellulase (1 ten thousand U/g) to obtain enzymolysis liquid;
discharging the enzymatic hydrolysate into a three-phase drum centrifuge, collecting DHA crude oil into a collection tank, and discharging the fungus meal into a waste material tank for later use;
(3) mixing the rest 50% of fermentation liquor, the bacterial meal provided by the waste tank, the glucose solution with the mass fraction of 50% provided by the carbon source supplement tank and the sodium glutamate supplemented by the nitrogen source supplement tank for continuous culture; the nitrogen content of the bacterial meal in the waste tank is 0.02g/g, the glucose concentration in the mixed fermentation liquor is 50% of the carbon source concentration in the fermentation medium, and the nitrogen concentration reaches 80% of the initial nitrogen content. And (5) continuing fermenting for 60h after mixing, repeating the step (2), and discharging materials for 8 times in total, and then finishing the fermentation. In the whole fermentation period, the sugar consumption of the schizochytrium limacinum is normal, which indicates that the fermentation system is not infected with bacteria in the fermentation process.
The amount of fermentation medium used in each time by using the method is 36L, wherein the density of the medium is calculated according to 1kg/L, 18L is discharged averagely in one time, 36L is discharged in the last time, the amount of crude oil produced in 8 times is 9.720kg, and the average DHA yield (calculated by the amount of the fermentation medium) is 6%.
Example 2
The embodiment provides a method for producing DHA by schizochytrium limacinum, which comprises the following steps:
(1) activation, propagation and fermentation of schizochytrium: transferring the preserved schizochytrium limacinum to a seeding tank for propagation after the culture of the activated culture medium, wherein the propagation temperature is 28 ℃, and the propagation time is 72 hours to obtain seed liquid; inoculating the seed solution into a fermentation tank, wherein the fermentation tank contains 40L of fermentation liquor after inoculation is completed, and continuously fermenting for 72 hours at 30 ℃;
(2) discharging 28L of fermentation liquor into an enzymolysis tank, inactivating, cooling to 55 ℃, adjusting the pH to 7.0, and adding protease (50 ten thousand U/g) with the final concentration of 0.7% and cellulase (1 ten thousand U/g) with the final concentration of 0.3% for enzymolysis for 10h to obtain enzymolysis liquid;
discharging the enzymatic hydrolysate into a three-phase drum centrifuge, collecting DHA crude oil into a collection tank, and discharging the fungus meal into a waste material tank for later use;
(3) mixing the rest 30% of fermentation liquor, the bacterial meal provided by the waste tank, the glucose solution with the mass fraction of 50% provided by the carbon source supplementing tank and the sodium glutamate supplemented by the nitrogen source supplementing tank for continuous culture; the nitrogen content of the bacterial meal in the waste tank is 0.02g/g, the glucose concentration in the mixed fermentation liquor is 40% of the carbon source concentration in the fermentation medium, and the nitrogen concentration reaches 80% of the nitrogen content in the initial nitrogen source. And (5) continuing fermentation for 72h after mixing, repeating the step (2), and discharging materials for 8 times in total, and then finishing the fermentation. In the whole fermentation period, the sugar consumption of the schizochytrium limacinum is normal, which indicates that the fermentation system is not infected with bacteria in the fermentation process.
The amount of fermentation broth used each time with this method was 40L, wherein the density of the medium was calculated as 1kg/L, the volume of single discharge was 28L, the volume of last discharge was 40L, the amount of crude oil produced in 8 times was 10.62kg, and the average DHA yield (calculated as the amount of fermentation medium) was 4.5%.
Example 3
The embodiment provides a method for producing DHA by schizochytrium limacinum, which comprises the following steps:
(1) activation, propagation and fermentation of schizochytrium: culturing the preserved schizochytrium limacinum in an activated culture medium, transferring the culture medium to a seed tank for propagation at the temperature of 25 ℃ for 48 hours to obtain a seed solution, inoculating the seed solution to a fermentation tank, inoculating the seed solution, and continuing to ferment for 96 hours at the temperature of 25 ℃, wherein the fermentation tank contains 36L of fermentation liquor;
(2) discharging 14.4L fermentation liquor into an enzymolysis tank after fermentation, cooling to 60 ℃ after inactivation, and then adding protease (50 ten thousand U/g) with the final concentration of 0.6% and cellulase (1 ten thousand U/g) for enzymolysis for 10h to obtain enzymolysis liquid; discharging the enzymatic hydrolysate into a three-phase drum centrifuge, collecting DHA crude oil into a collection tank, and discharging the fungus meal into a waste material tank for later use;
(3) mixing the rest 60% of fermentation liquor, the bacterial meal provided by the waste tank, the glucose solution with the mass fraction of 50% provided by the carbon source supplementing tank and the sodium glutamate supplemented by the nitrogen source supplementing tank for continuous culture; the nitrogen content of the bacterial meal in the waste tank is 0.02g/g, the glucose concentration in the mixed fermentation liquor is 50% of the carbon source concentration in the fermentation medium, and the nitrogen concentration reaches 80% of the nitrogen content in the initial nitrogen source. And (5) continuing fermentation for 72h after mixing, repeating the step (2), discharging materials for 9 times in total, and then finishing the fermentation. In the whole fermentation period, the sugar consumption of the schizochytrium limacinum is normal, which indicates that the fermentation system is not infected with bacteria in the fermentation process.
The amount of fermentation broth used in each time was 36L using this method, wherein the density of the medium was calculated as 1kg/L, 14.4L was discharged in average in a single discharge, the volume of the last discharge was 36L, the amount of crude oil produced in 9 discharges was 9.828kg, and the average DHA yield (calculated as the amount of fermentation medium) was 6.5%.
Example 4
The embodiment provides a method for producing DHA by schizochytrium limacinum, which comprises the following steps:
(1) activation, propagation and fermentation of schizochytrium: culturing the preserved Schizochytrium limacinum in an activated culture medium, transferring to a seed tank for propagation at 29 deg.C for 60 hr to obtain seed solution, inoculating the seed solution into a fermentation tank containing 36L fermentation broth, and continuously fermenting at 29 deg.C for 72 hr;
(2) discharging 7.2L fermentation liquor after fermentation into an enzymolysis tank, inactivating, cooling to 50 ℃, and adding protease (50 ten thousand U/g) with the final concentration of 0.6% and cellulase (1 ten thousand U/g) for enzymolysis for 10h to obtain enzymolysis liquid; discharging the enzymatic hydrolysate into a three-phase drum centrifuge, collecting DHA crude oil into a collection tank, and discharging the fungus meal into a waste material tank for later use;
(3) mixing the rest 80% of fermentation liquor, the bacterial meal provided by the waste tank, the glucose solution with the mass fraction of 50% provided by the carbon source supplementing tank and the sodium glutamate supplemented by the nitrogen source supplementing tank for continuous culture; the nitrogen content of the bacterial meal in the waste tank is 0.02g/g, the glucose concentration in the mixed fermentation liquor is 50% of the carbon source concentration in the fermentation medium, and the nitrogen concentration reaches 80% of the nitrogen content in the initial nitrogen source. And (5) continuing fermenting for 60h after mixing, repeating the step (2), and discharging materials for 8 times in total, and then finishing the fermentation. In the whole fermentation period, the sugar consumption of the schizochytrium limacinum is normal, which indicates that the fermentation system is not infected with bacteria in the fermentation process.
The amount of fermentation liquor used by the method every time is 36L, the average single discharge is 7.2L, the last discharge is 36L, the crude oil yield is 3.456kg in 8 times, and the average yield is 4%.
Comparative example 1
Compared with the embodiment 1, a nitrogen source supplement tank and a carbon source supplement tank are not arranged in a fermentation system for producing DHA by schizochytrium fermentation, a fermentation tank needs to be connected with a sterile pipeline to supplement a fermentation culture medium, and wastewater and bacterial meal in a waste tank are directly discharged to a waste treatment device.
Compared with the method of example 1, the method for producing DHA by utilizing the schizochytrium through the fermentation system has the difference that fresh culture medium outside the fermentation system is added into the residual fermentation liquid, and the wastewater and the bacterial meal are directly discharged outside the fermentation system for treatment. Thus, 18L of fermentation broth was discharged from the fermentor and 18L of fresh fermentation medium was required. The rest of the culture conditions, the medium composition, the enzymatic hydrolysis conditions, etc. were the same as in example 1.
The amount of fermentation medium used in each case was 36L using this method, wherein the density of the medium was 1kg/m3And calculating that the average crude oil yield is 0.052kg/L times when 18L of materials are discharged in one time and 36L of materials are discharged in the last time, the crude oil yield is 8.42kg when 8 times are produced, and the yield (calculated by the using amount of the fermentation culture medium) is 5.0 percent.
It can be known from the embodiment 1 and the comparative example 1 that the single crude oil yield of the method for producing DHA by schizochytrium provided by the invention is high, which indicates that the supplementation of the bacterial meal in the fermentation medium can improve the stability of the schizochytrium and delay the bacterial colony aging, so the total yield is high, and meanwhile, the bacterial meal obtained in the embodiment 1 is re-added into the fermentation broth as a nutrient component, so the bacterial meal is recycled, the cost can be reduced, the steps of treating wastes are reduced, the time for cleaning the fermentation system in the middle of fermentation is saved, and the method is suitable for industrial production. As is clear from the comparison between examples 1 and 2 and example 4, the yield of DHA is ultimately affected when a single discharge of more (70% in example 2) or less (20% in example 4) fermentation broth from the fermentor is performed, and therefore, the control of reasonable amount of feed is also a key factor in improving yield.
In conclusion, the method for producing DHA by using schizochytrium provided by the invention can continuously provide nutrient substances for the schizochytrium, reduce the substance exchange with the outside and the risk of bacterial contamination, and simultaneously improve the production efficiency of the traditional semi-continuous production and the yield of DHA. In addition, the reaction system can repeatedly utilize the bacterial dregs, thereby saving the production cost, saving the resources and achieving the purpose of protecting the environment.
The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.
Claims (10)
1. A method for producing DHA by fermentation of schizochytrium limacinum is characterized by comprising the following steps:
(1) activating, expanding culture and fermenting schizochytrium to obtain fermentation liquor;
(2) discharging the fermentation liquor part into an enzymolysis tank for enzymolysis, transferring the obtained enzymolysis liquid to separation equipment, extracting DHA crude oil, and recovering the bacterial meal for later use;
(3) supplementing part of culture medium components and the recycled bacterial dregs to the residual fermentation liquor, continuing to ferment, and repeating the step (2) until the fermentation period is finished.
2. The method according to claim 1, wherein the partial emptying in the step (2) is performed by emptying 20-70% of fermentation liquor of the volume of the fermentation tank into the enzymolysis tank, preferably 40-50%.
3. The method of claim 1 or 2, wherein the medium components in step (3) comprise a carbon source;
preferably, the carbon source is selected from any one or a combination of at least two of glucose, glycerol, fructose, xylose, sucrose, maltose, a starch saccharification liquid or a lignocellulose saccharification liquid, and is preferably glucose;
preferably, the concentration of the carbon source in the supplemented fermentation broth reaches 20-50% of the mass concentration of the carbon source in the fermentation medium;
preferably, the continuous fermentation time in the step (3) is 60-72 h;
preferably, the fermentation period in the step (3) is 490-745 h;
preferably, 8-10 times of emptying are carried out in the fermentation period.
4. The method according to any one of claims 1 to 3, wherein the medium components in step (3) further comprise a nitrogen source;
preferably, the nitrogen source is selected from any one or a combination of at least two of peanut cake powder, soybean cake powder, cottonseed cake powder, corn steep liquor, yeast extract, yeast powder, fish meal, silkworm chrysalis meal, peptone, bran or sodium glutamate, and preferably sodium glutamate and/or yeast extract;
preferably, the nitrogen source and the mushroom dregs are supplemented into the residual fermentation liquor together;
preferably, the mass concentration of nitrogen in the fermentation broth after the supplementation reaches 60-80% of the mass concentration of nitrogen in the fermentation medium.
5. The method according to any one of claims 1 to 4, wherein the temperature of the propagation in the step (1) is 25 to 30 ℃;
preferably, the time for the propagation in the step (1) is 48-96 h;
preferably, the inoculation amount in the fermentation in the step (1) is 10-30% of the volume of the fermentation medium, and is preferably 16-30%;
preferably, the fermentation time in step (1) is 72-96 h;
preferably, the fermentation temperature in the step (1) is 25-30 ℃.
6. The method according to any one of claims 1 to 5, wherein the enzyme used in the enzymolysis in step (2) is selected from any one or a combination of at least two of protease, cellulase, helicase and chitinase, preferably a combination of protease and cellulase;
preferably, the pH of the enzymolysis liquid in the enzymolysis in the step (2) is 6-9;
preferably, the enzymolysis temperature in the step (2) is 40-70 ℃;
preferably, the volume concentration of the enzyme in the fermentation liquor during enzymolysis in the step (2) is 0.08-0.20%;
preferably, the enzymolysis time in the step (2) is 5-10 h.
7. The method according to any one of claims 1 to 6, wherein the medium used in the activation in step (1) is an activation medium;
preferably, the culture medium used in the expanding culture of the step (1) is a seed culture medium;
preferably, the culture medium used in the fermentation in step (1) is a fermentation medium;
preferably, the components of the activation medium comprise 10-20g/L of carbon source, 20-40 g/L of nitrogen source and 10-25 g/L of inorganic salt;
preferably, the components of the seed culture medium comprise 20-40 g/L of carbon source, 20-40 g/L of nitrogen source and 5-20 g/L of inorganic salt;
preferably, the fermentation medium comprises 20-40 g/L of carbon source, 15-35 g/L of nitrogen source and 5-20 g/L of inorganic salt.
8. The method according to any one of claims 1 to 7, wherein the method uses a fermentation system comprising: the device comprises a seed tank, a fermentation tank, an enzymolysis tank, a separation device, a waste material tank, a material supplementing tank and a collection tank, wherein the material supplementing tank comprises a nitrogen source material supplementing tank and a carbon source material supplementing tank;
the seed tank, the fermentation tank, the enzymolysis tank and the separation equipment are sequentially connected, the collection tank is connected with the separation equipment, the waste tank and the fermentation tank are sequentially connected, the nitrogen source supplement tank is connected with the fermentation tank, and the carbon source supplement tank is connected with the fermentation tank.
9. The method of any one of claims 1 to 8, wherein the seed tank and the fermentor in the fermentation system are connected by a seed transfer station;
preferably, the fermentation tank, the enzymolysis tank, the separation equipment, the waste material tank, the feed supplement tank and the collection tank are connected through sterile pipelines;
preferably, the separation device is a centrifuge.
10. A method according to any one of claims 1-9, characterized by the steps of:
(1) activating schizochytrium limacinum, transferring the activated schizochytrium limacinum to a seed pot, and culturing to obtain a seed solution, wherein the propagation time is 48-96 hours, and the propagation temperature is 25-30 ℃; transferring the seed liquid into a fermentation tank according to the inoculation amount of 10-30% for fermentation to obtain fermentation liquid, wherein the fermentation time is 72-96h, and the fermentation temperature is 25-30 ℃;
(2) discharging 20-70% of the fermentation liquor into an enzymolysis tank, carrying out enzymolysis for 5-10 h by using protease and cellulase, transferring the obtained enzymolysis liquid into separation equipment, extracting DHA crude oil, and discharging the bacterial meal into a waste tank for recycling for later use;
(3) and (3) supplementing a carbon source and a nitrogen source to the residual fermentation liquor through a supplementing tank, supplementing bacterial dregs through a waste tank, continuously fermenting for 60-72 hours, and repeating the step (2) until the fermentation period is finished, wherein the concentration of the carbon source in the fermentation liquor reaches 20-50% of the mass concentration of the carbon source in the fermentation medium after the carbon source is supplemented, the concentration of nitrogen in the culture medium reaches 60-80% of the concentration of nitrogen in the fermentation medium after the nitrogen source and the bacterial dregs are supplemented, the fermentation period is 490-745 hours, and discharging is carried out for 8-10 times in the fermentation period.
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