CN110845557A - Method for extracting anthocyanin in three small berry compound fruit pulps under assistance of microwaves - Google Patents
Method for extracting anthocyanin in three small berry compound fruit pulps under assistance of microwaves Download PDFInfo
- Publication number
- CN110845557A CN110845557A CN201911107773.9A CN201911107773A CN110845557A CN 110845557 A CN110845557 A CN 110845557A CN 201911107773 A CN201911107773 A CN 201911107773A CN 110845557 A CN110845557 A CN 110845557A
- Authority
- CN
- China
- Prior art keywords
- anthocyanin
- powder
- extraction
- pulp
- microwave
- 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
- 150000004636 anthocyanins Chemical class 0.000 title claims abstract description 103
- 235000010208 anthocyanin Nutrition 0.000 title claims abstract description 102
- 229930002877 anthocyanin Natural products 0.000 title claims abstract description 102
- 239000004410 anthocyanin Substances 0.000 title claims abstract description 102
- 238000000034 method Methods 0.000 title claims abstract description 33
- 235000021028 berry Nutrition 0.000 title claims abstract description 30
- 150000001875 compounds Chemical class 0.000 title claims abstract description 15
- 235000013399 edible fruits Nutrition 0.000 title claims description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 86
- 238000000605 extraction Methods 0.000 claims abstract description 71
- 239000000843 powder Substances 0.000 claims abstract description 37
- 239000007788 liquid Substances 0.000 claims abstract description 28
- 239000002131 composite material Substances 0.000 claims abstract description 26
- 240000000851 Vaccinium corymbosum Species 0.000 claims abstract description 15
- 235000003095 Vaccinium corymbosum Nutrition 0.000 claims abstract description 15
- 235000017537 Vaccinium myrtillus Nutrition 0.000 claims abstract description 15
- 235000021014 blueberries Nutrition 0.000 claims abstract description 15
- 240000007651 Rubus glaucus Species 0.000 claims abstract description 14
- 235000011034 Rubus glaucus Nutrition 0.000 claims abstract description 14
- 235000009122 Rubus idaeus Nutrition 0.000 claims abstract description 14
- 238000002156 mixing Methods 0.000 claims abstract description 14
- 238000004108 freeze drying Methods 0.000 claims abstract description 11
- 235000000177 Indigofera tinctoria Nutrition 0.000 claims abstract description 10
- 229940097275 indigo Drugs 0.000 claims abstract description 10
- COHYTHOBJLSHDF-UHFFFAOYSA-N indigo powder Natural products N1C2=CC=CC=C2C(=O)C1=C1C(=O)C2=CC=CC=C2N1 COHYTHOBJLSHDF-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000000047 product Substances 0.000 claims abstract description 10
- 239000000706 filtrate Substances 0.000 claims abstract description 8
- 238000004537 pulping Methods 0.000 claims abstract description 7
- 239000011347 resin Substances 0.000 claims abstract description 7
- 229920005989 resin Polymers 0.000 claims abstract description 7
- 238000007873 sieving Methods 0.000 claims abstract description 7
- 238000002791 soaking Methods 0.000 claims abstract description 7
- 230000008569 process Effects 0.000 claims abstract description 6
- 238000012545 processing Methods 0.000 claims abstract description 5
- 238000001035 drying Methods 0.000 claims abstract description 3
- 241000205585 Aquilegia canadensis Species 0.000 claims abstract 3
- 238000005119 centrifugation Methods 0.000 claims abstract 2
- 238000005374 membrane filtration Methods 0.000 claims abstract 2
- 238000000874 microwave-assisted extraction Methods 0.000 claims description 8
- 239000002994 raw material Substances 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 7
- 239000003480 eluent Substances 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 5
- 230000008901 benefit Effects 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 2
- 238000007710 freezing Methods 0.000 claims description 2
- 230000008014 freezing Effects 0.000 claims description 2
- 230000020477 pH reduction Effects 0.000 claims description 2
- 238000000746 purification Methods 0.000 claims description 2
- 238000001704 evaporation Methods 0.000 claims 1
- 230000008020 evaporation Effects 0.000 claims 1
- 239000007858 starting material Substances 0.000 claims 1
- 238000010298 pulverizing process Methods 0.000 abstract 1
- 230000004044 response Effects 0.000 description 15
- 230000003993 interaction Effects 0.000 description 12
- 230000000694 effects Effects 0.000 description 11
- 239000002904 solvent Substances 0.000 description 9
- 241001570521 Lonicera periclymenum Species 0.000 description 7
- 235000016813 Lonicera caerulea var edulis Nutrition 0.000 description 5
- 240000007893 Lonicera caerulea var. edulis Species 0.000 description 5
- 239000000049 pigment Substances 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 4
- 230000000975 bioactive effect Effects 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 230000036541 health Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 235000016709 nutrition Nutrition 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- 239000013543 active substance Substances 0.000 description 3
- 238000003287 bathing Methods 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 210000002421 cell wall Anatomy 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 description 2
- 238000000944 Soxhlet extraction Methods 0.000 description 2
- 230000032683 aging Effects 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 description 2
- BJRNKVDFDLYUGJ-RMPHRYRLSA-N hydroquinone O-beta-D-glucopyranoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C(O)C=C1 BJRNKVDFDLYUGJ-RMPHRYRLSA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000005728 strengthening Methods 0.000 description 2
- 239000011701 zinc Substances 0.000 description 2
- 229910052725 zinc Inorganic materials 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- AFSDNFLWKVMVRB-UHFFFAOYSA-N Ellagic acid Chemical compound OC1=C(O)C(OC2=O)=C3C4=C2C=C(O)C(O)=C4OC(=O)C3=C1 AFSDNFLWKVMVRB-UHFFFAOYSA-N 0.000 description 1
- ATJXMQHAMYVHRX-CPCISQLKSA-N Ellagic acid Natural products OC1=C(O)[C@H]2OC(=O)c3cc(O)c(O)c4OC(=O)C(=C1)[C@H]2c34 ATJXMQHAMYVHRX-CPCISQLKSA-N 0.000 description 1
- 229920002079 Ellagic acid Polymers 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 229940123457 Free radical scavenger Drugs 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 description 1
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 229960000271 arbutin Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 210000003792 cranial nerve Anatomy 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 235000004132 ellagic acid Nutrition 0.000 description 1
- 229960002852 ellagic acid Drugs 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 229940074391 gallic acid Drugs 0.000 description 1
- 235000004515 gallic acid Nutrition 0.000 description 1
- 235000004280 healthy diet Nutrition 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 description 1
- 238000002803 maceration Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- FAARLWTXUUQFSN-UHFFFAOYSA-N methylellagic acid Natural products O1C(=O)C2=CC(O)=C(O)C3=C2C2=C1C(OC)=C(O)C=C2C(=O)O3 FAARLWTXUUQFSN-UHFFFAOYSA-N 0.000 description 1
- 239000002366 mineral element Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000000974 natural food coloring agent Substances 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- BJRNKVDFDLYUGJ-UHFFFAOYSA-N p-hydroxyphenyl beta-D-alloside Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=C(O)C=C1 BJRNKVDFDLYUGJ-UHFFFAOYSA-N 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 229960001285 quercetin Drugs 0.000 description 1
- 235000005875 quercetin Nutrition 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000000191 radiation effect Effects 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 238000000194 supercritical-fluid extraction Methods 0.000 description 1
- 239000001040 synthetic pigment Substances 0.000 description 1
- 229920001864 tannin Polymers 0.000 description 1
- 235000018553 tannin Nutrition 0.000 description 1
- 239000001648 tannin Substances 0.000 description 1
- 238000002137 ultrasound extraction Methods 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/06—Benzopyran radicals
- C07H17/065—Benzo[b]pyrans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B61/00—Dyes of natural origin prepared from natural sources, e.g. vegetable sources
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B67/00—Influencing the physical, e.g. the dyeing or printing properties of dyestuffs without chemical reactions, e.g. by treating with solvents grinding or grinding assistants, coating of pigments or dyes; Process features in the making of dyestuff preparations; Dyestuff preparations of a special physical nature, e.g. tablets, films
- C09B67/0096—Purification; Precipitation; Filtration
Abstract
The invention discloses a method for extracting anthocyanin in three small berry compound pulp by microwave assistance, belongs to the technical field of extraction of effective components of agricultural products, and particularly relates to a key process which comprises the following steps: selecting three small berries with uniform size, pulping, mixing pulp of indigo honeysuckle, raspberry and wild blueberry according to a ratio of 2:4:1, freeze drying, pulverizing into powder, and sieving; mixing the composite pulp powder with 60% volume fraction ethanol (acidified with 0.1% HCl) at a solid-to-liquid ratio of 1:45, soaking for 30min, and treating at microwave power of 250W for 77 s; then ice-bath is carried out for 5min, centrifugation is carried out for 10min at 4 ℃ and 7000rpm, membrane filtration of 0.45 mu m is carried out, filtrate is evaporated and concentrated and then is dried, and crude anthocyanin powder is obtained; and re-dissolving the crude anthocyanin, purifying by using activated D101 macroporous resin, eluting by using 60% ethanol, and drying to obtain a pure anthocyanin product. The method is suitable for various small berries, and can increase the utilization rate of the small berries and reduce the processing cost.
Description
Technical Field
A method for extracting anthocyanin in three small berry composite fruit pulps by microwave assistance belongs to the technical field of extraction of effective components of agricultural products, and particularly relates to a processing technology for extracting anthocyanin by microwave assistance, so that a higher extraction rate is obtained finally, and the cost is saved. The effective extraction method can maximize the yield of the target and minimize the degradation amount of the target on the basis of an environment-friendly technology.
Background
The small berries are succulent and fleshy single fruits, and berry fruit trees are various and are mostly distributed in northern areas of China, especially in the northeast area. The raspberry contains a large amount of ellagic acid, tannin, quercetin, gallic acid, anthocyanin, etc., and has effects of resisting oxidation, delaying aging, relieving fatigue and resisting cancer. The indigo honeysuckle is rich in nutrition, is rich in saccharide, amino acid, polyphenol, anthocyanin and the like, and has the effects of resisting virus, resisting cancer, improving hypertension, resisting fatigue, resisting oxidation and the like. The wild blueberry fruit is rich in VE, VA, VB, SOD, arbutin, protein, anthocyanin, edible fiber, K, Fe, Zn, Ca and other mineral elements besides conventional sugar, acid and Vc. The blueberry is a nutritional health-care fruit with high zinc, calcium, ferrum, copper and vitamins. It has the functions of nourishing, health care, preventing cranial nerve from ageing, strengthening heart, resisting cancer, softening blood vessel, strengthening immunity, etc. The three small berries all contain rich antioxidant components, and have high nutritional, health promoting and medicinal values.
Indigo honeysuckle, raspberry and wild blueberry are used as novel wild berries and have good edible value. The lonicera edulis, the raspberry and the wild blueberry all contain a large amount of anthocyanin, are flavonoid substances, are formed by condensing anthocyanin and sugar through glycosidic bonds, have high antioxidant activity, are the strongest free radical scavenger discovered at present, have the functions of resisting oxidation, cancer, mutation and the like, and have potential benefits on health. Anthocyanins have attracted considerable attention as natural food colorants due to the safety problems of synthetic pigments.
Extraction is a separation technique that can affect the yield, quality and composition of a target. Various extraction techniques can be used for extraction of anthocyanins, and generally these techniques can be divided into conventional techniques and modern techniques. Traditional extraction methods include maceration and soxhlet extraction. In recent years, new techniques have been used to extract bioactive compounds, mainly ultrasound-assisted extraction, microwave-assisted extraction, ultra-high pressure extraction, subcritical and supercritical fluid extraction, and the like. Most of the traditional extraction technologies select proper conditions such as solvents, temperatures, time and the like for extraction according to different polarities of compounds, and target products are separated. Although the method has the characteristics of simple equipment requirement, easy operation, cost saving and the like. But are time consuming, require relatively large amounts of high purity, expensive solvents, have low solvent recovery, and sometimes degrade the active compound. The microwave-assisted extraction has the advantages of less time consumption, low energy consumption, less material consumption and high extraction rate, can effectively protect functional components in raw materials, and belongs to the field of safe pollution-free green food processing. The principle is that the temperature and pressure generated by microwave energy in the microwave radiation process are utilized to accelerate the rupture of cell walls, so that active substances flow into the extraction solvent, and the yield of the active substances is improved.
At present, a great deal of research is carried out on the extraction of bioactive substances by scholars at home and abroad, and the conclusion is that the microwave-assisted extraction has the highest efficiency in all methods. The method takes the compound fruit pulp of the lonicera edulis, the raspberry and the wild blueberry in a ratio of 2:4:1 as raw materials, optimizes the condition of extracting anthocyanin from the compound fruit pulp, and provides a foundation for the utilization of small berries and the deep research of anthocyanin.
In the extraction of anthocyanin from composite pulp, microwave treatment is a very critical link, and the microwave-assisted extraction mechanism comprises three successive steps, firstly, separating solute from active sites of a sample matrix at elevated temperature and pressure; second, diffusion of the solvent in the sample matrix; third, the solute is released from the sample matrix into the solvent. The technology can extract bioactive compounds more quickly, reduces the use of organic solvents, and is an environment-friendly method.
At present, the research of extracting bioactive substances on the raw materials of single substances is more, and the research on composite raw materials is less. The method optimizes the condition of extracting anthocyanin in the composite pulp of three small berries by using microwaves, increases the utilization rate of lonicera edulis, raspberry and wild blueberry, enriches the market of natural pigment anthocyanin and meets the requirements of consumers.
Disclosure of Invention
The technical problem is as follows: indigo honeysuckle, raspberry and wild blueberry all have extremely high nutritional value and medicinal health care value, but the shelf life of small berries is short, anthocyanin extracted from compound pulp can increase the utilization rate of the small berries, people have higher and higher requirements on healthy diet, many pigments on the market are synthesized, and the development of natural and healthy pigments is a great trend.
The purpose of the invention is as follows: based on the defects of time and material consumption and the like in the traditional method for extracting anthocyanin, the method for extracting anthocyanin from three small berry compound fruit pulps under the assistance of microwave is developed, and has the advantages of simple requirement on equipment, short extraction time, high extraction rate, excellent quality, safety, greenness and no pollution.
The technical scheme is as follows: a method for extracting anthocyanin from three small berry compound fruit pulps under the assistance of microwave comprises the following specific steps:
(1) pretreatment of raw materials: selecting three small berries with uniform size and color, no rot, no immature green fruit and the like, pulping, fully and uniformly mixing pulp of indigo honeysuckle, raspberry and wild blueberry according to a ratio of 2:4:1, putting the composite pulp in a freeze dryer, crushing the pulp into powder by a crusher after freeze drying, sieving the powder by a 60-mesh sample sieve, and storing the powder in a dryer in a sealed bag for later use;
(2) microwave-assisted treatment: mixing the composite pulp powder obtained in the step (1) with 60% volume fraction ethanol (0.1% HCL acidification) according to a solid-to-liquid ratio of 1:45, placing the mixture in a triangular flask, soaking for 30min, and then treating for 77s under the condition that the microwave power is 250W;
(3) extracting crude anthocyanin: ice-cooling for 5min, centrifuging at 4 deg.C and 7000rpm for 10min, filtering with 0.45 μm membrane, concentrating the filtrate with rotary evaporator, and vacuum freeze drying to obtain crude anthocyanin powder;
(4) and (3) purification: redissolving the 3 times volume of water of the crude anthocyanin obtained in the step (3), purifying by using activated D101 macroporous resin, eluting by using 60 percent ethanol, collecting eluent, and freezing and drying to obtain a pure anthocyanin product.
The lonicera edulis, raspberry and wild blueberry compound beverage has the following beneficial effects:
1. the extraction raw material adopted by the invention is the composite fruit pulp, three kinds of anthocyanin of small berries are extracted, and the invention makes a contribution to meeting the requirements of consumers on the diversification of natural pigments in the market.
2. According to the invention, the raw material is crushed by a high-speed crusher and is subjected to vacuum freeze drying, so that the loss of anthocyanin is reduced, and the extraction efficiency is indirectly improved.
3. The method adopts microwave-assisted extraction, greatly shortens the extraction time compared with the traditional extraction methods such as Soxhlet extraction, hot reflux extraction and the like, has simple equipment requirement and simple operation, avoids the anthocyanin loss caused in the operation process, has high anthocyanin extraction rate, avoids environmental pollution at the same time, and has important guiding significance for the development and utilization of small berries and natural pigments.
Drawings
FIG. 1 Effect of ethanol volume fraction on anthocyanin extraction amount
Weighing 1.0g of the pretreated composite pulp powder, and researching the influence of the volume fraction of the ethanol on the extraction amount of the anthocyanin under the conditions of the microwave time of 90s, the microwave power of 300W and the material-liquid ratio of 1:40g/m L, wherein the result is shown in figure 1. As can be seen from FIG. 1, the concentration of ethanol is in direct proportion to the extraction amount of anthocyanin from 40% to 60%, and the highest concentration can reach 28.04 mg/g. When the volume fraction exceeds 60%, the volume fraction is continuously increased, and the extraction amount of anthocyanin is reduced. The anthocyanin molecules are polar, and the polarity of the composite solvent is increased continuously with the addition of water into the ethanol, so that the extraction amount of the anthocyanin is increased along with the increase of the concentration of the ethanol according to the principle of 'similarity and compatibility'. When the ethanol concentration exceeds 60%, the anthocyanin extraction amount is reduced. This is probably because water and low concentrations of ethanol easily enter cells, but high concentrations of ethanol cause denaturation of proteins, so that solubilization of anthocyanins is hindered, thereby affecting extraction yield. Therefore, ethanol volume fraction 50-70% was selected as the parameter range of the response surface.
FIG. 2 Effect of microwave time on anthocyanin extraction amount
Weighing 1.0g of the pretreated composite pulp powder, and researching the influence of the microwave treatment time on the anthocyanin extraction amount under the conditions of 60% of ethanol volume fraction containing 0.1% of HCl, 300W of microwave power and 1:40g/m L of material-liquid ratio, wherein the result is shown in figure 2. As is clear from fig. 2, the anthocyanin extraction amount did not increase with the increase of the microwave time. The time period of 30-90s is increased, up to 28.34 mg/g. The reason may be that the plant cell wall is broken and cracked, the cell structure is destroyed, and the release of anthocyanin in the cell is promoted; and the time period of more than 90s is reduced, which is probably because the temperature of the system is increased along with the prolonging of the processing time, the heat-sensitive anthocyanin active ingredients are damaged and degraded, and the extraction amount of anthocyanin is reduced. Therefore, the microwave time of 60-120s is selected as the parameter range of the response surface.
FIG. 3 influence of microwave power on anthocyanin extraction amount
Weighing 1.0g of the pretreated composite pulp powder, and researching the influence of microwave power on anthocyanin extraction amount under the conditions of ethanol volume fraction containing 0.1% HCl of 60%, microwave time of 90s and material-liquid ratio of 1:40g/m L, wherein the result is shown in figure 3. As can be seen from FIG. 3, the thermal effect and radiation effect of the microwave are correspondingly enhanced by increasing the microwave power, so as to accelerate the rupture of cell walls, facilitate the release of active substances and promote the dissolution of anthocyanin. The extraction amount of anthocyanin reaches 29.44mg/g at the maximum when the power is 300W. When the microwave power exceeds 300W, the extraction amount of anthocyanin is reduced with the increase of microwave power, and it is possible that the increase of microwave power causes the system temperature to be too high, so that the anthocyanin structure is damaged, and impurities are dissolved out, so that the extraction amount of anthocyanin is reduced. Therefore, the microwave power of 200-400W is selected as the parameter range of the response surface.
FIG. 4 Effect of feed liquid ratio on anthocyanin extraction amount
Weighing 1.0g of the pretreated composite pulp powder, and researching the influence of the feed liquid on the extraction amount of anthocyanin under the conditions of 60% of ethanol volume fraction containing 0.1% of HCl, 90s of microwave time and 300W of microwave power, wherein the result is shown in figure 4. As can be seen from fig. 4, the extraction amount of anthocyanin shows a tendency of increasing first and then decreasing with the increase of the feed-to-liquid ratio, and when the feed-to-liquid ratio is less than 1:40, the extraction amount of anthocyanin increases with the increase of the feed-to-liquid ratio because the anthocyanin component is easily dissolved from the material with the increase of the solvent dosage, and the extraction amount of anthocyanin reaches a maximum of 30.04mg/g at the feed-to-liquid ratio of 1: 40; when the amount of the solvent is further increased, the extraction amount of anthocyanin does not increase, but tends to decrease. Therefore, the ratio of the material to the liquid is 1:30-1:50, which is used as the parameter range of the response surface.
FIG. 5-10 response surface of interaction of various factors on anthocyanin extraction from composite pulp
On the basis of the single-factor test result, a response surface method is used for optimizing extraction parameters, ethanol volume fraction (A), microwave time (B), microwave power (C) and feed-liquid ratio (D) are selected as test factors, and response surface test optimization is carried out by taking anthocyanin extraction amount as a response value, and the result is shown in figures 5-10. A quadratic polynomial regression equation between response variables (ethanol volume fraction, microwave time, microwave power and feed-liquid ratio) and response values (anthocyanin extraction amount): the extraction amount (mg/g) of anthocyanin is 31.00+0.30A +0.044B-0.072C +0.89D-0.063AB +0.23AC-0.98AD +1.28BC-0.74BD-0.27CD-1.95A2-1.03B2-0.79C2-1.54D2. Regression models were highly significant (P < 0.0001), lacked fitting to verify the adequacy of the model and were not significant (P-0.0679 > 0.05), R2The value of (0.9617) is reasonably close to 1, indicating a high correlation between observed and predicted values, and the model can be well fitted to experimental data. According to the F value, the influence of various factors on the extraction amount of the loniceraedulis anthocyanin is shown in the following sequence: the ratio of the feed liquid (D) > the volume fraction of ethanol (A) > the microwave power (C) > the microwave time (B).
FIG. 5 is an interaction graph (A) and a contour graph (B) of ethanol volume fraction and microwave time, and it can be seen from FIG. 5 that the response curve is steeper, indicating that two factors have a significant effect on anthocyanin extraction amount. As can be seen from the contour diagram, the change along the axial contour line of the volume fraction of the ethanol is more intensive than the microwave time, which shows that compared with the microwave time, the volume fraction of the ethanol has obvious influence, the contour line is in a round shape, which shows that the interaction of the two is stronger, and the extraction amount of anthocyanin is obviously influenced. FIG. 6 is an interaction graph (A) and a contour graph (B) of ethanol volume fraction and microwave power, and it can be seen from FIG. 6 that the response curve is steeper, indicating that both factors have a significant effect on anthocyanin extraction. As can be seen from the contour diagram, the change along the axial contour line of the volume fraction of the ethanol is denser than the microwave power, which shows that the volume fraction of the ethanol has a remarkable influence compared with the microwave power, and the contour line is in a circular shape, which shows that the ethanol has a strong interaction and a remarkable influence on the extraction amount of anthocyanin. FIG. 7 is an interaction graph (A) and a contour graph (B) of ethanol volume fraction and feed-to-liquid ratio, and it can be seen from FIG. 7 that the response curve is steeper, indicating that both factors have a significant effect on anthocyanin extraction. The contour line graph shows that the change along the axial contour line of the material-liquid ratio is denser than that of the ethanol volume fraction, which shows that the material-liquid ratio has obvious influence compared with the ethanol volume fraction, and the contour line is elliptic, which shows that the interaction between the material-liquid ratio and the ethanol volume fraction is stronger and has obvious influence on anthocyanin extraction amount. FIG. 8 is an interaction graph (A) and a contour graph (B) of microwave time and microwave power, and it can be seen that the response curve is steeper, indicating that two factors have a significant effect on anthocyanin extraction amount. According to the contour diagram, the change of the contour line along the microwave power axial direction is more intensive than the microwave time number, which shows that the influence of the microwave power is obvious compared with the microwave time, and the contour line is elliptic, which shows that the interaction of the contour line and the microwave power axial direction is stronger and the influence on the extraction amount of anthocyanin is obvious. FIG. 9 is an interaction graph (A) and a contour graph (B) of microwave time and liquid-liquid ratio, FIG. 10 is an interaction graph (A) and a contour graph (B) of microwave power and liquid-liquid ratio, and it can be seen from FIGS. 9 and 10 that the response curve is steeper, indicating that both factors have a significant effect on anthocyanin extraction amount. As can be seen from the contour map, the contours are uniformly distributed along the axial contours of the anthocyanin and the anthocyanin, and the contours are elliptical, which shows that the interaction between the contours is strong and the extraction amount of the anthocyanin is obviously influenced.
The optimal technological parameter combination for extracting anthocyanin by microwave assistance is that the volume fraction of ethanol is 59.39%, the microwave time is 76.47s, the microwave power is 250.56W, the feed-liquid ratio is 1:44.57g/mL, and the extraction amount of anthocyanin under the condition is 31.20 mg/g. Considering the actual operation conditions, the experimental parameters of the microwave-assisted extraction of the composite pulp anthocyanin are adjusted, the adjusted process parameters are that the volume fraction of ethanol is 60%, the microwave time is 77s, the microwave power is 250W, and the feed-liquid ratio is 1:45g/mL, the process conditions are repeated for three times, and the actually measured extraction amount of the anthocyanin is 30.42mg/g, which is close to the predicted value. The model can be used for better measuring the anthocyanin extraction amount of the composite fruit pulp.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples, which are carried out in the light of the technical solutions of the present invention, and it should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.
Example 1
Selecting three small berries with uniform size and color, no rot, no immature green fruit and the like, pulping, fully and uniformly mixing pulp of indigo honeysuckle, raspberry and wild blueberry according to a ratio of 2:4:1, putting the composite pulp in a freeze dryer, crushing the pulp into powder by a crusher after freeze drying, sieving the powder by a 60-mesh sample sieve, and storing the powder in a dryer in a sealed bag for later use; mixing the obtained composite pulp powder with 70% volume fraction ethanol (0.1% HCl acidified) at a solid-to-liquid ratio of 1:40, placing in a triangular flask, soaking for 30min, and treating for 70s under the condition of microwave power of 250W; then ice-bathing for 5min, centrifuging at 4 deg.C 7000rpm for 10min, filtering with 0.45 μm membrane, concentrating the filtrate with rotary evaporator, and vacuum freeze drying to obtain crude anthocyanin powder; and re-dissolving the obtained crude anthocyanin 4 times volume of water, purifying by using activated D101 macroporous resin, eluting by using 60% ethanol, collecting eluent, and freeze-drying to obtain a pure anthocyanin product.
Example 2
Selecting three small berries with uniform size and color, no rot, no immature green fruit and the like, pulping, fully and uniformly mixing pulp of indigo honeysuckle, raspberry and wild blueberry according to a ratio of 2:4:1, putting the composite pulp in a freeze dryer, crushing the pulp into powder by a crusher after freeze drying, sieving the powder by a 60-mesh sample sieve, and storing the powder in a dryer in a sealed bag for later use; mixing the obtained composite pulp powder with 50% volume fraction ethanol (0.1% HCl acidified) at a solid-to-liquid ratio of 1:30, placing in a triangular flask, soaking for 30min, and treating for 70s under the condition of microwave power of 300W; then ice-bathing for 5min, centrifuging at 4 deg.C 7000rpm for 15min, filtering with 0.45 μm membrane, concentrating the filtrate with rotary evaporator, and vacuum freeze drying to obtain crude anthocyanin powder; and redissolving the obtained crude anthocyanin with 3 times of volume of water, purifying by using activated D101 macroporous resin, eluting by using 60% ethanol, collecting eluent, and freeze-drying to obtain a pure anthocyanin product.
Example 3
Selecting three small berries with uniform size and color, no rot, no immature green fruit and the like, pulping, fully and uniformly mixing pulp of indigo honeysuckle, raspberry and wild blueberry according to a ratio of 2:4:1, putting the composite pulp in a freeze dryer, crushing the pulp into powder by a crusher after freeze drying, sieving the powder by a 60-mesh sample sieve, and storing the powder in a dryer in a sealed bag for later use; mixing the obtained composite pulp powder with 70% volume fraction ethanol (0.1% HCl acidified) at a solid-to-liquid ratio of 1:50, placing in a triangular flask, soaking for 20min, and treating for 120s under the condition of 200W microwave power; then ice-bathing for 5min, centrifuging at 4 deg.C and 5000rpm for 10min, filtering with 0.45 μm membrane, concentrating the filtrate with rotary evaporator, and vacuum freeze drying to obtain crude anthocyanin powder; and redissolving the obtained crude anthocyanin with 3 times of volume of water, purifying by using activated D101 macroporous resin, eluting by using 60% ethanol, collecting eluent, and freeze-drying to obtain a pure anthocyanin product.
The above-described embodiments are merely examples for easy understanding, and it should be noted that, for those skilled in the art, several modifications can be made in the technical solution of the present invention and the description of the preferred embodiments thereof without departing from the principle of the present invention, and these modifications should also fall into the protection scope of the present invention. It is to be understood that the examples should not be construed as limiting the scope of the invention. Also, any person skilled in the art can make various possible equivalent changes or exchanges according to the description of the technical solution of the present invention and the implementation examples thereof, but all such changes or exchanges shall fall within the protection scope of the claims of the present invention.
Claims (6)
1. The method for extracting anthocyanin from three small berry compound pulp by microwave assistance is characterized in that compared with the traditional method, the method has the advantages of simple requirement on equipment, short extraction time, high extraction rate, excellent quality, safety, greenness and no pollution, and the extraction of anthocyanin can increase the utilization rate of small berries and reduce the processing cost. Selecting three small berries with uniform size and color, no rot, no immature green fruit and the like, pulping, fully and uniformly mixing pulp of indigo honeysuckle, raspberry and wild blueberry according to a ratio of 2:4:1, putting the composite pulp in a freeze dryer, crushing the pulp into powder by a crusher after freeze drying, sieving the powder by a 60-mesh sample sieve, and storing the powder in a dryer in a sealed bag for later use; mixing the obtained composite pulp powder with 60% volume fraction ethanol (0.1% HCl acidified) at a solid-to-liquid ratio of 1:45, placing in a triangular flask, soaking for 30min, and treating for 77s under the condition of microwave power of 250W; then ice-bath is carried out for 5min, centrifugation is carried out for 10min at 7000rpm at 4 ℃, membrane filtration of 0.45 mu m is carried out, and after the filtrate is concentrated by a rotary evaporator, vacuum freeze drying is carried out to obtain crude anthocyanin powder; and redissolving the obtained crude anthocyanin with 3 times of volume of water, purifying by using activated D101 macroporous resin, eluting by using 60% ethanol, collecting eluent, and freeze-drying to obtain a pure anthocyanin product. The method has high extraction efficiency, and can fully extract and utilize anthocyanin in berries.
2. The method for microwave-assisted extraction of anthocyanin from three small berry compound fruit pulps as claimed in claim 1, which is characterized by comprising the following steps:
(1) pretreatment of raw materials: selecting three small berries with uniform size and color, no rot, no immature green fruit and the like, pulping, fully and uniformly mixing pulp of indigo honeysuckle, raspberry and wild blueberry according to a ratio of 2:4:1, putting the composite pulp in a freeze dryer, crushing the pulp into powder by a crusher after freeze drying, sieving the powder by a 60-mesh sample sieve, and storing the powder in a dryer in a sealed bag for later use;
(2) microwave-assisted treatment: mixing the composite pulp powder obtained in the step (1) with 60% volume fraction ethanol (0.1% HCL acidification) according to a solid-to-liquid ratio of 1:45, placing the mixture in a triangular flask, soaking for 30min, and then treating for 77s under the condition that the microwave power is 250W;
(3) extracting crude anthocyanin: ice-cooling for 5min, centrifuging at 4 deg.C and 7000rpm for 10min, filtering with 0.45 μm membrane, concentrating the filtrate with rotary evaporator, and vacuum freeze drying to obtain crude anthocyanin powder;
(4) and (3) purification: redissolving the 3 times volume of water of the crude anthocyanin obtained in the step (3), purifying by using activated D101 macroporous resin, eluting by using 60 percent ethanol, collecting eluent, and freezing and drying to obtain a pure anthocyanin product.
3. The process of claim 2, wherein the three small berry pulps of step (1) are thoroughly mixed in proportions and freeze-dried to reduce anthocyanin loss from the starting material, and are finally screened to ensure homogeneity.
4. The method of claim 2, wherein the extraction in step (2) is performed under conditions strictly conducive to microwave-assisted extraction, thereby ensuring maximum extraction and maximum extraction yield.
5. The method of claim 2, wherein the filtrate in step (3) is concentrated by evaporation and then freeze-dried.
6. The method of claim 2, wherein in step (4) it must be reconstituted sufficiently to elute with 60% ethanol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911107773.9A CN110845557A (en) | 2019-11-13 | 2019-11-13 | Method for extracting anthocyanin in three small berry compound fruit pulps under assistance of microwaves |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911107773.9A CN110845557A (en) | 2019-11-13 | 2019-11-13 | Method for extracting anthocyanin in three small berry compound fruit pulps under assistance of microwaves |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110845557A true CN110845557A (en) | 2020-02-28 |
Family
ID=69600828
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911107773.9A Pending CN110845557A (en) | 2019-11-13 | 2019-11-13 | Method for extracting anthocyanin in three small berry compound fruit pulps under assistance of microwaves |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110845557A (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101941998A (en) * | 2010-07-14 | 2011-01-12 | 包怡红 | Method for extracting and measuring anthocyanin with special biological activity |
| CN102731462A (en) * | 2011-12-25 | 2012-10-17 | 大兴安岭林格贝有机食品有限责任公司 | Method for purifying anthocyanosides in blueberry |
| CN103408617A (en) * | 2013-08-22 | 2013-11-27 | 吉林大学 | Preparation method of acanthopanax sessiliflorus berry anthocyanin with antioxidant activity |
| CN104447911A (en) * | 2014-11-12 | 2015-03-25 | 黑龙江省科学院自然与生态研究所 | Method for extracting vaccinium uliginosum anthocyanin |
| CN105885469A (en) * | 2016-06-14 | 2016-08-24 | 吉林大学 | Process for extracting lonicera caerulea anthocyanins through acidized ethanol under cooperative assistance of ultrasonic waves-microwaves |
| CN107501365A (en) * | 2017-07-17 | 2017-12-22 | 长沙湘资生物科技有限公司 | A kind of method that raspberry glycosides and anthocyanin are extracted from raspberry |
-
2019
- 2019-11-13 CN CN201911107773.9A patent/CN110845557A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101941998A (en) * | 2010-07-14 | 2011-01-12 | 包怡红 | Method for extracting and measuring anthocyanin with special biological activity |
| CN102731462A (en) * | 2011-12-25 | 2012-10-17 | 大兴安岭林格贝有机食品有限责任公司 | Method for purifying anthocyanosides in blueberry |
| CN103408617A (en) * | 2013-08-22 | 2013-11-27 | 吉林大学 | Preparation method of acanthopanax sessiliflorus berry anthocyanin with antioxidant activity |
| CN104447911A (en) * | 2014-11-12 | 2015-03-25 | 黑龙江省科学院自然与生态研究所 | Method for extracting vaccinium uliginosum anthocyanin |
| CN105885469A (en) * | 2016-06-14 | 2016-08-24 | 吉林大学 | Process for extracting lonicera caerulea anthocyanins through acidized ethanol under cooperative assistance of ultrasonic waves-microwaves |
| CN107501365A (en) * | 2017-07-17 | 2017-12-22 | 长沙湘资生物科技有限公司 | A kind of method that raspberry glycosides and anthocyanin are extracted from raspberry |
Non-Patent Citations (3)
| Title |
|---|
| 李凤凤，等: "响应面优化微波辅助提取蓝靛果花色苷工艺及其抗氧化活性", 《食品工业科技》 * |
| 李蕊: "野生浆果花色苷提取纯化、结构鉴定及体外抗氧化活性分析", 《东北林业大学硕士研究生学位论文》 * |
| 田福: "三种野生浆果花色苷的提取、纯化及抗氧化活性研究", 《东北林业大学硕士研究生学位论文》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101948897B (en) | Method for extracting shinyleaf yellowhorn oil, shinyleaf yellowhorn polysaccharide and shinyleaf yellowhorn protolysate peptide from shinyleaf yellowhorn | |
| CN101704867A (en) | Method for preparing naringin or hesperidin | |
| CN103342692A (en) | Method for extracting blueberry anthocyanin | |
| US10844226B2 (en) | Methods for obtaining natural colourants from plant based materials | |
| CN105030878A (en) | Method for extracting multiple active ingredients of blueberry fruits | |
| CN103483304A (en) | Technological method for extracting anthocyanidin from lycium ruthenicum murr | |
| CN106632205A (en) | Method for extracting proanthocyanidins from Yunnan pine bark | |
| US20050095332A1 (en) | Extraction of phenolic antioxidants | |
| CN107058438A (en) | A kind of method that moringa seeds protein peptides are extracted from moringa seeds | |
| CN108530411A (en) | A method of the extraction and purification anthocyanidin from wild cherry certain kind of berries slag | |
| CN108410205A (en) | A kind of high-active tawny agent and preparation method thereof | |
| CN110746515A (en) | Lycium barbarum polysaccharide, lycium barbarum red element and lycium barbarum polypeptide prepared by synchronous separation and preparation method thereof | |
| CN109456420A (en) | A method of quick room temperature prepares high degree of gelation pectin from shaddock fresh hide | |
| CN1100795C (en) | Preparation of extracted active component of Lycium chinense | |
| CN1687238A (en) | Technique for extracting edible natural red color element from psammophyte of nitraria | |
| CN110669035A (en) | Method for preparing anthocyanin from indigo fruit | |
| CN110845557A (en) | Method for extracting anthocyanin in three small berry compound fruit pulps under assistance of microwaves | |
| CN112442136A (en) | Method for extracting functional components from tremella | |
| CN108185239B (en) | Blackberry and sea-buckthorn compound health-care stock solution and preparation method thereof | |
| CN110938054A (en) | Method for preparing indigo fruit anthocyanin from indigo fruit | |
| CN114904294A (en) | Preparation method of high-yield tea flavone | |
| CN112717009B (en) | Preparation method of gardenia extract | |
| RU2505076C2 (en) | Gogi berries extract production method | |
| CN111543633A (en) | Novel method for extracting okra seed polyphenol compound | |
| CN109805404A (en) | A kind of preparation method and application of the fluffy extract of alkali |
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 | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20200228 |
|
| WD01 | Invention patent application deemed withdrawn after publication |