CN105061587A - Ultrasonic wave aided extraction method suitable for high-viscosity collagen - Google Patents
Ultrasonic wave aided extraction method suitable for high-viscosity collagen Download PDFInfo
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- CN105061587A CN105061587A CN201510530640.8A CN201510530640A CN105061587A CN 105061587 A CN105061587 A CN 105061587A CN 201510530640 A CN201510530640 A CN 201510530640A CN 105061587 A CN105061587 A CN 105061587A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
Abstract
The invention discloses an ultrasonic wave aided extraction method suitable for high-viscosity collagen. The ultrasonic wave aided extraction method comprises the following steps that firstly, materials with extraction to be carried out is steeped in an acid solution to be expanded, then the acid solution with the materials with extraction to be carried out steeped is put into a material storage stirrer to carry out stirring, and a uniform mixing solution is formed; secondly, the mixing solution is sent into an ultrasonic reactor through a feeding pump, and pulse type ultrasonic processing is carried out on the solution through the ultrasonic reactor; thirdly, the mixing solution with the ultrasonic processing carried out is pumped out of the bottom of the ultrasonic reactor through a circulation pump, and the mixing solution is pumped into a heat exchanger to be cooled; fourthly, the cooled mixing liquid comes out of the heat exchanger and then is sent to the ultrasonic reactor to carry out ultrasonic processing further; fifthly, the operation in the third step and the operation in the fourth step are repeated, circulation is carried out in this way for many times, and the extraction of the collagen is completed. By means of the ultrasonic wave aided extraction method, the extraction efficiency of the collagen can be improved, and the biological activity of the collagen can be maintained.
Description
Technical field
The present invention relates to the extractive technique of thermo-sensitivity, high-viscosity material, specifically a kind of ultrasonically auxiliary extracting method being applicable to high viscosity collagen protein.
Background technology
Ultrasonic wave refers to that frequency is 2 × 10
4~ 1 × 10
7the sound wave of Hz, can produce the physical actions such as cavitation, microjet, heat effect.Cavatition refers to the physics, the chemical action that produce in the process such as formation, growth, vibration, collapse that ultrasonic cavitation is steeped; Microjet effect mainly cavitation bubble intense impact effect of supervening when collapsing; Ultrasonic energy is converted to heat energy by the mechanical vibration that heat effect and medium produce by absorbing ultrasonic wave.Ultrasonic cavatition can destroy cytolemma, the cell walls of crude substance, and this not only can improve the speed that solvent enters cell, also can promote that in cell, effective constituent is fast to dissolution with solvents simultaneously; And microjet interaction energy promotes the diffusion of material, release fully mixing with solvent, thus improve extraction efficiency and extraction degree; But ultrasonic heat effect can make the temperature of material organization internal raise instantaneously, its heat effect produced may destroy the structure of biologically active substance to a certain extent, finally causes biologically active substance to lose its original physiological function.
Collagen protein is the class formation albumen in animal tissues, and it has the features such as viscosity is large, solubleness is low, denaturation temperature is low, and thus the traditional extraction process of collagen protein is long-time under low temperature immersion or stir process.But there is a series of shortcoming in these extracting method, such as energy consumption large (accounting for 70% of total energy consumption in leaching process), high CO
2discharge, water consumption consume solvent and produce a large amount of bazardous wastes etc.Ultrasound assisted extraction is a kind of extracting method of green high-efficient, and it can not only reduce the usage quantity of solvent, and can improve extraction yield, the shortening extraction time of material.But existing ultrasound assisted extraction equipment, for the collagen protein comparatively large for viscosity, thermostability is lower, the heat energy that ultrasonic wave produces is enough to the triple-helix structure of collagen protein to be destroyed, and causes collagen protein to lose its distinctive function.Meanwhile, because ultrasonic wave propagates in media as well with the form of compressional wave, ultrasonic wave can decay in the process propagated, and absorption is the major cause of acoustic attenuation, and ultrasonic energy is converted into heat energy in the tissue in communication process, and sound wave is decayed.The viscosity of material is larger, and the trend of this decay is also larger.For the larger material of viscosity, ultrasonication decay is serious, and will cause the insufficient of material ultrasonication, ultrasound assisted extraction effect is undesirable.
Summary of the invention
The object of the invention is to the deficiency overcoming the existence of above prior art, provide a kind of ultrasonically auxiliary extracting method being applicable to high viscosity collagen protein.The heat energy that this ultrasonically auxiliary extracting method can prevent ultrasonic wave from producing destroys the triple-helix structure of collagen protein, and improves extraction efficiency.
In order to achieve the above object, the present invention is by the following technical solutions: a kind of ultrasonically auxiliary extracting method being applicable to high viscosity collagen protein, is characterized in that, comprise the following steps:
1) material to be extracted is soaked in acidic solution, after making mass expanded to be extracted, then the acidic solution immersed with material to be extracted is placed in storing agitator and stirs, form uniform mixed solution;
2) send in ultrasonic reactor by fresh feed pump by mixed solution, ultrasonic reactor carries out the ultrasonication of pulsed to this solution;
3) recycle pump extracts from the bottom of ultrasonic reactor the mixed solution having carried out ultrasonication out, and this mixed solution is pumped to heat exchanger and lowers the temperature;
4) the mixed solution automatic heat-exchanger after cooling out after, then be sent back in ultrasonic reactor and carry out ultrasonication further;
5) repeating step 3) and step 4) operation, so circulation is repeatedly, thus completes the extraction of collagen protein.
Preferably, described ultrasonic reactor comprises producer, controller, reaction vessel and insulating container, described reaction vessel is placed in insulating container, and one end of described producer is through insertion reaction container after insulating container, and the other end of described producer is connected with controller; The upper end of described reaction vessel is connected with storing agitator by fresh feed pump, and the lower end of described reaction vessel is connected with the hot-side channel entrance of heat exchanger by recycle pump; The hot-side channel outlet of described heat exchanger is connected with the upper end of reaction vessel, and meanwhile, the cold side channel of described heat exchanger is connected with refrigerator.
Preferably, described ultrasonic reactor also comprises the first pipeline, second pipe, the 3rd pipeline and the 4th pipeline, described fresh feed pump is connected with the upper end of reaction vessel by the first pipeline, the lower end of described reaction vessel is connected with recycle pump by second pipe, described recycle pump is connected with the hot-side channel entrance of heat exchanger by the 3rd pipeline, and the hot-side channel outlet of described heat exchanger is connected with the upper end of reaction vessel by the 4th pipeline; Described first pipeline, reaction vessel and the 3rd pipeline are equipped with temperature sensor.
Preferably, the volume being positioned at the mixed solution of reaction vessel is 1.2L ~ 2L; And producer is sonde-type producer, in the probe insertion reaction container of this sonde-type producer, the degree of depth of mixed solution liquid level is 1.5cm ~ 2.5cm.
Preferably, flow during described recycle pump extraction mixed solution is 150ml/min ~ 3000ml/min.
Preferably, described first pipeline, second pipe, the 3rd pipeline and the 4th pipeline are all enclosed with thermal insulation layer.
Preferably, described first pipeline is provided with under meter.
Preferably, in step 1) in, the volume ratio of the material to be extracted in mixed solution and the acidic solution in mixed solution is 1:(8 ~ 30).
Preferably, in step 3) in, the temperature that described heat exchanger controls mixed solution is-4 DEG C ~ 50 DEG C.
Preferably, described acidic solution is acetic acid, citric acid, lactic acid or hydrochloric acid.
The present invention, relative to prior art, has following advantage and effect:
1, the present invention adopts ultrasonically auxiliary extracting method to replace traditional long-time at low temperatures method of soaking or stirring extraction collagen protein, which increases the extraction efficiency of collagen protein and improves the solubility rate of collagen protein.
2, the present invention adopts ultrasonically auxiliary extracting method to extract collagen protein, the equipment used in its ultrasonically auxiliary extracting method has ultrasonic reactor, this ultrasonic reactor and heat exchanger form a recycle system, to this prevent in ultrasonic wave heat effect to the destruction of the triple-helix structure of collagen protein, thus avoid collagen protein and lose its distinctive bioactive functions; Meanwhile, this recycle system also reduces the impact of viscosity on ultrasound assisted extraction of material, therefore can carry out sufficient ultrasound assisted extraction effect to material, thus further increase the extraction efficiency of collagen protein.
3, when the amount of material to be extracted is more, ultrasonic reactor in the present invention also can be set up multiple, this multiple ultrasonic reactor series connection, and multiple ultrasonic reactor and heat exchanger form the recycle system, thus can ensure effectively to extract collagen protein, and improve the extraction efficiency of collagen protein.
4, the present invention can not destroy thermostability and the denaturation temperature of collagen protein, therefore collagen protein generation can not be made to become, and ensures that collagen protein can maintain original bioactive functions.
5, few than traditional extraction process of acidic solution that consume of the present invention, decreases extraction cost.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of the equipment that extracting method of the present invention uses.
Fig. 2 is that traditional extraction process and extracting method of the present invention are to the property representation figure of collagen protein solubleness.
Fig. 3 is that traditional extraction process and extracting method of the present invention are to the property representation figure of collagen protein viscosity.
Fig. 4 is traditional extraction process and will the textural property phenogram of collagen protein under invention extracting method.
Fig. 5 is traditional extraction process and will the gel electrophoresis figure of collagen protein under invention extracting method.
Embodiment
For ease of it will be appreciated by those skilled in the art that the present invention is described in further detail below in conjunction with drawings and Examples.
Embodiment 1:
As shown in Figure 1, be originally applicable to the ultrasonically auxiliary extracting method of high viscosity collagen protein, it is characterized in that, comprise the following steps:
1) material to be extracted is soaked in acidic solution, after making mass expanded to be extracted, then the acidic solution immersed with material to be extracted is placed in storing agitator 1 and stirs, form uniform mixed solution;
2) send in ultrasonic reactor 3 by fresh feed pump 2 by mixed solution, ultrasonic reactor 3 carries out the ultrasonication of pulsed to this solution;
3) recycle pump 4 extracts from the bottom of ultrasonic reactor 3 mixed solution having carried out ultrasonication out, and this mixed solution is pumped to heat exchanger 5 and lowers the temperature;
4) after the mixed solution automatic heat-exchanger after cooling goes out 5, then be sent back in ultrasonic reactor 4 and carry out ultrasonication further;
5) repeating step 3) and step 4) operation, so circulation is repeatedly, thus completes the extraction of collagen protein.
Described ultrasonic reactor 3 comprises producer 301, controller 302, reaction vessel 303 and insulating container 304, described reaction vessel 303 is placed in insulating container 304, one end of described producer 301 is through insertion reaction container 303 after insulating container 304, and the other end of described producer 301 is connected with controller 302; The upper end of described reaction vessel 303 is connected with storing agitator 1 by fresh feed pump 2, and the lower end of described reaction vessel 303 is connected with the hot-side channel entrance of heat exchanger 5 by recycle pump 4; The hot-side channel outlet of described heat exchanger 5 is connected with the upper end of reaction vessel 303, and meanwhile, the cold side channel of described heat exchanger 5 is connected with refrigerator 6.Concrete, the mixed solution stirred at storing agitator 1 be fed pump take out 2 get after, then enter reaction vessel 303 by the first pipeline 305, and the volume being positioned at the mixed solution of reaction vessel 303 is 2L, then producer produces pulse type supersonic, thus carries out ultrasonication to these mixed solutions.Then, be recycled pump 4 by the mixed solution of ultrasonication and extract out, then entered in the hot-side channel of heat exchanger 5 by the 3rd pipeline 307 and lower the temperature.And the mixed solution after cooling adds from the 4th pipeline 308 and flows to reaction vessel 303.
Described ultrasonic reactor 3 also comprises the first pipeline 305, second pipe 306, the 3rd pipeline 307 and the 4th pipeline 308, described fresh feed pump 2 is connected with the upper end of reaction vessel 303 by the first pipeline 305, the lower end of described reaction vessel 303 is connected with recycle pump 4 by second pipe 306, described recycle pump 4 is connected with the hot-side channel entrance of heat exchanger 5 by the 3rd pipeline 307, and the hot-side channel outlet of described heat exchanger 5 is connected with the upper end of reaction vessel 303 by the 4th pipeline 308; Described first pipeline 305, reaction vessel 303 and the 3rd pipeline 307 are equipped with temperature sensor 7.
The volume being positioned at the mixed solution of reaction vessel 304 is 2L, and suitable volume to adapt to the power of corresponding producer 301, thus ensures the efficiency extracting collagen protein; And producer 301 is sonde-type producer, for ensureing ultrasonication effect, in the probe insertion reaction container 304 of this sonde-type producer, the degree of depth of mixed solution liquid level is 2cm.
For ensureing extraction efficiency, reduce energy waste, described first pipeline 305, second pipe 306, the 3rd pipeline 307 and the 4th pipeline 308 are all enclosed with thermal insulation layer.
Described first pipeline 305 is provided with under meter 8.The mixed solution of respective amount can join in reaction vessel by under meter 8 accurately.
The parameter setting of producer is 20kHz, 1000W.Make material to be extracted be immersed in the acetum of 0.5mol/L, namely acidic solution is acetic acid, and material to be extracted is fish-skin, and in mixed solution, the mass ratio of fish-skin and acetum is 1:20, and the volume being positioned at the mixed solution of reaction vessel is 2L; Then reactor carries out the time of pulsating ultrasound reaction is 10min ~ 30min; And flow during recycle pump extraction mixed solution is 350ml/min.Refrigerator makes the temperature of heat exchanger cold side channel be 0 DEG C and 10 DEG C, is mixed into 4 DEG C and 15 DEG C with what ensure mixed solution in reaction vessel.The temperature of mixed solution be 4 DEG C and 15 DEG C time, the solubility rate that extracting method of the present invention extracts collagen protein is respectively: be 78.5% and 89.8%; And the temperature of mixed solution is 4 DEG C, it is 38.2% that traditional extraction process extracts collagen protein solubility rate.It can thus be appreciated that, adopt extracting method of the present invention to extract the solubility rate of collagen protein far away higher than traditional extracting method.
Embodiment 2
Originally the ultrasonically auxiliary extracting method being applicable to high viscosity collagen protein except following technical characteristic with embodiment 1:
The parameter setting of producer is 20kHz, 600W.Make material to be extracted be immersed in the acetum of 0.5mol/L, namely acidic solution is acetic acid, and material to be extracted is fish-skin, and in mixed solution, the mass ratio of fish-skin and acetum is 1:20, and the volume being positioned at the mixed solution of reaction vessel is 1.2L; Then reactor carries out the time of pulsating ultrasound reaction is 10min ~ 30min; And flow during recycle pump extraction mixed solution is 150ml/min.Refrigerator makes the temperature of heat exchanger cold side channel be 0 DEG C, is mixed into 4 DEG C with what ensure mixed solution in reaction vessel.Then measure the stripping quantity of collagen protein under the different ultrasound assisted extraction time by Lowry method and measure the denaturation temperature of collagen protein after purifying by DSC (differential scanning calorimeter) method.Determination data is as table 1:
Table 1
| Sonication treatment time (min) | Stripping quantity (concentration mg/mL) | Denaturation temperature (DEG C) |
| 10 | 6.20 | 34.8 |
| 20 | 8.64 | 34.5 |
| 30 | 10.51 | 33.2 |
Can be obtained by table 1, the collagen content using the ultrasonically auxiliary extracting method described in this patent to obtain increases with the increase of ultrasound assisted extraction time, after ultrasound assisted extraction 10min, the stripping quantity of collagen protein is 6.2mg/mL, after ultrasound assisted extraction 20min, the stripping quantity of collagen protein is 8.64mg/mL, and after ultrasound assisted extraction 30min, the stripping quantity of collagen protein is 10.51mg/mL.The denaturation temperature of the collagen protein recorded after purifying, all higher than 33 DEG C, apparently, increases the extraction yield of collagen protein under the prerequisite not destroying collagen structure and thermally-stabilised, denaturation temperature by this method.
Embodiment 3
Originally the ultrasonically auxiliary extracting method being applicable to high viscosity collagen protein except following technical characteristic with embodiment 1:
The parameter setting of producer is 20kHz, 900W.Make material to be extracted be immersed in the acetum of 0.5mol/L, namely acidic solution is acetic acid, and material to be extracted is fish-skin, and in mixed solution, the mass ratio of fish-skin and acetum is 1:20, and the volume being positioned at the mixed solution of reaction vessel is 1.8L; Then reactor carries out the time of pulsating ultrasound reaction is 30min; And flow during recycle pump extraction mixed solution is 200ml/min.Refrigerator makes the temperature of heat exchanger cold side channel be respectively 0 DEG C, 10 DEG C and 28 DEG C, is mixed into 4 DEG C, 15 DEG C and 30 DEG C with what ensure mixed solution in reaction vessel.Measure solubleness and the viscosity of collagen protein after ultrasound assisted extraction, use CD (circular dichroism spectrum) to detect the change of collagen protein secondary structure, use electrophoretic technique to analyze its primary structure, and the collagen protein extracted with 4 DEG C, 15 DEG C and 30 DEG C of traditional convention stir 12h respectively compares.Comparative result is as Fig. 2 to Fig. 5.
H-30 DEG C, H-15 DEG C and H-4 DEG C are illustrated respectively in the collagen protein of 30,15,4 DEG C of ultrasound assisted extraction and S-30 DEG C, S-15 DEG C and S-4 DEG C are illustrated respectively in the collagen protein that 30,15,4 DEG C of traditional convention stir extract physics, chemistry and textural property phenogram in Fig. 3 to Fig. 5.As shown in Figure 2, the solubleness of the collagen of fish skin extracted at different ultrasonic wave added temperature under different pH all than traditional convention stir extract large; As shown in Figure 3, the collagen protein viscosity of ultrasound assisted extraction also more traditional convention stir extract large; As shown in Figure 4, its distinctive triple-helix structure of collagen protein of 4 DEG C and 15 DEG C ultrasound assisted extraction does not change yet; As shown in Figure 5, under differing temps, its peptide chain structure of collagen protein of ultrasound assisted extraction does not change.Comprehensively above-mentioned, its yield of collagen protein using the method (ultrasound assisted extraction) to obtain is higher, secondary structure, the distinctive triple-helix structure of collagen protein all do not change, and under the constant prerequisite of collagen protein thermostability, denaturation temperature, its solvability and viscosity also improve to a certain extent significantly.
Above-mentioned embodiment is the preferred embodiments of the present invention, can not limit the present invention, and any of other does not deviate from technical scheme of the present invention and the substitute mode of the change made or other equivalence, is included within protection scope of the present invention.
Claims (10)
1. be applicable to a ultrasonically auxiliary extracting method for high viscosity collagen protein, it is characterized in that, comprise the following steps:
1) material to be extracted is soaked in acidic solution, after making mass expanded to be extracted, then the acidic solution immersed with material to be extracted is placed in storing agitator and stirs, form uniform mixed solution;
2) send in ultrasonic reactor by fresh feed pump by mixed solution, ultrasonic reactor carries out the ultrasonication of pulsed to this solution;
3) recycle pump extracts from the bottom of ultrasonic reactor the mixed solution having carried out ultrasonication out, and this mixed solution is pumped to heat exchanger and lowers the temperature;
4) the mixed solution automatic heat-exchanger after cooling out after, then be sent back in ultrasonic reactor and carry out ultrasonication further;
5) repeating step 3) and step 4) operation, so circulation is repeatedly, thus completes the extraction of collagen protein.
2. the ultrasonically auxiliary extracting method being applicable to high viscosity collagen protein according to claim 1, it is characterized in that: described ultrasonic reactor comprises producer, controller, reaction vessel and insulating container, described reaction vessel is placed in insulating container, one end of described producer is through insertion reaction container after insulating container, and the other end of described producer is connected with controller; The upper end of described reaction vessel is connected with storing agitator by fresh feed pump, and the lower end of described reaction vessel is connected with the hot-side channel entrance of heat exchanger by recycle pump; The hot-side channel outlet of described heat exchanger is connected with the upper end of reaction vessel, and meanwhile, the cold side channel of described heat exchanger is connected with refrigerator.
3. the ultrasonically auxiliary extracting method being applicable to high viscosity collagen protein according to claim 2, it is characterized in that: also comprise the first pipeline, second pipe, the 3rd pipeline and the 4th pipeline, described fresh feed pump is connected with the upper end of reaction vessel by the first pipeline, the lower end of described reaction vessel is connected with recycle pump by second pipe, described recycle pump is connected with the hot-side channel entrance of heat exchanger by the 3rd pipeline, and the hot-side channel outlet of described heat exchanger is connected with the upper end of reaction vessel by the 4th pipeline; Described first pipeline, reaction vessel and the 3rd pipeline are equipped with temperature sensor.
4. the ultrasonically auxiliary extracting method being applicable to high viscosity collagen protein according to claim 2, is characterized in that: the volume being positioned at the mixed solution of reaction vessel is 1.2L ~ 2L; And producer is sonde-type producer, in the probe insertion reaction container of this sonde-type producer, the degree of depth of mixed solution liquid level is 1.5cm ~ 2.5cm.
5. the ultrasonically auxiliary extracting method being applicable to high viscosity collagen protein according to claim 1, is characterized in that: flow during described recycle pump extraction mixed solution is 150ml/min ~ 3000ml/min.
6. the ultrasonically auxiliary extracting method being applicable to high viscosity collagen protein according to claim 3, is characterized in that: described first pipeline, second pipe, the 3rd pipeline and the 4th pipeline are all enclosed with thermal insulation layer.
7. the ultrasonically auxiliary extracting method being applicable to high viscosity collagen protein according to claim 3, is characterized in that: described first pipeline is provided with under meter.
8. the ultrasonically auxiliary extracting method being applicable to high viscosity collagen protein according to claim 1, is characterized in that: in step 1) in, the volume ratio of the material to be extracted in mixed solution and the acidic solution in mixed solution is 1:(8 ~ 30).
9. the ultrasonically auxiliary extracting method being applicable to high viscosity collagen protein according to claim 1, is characterized in that: in step 3) in, the temperature that described heat exchanger controls mixed solution is-4 DEG C ~ 50 DEG C.
10. the ultrasonically auxiliary extracting method being applicable to high viscosity collagen protein according to claim 1, is characterized in that: described acidic solution is acetic acid, citric acid, lactic acid or hydrochloric acid.
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Cited By (5)
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| CN106220727A (en) * | 2016-08-31 | 2016-12-14 | 江苏省农业科学院 | Response phase method is utilized to optimize the ultrasonic wave added acid extracting method of collagen protein |
| CN106366181A (en) * | 2016-09-14 | 2017-02-01 | 成都百奥诺维生物科技有限公司 | High-linearity temperature monitoring system for hydrolysis extraction of fishskin collagen |
| CN106432477A (en) * | 2016-09-14 | 2017-02-22 | 成都百奥诺维生物科技有限公司 | Temperature monitoring system for fish skin collagen extraction using hot water hydrolysis |
| CN106432476A (en) * | 2016-09-14 | 2017-02-22 | 成都百奥诺维生物科技有限公司 | Signal compensation type temperature monitoring system for extracting fish skin collagen through hydrolysis method |
| CN107014854A (en) * | 2017-03-29 | 2017-08-04 | 淄博黄河龙生物工程有限公司 | The durothermic detection method of collagen |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106220727A (en) * | 2016-08-31 | 2016-12-14 | 江苏省农业科学院 | Response phase method is utilized to optimize the ultrasonic wave added acid extracting method of collagen protein |
| CN106366181A (en) * | 2016-09-14 | 2017-02-01 | 成都百奥诺维生物科技有限公司 | High-linearity temperature monitoring system for hydrolysis extraction of fishskin collagen |
| CN106432477A (en) * | 2016-09-14 | 2017-02-22 | 成都百奥诺维生物科技有限公司 | Temperature monitoring system for fish skin collagen extraction using hot water hydrolysis |
| CN106432476A (en) * | 2016-09-14 | 2017-02-22 | 成都百奥诺维生物科技有限公司 | Signal compensation type temperature monitoring system for extracting fish skin collagen through hydrolysis method |
| CN107014854A (en) * | 2017-03-29 | 2017-08-04 | 淄博黄河龙生物工程有限公司 | The durothermic detection method of collagen |
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Application publication date: 20151118 |
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| RJ01 | Rejection of invention patent application after publication |