CN111394280A - Culture medium suitable for growth of bacillus licheniformis and application thereof - Google Patents
Culture medium suitable for growth of bacillus licheniformis and application thereof Download PDFInfo
- Publication number
- CN111394280A CN111394280A CN202010223315.8A CN202010223315A CN111394280A CN 111394280 A CN111394280 A CN 111394280A CN 202010223315 A CN202010223315 A CN 202010223315A CN 111394280 A CN111394280 A CN 111394280A
- Authority
- CN
- China
- Prior art keywords
- bacillus licheniformis
- culture medium
- growth
- fermentation
- steep liquor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N3/00—Spore forming or isolating processes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a culture medium suitable for growth of bacillus licheniformis and application thereof. The culture medium comprises the following components: corn steep liquor dry powder, soybean meal powder, molasses, ammonium sulfate, magnesium sulfate, manganese sulfate and dipotassium phosphate. Compared with the prior art, the method optimizes the type of the carbon and nitrogen source, mainly utilizes corn steep liquor dry powder to replace peptone in the aspect of the nitrogen source, and utilizes molasses to replace glucose in the aspect of the carbon source. The raw materials of the two are easy to obtain in the aspect of purchasing, and the cost is lower; meanwhile, compared with glucose, the molasses contains more growth factors for promoting bacteria growth, can meet the requirement of bacteria growth, and improves the bacteria quantity level on the original basis; and the corn steep liquor dry powder contains trace elements, so that the generation of bacillus licheniformis spores can be promoted, and the survival rate of thalli is remarkably improved.
Description
Technical Field
The invention relates to a culture medium, in particular to a culture medium suitable for growth of bacillus licheniformis and application thereof.
Background
Bacillus licheniformis is one of feed-grade strains approved for use in 2003 announced by Ministry of agriculture in China, and plays an important role in improving intestinal health, promoting animal growth and improving host immunity. The strain has strong stress resistance, high humidity and high pressure resistance and easy storage, can secrete protease, amylase, lipase, various amino acids and the like, is considered as the most ideal microbial additive, and has higher application value in the aspects of livestock breeding and the like.
In industrial production, the fermentation of the bacillus licheniformis mainly comprises solid fermentation and liquid fermentation, the solid fermentation is relatively stable, the bacterial quantity is limited to a certain extent, the liquid fermentation can greatly improve the bacterial quantity, and the high bacterial quantity is formed, but depends on a nutrient-rich culture medium.
Chinese patent CN102260637A discloses a liquid fermentation culture medium of Bacillus mucilaginosus PM13, which comprises 3.0-5.0 g/L of molasses, 1.0-3.0 g/L of starch, 5.0-10.0 g/L of soybean meal, 4.0-10.0 g/L of calcium carbonate, 1.0-3.0 g/L of dipotassium hydrogen phosphate, 1.0-3.0 g/L-5.0 g/L of magnesium sulfate heptahydrate, pH 7.0-8.0 and distilled water.
Chinese patent CN103525728B provides a process technology for forming spores by using industrial liquid of Bacillus mucilaginosus, and particularly relates to a submerged fermentation culture medium of Bacillus mucilaginosus applied to microbial fertilizers. The composite material comprises the following raw materials in percentage by weight: 0.9-1.5% of corn flour, 0.1-0.3% of soybean meal, 0.08-0.2% of dipotassium hydrogen phosphate and the balance of water. Mixing corn flour, soybean meal powder, dipotassium phosphate and water, stirring uniformly, sterilizing at 121 ℃ for 20-40 min, and cooling to 25-40 ℃ to obtain the culture medium. The pH value of the culture medium is about 6.0-7.5, and the colloidal spores grow well within the pH value range of 6.0-8.0, so that the culture medium can promote the growth of thalli and the formation of spores without using acid and alkali to adjust the pH value before sterilization, the operation is simplified, the product quality is improved, the production cost is reduced, and the method is more suitable for industrial large-scale production.
CN104862263A discloses a culture medium containing corn stalks, a preparation method thereof and a method for culturing bacillus subtilis (or lactobacillus plantarum), wherein the culture medium comprises corncobs, corn stalks, bean pulp and soy molasses; the preparation method adopts a mixing method. The culture method of the strain comprises the following steps: activating the strain, preparing seed liquid, and inoculating to culture medium. The cells were cultured with shaking in a 37 ℃ incubator for 24 hours. The medium contained peptone.
The cost of the culture medium for producing the currently used bacillus licheniformis is generally higher, and the production benefit obtained finally is limited to a certain extent. Therefore, the method for finding a culture medium formula which is low in cost and does not influence the bacterial quantity becomes one of the means for fermentation production application of the bacillus licheniformis and effectively reducing the cost.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a culture medium suitable for growth of bacillus licheniformis and application thereof.
The purpose of the invention can be realized by the following technical scheme:
the invention provides a culture medium suitable for growth of bacillus licheniformis, which comprises the following components of 3.5-5 g/L of corn steep liquor dry powder, 2-3 g/L of soybean meal, 0.8-1.5 g/L of molasses, 0.2-0.3 g/L of ammonium sulfate, 0.005-0.007 g/L of magnesium sulfate, 0.001-0.0015 g/L of manganese sulfate, and 0.3-0.4 g/L of dipotassium phosphate.
The corn steep liquor dry powder is prepared by taking fresh corn steep liquor as a raw material and carrying out low-temperature instantaneous heating and spray drying. The water-soluble protein of the corn steep liquor dry powder is well preserved, all characteristics of the corn steep liquor are kept, the content of the protein and the trace elements is high, and the corn steep liquor dry powder can be used as a nutritional element supplement such as water-soluble plant protein and water-soluble vitamins in the biological fermentation process. The invention mainly utilizes corn steep liquor dry powder to replace peptone as a nitrogen source. Meanwhile, the trace elements in the corn steep liquor dry powder can promote the generation of bacillus licheniformis spores, and the survival rate of the bacteria is obviously improved.
Molasses is a byproduct of sugar industry, mainly contains a large amount of fermentable sugar, is a good fermentation raw material, has high mineral content of about 8-10%, can resist acid and alkali and oxidation, and promotes the absorption and utilization of minerals by livestock and poultry; it also contains vitamins, minerals, mycoprotein, nucleic acid, surfactant, growth promoting factor (bioactive substance), etc. The invention utilizes molasses to replace glucose as a carbon source. Compared with glucose, molasses has more growth factors for promoting bacteria growth, can meet the requirement of bacteria growth, and improves the bacteria quantity level on the original basis.
Further, the culture medium also contains an antifoaming agent.
Furthermore, the content of the defoaming agent is 0.03-0.05 g/L. if the amount of the defoaming agent is too much, the growth of bacteria can be inhibited, and if the amount of the defoaming agent is too little, the defoaming effect can be influenced.
Further, the culture medium is a culture medium for liquid fermentation.
The invention further improves the bacterial load of the bacillus licheniformis by mainly optimizing the components of the carbon and nitrogen source in the culture medium. The optimized culture medium not only effectively improves the bacterial load of the bacillus licheniformis, but also reduces the cost of the culture medium and improves the production benefit.
The invention also provides an application of the culture medium, the culture medium is used for fermenting the bacillus licheniformis, and the method comprises the following steps:
inoculating the bacillus licheniformis stored on the inclined plane into a shake flask fermentation culture medium, and performing shake culture at 37 ℃ to obtain a fermentation seed solution;
and adding the fermentation seed liquid into a fermentation tank containing the culture medium for fermentation.
Further, the formula of the shake flask fermentation medium comprises 10 g/L of peptone, 5 g/L of yeast powder, 5 g/L of sodium chloride and 2 g/L of glucose.
Further, the conditions for fermentation in the fermenter were: temperature: 37 ℃; ventilating: 3-5m3H; pH: 7.2; rotating speed: 200 and 300 rpm.
Further, after fermentation, the bacterial load of the Bacillus licheniformis reaches 210-230 × 108CFU/m L, the spore rate reaches 95-97%, the spore number reaches 199-220 × 108CFU/mL。
Compared with the prior art, the invention has the following characteristics:
1. optimizes the kind of nitrogen source, and mainly utilizes corn steep liquor dry powder and soybean meal as organic nitrogen source to replace peptone. The corn steep liquor dry powder mainly comprises amino acids and polypeptides, and contains abundant vitamins, growth factors, and a small amount of reducing sugar and lactic acid, wherein abundant proteins can provide a nitrogen source for bacillus licheniformis, and the small amount of reducing sugar and lactic acid can provide a part of carbon source and organic acid, so that the cost of a culture medium can be reduced, and the generation of bacillus licheniformis spores is promoted. Ammonium sulfate is used as an inorganic nitrogen source for Bacillus licheniformis, and the ammonium sulfate exists in the raw material in the form of ammonium salt and can be directly decomposed into ammonium to be used by microorganisms. The soybean meal provides a nitrogen source for the growth of the bacterial strain, the material is easy to obtain, and the cost is low.
2. In the aspect of carbon source, molasses is used for replacing glucose, the molasses contains rich nutrient substances such as cane sugar, reducing sugar (glucose and fructose), amino acid, vitamins and trace elements, and compared with glucose, the molasses contains more bacteria growth promoting factors, can meet the requirement of thallus growth, and improves the bacteria quantity level on the original basis.
3. The corn steep liquor dry powder containing trace elements can promote the generation of bacillus licheniformis spores and obviously improve the survival rate of thalli. Meanwhile, phosphorus and potassium elements are all nutrients necessary for the growth of microorganisms, and dipotassium hydrogen phosphate is used as a culture medium, increases the phosphorus and potassium elements, is alkalescent, can be used as a buffering agent to balance acid generated by the bacillus licheniformis during fermentation, maintains the pH value suitable for the growth of the bacillus licheniformis, synergistically promotes the generation of bacillus licheniformis, and improves the survival rate of thalli.
Detailed Description
The present invention will be described in detail with reference to specific examples.
And (3) carrying out fermentation experiments on the bacillus licheniformis according to an experimental design scheme. Sampling and microscopic examination are carried out at regular time in the experimental process, and the growth condition and spore formation condition of the bacillus licheniformis thallus are observed. After the fermentation was completed, the amount of bacteria was measured by the dilution plate application method.
The different media formulations in examples 1-5 are shown in table 1.
TABLE 1 culture Medium formulation
| Ingredients (g/L) | Example 1 | Example 2 | Example 3 | Example 4 | Example 5 |
| Corn steep liquor dry powder | 4 | 3.5 | 4.5 | 5 | 4.5 |
| Soybean meal powder | 2.5 | 3 | 2 | 2 | 2.5 |
| Molasses for health protection | 1.5 | 0.9 | 1 | 1.5 | 0.8 |
| Ammonium sulfate | 0.25 | 0.3 | 0.25 | 0.2 | 0.2 |
| Magnesium sulfate | 0.005 | 0.006 | 0.005 | 0.007 | 0.006 |
| Dipotassium hydrogen phosphate | 0.3 | 0.35 | 0.35 | 0.4 | 0.3 |
| Manganese sulfate | 0.0015 | 0.001 | 0.001 | 0.0015 | 0.001 |
| Defoaming agent | 0.04 | 0.04 | 0.05 | 0.03 | 0.04 |
The formulation of the culture medium of the comparative example is shown in Table 2.
TABLE 2 culture Medium formulation of comparative examples
Bacillus licheniformis was cultured using the fermentation media of lichen of examples 1-5 and comparative example as follows:
inoculating the bacillus licheniformis preserved on the inclined plane into a shaking flask fermentation medium of 100m L, wherein the formula of the shaking flask fermentation medium comprises peptone of 10 g/L, yeast powder of 5 g/L, sodium chloride of 5 g/L and glucose of 2 g/10, and shaking-culturing the bacillus licheniformis at 37 ℃ and 200rpm for 24 hours to serve as fermentation seed liquid;
fermenting the seed liquid in a 50L fermenter containing the culture medium at 37 deg.C under 3-5m of air3H; pH: 7.2; rotating speed: 200 and 300 rpm.
The results of culturing Bacillus licheniformis using the fermentation media for lichen of examples 1-5 and comparative example are shown in Table 3.
TABLE 3
Note: calculating the spore rate: after fermentation is finished, measuring the total bacterial quantity by adopting a flat plate coating dilution counting method; after 20min after 80 ℃ water bath, plate coating, dilution and counting are carried out to detect the spore generation amount. The spore rate is calculated by the following steps: spore productivity ═ 100% spore formation/total viable count.
In this experimental study, it was found that the fermentation medium used in this experiment was used to make the lichen sporeThe bacterial load of bacillus reaches 205-8CFU/m L, the spore rate reaches 95-97 percent, and the spore number 199, 220 and 220 × 108CFU/m L, the viable count, the total amount of spores and the formation rate are greatly improved on the premise of reducing the cost.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (9)
1. The culture medium suitable for growth of the bacillus licheniformis is characterized by comprising 3.5-5 g/L of corn steep liquor dry powder, 2-3 g/L of soybean meal, 0.8-1.5 g/L of molasses, 0.2-0.3 g/L of ammonium sulfate, 0.005-0.007 g/L of magnesium sulfate, 0.001-0.0015 g/L of manganese sulfate and 0.3-0.4 g/L of dipotassium hydrogen phosphate.
2. The culture medium for the growth of bacillus licheniformis according to claim 1, wherein the corn steep liquor dry powder is prepared by using fresh corn steep liquor as raw material and performing low-temperature instantaneous heating and spray drying.
3. The culture medium suitable for growth of bacillus licheniformis according to claim 1, characterized in that the culture medium further comprises an antifoaming agent.
4. A culture medium suitable for the growth of Bacillus licheniformis according to claim 3 wherein the amount of antifoaming agent is 0.03-0.05 g/L.
5. The medium suitable for growth of bacillus licheniformis according to claim 1 characterized in that the medium is a liquid fermentation medium.
6. The use of a medium according to claim 1 for the fermentation of bacillus licheniformis by:
inoculating the bacillus licheniformis stored on the inclined plane into a shake flask fermentation culture medium, and performing shake culture at 37 ℃ to obtain a fermentation seed solution;
fermenting the seed broth by adding the seed broth to a fermentor containing the medium of claim 1.
7. The culture medium suitable for the growth of the bacillus licheniformis according to the claim 6, wherein the formulation of the shake flask fermentation medium is peptone 10 g/L, yeast powder 5 g/L, sodium chloride 5 g/L, glucose 2 g/L.
8. A culture medium suitable for the growth of Bacillus licheniformis according to claim 6, characterized in that the fermentation is carried out under the following conditions: temperature: 37 ℃; ventilating: 3-5m3H; pH: 7.2; rotating speed: 200 and 300 rpm.
9. The medium for growing Bacillus licheniformis according to claim 6, wherein the fermentation is performed in such an amount that the bacterial load of Bacillus licheniformis reaches 205-8CFU/m L, the spore rate reaches 95-97 percent, and the spore number 199, 220 and 220 × 108CFU/mL。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010223315.8A CN111394280A (en) | 2020-03-26 | 2020-03-26 | Culture medium suitable for growth of bacillus licheniformis and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010223315.8A CN111394280A (en) | 2020-03-26 | 2020-03-26 | Culture medium suitable for growth of bacillus licheniformis and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111394280A true CN111394280A (en) | 2020-07-10 |
Family
ID=71427681
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010223315.8A Pending CN111394280A (en) | 2020-03-26 | 2020-03-26 | Culture medium suitable for growth of bacillus licheniformis and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111394280A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109468259A (en) * | 2018-10-24 | 2019-03-15 | 亚太星原农牧科技海安有限公司 | A kind of culture medium for promoting gemma to generate |
| CN114703087A (en) * | 2022-02-09 | 2022-07-05 | 上海圣珑环境修复材料有限公司 | Bacillus licheniformis fermentation culture medium, biological preparation and application of biological preparation |
| CN115466700A (en) * | 2022-10-10 | 2022-12-13 | 山东天润和生物工程有限公司 | Novel fermentation medium formula of bacillus licheniformis and culture method thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6010898A (en) * | 1997-09-13 | 2000-01-04 | Soonchundang Pharmaceuticals Co., Ltd. | Liquid cultivation of strains of Bacillus polyfermenticus |
| CN103232959A (en) * | 2013-04-23 | 2013-08-07 | 保龄宝生物股份有限公司 | Preparation method and application of feeding bacillus licheniformis inoculant |
| CN103525728A (en) * | 2013-10-10 | 2014-01-22 | 合肥市钱鑫生物科技发展有限公司 | Submerged fermentation culture medium of bacillus mucilaginosus applied to microbial fertilizers |
| CN109468259A (en) * | 2018-10-24 | 2019-03-15 | 亚太星原农牧科技海安有限公司 | A kind of culture medium for promoting gemma to generate |
| CN110305812A (en) * | 2019-07-05 | 2019-10-08 | 山东苏柯汉生物工程股份有限公司 | A kind of the lichen bacillus ferments culture process |
-
2020
- 2020-03-26 CN CN202010223315.8A patent/CN111394280A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6010898A (en) * | 1997-09-13 | 2000-01-04 | Soonchundang Pharmaceuticals Co., Ltd. | Liquid cultivation of strains of Bacillus polyfermenticus |
| CN103232959A (en) * | 2013-04-23 | 2013-08-07 | 保龄宝生物股份有限公司 | Preparation method and application of feeding bacillus licheniformis inoculant |
| CN103525728A (en) * | 2013-10-10 | 2014-01-22 | 合肥市钱鑫生物科技发展有限公司 | Submerged fermentation culture medium of bacillus mucilaginosus applied to microbial fertilizers |
| CN109468259A (en) * | 2018-10-24 | 2019-03-15 | 亚太星原农牧科技海安有限公司 | A kind of culture medium for promoting gemma to generate |
| CN110305812A (en) * | 2019-07-05 | 2019-10-08 | 山东苏柯汉生物工程股份有限公司 | A kind of the lichen bacillus ferments culture process |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109468259A (en) * | 2018-10-24 | 2019-03-15 | 亚太星原农牧科技海安有限公司 | A kind of culture medium for promoting gemma to generate |
| CN109468259B (en) * | 2018-10-24 | 2022-04-05 | 亚太星原农牧科技海安有限公司 | Culture medium for promoting spore generation |
| CN114703087A (en) * | 2022-02-09 | 2022-07-05 | 上海圣珑环境修复材料有限公司 | Bacillus licheniformis fermentation culture medium, biological preparation and application of biological preparation |
| CN115466700A (en) * | 2022-10-10 | 2022-12-13 | 山东天润和生物工程有限公司 | Novel fermentation medium formula of bacillus licheniformis and culture method thereof |
| CN115466700B (en) * | 2022-10-10 | 2024-06-11 | 山东天润和生物工程有限公司 | Novel bacillus licheniformis fermentation medium formula and culture method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104996722B (en) | A kind of method of the step combined ferment feed of multi-cultur es two | |
| CN111394280A (en) | Culture medium suitable for growth of bacillus licheniformis and application thereof | |
| CN103468594B (en) | Candidautilis strain and application thereof | |
| CN104757267A (en) | Apple pomace microbial culture starter and method for producing biological feed by apple pomace microbial culture starter | |
| CN109468259B (en) | Culture medium for promoting spore generation | |
| CN104664154A (en) | Yeast culture and preparation method thereof | |
| CN113243450A (en) | Method for improving quality of mushroom bran feed through multi-strain mixed fermentation | |
| CN115181707A (en) | Bacillus subtilis and liquid-solid two-phase fermentation method thereof | |
| CN110846362B (en) | Neomycin sulfate fermentation clean production method | |
| CN104388341A (en) | Bacillus mucilaginosus strain and application thereof in seaweed degradation | |
| CN101451107B (en) | Method for large scale preparing Gliocladium chlamydospore | |
| CN102127515B (en) | Screening and application of L-proline high-producing Brevundimonas sp. (JNPP-1) | |
| CN113817635A (en) | Method for culturing bacillus by using soybean whey wastewater | |
| CN109699812A (en) | Solid state fermentation produces feeding saccharomyces cerevisiae-lactobacillus plantarum product mix method | |
| CN102787153B (en) | Method for producing enramycin by microbial fermentation supplement feed | |
| CN104497174A (en) | Method for coagulating natural latex by using compound microorganisms | |
| CN102392008B (en) | Bioprotein capable of replacing protein raw material and preparation method of bioprotein | |
| CN113875975B (en) | Fermentation process for preparing metaplasia by using wheat processing byproducts | |
| CN105994940B (en) | Bioactive protein feed and preparation method thereof | |
| CN113046257B (en) | Fermentation culture method of bacillus pumilus | |
| CN116355771A (en) | Yeast protein with antibacterial function and preparation method thereof | |
| CN112852680A (en) | Liquid fermentation method of bacillus coagulans with high spore number | |
| CN113528599A (en) | Production method of efficient chelating enzyme peptide | |
| CN102533604A (en) | Brevibacterium flavum and application thereof as well as method for preparing lysine through fermentation | |
| RU2658977C1 (en) | Method for producing protein fodder additive |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200710 |
|
| RJ01 | Rejection of invention patent application after publication |