CN111066961A - Natural long-chain fatty acid lutein ester feed additive with high bioavailability - Google Patents
Natural long-chain fatty acid lutein ester feed additive with high bioavailability Download PDFInfo
- Publication number
- CN111066961A CN111066961A CN202010052574.9A CN202010052574A CN111066961A CN 111066961 A CN111066961 A CN 111066961A CN 202010052574 A CN202010052574 A CN 202010052574A CN 111066961 A CN111066961 A CN 111066961A
- Authority
- CN
- China
- Prior art keywords
- lutein
- lutein ester
- fatty acid
- feed additive
- ester
- 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.)
- Withdrawn
Links
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/105—Aliphatic or alicyclic compounds
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K40/00—Shaping or working-up of animal feeding-stuffs
- A23K40/30—Shaping or working-up of animal feeding-stuffs by encapsulating; by coating
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Polymers & Plastics (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Animal Husbandry (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
A natural long-chain fatty acid lutein ester feed additive with high bioavailability is characterized in that marigold extract is used as a raw material, the feed additive is prepared by utilizing the principle that emulsified lutein ester cannot be saponified and fatty glyceride is easily saponified, the main effective component is natural long-chain fatty acid lutein ester, the lutein ester content is 0.5-20%, and fatty glyceride is not contained or less contained. Experimental results prove that the natural fatty acid lutein ester which is subjected to micro-emulsification to obtain high dispersion degree and further subjected to saponification and grease coating removal has a good coloring effect, and the colored natural lutein ester has better thermal stability than natural lutein and lutein acetate ester and has high use value.
Description
Technical Field
The invention relates to a natural lutein ester feed additive preparation technology, in particular to a saponification emulsification processing and application technology of marigold extract, belonging to the field of feed.
Background
The lutein feed additive is widely applied to the breeding feed of meat poultry, laying poultry and aquatic products. At present, lutein sold in the market is almost extracted from marigold. Generally, lutein and oil in plant tissues coexist in symbiosis, and lutein exists in marigold flowers in the form of lutein ester as a main component. In order to improve the bioavailability of lutein, the traditional practice in the feed field is to perform saponification reaction with strong base (potassium hydroxide, sodium hydroxide, etc.) and lutein ester together with oil to produce a mixture mainly composed of free lutein, fatty acid salt and glycerol, which is called saponification cream in the industry. The lutein in the saponified paste exists in a free form, and the bioavailability is greatly improved due to no wrapping of viscous grease. At the same time, however, the stability is greatly reduced after the lutein ester is converted into the free state. The free lutein is unstable to light, heat and oxygen, is sensitive to acid and alkali environments, and is rapidly degraded in the gastrointestinal environment of animals.
In order to increase the bioavailability while maintaining the stability of lutein as much as possible, the industry is constantly seeking countermeasures. One technique is to subject the natural lutein ester to a multi-step process to convert the lutein long chain fatty acid ester to lutein acetate. The technology keeps the stability of the ester and has better capability of being absorbed and utilized, thereby being a good scheme. However, the cost of the scheme is greatly increased, and the saponification is required, and the saponified paste obtained by saponification is further purified into lutein crystals to be capable of continuously preparing the lutein acetate through acylation reaction.
Researchers at the technical center of Lidaer prepared and compared the bioavailability of lutein acetate (40% by lutein) and lutein natural ester (40% by lutein, the natural fatty acids of which are mainly stearic acid and palmitic acid) as purified as possible, and found that there was no obvious difference in coloration at the same lutein dosage. Experiments on eggs and broilers prove that the short chain fatty acid lutein ester is not higher than the long chain fatty acid lutein ester. In the state of unpurified marigold ointment, compared with lutein acetate with the same lutein amount, the bioavailability difference exists, and the difference is obvious particularly for laying hens. This suggests that the contributors to the bioavailability of lutein come from the oil encapsulating the long chain fatty acid lutein ester, not the fatty acid itself.
Based on the discovery, a method for preparing natural lutein ester ultramicro emulsion by using marigold extract as a raw material and eliminating grease in the extract through saponification and keeping natural lutein ester to the maximum is researched and obtained. The prepared natural lutein ester ultramicro emulsion can be further spray-dried to obtain stable natural lutein ester microcapsule particles or powder. The natural lutein ester in the microemulsion or microcapsule has no interference of grease, and has native lutein ester ultramicro particle form, so that the microemulsion or microcapsule has very high bioavailability.
There are many methods for saponifying lutein ester (marigold extract).
The traditional saponification method is that lutein oil is heated to 60-80 ℃ in alkali and alcohol, lutein monomers (lutein and a small amount of zeaxanthin) are obtained by saponification, and the oil part is also converted into corresponding fatty acid salt and glycerol.
The method for saponifying in the water-poor phase with low water content can generate lutein in crystalline form, and the lutein can be directly prepared into preparation, so that the lutein with large crystal size is difficult to be absorbed by intestinal tract of animals, and the bioavailability and final coloring effect are affected. If the saponified paste is washed by alcohol water to prepare lutein crystals, and a large amount of organic solvent such as dichloromethane is further used for dissolving the crystals for emulsification, and gelatin, white sugar, lignosulfonate, dextrin and the like are used as wall materials to prepare microcapsules, very small lutein particle size can be obtained, and the biological utilization effect is very good.
CN 105188408B discloses a saponification method in a high-temperature tubular reactor, which is stated to prepare a saponified solution with lutein particles smaller than 0.5 micron, but the method has complex process equipment and needs reaction conditions of high temperature and nitrogen introduction, the prepared lutein exists in a free state, and the saponified free lutein is also unstable and easy to oxidize. The lutein has low solubility due to no addition of emulsifier, and has certain influence on application.
CN101869261A discloses a method for preparing water-soluble emulsion of lutein ester, which is to dilute lutein extract with vegetable oil and then use emulsifier to prepare oil-in-water emulsion. The method retains oil and fat of marigold extract, so that even if prepared into emulsion, the content is low, which is not beneficial for processing and utilization, and on the other hand, the bioavailability is not improved due to the continuous existence of original oil and fat. In the case of animal feed additives, the inability to increase bioavailability means that it cannot compete with short chain fatty acid esters such as saponified paste or lutein acetate.
CN 109645235 a discloses a method for preparing a water-soluble emulsion of lutein ester with polyethylene glycol glycerol ricinoleate as an emulsifier. The method also directly prepares lutein ointment into emulsion, and does not saponify oil in the ointment. Therefore, the bioavailability is low, and the compound is still not suitable for being used as a feed additive.
Disclosure of Invention
The invention utilizes the property that alkali dissolved in water can react with fatty glyceride with certain solubility in water but can not react with fatty acid lutein ester which is slightly soluble in water but strongly encapsulated in an oil-in-water core when marigold extract (natural oil mixture of lutein ester) is subjected to saponification reaction in an oil-in-water emulsion, fatty glyceride is converted into fatty acid salt and glycerol, so that the newly-generated fatty acid soap and a main emulsifier form a wall material together, and newly-dissociated lutein ester molecules are dispersed and isolated, thereby obtaining the highly-dispersed lutein ester dispersion. The lutein ester dispersion is characterized in that the lutein ester of the lutein ester dispersion is not coated by grease, and fatty glyceride is converted into fatty acid salt to become a part of an emulsification system. The lutein ester feed additive with high stability and high bioavailability is prepared by further spray drying.
A production method of a lutein ester feed additive with high bioavailability is characterized by comprising the following steps:
(1) adding common antioxidant into marigold ointment, and heating to 50-70 deg.C for melting;
(2) dissolving emulsifier and defoamer in water phase, heating to 60-80 deg.C, adding melted marigold ointment under high speed shearing condition, and making into emulsion;
(3) adding strong alkali aqueous solution, keeping the temperature and performing saponification reaction for 0.5-5 h;
(4) after the saponification reaction is finished, adding wall materials such as lignosulfonate, dextrin, water-soluble starch, maltodextrin and the like, and dissolving for 0.5-2 h;
(5) after the dissolution is finished, adding water to adjust the viscosity to be proper, and carrying out spray granulation and drying while the solution is hot to obtain a microcapsule product wrapped with the flowing agent.
Or directly adding water into the saponification solution obtained in the step (3) for dilution to directly obtain an aqueous product.
The invention prepares submicron-grade natural fatty acid lutein ester emulsion (dispersoid) by an emulsification-saponification process by means of a hydrophilic lipophilic guiding principle of an oil-in-water emulsion system and a principle that natural long-chain fatty acid lutein ester is strongly hydrophobic and cannot contact inorganic base dissolved in a water phase, but fatty acid glyceride is partially distributed on an oil-water interface due to certain hydrophilicity so as to be saponified by the inorganic base in the water phase, and the submicron-grade natural fatty acid lutein ester emulsion (dispersoid) is further processed into corresponding microcapsule preparation or aqueous agent product.
The specific process is as follows:
adding fatty glyceride (marigold ointment) containing natural lutein ester and emulsifiers such as gelatin, sodium lignosulfonate, potassium lignosulfonate and sucrose ester into a proper amount of water for emulsification to obtain an oil-in-water emulsion with fatty lutein ester-fatty glyceride as an internal phase, then adding a strong alkali aqueous solution into an emulsion system to saponify plant oil slightly soluble in water, wherein the fatty lutein ester is kept in a stable state in the process, and finally, the fatty lutein ester is kept to obtain a submicron oil-in-water emulsion (or dispersion) with the fatty lutein ester as the internal phase. Based on the submicron dispersed natural fatty acid lutein ester emulsion, water-soluble carriers such as lignosulfonate, dextrin, water-soluble starch, maltodextrin and the like are added to serve as wall materials, and then a spray drying process is carried out to obtain the natural fatty acid lutein ester microcapsule feed additive with high dispersity and no grease coating.
The follow-up detection of High Performance Liquid Chromatography (HPLC) proves that no free lutein is generated even if saponification reaction is carried out for 5h at 80 ℃, which indicates that lutein fatty acid ester still retains the original state of natural fatty acid lutein ester. The microcapsule feed additive of natural fatty acid lutein ester has high dispersity, and 90% of particles are in a submicron range of 500-1000 μm when the particle size distribution of an aqueous solution of the microcapsule feed additive is measured by a Malvern laser particle sizer. Although it is acknowledged that a high degree of dispersion helps to improve the bioavailability of carotenoids, it is still surprising that the formulations of long chain fatty acid lutein esters obtained in the present invention exhibit bioavailability higher than lutein acetate and free lutein in various animal feeding experiments.
Compared with the traditional marigold ointment single-phase saponification process, the emulsification-saponification process reduces the viscosity of the oil, improves the contact area of the reaction and improves the reaction speed. And the lutein ester is prevented from forming larger crystals by saponification reaction in the emulsion, so that a stable emulsified product is obtained, and the utilization rate of animals is improved. The microcapsule technology is used for preparing particle products or water aqua, so that oxygen can be well isolated, and the stability of the products is improved. As lutein ester, even if the lutein ester is not further processed into microcapsules, the diluted emulsion is used as a product, the stability is higher, tests show that the lutein ester has better bioavailability, is colored and durable in color block, has similar coloring power of fresh eggs and greatly improves the color retention after processing compared with the emulsion product of free lutein in experiments of laying hens and broilers.
The lutein grease raw material used in the invention is commercial marigold extract, and the lutein content is about 15 percent generally. The antioxidant is selected from one or more of BHT \ BHA \ tocopherol \ ethoxyquinoline, and the addition amount is 0-3%. The emulsifier is selected from gelatin, sucrose ester, lignosulfonate, etc. Dissolving emulsifier and defoamer in water phase, heating to 60-80 deg.C, adding melted marigold ointment under high speed shearing condition, and making into emulsion. Adding strong alkali water solution, and performing saponification reaction for 0.5-5 h. And after the saponification reaction is finished, adding wall materials such as dextrin, water-soluble starch, maltodextrin and the like, and dissolving for 0.5-2 h. After the dissolution is finished, adding water to adjust the viscosity to be proper, and carrying out spray granulation and drying while the solution is hot to obtain a microcapsule product wrapped with the flowing agent.
Or directly adding water into the saponification solution obtained in the step (3) for dilution to directly obtain an aqueous product.
The invention utilizes the principle of hydrophilic and oleophilic guiding of oil-in-water emulsion system, utilizes the strong hydrophobicity of lutein ester which can not contact with inorganic base dissolved in water phase and can not be saponified, but the principle that fatty glyceride partially exists on an oil-water interface due to certain hydrophilicity and can be saponified by inorganic base in a water phase is that fatty glyceride (marigold ointment) containing natural lutein ester is added into a proper amount of water together with emulsifiers such as gelatin, sodium lignosulfonate, potassium lignosulfonate and sucrose ester for emulsification to obtain an oil-in-water emulsion with fatty lutein ester-fatty glyceride as an inner phase, then saponifying the slightly water-soluble vegetable oil and fat in the emulsion system through aqueous phase saponification, the fatty acid lutein ester keeps a stable emulsification dispersion state continuously, and a submicron oil-in-water emulsion (or dispersion) with fatty acid lutein ester as an inner phase is obtained. Based on the natural fatty acid lutein ester emulsion with submicron dispersion, a water-soluble carrier is added to prepare a spray drying working solution, and then a spray drying process is carried out to obtain the natural fatty acid lutein ester microcapsule feed additive with high dispersion degree and no grease coating. This microencapsulated feed additive of natural fatty acid lutein esters surprisingly demonstrated in multiple animal feeding trials to have a bioavailability higher than lutein acetate and free lutein.
Compared with the traditional marigold ointment single-phase saponification process, the emulsification-saponification process reduces the viscosity of the oil, is beneficial to the contact of alkali and the oil, and improves the reaction speed. The emulsion is saponified to prevent lutein ester from forming large crystal, obtain stable emulsified product and raise animal utilization. The microcapsule technology is used for preparing particle products or water aqua, so that oxygen can be well isolated, and the stability of the products is improved.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
Detailed description of the preferred embodiment 1
Preparing natural long-chain fatty acid lutein ester microemulsion:
(1) adding 1 kg BHT into 100 kg marigold ointment containing 15.8% xanthophyll, heating to 70 deg.C, and stirring;
(2) adding 6 kg of sodium lignosulphonate into 500 kg of water, stirring and dissolving completely, heating to 80 ℃, adding 1 kg of silicone oil emulsion defoaming agent, starting a high-speed shearing emulsifying machine, adding the hot marigold ointment in the step (1), and shearing and emulsifying at high speed for 0.5 h;
(3) adding an alkali solution consisting of 20 kg of potassium hydroxide and 20 kg of water, keeping the temperature at 80-90 ℃, carrying out saponification reaction for 1 h, converting the solution from brownish black to bright yellow, cooling to 50 ℃, adding 100 kg of sodium lignosulfonate 150 kg of maltodextrin, and stirring to completely dissolve the maltodextrin to obtain 890 kg of the stable microemulsion of the natural long-chain fatty acid lutein ester.
Example 2
Preparation of a microparticle formulation of long chain fatty acid lutein ester by spray drying:
100 kg of the emulsion prepared in example 1 is conveyed by a peristaltic pump, enters a drying tower in a high-speed centrifugal atomization mode, hot air carrying corn starch enters the drying tower from the periphery of a spray head, and the inlet end hot air is controlled at 120 ℃ to 150 ℃. The spray drying process finally forms high-fluidity particles coated with corn starch on the surface, and 96 kilograms of particle preparation products of 20-60 meshes of long-chain fatty acid lutein esters are obtained after screening.
Example 3
Mixing 100 g of marigold extract containing 16.0% xanthophyll with 1 g of BHA, heating to 70 deg.C for melting, and stirring to obtain oil phase. 20 g of potassium lignosulphonate was dissolved completely in 500 g of water, heated to 80 ℃ and 1 g of silicone oil emulsion defoamer was added as the aqueous phase. Starting a high-speed emulsifying machine, adding the oil phase into the water phase, carrying out high-speed shearing emulsification for 0.4 h, adding an alkali solution consisting of 18 g of potassium hydroxide and 20 g of water, keeping the temperature for reaction for 1 h, converting the solution from brown black to orange red, adding 340 g of water, uniformly stirring, and preparing into a liquid preparation with the lutein content of 1.60%.
Example 4
The liquid formulation of example 3 was mixed with free lutein (crystals) of the same content and lutein acetate emulsion with chicken feed to make a daily ration with 20ppm lutein for a coloration experiment in layer chicken:
30 laying hens in each experimental group are continuously and normally fed with the daily ration for 30 days. From the eggs laid by the laying hens in each group on day 30, 10 eggs were randomly selected and the average value of the group was determined, and the results were as follows:
| sample name corresponding to 20ppm daily ration | Fresh egg yolk, chroma (mean value) | Fried yolk, chroma (average) |
| 1.60% natural fatty acid lutein ester emulsion | 11 | 8 |
| 1.60% free lutein emulsion | 9 | 6 |
| 1.60% lutein acetate emulsion | 10 | 7 |
Experimental results prove that the natural fatty acid lutein ester which is subjected to micro-emulsification to obtain high dispersion degree and further subjected to saponification and grease coating removal has a good coloring effect, and the colored natural lutein ester has better thermal stability than natural lutein and lutein acetate ester and has high use value.
Claims (3)
1. A natural long-chain fatty acid lutein ester feed additive with high bioavailability is characterized in that marigold extract is used as a raw material, the feed additive is prepared by utilizing the principle that emulsified lutein ester cannot be saponified and fatty glyceride is easy to saponify, the main effective component is natural long-chain fatty acid lutein ester, the lutein ester content is 0.5-20%, fatty glyceride is not contained or less contained, and the feed additive has good water dispersibility.
2. A lutein ester feed additive according to claim 1, prepared by the method comprising the following steps: (1) adding common antioxidant into marigold ointment, and heating to 50-70 deg.C for melting; (2) dissolving emulsifier and defoamer in water phase, heating to 60-80 deg.C, adding melted marigold ointment under high speed shearing condition, and making into emulsion; (3) adding strong alkali aqueous solution, keeping the temperature and performing saponification reaction for 0.5-5 h; (4) after the saponification reaction is finished, adding wall materials such as lignosulfonate, dextrin, water-soluble starch, maltodextrin and the like, and dissolving for 0.5-2 h; (5) after the dissolution is finished, adding water to adjust the viscosity to be proper, and carrying out spray granulation and drying while the solution is hot to obtain a microcapsule product wrapped with the flowing agent.
3. The lutein ester feed additive according to claim 1 for use in feed additive, having good coloring and high stability.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010052574.9A CN111066961A (en) | 2020-01-17 | 2020-01-17 | Natural long-chain fatty acid lutein ester feed additive with high bioavailability |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010052574.9A CN111066961A (en) | 2020-01-17 | 2020-01-17 | Natural long-chain fatty acid lutein ester feed additive with high bioavailability |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111066961A true CN111066961A (en) | 2020-04-28 |
Family
ID=70324026
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010052574.9A Withdrawn CN111066961A (en) | 2020-01-17 | 2020-01-17 | Natural long-chain fatty acid lutein ester feed additive with high bioavailability |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111066961A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101269310A (en) * | 2008-05-06 | 2008-09-24 | 大连赛诺微囊技术有限公司 | Method for preparing cold water dispersion type microcapsule powder |
| CN108822947A (en) * | 2018-04-19 | 2018-11-16 | 广州市浪奇实业股份有限公司 | A kind of soap-base lubricant |
| CN108813616A (en) * | 2018-07-05 | 2018-11-16 | 河北东之星生物科技股份有限公司 | A method of preparing lutein water-soluble microemulsion |
-
2020
- 2020-01-17 CN CN202010052574.9A patent/CN111066961A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101269310A (en) * | 2008-05-06 | 2008-09-24 | 大连赛诺微囊技术有限公司 | Method for preparing cold water dispersion type microcapsule powder |
| CN108822947A (en) * | 2018-04-19 | 2018-11-16 | 广州市浪奇实业股份有限公司 | A kind of soap-base lubricant |
| CN108813616A (en) * | 2018-07-05 | 2018-11-16 | 河北东之星生物科技股份有限公司 | A method of preparing lutein water-soluble microemulsion |
Non-Patent Citations (5)
| Title |
|---|
| 原毅: "《天然使用色素的生产和应用》", 31 March 2004 * |
| 宋洪波等: "《食品物性学》", 31 July 2016 * |
| 惠伯棣: "《类胡萝卜素化学及生物化学》", 31 January 2005 * |
| 毕灵玲: "《有机化学实验》", 28 February 2013 * |
| 荣瑞芬、闫文杰: "《食品科学与工程综合实验指导》", 31 December 2018 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7205835B2 (en) | Fat-soluble nutrient microcapsules and method for producing the same | |
| US6863914B1 (en) | Stable, aqueous dispersions and stable, water-dispersible dry powders of xanthophylls, and production and use of the same | |
| US20230063291A1 (en) | Stable carotenoid microcapsule having high bioavailability and preparation method therefor | |
| US5863953A (en) | Liquid, oil-miscible carotenoid preparations | |
| AU2005279201B2 (en) | Method for pigment solubilisation, a pigment composition and its use | |
| US5668183A (en) | Water dispersible compositions | |
| RU2145211C1 (en) | Method of manufacturing active principle-based spherical particles | |
| CN101396068B (en) | Carotenoid preparation and use thereof and feedstuff containing the preparation and preparation method thereof | |
| EP1846153B1 (en) | Microcapsules and emulsions containing low bloom gelatin and methods of making and using thereof | |
| US3655406A (en) | Carotenoid compositions | |
| EP2062570B1 (en) | A method for obtaining stable micro-emulsions of derivatives of oxycarotenoids of short-chain organic acids, micro-emulsions obtained, and formulation which contains these | |
| JPS62240364A (en) | Production of fine granular carotinoid product dispersible in water | |
| CN103284290B (en) | Good microencapsulation method for carotenoid | |
| JP2001506685A (en) | Stable aqueous dispersions and stable water-dispersible dry powders of xanthophylls, their production and use | |
| JP3595300B2 (en) | Preparation of dry powder of one or more oxidized carotenoids | |
| JPH05304903A (en) | Stable liquid product containing fat-soluble material | |
| EP0347751A1 (en) | Stable compositions dispersible in cold water | |
| CN101921495B (en) | Method for preparing lutein oleoresin microcapsule | |
| WO2004057980A1 (en) | Homogeneous solid granules containing carotenoids | |
| CN100421650C (en) | Water-dispersed carotenoid powder preparation method | |
| CN102341006A (en) | Powdered compositions of astaxanthin derivatives II | |
| CN101754693A (en) | The dispersible carotenoid formulation of water | |
| CN111066961A (en) | Natural long-chain fatty acid lutein ester feed additive with high bioavailability | |
| CN103315303A (en) | Preparation method of maize yellow pigment microcapsules | |
| CA2028213A1 (en) | Dry solid compositions containing lipid |
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 | ||
| WW01 | Invention patent application withdrawn after publication | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20200428 |