CN102181509A - Method for producing vernine - Google Patents
Method for producing vernine Download PDFInfo
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- CN102181509A CN102181509A CN2011100680393A CN201110068039A CN102181509A CN 102181509 A CN102181509 A CN 102181509A CN 2011100680393 A CN2011100680393 A CN 2011100680393A CN 201110068039 A CN201110068039 A CN 201110068039A CN 102181509 A CN102181509 A CN 102181509A
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Abstract
The invention belongs to the field of biomedicine, and particularly discloses a method for producing vernine. The method for producing the vernine comprises the steps of preparing a seed culture medium, culturing seeds, preparing a fermentation culture medium, inoculating and fermenting, and performing fed-batch fermentation. The method has the advantages that: the initial glucose concentration of the fermentation and the fermentation time are reduced to ensure that the environments which are used from seed culture to the growth of fermented thalli are not changed greatly so as to be favorable for the growth of the thalli; glucose and yeast powder are continuously added at an earlier stage to ensure that the concentration of the thalli is highest under the condition of keeping the concentration of a substrate unchanged in the growth process of the thalli; and xanthine is immediately added after the thalli are finished so as to promote the thalli to produce the vernine, so that the yield of the vernine is improved. Finally, the concentration of the vernine in zymotic fluid is improved from 30g/l to 36g/l; and the concentration of the vernine in the zymotic fluid is improved by 20 percent.
Description
Technical field
The invention belongs to biomedicine field, especially a kind of production method of guanosine.
Background technology
The purposes of guanosine is very extensive, it is the important intermediate of food and medicine product, can be used for the synthesised food freshener---5 '-Sodium guanylate and ucleosides antiviral such as ribavirin, acyclovir etc. also are the main raw materials that is used to make acycloguanosine (Acyclovir), ribavirin (ATC), Sodium Guanosine Triphosphate medicines such as (GTP).
The fermentation of bacillus subtilis method is mainly adopted in present domestic guanosine production.The guanosine fermentation can be adopted forms such as batch fermentation and fed-batch fermentation.Wherein fed-batch fermentation is removed substrate and is suppressed owing to can reduce concentration of substrate and fermentation viscosity in the tunning, improves the nutritive ingredient utilization ratio, thereby helps the generation of guanosine.In actual production process, adopted by most of producer.
Existing bacterial classification produces the glycosides amount and is about every liter 30 gram under the situation of fed-batch fermentation, and the yield of product is lower.
Summary of the invention
The present invention is directed to the low shortcoming of yield that fed-batch fermentation produces guanosine in the above-mentioned technology, propose a kind of production method of guanosine, this method can improve the yield that produces guanosine, makes that producing the glycosides amount is to rise to about 36 grams about every liter 30 gram.
The technical scheme that realizes the object of the invention is as follows:
The production method of guanosine comprises the preparation seed culture medium, cultivates seed, prepares fermention medium, inoculation fermentation and fed-batch fermentation, and its concrete steps are as follows:
A, preparation seed culture medium, in water, add glucose 2-4%, monosodium glutamate 0.3-1%, peptone 0.5-1.5%, yeast extract powder 0.5-1.5%, NaCl 0.2-0.3%, urea 0.1-0.3%, corn steep liquor 0.5-1.5% by weight percentage, regulate pH7.0-7.2 with NaOH and hydrochloric acid then, and 110 ℃-120 ℃ the sterilization 10-20 minute, obtain seed culture medium;
B, cultivation seed are produced bacterium with guanosine and are inserted in the seed culture medium, and inoculum size is 8%-15%, under 33 ℃-35 ℃ temperature, keep the pH value being cultured to logarithmic phase in the automatic control fermentor tank at 5L under 6.5 the condition then;
C, preparation fermention medium, in water, add glucose 4.5-5.5%, monosodium glutamate 0.8-1.2%, ammonium sulfate 0.7-0.9%, yeast powder 0.8-1.2%, sal epsom 0.3-0.45%, potassium primary phosphate 0.15-0.25%, calcium chloride 0.15-0.25%, soya-bean cake hydrolyzed solution 1.5-3.3%, corn steep liquor 1.5-1.9% by weight percentage, defoamer 0.03-0.05%, regulate pH7.0-7.2 with NaOH and hydrochloric acid, and 110 ℃-120 ℃ the sterilization 10-20 minute, obtain fermention medium;
D, inoculation fermentation insert the 50L that contains fermention medium with the seed cultivated among the step b by the inoculum size of 8%-15% and control in the fermentor tank automatically and ferment, and the time is 8h, and temperature is that 0-4h is 33.9-34.1 ℃, and 4-8h is 34.9-35.1 ℃;
E, fed-batch fermentation, in the d step, after the 8h temperature is controlled at 35.9-36.1 ℃, add ammoniacal liquor by stream pH is controlled at 6.5, dissolved oxygen is controlled at 40%, begin stream and add the aqueous solution of glucose, yeast powder, soya-bean cake hydrolyzed solution, wherein the weight of glucose, yeast powder, soya-bean cake hydrolyzed solution is respectively 10%, 0.6%, 2% of the total fermented liquid weight behind the inoculation fermentation in the d step, it is 16h that stream adds the time, begin slow stream after the end and add the production of xanthoglobulin promotion guanosine, after 24 hours completed product run.
The concentration of the aqueous solution of described glucose is 60%-70%.
Advantage of the present invention and positively effect are 1, pass through to reduce fermentation initial glucose concentration and leavening temperature, make from seed culture and do not produce big variation to zymophyte bulk-growth environment, help thalli growth;
2, by early stage glucose and yeast powder Continuous Flow add, in the thalli growth process, keep making cell concentration reach maximum under the constant situation of concentration of substrate;
3, begin stream immediately after thalline is finished and add xanthoglobulin, promote thalline to produce guanosine, guanosine output is improved.Guanosine concentration is brought up to more than the 36g/l from 30g/l, and guanosine concentration has improved 20% in the fermented liquid.
Embodiment
Implement example 1, the production method of guanosine comprises the preparation seed culture medium, cultivates seed, prepares fermention medium, inoculation fermentation and fed-batch fermentation, and its concrete steps are as follows:
A, preparation seed culture medium, in water, add glucose 2%, monosodium glutamate 0.3-0.6%, peptone 0.5%, yeast extract powder 0.5%, NaCl 0.2%, urea 0.1%, corn steep liquor 0.5% by weight percentage, regulate pH7.0 with NaOH and hydrochloric acid then, and 110 ℃ the sterilization 10 minutes, obtain seed culture medium;
B, cultivation seed are produced bacterium with guanosine and are inserted in the seed culture medium, and inoculum size is 8%-10%, under 33 ℃ temperature, keep the pH value being cultured to logarithmic phase in the automatic control fermentor tank at 5L under 6.5 the condition then;
C, preparation fermention medium, in water, add glucose 4.5%, monosodium glutamate 0.8%, ammonium sulfate 0.7%, yeast powder 0.8%, sal epsom 0.3%, potassium primary phosphate 0.15%, calcium chloride 0.15%, soya-bean cake hydrolyzed solution 1.5%, corn steep liquor 1.5% by weight percentage, defoamer 0.03%, regulate pH7.0-7.2 with NaOH and hydrochloric acid, and 110 ℃ the sterilization 10 minutes, obtain fermention medium;
D, inoculation fermentation insert the 50L that contains fermention medium with the seed cultivated among the step b by 8% inoculum size and control in the fermentor tank automatically and ferment, and the time is 8h, and temperature is that 0-4h is 33.9 ℃, and 4-8h is 34.9 ℃;
E, fed-batch fermentation, in the d step, after the 8h temperature is controlled at 35.9 ℃, add ammoniacal liquor by stream pH is controlled at 6.5, dissolved oxygen is controlled at 40%, begin stream and add the aqueous solution of glucose, yeast powder, soya-bean cake hydrolyzed solution, wherein the weight of glucose, yeast powder, soya-bean cake hydrolyzed solution is respectively 10%, 0.6%, 2% of the total fermented liquid weight behind the inoculation fermentation in the d step, and it is 16h that stream adds the time, begin slow stream after the end and add the production of xanthoglobulin promotion guanosine, after 24 hours completed product run.
Implement example 2, the production method of guanosine comprises the preparation seed culture medium, cultivates seed, prepares fermention medium, inoculation fermentation and fed-batch fermentation, and its concrete steps are as follows:
A, preparation seed culture medium, in water, add glucose 4%, monosodium glutamate 0.6%, peptone 1.5%, yeast extract powder 1.5%, NaCl 0.3%, urea 0.3%, corn steep liquor 1.5% by weight percentage, regulate pH7.2 with NaOH and hydrochloric acid then, and 120 ℃ the sterilization 20 minutes, obtain seed culture medium;
B, cultivation seed are produced bacterium with guanosine and are inserted in the seed culture medium, and inoculum size is 15%, under 35 ℃ temperature, keep the pH value being cultured to logarithmic phase in the automatic control fermentor tank at 5L under 6.5 the condition then;
C, preparation fermention medium, in water, add glucose 5.5%, monosodium glutamate 1.2%, ammonium sulfate 0.9%, yeast powder 1.2%, sal epsom 0.45%, potassium primary phosphate 0.25%, calcium chloride 0.25%, soya-bean cake hydrolyzed solution 3.3%, corn steep liquor 1.9% by weight percentage, defoamer 0.05%, regulate pH7.2 with NaOH and hydrochloric acid, and 120 ℃ the sterilization 20 minutes, obtain fermention medium;
D, inoculation fermentation insert the 50L that contains fermention medium with the seed cultivated among the step b by 15% inoculum size and control in the fermentor tank automatically and ferment, and the time is 8h, and temperature is that 0-4h is 34.1 ℃, and 4-8h is 35.1 ℃;
E, fed-batch fermentation, in the d step, after the 8h temperature is controlled at 36.1 ℃, add ammoniacal liquor by stream pH is controlled at 6.5, dissolved oxygen is controlled at 40%, begin stream and add the aqueous solution of glucose, yeast powder, soya-bean cake hydrolyzed solution, wherein the weight of glucose, yeast powder, soya-bean cake hydrolyzed solution is respectively 10%, 0.6%, 2% of the total fermented liquid weight behind the inoculation fermentation in the d step, and it is 16h that stream adds the time, begin slow stream after the end and add the production of xanthoglobulin promotion guanosine, after 24 hours completed product run.
Implement example 3, the production method of guanosine comprises the preparation seed culture medium, cultivates seed, prepares fermention medium, inoculation fermentation and fed-batch fermentation, and its concrete steps are as follows:
A, preparation seed culture medium, in water, add glucose 2%, monosodium glutamate 0.5%, peptone 1%, yeast extract powder 1%, NaCl 0.25%, urea 0.2%, corn steep liquor 1% by weight percentage, regulate between the pH7.0-7.2 with NaOH and hydrochloric acid then, and 115 ℃ the sterilization 15 minutes, obtain seed culture medium;
B, cultivation seed are produced bacterium with guanosine and are inserted in the seed culture medium, and inoculum size is 8%, under 34 ℃ temperature, keep the pH value being cultured to logarithmic phase in the automatic control fermentor tank at 5L under 6.5 the condition then;
C, preparation fermention medium, in water, add glucose 5%, monosodium glutamate 1%, ammonium sulfate 0.8%, yeast powder 1%, sal epsom 0.3%, potassium primary phosphate 0.15%, calcium chloride 0.15%, soya-bean cake hydrolyzed solution 1.5%, corn steep liquor 1.7% by weight percentage, defoamer 0.04%, regulate pH7.0-7.2 with NaOH and hydrochloric acid, and 115 ℃ the sterilization 15 minutes, obtain fermention medium;
D, inoculation fermentation insert the 50L that contains fermention medium with the seed cultivated among the step b by 10% inoculum size and control in the fermentor tank automatically and ferment, and the time is 8h, and temperature is that 0-4h is 34 ℃, and 4-8h is 35 ℃;
E, fed-batch fermentation, in the d step, after the 8h temperature is controlled at 36 ℃, add ammoniacal liquor by stream pH is controlled at 6.5, dissolved oxygen is controlled at 40%, begin stream and add the aqueous solution of glucose, yeast powder, soya-bean cake hydrolyzed solution, wherein the weight of glucose, yeast powder, soya-bean cake hydrolyzed solution is respectively 10%, 0.6%, 2% of the total fermented liquid weight behind the inoculation fermentation in the d step, and it is 16h that stream adds the time, begin slow stream after the end and add the production of xanthoglobulin promotion guanosine, after 24 hours completed product run.
Implement example 4, the production method of guanosine comprises the preparation seed culture medium, cultivates seed, prepares fermention medium, inoculation fermentation and fed-batch fermentation, and its concrete steps are as follows:
A, preparation seed culture medium, in water, add glucose 5%, monosodium glutamate 1%, peptone 1%, yeast extract powder 1%, NaCl 0.25%, urea 0.2%, corn steep liquor 1% by weight percentage, regulate between the pH7.0-7.2 with NaOH and hydrochloric acid then, and 115 ℃ the sterilization 15 minutes, obtain seed culture medium;
B, cultivation seed are produced bacterium with guanosine and are inserted in the seed culture medium, and inoculum size is 10%, under 34 ℃ temperature, keep the pH value being cultured to logarithmic phase in the automatic control fermentor tank at 5L under 6.5 the condition then;
C, preparation fermention medium, in water, add glucose 5%, monosodium glutamate 1%, ammonium sulfate 0.8%, yeast powder 1%, sal epsom 0.4%, potassium primary phosphate 0.2%, calcium chloride 0.2%, soya-bean cake hydrolyzed solution 1.5%, corn steep liquor 1.7% by weight percentage, defoamer 0.04%, regulate pH7.0-7.2 with NaOH and hydrochloric acid, and 115 ℃ the sterilization 15 minutes, obtain fermention medium;
D, inoculation fermentation insert the 50L that contains fermention medium with the seed cultivated among the step b by 12% inoculum size and control in the fermentor tank automatically and ferment, and the time is 8h, and temperature is that 0-4h is 34 ℃, and 4-8h is 35 ℃;
E, fed-batch fermentation, in the d step, after the 8h temperature is controlled at 36 ℃, add ammoniacal liquor by stream pH is controlled at 6.5, dissolved oxygen is controlled at 40%, begin stream and add the aqueous solution of glucose, yeast powder, soya-bean cake hydrolyzed solution, wherein the weight of glucose, yeast powder, soya-bean cake hydrolyzed solution is respectively 10%, 0.6%, 2% of the total fermented liquid weight behind the inoculation fermentation in the d step, and it is 16h that stream adds the time, begin slow stream after the end and add the production of xanthoglobulin promotion guanosine, after 24 hours completed product run.
Implement example 5, the production method of guanosine comprises the preparation seed culture medium, cultivates seed, prepares fermention medium, inoculation fermentation and fed-batch fermentation, and its concrete steps are as follows:
A, preparation seed culture medium, in water, add glucose 5%, monosodium glutamate 1%, peptone 1%, yeast extract powder 1%, NaCl 0.25%, urea 0.2%, corn steep liquor 1% by weight percentage, regulate between the pH7.0-7.2 with NaOH and hydrochloric acid then, and 115 ℃ the sterilization 15 minutes, obtain seed culture medium;
B, cultivation seed are produced bacterium with guanosine and are inserted in the seed culture medium, and inoculum size is 15%, under 34 ℃ temperature, keep the pH value being cultured to logarithmic phase in the automatic control fermentor tank at 5L under 6.5 the condition then;
C, preparation fermention medium, in water, add glucose 5%, monosodium glutamate 1%, ammonium sulfate 0.8%, yeast powder 1%, sal epsom 0.4%, potassium primary phosphate 0.2%, calcium chloride 0.2%, soya-bean cake hydrolyzed solution 1.5%, corn steep liquor 1.7% by weight percentage, defoamer 0.04%, regulate pH7.0-7.2 with NaOH and hydrochloric acid, and 115 ℃ the sterilization 15 minutes, obtain fermention medium;
D, inoculation fermentation insert the 50L that contains fermention medium with the seed cultivated among the step b by 8% inoculum size and control in the fermentor tank automatically and ferment, and the time is 8h, and temperature is that 0-4h is 34 ℃, and 4-8h is 35 ℃;
E, fed-batch fermentation, in the d step, after the 8h temperature is controlled at 36 ℃, add ammoniacal liquor by stream pH is controlled at 6.5, dissolved oxygen is controlled at 40%, begin stream and add the aqueous solution of glucose, yeast powder, soya-bean cake hydrolyzed solution, wherein the weight of glucose, yeast powder, soya-bean cake hydrolyzed solution is respectively 10%, 0.6%, 2% of the total fermented liquid weight behind the inoculation fermentation in the d step, and it is 16h that stream adds the time, begin slow stream after the end and add the production of xanthoglobulin promotion guanosine, after 24 hours completed product run.
In the above-mentioned concentration that each implements the aqueous solution of glucose in the example is 60%-70%, usually adopt 55%, 60%, 65%, 67%, 70% or 75% glucose water-soluble, but wherein the gross weight of glucose be the fermented liquid behind the inoculation fermentation gross weight 10%.
Claims (2)
1. the production method of guanosine comprises the preparation seed culture medium, cultivates seed, prepares fermention medium, inoculation fermentation and fed-batch fermentation, and its concrete steps are as follows:
A, preparation seed culture medium, in water, add glucose 2-4%, monosodium glutamate 0.3-1%, peptone 0.5-1.5%, yeast extract powder 0.5-1.5%, NaCl 0.2-0.3%, urea 0.1-0.3%, corn steep liquor 0.5-1.5% by weight percentage, regulate pH7.0-7.2 with NaOH and hydrochloric acid then, and 110 ℃-120 ℃ the sterilization 10-20 minute, obtain seed culture medium;
B, cultivation seed are produced bacterium with guanosine and are inserted in the seed culture medium, and inoculum size is 8%-15%, under 33 ℃-35 ℃ temperature, keep the pH value being cultured to logarithmic phase in the automatic control fermentor tank at 5L under 6.5 the condition then;
C, preparation fermention medium, in water, add glucose 4.5-5.5%, monosodium glutamate 0.8-1.2%, ammonium sulfate 0.7-0.9%, yeast powder 0.8-1.2%, sal epsom 0.3-0.45%, potassium primary phosphate 0.15-0.25%, calcium chloride 0.15-0.25%, soya-bean cake hydrolyzed solution 1.5-3.3%, corn steep liquor 1.5-1.9% by weight percentage, defoamer 0.03-0.05%, regulate pH7.0-7.2 with NaOH and hydrochloric acid, and 110 ℃-120 ℃ the sterilization 10-20 minute, obtain fermention medium;
D, inoculation fermentation insert the 50L that contains fermention medium with the seed cultivated among the step b by the inoculum size of 8%-15% and control in the fermentor tank automatically and ferment, and the time is 8h, and temperature is that 0-4h is 33.9-34.1 ℃, and 4-8h is 34.9-35.1 ℃;
E, fed-batch fermentation, in the d step, after the 8h temperature is controlled at 35.9-36.1 ℃, add ammoniacal liquor by stream pH is controlled at 6.5, dissolved oxygen is controlled at 40%, begin stream and add the aqueous solution of glucose, yeast powder, soya-bean cake hydrolyzed solution, wherein the weight of glucose, yeast powder, soya-bean cake hydrolyzed solution is respectively 10%, 0.6%, 2% of the total fermented liquid weight behind the inoculation fermentation in the d step, it is 16h that stream adds the time, begin slow stream after the end and add the production of xanthoglobulin promotion guanosine, after 24 hours completed product run.
2. the production method of guanosine according to claim 1, it is characterized in that: the concentration of the aqueous solution of described glucose is 60%-70%.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102304553A (en) * | 2011-09-20 | 2012-01-04 | 中国科学院微生物研究所 | Method for producing L-threonine |
| CN102864193A (en) * | 2012-08-30 | 2013-01-09 | 广东肇庆星湖生物科技股份有限公司 | Production process of guanosine |
| CN112301071A (en) * | 2020-11-04 | 2021-02-02 | 赤峰蒙广生物科技有限公司 | Method for producing adenine by fermentation method |
| CN112522351A (en) * | 2020-12-28 | 2021-03-19 | 广东肇庆星湖生物科技股份有限公司 | Synthetic method of guanine |
-
2011
- 2011-03-22 CN CN2011100680393A patent/CN102181509A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102304553A (en) * | 2011-09-20 | 2012-01-04 | 中国科学院微生物研究所 | Method for producing L-threonine |
| CN102864193A (en) * | 2012-08-30 | 2013-01-09 | 广东肇庆星湖生物科技股份有限公司 | Production process of guanosine |
| CN112301071A (en) * | 2020-11-04 | 2021-02-02 | 赤峰蒙广生物科技有限公司 | Method for producing adenine by fermentation method |
| CN112522351A (en) * | 2020-12-28 | 2021-03-19 | 广东肇庆星湖生物科技股份有限公司 | Synthetic method of guanine |
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Application publication date: 20110914 |