CN106442817A - Method for detecting mold in food - Google Patents
Method for detecting mold in food Download PDFInfo
- Publication number
- CN106442817A CN106442817A CN201611083083.0A CN201611083083A CN106442817A CN 106442817 A CN106442817 A CN 106442817A CN 201611083083 A CN201611083083 A CN 201611083083A CN 106442817 A CN106442817 A CN 106442817A
- Authority
- CN
- China
- Prior art keywords
- mobile phase
- sample
- acetonitrile
- centrifugation
- extracting solution
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
Abstract
The invention discloses a method for detecting mold in food. The method includes the steps: weighing 1-5g of video samples to be detected by a standard weighing appliance; vibrating the samples by 20ml of 80% acetonitrile-water solution containing 0.1% of methanoic acid and extracting supernate; placing the solution into a centrifuge for centrifuging for 20-40 minutes at the speed of 1000-2000 revolutions per minute to obtain extracting solution A. After the food is pretreated by an improved QuEChERS method, 26 extracted mold toxins are simultaneously detected by super-efficient liquid chromatography tandem mass spectrometry. The samples are extracted twice by the acid-containing acetonitrile-water solution with different concentrations, the sample extracting solution is salted out, degreased, purified, concentrated and re-dissolved, the mold is detected by a multi-reaction monitoring mode of super-efficient liquid chromatography tandem mass spectrometry, detection efficiency is improved, detection cost is saved, sensitivity and adding standard recovery rate are high, repeatability is good, and the method can be expanded to detect other samples with high fat content.
Description
Technical field
The present invention relates to a kind of detection method, the detection method of mycete in specifically a kind of food.
Background technology
Mycotoxin is a global problem, according to food and agricultural organization estimation, the whole world have every year about 5～7% grain,
The agricultural product such as feedstuff are encroached on by mycete, and 25% corn is by known mycotoxin contamination, and causes hundreds billion of every year dollars
Loss.The nineties in 20th century, it is higher than 1,000,000,000 dollars that Canadian feedstuff industry causes estimated amount of damage by mycotoxin contamination, and the U.S.
Then up to 2,500,000,000 dollars, the mixed feed that the Asian-Pacific area produces simultaneously is subject to mycotoxin contamination to be about 1.6 hundred million tons, causes every year
The loss that up to 10,000,000,000 dollars of the whole world.
Mycotoxin be by some funguses as aspergillosiss, Fusarium spp., penicillium etc. produced two grades after growth and maturity
Metabolite, these toxin all can produce in multiple links such as the results of corn, storage, processing, transports.The mycete being currently known
Toxin has more than 400 kinds, mainly includes aflatoxin, 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone, vomitoxin etc..These toxin are high temperature resistant, resistance to plus
Various process during work, virulence is strong, very harmful to humans and animals.As aflatoxin people and animals are had strong pathogenic
And carcinogenecity, it is the mycotoxin that current toxicity endangers the most by force most serious.6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone (ZEA) is a kind of reproductive system poison
Element, can cause estrogen excessive, lead to animals femaleization to be poisoned, the particularly effect to pig is especially pronounced, can lead to pudendum
There is pathological changes, mammary gland swelling, and have ovarian cyst to occur, can also result in calf sexual precosity, milk cow colpitis, oestrus extend
Deng.Vomitoxin is declined with cow feeding amount and milk yield decline is closely related.Vomitoxin has immunosuppressive action, in milk
In cattle body, performance becomes apparent from.
Mycotoxin is widely present in the food that corn and cereal materialses are made.Mycotoxin in food is exceeded to ask
Topic is increasingly serious, especially cereals grain, and the corn easily going mouldy is food such as vegetable oil, flour, rice flour etc. of raw material
Mycotoxin exceeded serious.It is enriched with more mycotoxin in addition with the livestock products such as milk.Health is caused hinder
Evil, such as：The growth of impact child, increases carcinogenic probability etc..
The detection technique for mycotoxin residual quantity in food commonly used at present mainly has enzyme linked immunological kit
（ELISA）, thin layer chromatography（TLC）, high performance liquid chromatography（HPLC）And Ultra Performance Liquid Chromatography tandem mass spectrometry（UPLC-
MS/MS）Deng.Euzymelinked immunosorbent assay (ELISA) principle is to be combined with multienzyme complex using antibody, then to be detected by colour developing；Thin layer chromatography
It is according to the Rf value with the suitable tester chromatogram of gained by the same method（Rf）Compare, the method carrying out assay,
Both of which is semi-quantitative method, is used for a kind of or a few toxin preliminary examination；High performance liquid chromatography is to adopt
High pressure transfusion system, pumps into having the mobile phase such as the single solvent of opposed polarity or the mixed solvent of different proportion, buffer
Equipped with the chromatographic column of fixing phase, after each composition is by separation in post, enters detector and detected, thus realizing sample is divided
Analysis, this standard measure is accurate, but during for detecting multi-component mycotoxin, and incomplete is purified to sample substrate simultaneously, mesh
Mark compound cannot realize baseline separation on a column it is impossible to carry out accurate quantitative analysis to target compound.
Content of the invention
It is an object of the invention to provide in a kind of food mycete detection method, with solve in above-mentioned background technology propose
Problem.
For achieving the above object, the present invention provides following technical scheme：
In a kind of food, the detection method of mycete, comprises the steps of；Weighing utensil using standard weighs 1-5g video to be measured
Sample, the acetonitrile-aqueous solution first containing the 80% of volume ratio 0.1% formic acid with 20ml carries out mechanical shaking extraction supernatant to sample, then
Put in centrifuge turn through 1000-2000/min speed carries out the centrifugally operated of 20-40min, obtains extracting solution A, then to extraction
Residue afterwards with 3-7ml contain volume ratio 0.1% formic acid volume ratio 20% acetonitrile-aqueous solution carry out mechanical shaking extraction 30min, from
The heart, obtains extracting solution 2, merges described extracting solution 1 and described extracting solution 2, obtains sample extracting solution；Described sample extracting solution adds
1.0g sodium citrate, 4.0g magnesium sulfate, 1.0g sodium chloride, 0.5g DisodiumHydrogen Citrate are saltoutd, and shift supernatant after centrifugation,
It is added thereto to 20ml normal hexane, vortex vibration 1min removes fat；Sample centrifugation by saltouing, after grease removal, transfer lower floor second
To centrifuge tube, vortex vibration after adding 150mgC18 and 900mg magnesium sulfate is purified nitrile layer, and centrifugation is washed with 5mL acetonitrile
Wash, centrifugation, merge the described supernatant purifying and wash twice after centrifugation, carry out concentrated in vacuo；First 1.5mL methanol is used to redissolve residual
Slag, then redissolve residue with the water more than or equal to 1mL, merge the redissolution liquid redissolving gained twice, filtered with 0.22um filter membrane
Obtain sample analysis liquid afterwards；Analyze the multiple mycotoxins in liquid using Ultra Performance Liquid Chromatography tandem mass spectrum method detection sample.
As the further scheme of the present invention：Described high-efficient liquid phase chromatogram condition is：Mobile phase A is the first containing 0.1% formic acid
Alcohol, Mobile phase B is the water containing 0.1% formic acid, and the volume ratio of mobile phase A and Mobile phase B is：70-20:30-80, gradient elution, or
Person's mobile phase A is methanol, and Mobile phase B is water, and the volume ratio of mobile phase A and Mobile phase B is：90-10:10-90, gradient elution；
Chromatographic column is built with silica gel as filler with octadecylsilane.
As the further scheme of the present invention：Described Mass Spectrometry Conditions are：Using electric spray ion source（ESI）, cation mould
Formula（ESI+）And negative ion mode（ESI-）After being scanned, using multiple-reaction monitoring pattern（MRM）Detection.
As the further scheme of the present invention：Described high-efficient liquid phase chromatogram condition is：Mobile phase is containing 0.1% formic acid
During the water of methanol-containing 0.1% formic acid, corresponding mass spectrum ionization pattern is positive ion mode；When mobile phase is methanol-water, institute is right
The ionization pattern answered is negative ion mode.
Compared with prior art, the invention has the beneficial effects as follows：The present invention adopts improved QuEChERS method to food
After carrying out pre-treatment, using Ultra Performance Liquid Chromatography tandem mass spectrometry, the 26 kinds of mycotoxins extracting are detected simultaneously.
With the acetonitrile solution containing sour variable concentrations, sample is extracted twice, sample extracting solution is carried out saltout, grease removal, net
Change, concentrate, redissolve, the multiple-reaction monitoring pattern using high performance liquid chromatography tandem mass spectrum is detected, improves detection efficiency,
Save testing cost, sensitivity is high, reproducible, recovery of standard addition is high, can expand to other high lipid content samples simultaneously
Detection.
Specific embodiment
With reference to specific embodiment, the technical scheme of this patent is described in more detail.
In a kind of food of detection simultaneously, the method for multiple mycotoxins is it is characterised in that the method comprises the following steps：
（1）Food samples to be measured are extracted, successively with the acetonitrile-aqueous solution containing sour variable concentrations, sample is shaken at twice
Swing extraction, centrifugation, merge extracting solution twice, obtain sample extracting solution；（2）Sample extracting solution is carried out saltout, grease removal, in described sample
Magnesium sulfate and sodium chloride is added to be saltoutd in product extracting solution, centrifugation, gained supernatant adds normal hexane, vortex vibrates,
Grease removal；（3）Sample to saltouing, after grease removal carries out purifying, concentrates, redissolves, and the sample centrifugation by saltouing, after grease removal, in gained
Lower floor acetonitrile layer add C18 (octadecylsilane chemically bonded silica) and the vibration of magnesium sulfate vortex to be purified, be centrifuged, to gained
Supernatant carry out concentrated in vacuo after, first with methanol, residue is redissolved, then with water, remaining residue is redissolved, merge redissolution institute twice
The redissolution liquid obtaining, carries out membrane filtration, obtains sample analysis liquid；（4）Detect sample using Ultra Performance Liquid Chromatography tandem mass spectrum method
Product analyze the multiple mycotoxins in liquid.
Described step（1）In the acetonitrile-aqueous solution containing sour variable concentrations be acetonitrile containing 0.1% (volume ratio) formic acid-
Aqueous solution, the acetonitrile-aqueous solution of variable concentrations is 70-90 for volume ratio：The acetonitrile-aqueous solution of 30-10 and volume ratio are 10-
40：The acetonitrile-aqueous solution of 90-60, the acetonitrile-aqueous solution of variable concentrations is 80 for volume ratio：20 acetonitrile-aqueous solution and volume
Than for 20：80 acetonitrile-aqueous solution.Described step（2）In saltout, grease removal includes：Sulfur is added in described sample extracting solution
Sour magnesium, sodium chloride, sodium citrate, DisodiumHydrogen Citrate are saltoutd, centrifugation, add normal hexane, vortex in gained supernatant
Vibration, grease removal.
The operation principle of the present invention：In the positive-ion mode, the mycotoxin that can detect in food includes：New eggplant disease reaping hook
Bacterium enol (NEO), aflatoxin（AFB1、AFB2、AFG1、AFG2、AFM1、AFM2）, fumonisin（FB1、FB2、FB3）、
Diacetyl sugarcane grass Fusarium spp. enol (DAS), aspergillus versicolor（ST）, HT2 toxin（HT2）, gliotoxin (GLT), T2 toxin（T2）、
Amebacilin (FUM).
In the negative ion mode, the mycotoxin that can detect in food includes：Myrothecium verrucaria is former（VER）, Aspergillus ochraceus A
（OTA）, 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone（ZEN）, the rotten bacterium enol of deoxidation reaping hook snow（DON）, Fusarium spp. ketenes（FUS-X）, go epoxy-deoxidation
The rotten bacterium enol of reaping hook snow（DOM-1）, mycophenolic acid（MPA）, Microcystin（PAX）, the rotten bacterium enol of 3- acetyl group deoxidation reaping hook snow
（3-ADON）, the rotten bacterium enol of 15- acetyl group deoxidation reaping hook snow（15-ADON）Mycotoxin adopts many reaction monitorings pattern（MRM）
Contratoxin carries out qualitative and quantitative.Decree 2002/657/EU being required with regard to analysis method according to European Union, using liquid chromatograph matter
It is necessary to reach 4 points of requirement, this method selects 1 parent ion to Spectral Analysis（2 points）, 2 daughter ions（1 point）To targeted
Compound carries out qualitative, meets 4 points of requirement.Quantitative manner employs substrate curve quantitation, and every curve is made up of 5 points,
Linearly dependent coefficient（r2）More than 0.995, meet quantitative requirement.
Test analysis equipment of the present embodiment is：Agilent1290 Ultra Performance Liquid Chromatography instrument（It is furnished with quaternary
Pump；Degasser and automatic sampler）, 6460 mass detectors（It is furnished with electric spray ion source）.
Claims (4)
1. in a kind of food the detection method of mycete it is characterised in that comprising the steps of；Weighing utensil using standard weighs
1-5g video to be measured sample, first contains the 80% of volume ratio 0.1% formic acid acetonitrile-aqueous solution and carries out vibration to sample and carry with 20ml
Take supernatant, place in centrifuge turn through 1000-2000/min speed carries out the centrifugally operated of 20-40min, obtains extracting solution
A, then the acetonitrile-aqueous solution of the volume ratio 20% that the residue after extracting contains volume ratio 0.1% formic acid with 3-7ml is carried out by vibration carries
Take 30min, centrifugation, obtain extracting solution 2, merge described extracting solution 1 and described extracting solution 2, obtain sample extracting solution；Carry in described sample
Take and in liquid, add 1.0g sodium citrate, 4.0g magnesium sulfate, 1.0g sodium chloride, 0.5g DisodiumHydrogen Citrate to be saltoutd, after centrifugation
Transfer supernatant, is added thereto to 20ml normal hexane, and vortex vibration 1min removes fat；Sample centrifugation by saltouing, after grease removal,
To centrifuge tube, vortex vibration after adding 150mgC18 and 900mg magnesium sulfate is purified transfer lower floor acetonitrile layer, and centrifugation is used
5mL acetonitrile washs, centrifugation, merges the described supernatant purifying and wash twice after centrifugation, carries out concentrated in vacuo；First use 1.5mL
Methanol redissolves residue, then redissolves residue with the water more than or equal to 1mL, merges the redissolution liquid redissolving gained twice, is filtered with 0.22um
Film obtains sample analysis liquid after being filtered；Analyzed multiple in liquid using Ultra Performance Liquid Chromatography tandem mass spectrum method detection sample
Mycotoxin.
2. in a kind of food according to claim 1 the detection method of mycete it is characterised in that described high performance liquid chromatography
Condition is：Mobile phase A is the methanol containing 0.1% formic acid, and Mobile phase B is the water containing 0.1% formic acid, the body of mobile phase A and Mobile phase B
Long-pending ratio is：70-20:30-80, gradient elution, or mobile phase A are methanol, and Mobile phase B is water, mobile phase A and Mobile phase B
Volume ratio is：90-10:10-90, gradient elution；Chromatographic column is built with silica gel as filler with octadecylsilane.
3. in a kind of food according to claim 1 the detection method of mycete it is characterised in that described Mass Spectrometry Conditions are：
Using electric spray ion source（ESI）, positive ion mode（ESI+）And negative ion mode（ESI-）After being scanned, reacted using more
Monitoring pattern（MRM）Detection.
4. in a kind of food according to claim 1 the detection method of mycete it is characterised in that described high-efficient liquid phase color
Spectral condition is：When mobile phase is the water of methanol containing 0.1% formic acid-containing 0.1% formic acid, corresponding mass spectrum ionization pattern be just from
Subpattern；When mobile phase is methanol-water, corresponding ionization pattern is negative ion mode.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611083083.0A CN106442817A (en) | 2016-11-30 | 2016-11-30 | Method for detecting mold in food |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611083083.0A CN106442817A (en) | 2016-11-30 | 2016-11-30 | Method for detecting mold in food |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106442817A true CN106442817A (en) | 2017-02-22 |
Family
ID=58224217
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611083083.0A Pending CN106442817A (en) | 2016-11-30 | 2016-11-30 | Method for detecting mold in food |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106442817A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106950328A (en) * | 2017-03-27 | 2017-07-14 | 中山市食品药品检验所 | A kind of method for detecting mycotoxin in fermented tea |
| CN107219311A (en) * | 2017-05-26 | 2017-09-29 | 上海烟草集团有限责任公司 | A kind of method of aspertoxin in quick measure smokeless tobacco |
| CN109781889A (en) * | 2019-02-14 | 2019-05-21 | 山东省食品药品检验研究院 | A kind of measuring method of 24 kinds of mycotoxins in nourishing rice flour for babies |
| CN114137105A (en) * | 2021-11-15 | 2022-03-04 | 北京奶牛中心 | Method for analyzing content of melatonin in sample by liquid chromatography-tandem mass spectrometry |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2708604A1 (en) * | 2012-09-14 | 2014-03-19 | Centre Scientifique Et Technique Du Batiment (CSTB) | Method for determining a risk of aspergillus contamination based on the detection of microbial volatile organic compounds |
| CN104142375A (en) * | 2014-08-20 | 2014-11-12 | 中华人民共和国潍坊出入境检验检疫局 | Methods for constructing fingerprint by using mildew metabolites and authenticating mildew strains according to fingerprint |
| CN104515821A (en) * | 2014-11-25 | 2015-04-15 | 山东省农业科学院农业质量标准与检测技术研究所 | Corn kernel kumonisins rapid determination method |
| CN104535664A (en) * | 2014-03-31 | 2015-04-22 | 中华人民共和国北京出入境检验检疫局 | Method for simultaneously detecting a plurality of mycotoxins in sesame paste |
| CN106018648A (en) * | 2016-07-13 | 2016-10-12 | 云南省烟草烟叶公司 | Method for detecting concentrations of 12 mycotoxins in mildewed tobacco |
-
2016
- 2016-11-30 CN CN201611083083.0A patent/CN106442817A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2708604A1 (en) * | 2012-09-14 | 2014-03-19 | Centre Scientifique Et Technique Du Batiment (CSTB) | Method for determining a risk of aspergillus contamination based on the detection of microbial volatile organic compounds |
| CN104535664A (en) * | 2014-03-31 | 2015-04-22 | 中华人民共和国北京出入境检验检疫局 | Method for simultaneously detecting a plurality of mycotoxins in sesame paste |
| CN104142375A (en) * | 2014-08-20 | 2014-11-12 | 中华人民共和国潍坊出入境检验检疫局 | Methods for constructing fingerprint by using mildew metabolites and authenticating mildew strains according to fingerprint |
| CN104515821A (en) * | 2014-11-25 | 2015-04-15 | 山东省农业科学院农业质量标准与检测技术研究所 | Corn kernel kumonisins rapid determination method |
| CN106018648A (en) * | 2016-07-13 | 2016-10-12 | 云南省烟草烟叶公司 | Method for detecting concentrations of 12 mycotoxins in mildewed tobacco |
Non-Patent Citations (2)
| Title |
|---|
| 庄倩 等: "高效液相色谱-串联质谱法同时测定饲料中9种霉菌毒素及其代谢物", 《分析科学学报》 * |
| 王瑞国 等: "液相色谱-串联质谱法测定饲料原料中 26 种霉菌毒素", 《分析化学》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106950328A (en) * | 2017-03-27 | 2017-07-14 | 中山市食品药品检验所 | A kind of method for detecting mycotoxin in fermented tea |
| CN107219311A (en) * | 2017-05-26 | 2017-09-29 | 上海烟草集团有限责任公司 | A kind of method of aspertoxin in quick measure smokeless tobacco |
| CN109781889A (en) * | 2019-02-14 | 2019-05-21 | 山东省食品药品检验研究院 | A kind of measuring method of 24 kinds of mycotoxins in nourishing rice flour for babies |
| CN114137105A (en) * | 2021-11-15 | 2022-03-04 | 北京奶牛中心 | Method for analyzing content of melatonin in sample by liquid chromatography-tandem mass spectrometry |
| CN114137105B (en) * | 2021-11-15 | 2024-01-19 | 北京奶牛中心 | Method for analyzing melatonin content in sample by liquid chromatography-tandem mass spectrometry |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106442817A (en) | Method for detecting mold in food | |
| CN104535664B (en) | A kind of method simultaneously detecting multiple mycotoxin in sesame paste | |
| Fletouris et al. | Rapid determination of cholesterol in milk and milk products by direct saponification and capillary gas chromatography | |
| Capriotti et al. | Analytical strategies based on chromatography–mass spectrometry for the determination of estrogen-mimicking compounds in food | |
| Sørensen et al. | Determination of mycotoxins in bovine milk by liquid chromatography tandem mass spectrometry | |
| González-Curbelo et al. | Evolution and applications of the QuEChERS method | |
| Krska et al. | The state-of-the-art in the analysis of estrogenic mycotoxins in cereals | |
| Du et al. | Simultaneous analysis of tocopherols, cholesterol, and phytosterols using gas chromatography | |
| Shephard et al. | Developments in mycotoxin analysis: an update for 2011-2012 | |
| Göbel et al. | Simultaneous determination of aflatoxins, ochratoxin A, and zearalenone in grains by new immunoaffinity column/liquid chromatography | |
| Thompson et al. | High performance liquid chromatographic determination of vitamin A in margarine, milk, partially skimmed milk, and skimmed milk | |
| WO2022121942A1 (en) | Method for extracting canthaxanthin from egg yolk and purifying same, and solid phase extraction high performance liquid chromatography method for determining canthaxanthin in egg yolk | |
| CN106526041A (en) | Method for simultaneously detecting multiple mycotoxins | |
| Chen et al. | Development of a liquid chromatography–tandem mass spectrometry with ultrasound-assisted extraction and auto solid-phase clean-up method for the determination of Fusarium toxins in animal derived foods | |
| CN107144645A (en) | It is a kind of while detecting the method for a variety of mycotoxins | |
| Manetta | Aflatoxins: Their measure and analysis | |
| Ryu et al. | Validation of analytical methods for ethyl carbamate in nine food matrices | |
| Arroyo‐Manzanares et al. | Review of Sample Treatments and the State‐of‐the‐art of Analytical Techniques for Mycotoxins in Food | |
| Pospiech et al. | Immunohistochemical detection of soya protein-optimisation and verification of the method. | |
| Liu et al. | Simultaneous determination of five hormones in milk by automated online solid-phase extraction coupled to high-performance liquid chromatography | |
| Goryacheva et al. | Immunoaffinity pre-concentration combined with on-column visual detection as a tool for rapid aflatoxin M1 screening in milk | |
| Kovacs | Determination of cholesterol in pasta products using gas-liquid chromatography | |
| CN107957473A (en) | A kind of quick determination method of tetraodotoxin | |
| Bui | Sample preparation and liquid chromatographic determination of vitamin D in food products | |
| CN106908544A (en) | A kind of method of the toxin metabolic analysis of trace F 2 in sample for animal sources |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170222 |
|
| RJ01 | Rejection of invention patent application after publication |