CN112111480A - Method for prolonging shelf life of paecilomyces lilacinus by using double-layer embedding technology - Google Patents

Method for prolonging shelf life of paecilomyces lilacinus by using double-layer embedding technology Download PDF

Info

Publication number
CN112111480A
CN112111480A CN202011093827.3A CN202011093827A CN112111480A CN 112111480 A CN112111480 A CN 112111480A CN 202011093827 A CN202011093827 A CN 202011093827A CN 112111480 A CN112111480 A CN 112111480A
Authority
CN
China
Prior art keywords
paecilomyces lilacinus
shelf life
double
prolonging
water
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
Application number
CN202011093827.3A
Other languages
Chinese (zh)
Inventor
梁承�
陈成
邹林君
吴宁
江漫
罗培繁
闭革林
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanning Harworld Biotechnology Co ltd
Original Assignee
Nanning Harworld Biotechnology Co ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Nanning Harworld Biotechnology Co ltd filed Critical Nanning Harworld Biotechnology Co ltd
Priority to CN202011093827.3A priority Critical patent/CN112111480A/en
Publication of CN112111480A publication Critical patent/CN112111480A/en
Pending legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14Enzymes or microbial cells immobilised on or in an inorganic carrier
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/082Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • C12N11/087Acrylic polymers
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Inorganic Chemistry (AREA)
  • Dispersion Chemistry (AREA)
  • Molecular Biology (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

In order to solve the problems of short shelf life, low spore survival rate, high storage cost, inconvenience in transportation, large-area popularization and use and the like in the preservation of the paecilomyces lilacinus, the invention discloses a method for prolonging the shelf life of the paecilomyces lilacinus by using a double-layer embedding technology, particles with a double-layer structure are mainly formed by the paecilomyces lilacinus, a water-soluble high polymer material, an ultraviolet protective agent, a cross-linking agent, zeolite and water, and the paecilomyces lilacinus spores are wrapped by the inner-layer structure, so that nutrient substances can be provided for the paecilomyces lilacinus, and the survival rate of the paecil; the outer layer structure increases the mechanical strength, can more effectively protect microorganisms, prolongs the quality guarantee period of the paecilomyces lilacinus, and is convenient to transport and popularize and use in a large area.

Description

Method for prolonging shelf life of paecilomyces lilacinus by using double-layer embedding technology
Technical Field
The invention belongs to the technical field of biocontrol bacteria preservation, and particularly relates to a method for prolonging the quality guarantee period of paecilomyces lilacinus by using a double-layer embedding technology.
Background
The control of plant diseases has always been the major focus in agricultural production work. In agriculture, crop root knot nematode disease is caused along with the growth of crop planting time in the same place, in recent years, a large amount of crop yield reduction is caused by a plurality of plant diseases such as blight, root rot and the like caused by the plant root knot nematode, the yield reduction is generally 10% -30%, the serious yield reduction is more than 50% -70%, even no harvest is realized, and the annual loss caused by the root knot nematode disease in China is more than 700 million yuan according to incomplete statistics.
The paecilomyces lilacinus is inhabitation bacteria of various plant roots in soil and is an important natural enemy of plant parasitic nematodes, and the paecilomyces lilacinus is known to have a good effect on preventing and treating plant root-knot nematodes and simultaneously has a prevention and treatment effect on diseases such as fusarium oxysporum caused by various kinds of soil. The paecilomyces lilacinus has the characteristics of high efficiency, broad spectrum, long effect, safety, no pollution, no residue and the like, and cannot cause damage to the environment; in addition, paecilomyces lilacinus can also secrete plant hormones in the growth process, and has remarkable promotion and regulation effects on the growth of plants, so that the paecilomyces lilacinus is widely applied to development of efficient, stable and safe biopesticides.
The prevention and control effect of the paecilomyces lilacinus on plant pathogenic bacteria is mainly realized through spore action. Paecilomyces lilacinus exists in the form of spore, spore germinates to form hypha to invade host under proper temperature and proper nutrition condition to realize disease resistance, the hypha of Paecilomyces lilacinus surrounds the whole egg, the end of the hypha becomes thick, the surface layer of the egg shell is cracked due to the activity of exogenous metabolites and fungal chitinase, and then the fungus invades and replaces the shell. Therefore, the spore survival rate of the paecilomyces lilacinus determines the using effect of the product. However, the paecilomyces lilacinus spores are poor in stress resistance and easy to die, and the using effect of the paecilomyces lilacinus microbial inoculum is seriously influenced.
At present, the common preservation methods of paecilomyces lilacinus spores are as follows: 1. freeze-drying preservation is carried out, and the cost is high; 2. calcium alginate is embedded, so that the cost is high and the calcium alginate is not easy to use; 3. the spores are directly dried or absorbed into carriers such as river sand and the like for drying and low-temperature storage, so that the transportation and the use are not easy; 4. The paecilomyces lilacinus spores are directly exposed in soil environment with uncertain factors after being mixed with activated carbon and stored at normal temperature, so that the survival rate of the spores is low easily, and a method for prolonging the quality guarantee period of the paecilomyces lilacinus is urgently needed.
Disclosure of Invention
The invention aims to solve the problems of short shelf life, low spore survival rate, high storage cost, inconvenience in transportation, large-area popularization and use and the like in the storage of paecilomyces lilacinus, and provides a method for prolonging the shelf life of the paecilomyces lilacinus by using a double-layer embedding technology.
The invention relates to a method for prolonging the quality guarantee period of paecilomyces lilacinus by using a double-layer embedding technology, which comprises the following raw materials: paecilomyces lilacinus; water-soluble high molecular material, ultraviolet protective agent, cross-linking agent, zeolite and water; the ultraviolet ray protective agent is one or more of riboflavin, fluorescein and ascorbic acid.
Preferably, the method for prolonging the shelf life of the paecilomyces lilacinus by using the double-layer embedding technology specifically comprises the following steps:
the method comprises the following steps: mixing a water-soluble polymer material and water according to a mass ratio of 1: 1-50, heating and stirring for 15-30 minutes, and cooling to room temperature to obtain a colloidal solution;
step two: the mass ratio of the colloidal solution to the cross-linking agent is 5-15: 1-5, stirring uniformly to obtain an embedding solution;
step three: uniformly stirring and mixing the paecilomyces lilacinus fermentation liquor and the ultraviolet protective agent according to the mass ratio of 1-5: 0.1-1, uniformly dripping the mixture into embedding liquid at the speed of 30-60 drops/minute, stirring while dripping, and reacting for 5-12 hours to obtain embedded particles;
step four: and uniformly mixing zeolite and the embedded particles, standing for 10-20 hours to obtain outer-layer embedded particles, and exposing in a ventilated and cool environment for 4-10 days to dry in the shade.
Preferably, the method for prolonging the shelf life of the paecilomyces lilacinus by using the double-layer embedding technology is characterized in that the bacterial concentration of the paecilomyces lilacinus is 2.0-50.0 hundred million/ml.
Preferably, the method for prolonging the shelf life of the paecilomyces lilacinus by using the double-layer embedding technology is characterized in that the water-soluble high polymer material is any one or more of modified starch, chitosan and sodium alginate agar, and the cross-linking agent is any one of polyacrylamide and calcium salt.
Preferably, the method for prolonging the shelf life of the paecilomyces lilacinus by using the double-layer embedding technology is characterized in that the calcium salt is any one of calcium carbonate, calcium chloride and calcium sulfate, and gluconolactone is added to form a slow-release calcium source.
The invention at least comprises the following beneficial effects: the invention provides a method for prolonging the quality guarantee period of paecilomyces lilacinus by using a double-layer embedding technology, wherein an inner layer structure contains one or more of ultraviolet protective agents riboflavin, fluorescein and ascorbic acid, so that the protective effect on paecilomyces lilacinus spores is enhanced, and the quality guarantee period is prolonged; in addition, the paecilomyces lilacinus spores are wrapped by the inner-layer structure, so that nutrient substances can be provided for the paecilomyces lilacinus spores, and the influence on the survival rate of the paecilomyces lilacinus spores caused by direct exposure of the paecilomyces lilacinus spores in soil is avoided; the outer structure increases mechanical strength, can more effectively protect microorganisms, and is convenient to transport and popularize and use in a large area.
Detailed Description
Example 1
A method for prolonging the shelf life of Paecilomyces lilacinus by using a double-layer embedding technology comprises the following raw materials: paecilomyces lilacinus; water-soluble high molecular material, ultraviolet protective agent, cross-linking agent, zeolite and water; the ultraviolet protective agent is riboflavin, the water-soluble high polymer material is modified starch, and the cross-linking agent is calcium salt and mainly is a mixture of calcium carbonate and gluconolactone.
Preferably, the method for prolonging the shelf life of the paecilomyces lilacinus by using the double-layer embedding technology comprises the following steps:
the method comprises the following steps: mixing modified starch and water according to the mass ratio of 1:10, heating and stirring for 15 minutes, and cooling to room temperature to obtain a colloidal solution;
step two: the mass ratio of the colloidal solution to the calcium salt is 5: 1, uniformly stirring to obtain an embedding solution;
step three: uniformly stirring and mixing 2.0 hundred million/ml of paecilomyces lilacinus fermentation liquor and an ultraviolet ray protective agent riboflavin according to a mass ratio of 1:0.3, uniformly dripping 60 drops/min into embedding liquid while stirring, and reacting for 5 hours to obtain embedded particles;
step four: and uniformly mixing zeolite and the embedded particles, standing for 10 hours to obtain outer-layer embedded particles, and exposing in a ventilated and cool environment for 4 days to dry in the shade.
Example 2
A method for prolonging the shelf life of Paecilomyces lilacinus by using a double-layer embedding technology comprises the following raw materials: paecilomyces lilacinus; water-soluble high molecular material, ultraviolet protective agent, cross-linking agent, zeolite and water; the ultraviolet ray protective agent is ascorbic acid, the water-soluble polymer material is sodium alginate, and the cross-linking agent is calcium salt, mainly a mixture of calcium chloride and gluconolactone.
Preferably, the method for prolonging the shelf life of the paecilomyces lilacinus by using the double-layer embedding technology comprises the following steps:
the method comprises the following steps: mixing sodium alginate and water according to a mass ratio of 1:5, heating and stirring for 20 minutes, and cooling to room temperature to obtain a colloidal solution;
step two: the mass ratio of the colloidal solution to the cross-linking agent is 10: 3, uniformly stirring to obtain an embedding solution;
step three: mixing the paecilomyces lilacinus fermentation liquor with the thallus concentration of 10.0 hundred million/ml and the ultraviolet protective agent uniformly according to the mass ratio of 5: 1, uniformly dripping the mixture into embedding liquid at a speed of 45 drops/minute, stirring while dripping, and reacting for 12 hours to obtain embedded particles;
step four: and uniformly mixing zeolite and the embedded particles, standing for 20 hours to obtain outer-layer embedded particles, and exposing in a ventilated and cool environment for 5 days and drying in the shade.
Example 3
A method for prolonging the shelf life of Paecilomyces lilacinus by using a double-layer embedding technology comprises the following raw materials: paecilomyces lilacinus; water-soluble high molecular material, ultraviolet protective agent, cross-linking agent, zeolite and water; the ultraviolet ray protective agent is a mixture of ascorbic acid and riboflavin, the water-soluble polymer material is a mixture of sodium alginate and modified starch, and the cross-linking agent is calcium salt and mainly calcium sulfate.
Preferably, the method for prolonging the shelf life of the paecilomyces lilacinus by using the double-layer embedding technology comprises the following steps:
the method comprises the following steps: mixing a water-soluble polymer material and water according to a mass ratio of 1: 50, heating and stirring for 30 minutes, and cooling to room temperature to obtain a colloidal solution;
step two: the mass ratio of the colloidal solution to the cross-linking agent is 15: 3, uniformly stirring to obtain an embedding solution;
step three: uniformly stirring and mixing the paecilomyces lilacinus fermentation liquor with the thallus concentration of 50.0 hundred million/ml and the ultraviolet protective agent according to the mass ratio of 3:0.3, uniformly dripping into embedding liquid at the speed of 30 drops/minute, stirring while dripping, and reacting for 10 hours to obtain embedded particles;
step four: and uniformly mixing zeolite and the embedded particles, standing for 15 hours to obtain outer-layer embedded particles, and exposing in a ventilated and cool environment for 10 days to dry in the shade.
Example 4
And (3) storing the paecilomyces lilacinus embedded particles prepared in the step 1-3 at the temperature of 0-30 ℃, and determining the germination rate. The results are given in the table below.
Table 1: step 1 the prepared granules are preserved for 12 months under the condition of 0-30 ℃ to have spore germination rate.
Figure DEST_PATH_IMAGE001
The result shows that the method for prolonging the quality guarantee period of the paecilomyces lilacinus by using the double-layer embedding technology is stored at different temperatures, and the spore germination rate is more than 85%.
While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable to a variety of fields of endeavor for which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.

Claims (5)

1. A method for prolonging the shelf life of Paecilomyces lilacinus by using a double-layer embedding technology is characterized by comprising the following raw materials: paecilomyces lilacinus; water-soluble high molecular material, ultraviolet protective agent, cross-linking agent, zeolite and water; the ultraviolet ray protective agent is one or more of riboflavin, fluorescein and ascorbic acid.
2. The method for prolonging the shelf life of Paecilomyces lilacinus by using the double-layer embedding technology according to claim 1, comprising the following steps:
the method comprises the following steps: mixing a water-soluble polymer material and water according to a mass ratio of 1: 1-50, heating and stirring for 15-30 minutes, and cooling to room temperature to obtain a colloidal solution;
step two: the mass ratio of the colloidal solution to the cross-linking agent is 5-15: 1-5, stirring uniformly to obtain an embedding solution;
step three: uniformly stirring and mixing the paecilomyces lilacinus fermentation liquor and the ultraviolet protective agent according to the mass ratio of 1-5: 0.1-1, uniformly dripping the mixture into embedding liquid at the speed of 30-60 drops/minute, stirring while dripping, and reacting for 5-12 hours to obtain embedded particles;
step four: and uniformly mixing zeolite and the embedded particles, standing for 10-20 hours to obtain outer-layer embedded particles, and exposing in a ventilated and cool environment for 4-10 days to dry in the shade.
3. The method for prolonging the shelf life of the paecilomyces lilacinus by using the double-layer embedding technology as claimed in claim 2, wherein the concentration of the paecilomyces lilacinus is 2.0-50.0 hundred million/ml.
4. The method for prolonging the shelf life of paecilomyces lilacinus by using the double-layer embedding technology according to claims 1-2, wherein the water-soluble high polymer material is any one or more of modified starch, chitosan and sodium alginate agar, and the cross-linking agent is any one of polyacrylamide and calcium salt.
5. The method for prolonging the shelf life of Paecilomyces lilacinus according to claim 4, wherein the calcium salt is selected from calcium carbonate, calcium chloride, and calcium sulfate, and gluconolactone is added to form a slow-release calcium source.
CN202011093827.3A 2020-10-14 2020-10-14 Method for prolonging shelf life of paecilomyces lilacinus by using double-layer embedding technology Pending CN112111480A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202011093827.3A CN112111480A (en) 2020-10-14 2020-10-14 Method for prolonging shelf life of paecilomyces lilacinus by using double-layer embedding technology

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202011093827.3A CN112111480A (en) 2020-10-14 2020-10-14 Method for prolonging shelf life of paecilomyces lilacinus by using double-layer embedding technology

Publications (1)

Publication Number Publication Date
CN112111480A true CN112111480A (en) 2020-12-22

Family

ID=73797847

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202011093827.3A Pending CN112111480A (en) 2020-10-14 2020-10-14 Method for prolonging shelf life of paecilomyces lilacinus by using double-layer embedding technology

Country Status (1)

Country Link
CN (1) CN112111480A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113699142A (en) * 2021-08-30 2021-11-26 南宁汉和生物科技股份有限公司 Paecilomyces lilacinus embedding method

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101100646A (en) * 2007-06-25 2008-01-09 中国科学院微生物研究所 Paecilomyces lilacinus and application thereof
CN106376602A (en) * 2016-08-31 2017-02-08 江西天人生态股份有限公司 Paecilomyces lilacinus granule and preparation method thereof
CN111642520A (en) * 2020-07-16 2020-09-11 浙江省柑桔研究所 Paecilomyces lilacinus wettable powder and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101100646A (en) * 2007-06-25 2008-01-09 中国科学院微生物研究所 Paecilomyces lilacinus and application thereof
CN106376602A (en) * 2016-08-31 2017-02-08 江西天人生态股份有限公司 Paecilomyces lilacinus granule and preparation method thereof
CN111642520A (en) * 2020-07-16 2020-09-11 浙江省柑桔研究所 Paecilomyces lilacinus wettable powder and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
史怀;肖马云;刘波;: "淡紫拟青霉的固定化研究", 武夷科学, no. 00 *
张寅平: "《建筑环境传质学》", 中国建筑工业出版社, pages: 179 - 180 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113699142A (en) * 2021-08-30 2021-11-26 南宁汉和生物科技股份有限公司 Paecilomyces lilacinus embedding method

Similar Documents

Publication Publication Date Title
EP2676536A1 (en) Method for producing plant seed containing endophytic micro-organisms
CN102224784B (en) Chemical and biological combined soil remediation method of banana wasteland
CN110577916B (en) Composite microbial inoculant for preventing and controlling root-knot nematodes of crops and preparation method thereof
CN107652027A (en) A kind of complex composition of biological control crop droop and its application
CN110004082B (en) Bacterial strain suitable for nitrogen and phosphorus pollution regulation and control in facility agriculture and application
US11976014B2 (en) Special controlled-release bacterial fertilizer for peanuts and preparation method thereof
CN112111480A (en) Method for prolonging shelf life of paecilomyces lilacinus by using double-layer embedding technology
CN107668075A (en) A kind of coating bacteria agent for preventing and treating clubroot and preparation method thereof
CN107691209A (en) Straw biomass rice soilless breeding seeding disk
CN110679432A (en) Seedling culture medium and preparation method thereof
CN114763311A (en) Compound microbial agent for preventing and treating bacterial diseases of fruits and vegetables as well as preparation method and application of compound microbial agent
CN108314568A (en) A kind of slow release long-acting mixed fertilizer preventing graw mold of tomato and root rot
CN113812292A (en) Method for preventing and treating pear tree yellowing disease
CN106699462A (en) Organic fertilizer for flowers, and preparation method thereof
CN111296227A (en) Organic planting soil and preparation method thereof
CN112500217A (en) Mineral compound microbial agent and preparation method and application thereof
CN106631446B (en) Organic master batch and preparation method and application thereof
CN110679431A (en) Peach seed emergence substrate and seedling raising method
CN110330389A (en) It is a kind of using feces of livestock and poultry as organic fertilizer of raw material and preparation method thereof
Yanni et al. Rice performance and natural infection with blast (Pyricularia oryzae Cav.) under different algalization techniques and rates of fertilizer nitrogen
CN117088733B (en) Biological organic fertilizer for preventing and treating tomato soil borne diseases and preparation method thereof
CN108990571B (en) Matrix for maple cuttage
KR102660548B1 (en) Bacillus novalis KT24 strain having auxin producting ability, phosphate solubilizing ability, and nitrates or nitrites eliminating ability, and use thereof
CN112661547B (en) Composite biological decomposition agent and preparation method thereof
CN103319248B (en) Microorganism fertilizer for preventing and treating valsa mali of lettuce and preparation method thereof

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: 20201222

RJ01 Rejection of invention patent application after publication