CN104761617A - Preparation method and application of chickpea polypeptide part - Google Patents
Preparation method and application of chickpea polypeptide part Download PDFInfo
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Abstract
The invention relates to a preparation method and application of a chickpea polypeptide part. The chickpea polypeptide part is chickpea polypeptide powder prepared by crushing and degeasing chickpeas or chickpeas residues obtained after extracting small molecule compounds with an organic solvent, extracting with ethanol, purifying with C18 reversed-phase column chromatography, preparing total polypeptide part with an ultrafiltration membrane technology, refining with a modern spray drying technology or freeze drying technology. Through verification, the chickpea polypeptide powder part has antibacterial and antifungal activities, has wide antimicrobial activity and a certain antioxidant activity and hypoglycemic activity, has the effects of enhancing the humoral immunity and general immunity and is enriched in pure natural plant source polypeptides and proteins which are easily digested and absorbed by the human body. The chickpea polypeptide powder part has the characteristics that the chickpea polypeptide proteins are remained, and the aims of changing the wastes into valuable materials and fully utilizing the resources are achieved; and moreover, the chickpea polypeptide powder serves as natural antibacterial agents, food additives or medicines and can be applied to the food and medicine industries.
Description
Technical field
The present invention relates to a kind of preparation method and application thereof of garbanzo polypeptide position, it is the polypeptide position with anti-microbial activity utilizing garbanzo bean cotyledon, olecranon bean dregs to prepare for raw material, there is antibacterium, the biological activity such as antimycotic, at medicine, field of food, there is good application prospect.
Background technology
Antibiotic discovery was once a milestone in physianthropy history.But, the appearance of resistant organism is caused every now and then due to antibiotic abuse, and the frequency occurred exceedes the tempo of development of new antibiotic, many once highly effective antibiotic medicines were lost efficacy, to such an extent as to bring many new problems to the treatment of infectious diseases, the use of conventional antibiotic is faced with stern challenge.Investigation and application is nontoxic, nuisanceless, become development trend without the novel antibacterial preparation substitute antibiotics of resistance.
Plant will contact with the pathogenic bacteria of millions of kinds in environment every day in process of growth; all may suffer the invasion and attack of microorganism at any time; and plant does not resemble immunity system special Mammals, thus non-specific natural immunity system plays important provide protection to plant.It is found that, after biology is subject to infected by microbes, produces the micromolecule polypeptide (i.e. antibacterial peptide) with anti-microbial activity rapidly, in a large number and participate in immunity of organism.Antibacterial peptide is almost the important immune molecule had in all biologies, is the important component part of the non-specific natural immunity system of plant.In microorganism, plant, insect, arthropods, Amphibians, Mammals even human body, identify thousands of kinds of antibacterial peptides at present, this kind of antibacterial peptide is different from the polypeptide antibiotics that traditional microorganism (comprising bacterium, fungi and streptomycete etc.) produces in synthesis mechanism, amino acid composition and mechanism of action etc.Antibacterial peptide as conventional antibiotic possible surrogate with its characteristic be unsurpassed in excellence more and more become people pay close attention to focus.Antibacterial peptide (antibacterial peptide, ABP) also known as antimicrobial peptide (antimicrobial peptide, or peptide antibiotic (peptide antibiotics) AMP), that the one be extensively present in nature biotechnology has anti-mattress, antiviral, parasiticide, improve the active polypeptide of the biological functions such as immunity of organisms, it is the immunne response product of biological non-specific defense system, there is broad spectrum antibiotic activity, not only can suppress various bacteria or fungi, even can not injuring the tumour cell suppressed in Normocellular situation in animal body.Antibacterial peptide is the important component part of body natural immune system, and can play anti-infectious function before body generation specific immune response.At present, the research and development of antibacterial peptide has become one of focus of research both at home and abroad.Antibacterial peptide has broad-spectrum antimicrobial and other excellent characteristic, inseparable with it self constructional feature.Although antibacterial peptide is of a great variety, source is also different, and their primary structure (i.e. aminoacid sequence) has stronger conservative property.Antibacterial peptide relative molecular mass little (about <10KD).And Antimicrobial Peptides From Plants is generally the polypeptide of 40-90 amino-acid residue composition, is rich in halfcystine, there is several beta sheet and one section of α-helixstructure usually.The primary structure (i.e. aminoacid sequence) of the antibacterial peptide of different sources, different structure has stronger conservative property, and has following common feature:
1. N end is rich in polare Aminosaeren, and as Methionin (Lys), arginine (Arg) etc., in strong basicity, containing superfluous positive charge, make antibacterial peptide be cationic characteristic, this feature makes antibacterial peptide have surfactant activity; ; 2. C holds usual amidation, and in neutral hydrophilic, be rich in the nonpolar amino acids such as L-Ala (Ala), α-amino-isovaleric acid (Val), glycine (Gly), this may be relevant with the broad spectrum antibiotic activity of antibacterial peptide; 3. middle interconnecting piece divides proline rich (Pro), the most amidation of end, and it also directly affects the fungicidal activity of antibacterial peptide; 4. the amino acid of most of antibacterial peptide on second is tryptophane (Trp), and it plays vital effect to the height of the fungicidal activity of antibacterial peptide.
Conventional antibiotic is by eliminating microorganism growth or required function of surviving, and as obstructed the activity of the synthesis of bacterioprotein or change enzyme to reach sterilization object, and bacterium is just enough to tackle antibiotic this attack by changing a kind of gene.Antibacterial peptide then acts on bacterial cell membrane, causes the permeability of film to increase, penetrates with this, kill bacteria.Bacterium must change the structure of film, and the gene namely changing considerable part could defend the attack of antibacterial peptide, and this is almost impossible.Therefore, antibacterial peptide considerably reduces the possibility producing resistance, and this is also the large advantage compared with microbiotic.Antibacterial peptide is the small molecules small peptide of animal body secretion, is also one of body composition, therefore has the biological characteristics of safe without toxic side effect, thus avoid conventional antibiotic class medicine to the infringement of body immune system.Antibacterial peptide still can maintain vigour under 100 DEG C of heating 10min conditions, has comparatively strong adaptability, good water solubility to pH value.Antibacterial peptide does not almost have lethal effect to normal eukaryotic cell, only acts on the eukaryotic cell of prokaryotic cell prokaryocyte and generation pathology.Its reason is:
Due to higher animal cells film having a large amount of cholesterol and protein, particularly the former, hinder the hydrophobic surface of antibacterial peptide to insert in phospholipid bilayer.
The outer lipid of mammalian cell membrane is that it is specific in electroneutral both sexes phosphatide, mainly Yelkin TTS and sphingophospholipid; Bacterial cell membrane is then containing a large amount of phosphatide with negative charge (as phosphatidyl glycerol and Val), and its content can, more than 50%, make it be exposed to epicyte sometimes; In addition, gram-negative bacteria cell walls is primarily of the lipopolysaccharides composition of a large amount of negative charge of band.
May be the difference of the film outer structure due to microorganism and higher animal cells film: the sialic acid of higher animal cells film outer surface and the distance of film have
it may be combined with the positively charged district of antibacterial peptide and stop antibacterial peptide to play lethal effect close to cytolemma.
Highly developed cytoskeleton system is there is in mammiferous eukaryotic cell, microfilament wherein and microtubule and cytolemma internal layer have many binding sites, this structure is the primary factor maintaining cell special shape and osmotic pressure, highly developed cytoskeleton system, plays an important role in the susceptibility of antibacterial peptide lysis at mammalian cell.
Sanitas in current foodstuffs industry is mainly based on chemosynthesis agent such as Sorbic Acid, phenylformic acid and its esters, and this compounds easily exceeds standard and works the mischief to human body in the course of processing.Antibacterial peptide is used as food preservative freshness retaining agent not only to be had broad spectrum antibiotic activity, effectively kills deteriorative microorganism, and antibacterial peptide molecular weight, thermostability are strong, good water solubility, non-immunogenicity, have no side effect to people, antibacterial peptide can also be easily degraded by proteases in human body in addition, and the guarantee of dual safety makes antibacterial peptide demonstrate wide application prospect gradually in field of food industry.
Medicine industry is that antibacterial peptide is most widely used and the most promising industry, along with antibiotic long-term widespread use, many pathogeny bacterium create resistance to them, the use of Antibiotic Additive is the havoc microbial balance of animal intestinal then, and easily remain in animal body, have a strong impact on Animal product quality and human health, and there is broad-spectrum antimicrobial and have the antibacterial peptide of unique antimicrobial mechanism obviously to have clear superiority in applied research in this respect.Along with people's deepening continuously to the relation of antibacterial peptide structure and energy, antibacterial peptide mechanism of action and gene expression regulation thereof mechanism understanding, the part that antibacterial peptide is combined with pathogenic agent surface specific receptors is connected, completely likely design a collection of can specific action in efficient peptide antibiotics, antitumor drug, the antiviral of tumour, fungi or virus, make the mankind break away from the torment of this type of disease, bring immeasurable realistic meaning to the development of medical and health and disease treatment.
Cancer is a great problem in human diseases, and the chemotherapeutics used at present can kill cancer cells, but also kills normal cell in human body simultaneously, and side effect is very big.Antibacterial Toplink suppresses the growth of some tumour cell and harmless to human normal cell, very likely becomes anti-cancer agent that is nontoxic or low toxicity, and this brings hope to the exploitation of cancer therapy drug.
Antibacterial peptide is used as sanitas on makeup, and fish antibacterial peptide is not only nutritive ingredient, the more important thing is the antibacterial of it and beautification function, is used for makeup has unique advantage and market potential widely than Chemical Preservative.
The application of antibacterial peptide in agricultural and livestock industry: 1, antibacterial peptide has specificity as the effect of fodder additives normal antibiotics, and easily makes body develop immunity to drugs, causes the action effect of generation not good.Antibacterial peptide not only has broad-spectrum antibacterial action, and livestock and poultry are had to the function of growth promotion, health care and disease therapy, belong to have no side effect, noresidue, without causing a class environment-friendly type preparation of bacterial drug resistance, can be used as additive for farm animal feed replace or partly replace current feeding animals microbiotic used, reduce microbiotic to the dependence to medicine in the harm of animal body and minimizing animal cultivation.2, transgenic animal use for reference successfully insect antimicrobial peptide transgenic engineering, as transgenosis mosquito, transgenic Rhizoma Solani tuber osi, transgenic paddy rice etc., special antibacterial peptide gene is proceeded to livestock and poultry specific cells express by it, thus generation disease-resistant varieties, open the new approaches of a Develop the stockbreeding industry.3, play a significant role in Green Husbandry: due to a large amount of use of microbiotic in Production of Livestock and Poultry.Medicine serious generation that is residual and drug-resistant microorganism in livestock and poultry body makes the safety issue of animal food become increasingly conspicuous, the Sustainable development of people's health and whole aquaculture in this serious threat, brings huge loss also to China's livestock product export trade.Antibacterial peptide has broad spectrum antibiotic activity and quick sterilization, does not affect, not easily produces Resistant strain, act synergistically with conventional antibiotic by the strain of conventional antibiotic medicament-resistant mutation, in and intracellular toxin and play the characteristics such as useful effect in animal body.So in the animal husbandry development of green, safety, environmental protection, antibacterial peptide also plays an important role.
Antibacterial peptide is at the research and apply of plant field
:plant diseases is nearly hundreds of, and nearly all crop all can suffer harm in various degree within vegetative period, cultivates the fundamental way that disease-resistant varieties is controlling plant diseases.Antibacterial peptide has broad-spectrum sterilization effect and not easily causes pathogenic micro-organism to produce the advantages such as resistance.Thus, antibacterial peptide gene being proceeded to farm crop, is the available strategy of cultivating crop disease-resistant new variety.
Antioxidant and antioxidation polypeptide: along with people's living standard improves and living-pattern preservation, diabetes, cardiovascular disorder, especially hyperlipidaemia and the coronary heart disease caused by atherosclerosis, the health of the mankind in serious threat day by day, in the U.S., Europe and many Asian countries (comprising China), it has become the major cause of death.
Cause the Hazard Factor of atherosclerosis, cardiovascular disorder quite extensive, wherein, hyperlipidemia, hypertension, diabetes, low-level high density lipoprotein cholesterol (high density lipoprotein cholesterol, HDL-c), high-caliber low density lipoprotein cholesterol (low density lipoprotein chofesterol, LDL-c) etc. are main inducing.Large quantity research display, the enhancing of oxidative stress plays key effect at atherosclerotic initial period, and free radical and lipid peroxidation are its major causes.
Under general condition, anti-oxidative defense system can pass through enzyme (as superoxide dismutase and Selenoperoxidase) and non-enzymatic antioxidant (as antioxidant vitamins, trace element, coenzyme and cofactors) cleaning reaction material.But under certain condition, endogenous system of defense can not defend the free radical self produced, and then body can not be protected injury-free.This generates oxidative stress, oxidative stress refers to that in body, high reactivity molecule reduces as reactive oxygen species (ROS) and active nitrogen bunch (RNS) produce too much or eliminate, thus causes tissue injury.ROS comprises superoxide anion (O
2-), hydroxy radical qiao (OH), hydrogen peroxide (H
2o
2) etc., RNS comprises nitrogen protoxide (NO), nitrogen peroxide (NO
2), peroxynitrite base negatively charged ion (ONOO
-) etc.Body can produce a small amount of ROS and participate in eubolism in normal state, there is removing simultaneously, suppresses the system of free radical reaction, make too much free radical be eliminated or make free radical to reduce, keep the generation of free radical and the balance of removing in body; But under some pathological state, this mechanism is destroyed, interior free yl produces too much or removed slow, and interior free yl significantly increases.Free radical is very unstable, can react with various material in body, as caused unsaturated fatty acids generation peroxidatic reaction of lipid, the structure of damage film, they go back the materials such as attack carbohydrate, protein, lipid, nucleic acid, thus cause cell or tissue to damage and death.In order to the balance keeping the generation of free radical and between removing, suitable scavenging free radicals and active oxygen are one of most effective meanss of prevention organism various diseases.
Lipid oxidation is also the focus that foodstuffs industry and human consumer are concerned about, because lipid oxidation can produce many toxic gases and toxicant.Especially, in food, peroxidatic reaction of lipid affects Nutritive value of food, may produce some objectionable impuritiess.Oxygenant in air pollutant and tobacco can cause some harmful reactions in skin, or absorb by blood circulation, cause side reaction.In addition, ultraviolet radiation energy promotes the generation of a series of oxygenant.
Therefore, in the more than ten years in the past, the focus of human nutriology and biochemical research finds antioxidant from food composition, to delay peroxidatic reaction of lipid.Free-radical scavengers is exactly the preventative antioxidant of a class.Therefore, antioxidant protects body to serve vital role to preventing oxidative stress.Reasonable antioxidant is used to preserve food, and the food discoloration delaying to produce because of oxidizing reaction is with rotten.The antioxidant of synthesis is efficient, but has certain toxicity and harm.In human nutrition and biochemical field, the natural antioxidants of food source becomes the focus of research because it is efficient, toxicity is little.People would rather select natural antioxidants to be also reluctant to select the antioxidant of synthesis.
Antioxidant has scavenging free radicals and delays the generation of peroxidatic reaction of lipid.Antioxidant developed very fast in the last few years at home and abroad, and purposes is also more and more wider.Since Harman proposes free-radical theory, people recognize that the free radical that people's vivo oxidation produces is relevant with numerous disease with the aging of people.Therefore anti-oxidant not only for fat-containing food of antioxidant, and the exploitation being used for protective foods and makeup etc. as functional factor.The kind of antioxidant is a lot, the antioxidant of chemosynthesis, as BHA (BHA), Butylated Hydroxytoluene (BHT), Tert. Butyl Hydroquinone (TBHQ) and Tenox PG (PG), all be used to the formation preventing free radical in food, thus delay lipid oxidation.But synthetized oxidation preventive agent is due to its toxic side effect, and national governments control its addition one after another, prevents excessive interpolation.So people progressively turn to sight and extract natural antioxidants from each kind of plant, animal tissuess, find antioxidant that is efficient, low toxicity is the problem that Recent study person pay close attention to always, and particularly the native oxidant in search of food source has very important significance.
After Marcuse reported first antioxidation polypeptide, from food proteins, be separated to the polypeptide that some have resistance of oxidation, and their anti-oxidant activity is studied widely.Compare with enzyme antioxidant, antioxidation polypeptide has some advantages.That is exactly under different conditions, and anti-oxidation peptide structure is simple, more stable does not have toxic side effect.And they not only have anti-oxidant activity, also there is nutritive value and functional performance.
The present invention is found by research, containing abundant component polypeptides in garbanzo bean cotyledon, extracts polypeptide in olecranon bean cotyledon, and the garbanzo polypeptide powder that the nutritive ingredient exploitation enriched in conjunction with garbanzo has antimicrobial function has realistic meaning very much.
Summary of the invention:
The object of the invention is, a kind of preparation method and application thereof of garbanzo polypeptide position are provided, raw material in the method is that garbanzo is germinateed the olecranon bean dregs after except the organic solvent extraction micromolecular compound of the bean cotyledon after tooth or drying, adopt to pulverize and first shatter degreasing, utilize extraction using alcohol, C18 reversed phase column chromatography purifying, finally prepares total polypeptide position by ultrafiltration membrane technique, recycles the garbanzo polypeptide powder that modern spray drying technology or lyophilize are refined.This garbanzo polypeptide powder primarily of molecular weight is: 4002.5, 4151.3, 4037.43Da, 4191.8Da, 4276.8Da, 4249.63Da, 4346.2Da, 4426.8Da, 4459.3Da, 4531.55.8Da, 4614.15D4a, 5100.29Da, 5168.98Da, 5383.8.2Da, 5485.2Da, 5876.8Da, 6267.6Da, 6403.1Da, 7023.8Da, 7923.5.57Da, the intensive position coexisted of the multiple polypeptides of 7934.21Da and 7604.3Da etc. 20, this garbanzo polypeptide position is verified its antibacterium and fungi activity, still not there is antimicrobial acivity widely, and there is certain anti-oxidant and hypoglycemic activity, there is the function strengthening humoral immunization and general immunity simultaneously, be rich in human body easy to digest and absorb natural plant borne peptides and protein.The feature at this garbanzo polypeptide powder position be both remained olecranon all polypeptide protein while also realize turning waste into wealth and the object that makes full use of of resource; This garbanzo polypeptide powder, as natural antibacterial agent, food additive agent or medicine, can be used for food and medicine industry.
The preparation method at a kind of garbanzo polypeptide position of the present invention, the raw material in the method is that garbanzo is germinateed the olecranon bean dregs after except the organic solvent extraction micromolecular compound of the bean cotyledon after tooth or drying, and concrete operations follow these steps to carry out:
A, get garbanzo and to germinate the olecranon bean dregs after except the organic solvent extraction micromolecular compound of the bean cotyledon after tooth or drying, after pulverizing, be add sherwood oil at 1: 5 by feed liquid weight ratio, at room temperature stir 1-2 hour, repeatedly extract 3-4 time to not having grease to extract, suction filtration, merges precipitation, seasoning, to not having petroleum ether solvent residual, obtains the olecranon bean powder of degreasing;
B, add 50% ethanol by the olecranon bean powder of degreasing abundant in step a, feed liquid weight ratio is 1: 5, at room temperature stirs and extracts 2-3 hour, extracts 3-4 time at every turn, decompress filter, united extraction liquid, and separating sundries, obtains polypeptide and slightly extract solution for standby;
C, the polypeptide obtained in step b is slightly extracted solution, distilled water diluting to alcohol concn is utilized to be 10%-20%, the extraction solution of dilution, on used the ethanol of 10%-20%, flow velocity is 3 milliliters/MIN, under UV-280NM, carry out the anti-phase C18 silicagel column that stripping equilibria is good, and be 50%-80% ethanol elution by concentration, obtain thick polypeptide position:
D, the thick polypeptide position that step c is obtained utilize Rotary Evaporators be concentrated into residual without ethanolic soln after, adopt molecular weight cut-off to be the regenerated cellulose flat sheet membrane of 1KDa, pressure 0.15MPa in concentration process, temperature 25 DEG C, flow velocity 35m/s, cycles of concentration is 5-10 times, obtains garbanzo polypeptide enriched material, successively through centrifugal, spraying dry or lyophilize, sieve, ultraviolet sterilization, packaging, can obtain garbanzo polypeptide powder.
The purposes of the garbanzo polypeptide powder that described method obtains in preparation antibacterium, antimycotic bioactive medicine.
The garbanzo polypeptide powder that described method obtains is preparing the purposes in foodstuff additive.
The preparation method at a kind of garbanzo polypeptide position of the present invention and application thereof, the raw material of this garbanzo polypeptide powder be germinate except tooth by garbanzo after bean cotyledon or drying organic solvent extraction micromolecular compound after olecranon bean dregs, the physicochemical property of Binding peptide compounds, the intensive powder coexisted of polypeptide made in conjunction with modern reversed phase column chromatography and ultrafiltration membrane technique technique.The garbanzo polypeptide position obtained by method of the present invention is from the free polypeptide in garbanzo raw material, the Measures compare preparing polypeptide with other has and uses chemical reagent to the greatest extent less, do not need complicated extraction and isolation flow process, equipment is simple, is easy to amplify the advantages such as preparation; This garbanzo polypeptide powder is verified its antibacterium and fungi activity, this garbanzo polypeptide position does not still have antimicrobial acivity widely, and there is certain anti-oxidant and hypoglycemic activity, there is the function strengthening humoral immunization and general immunity simultaneously, be rich in the easy to digest and natural plant borne peptides that absorbs of human body and protein, both remained olecranon and all also realized turning waste into wealth while polypeptide protein and resource makes full use of.
Accompanying drawing explanation
Fig. 1 is the liquid phase figure at polypeptide position of the present invention;
Fig. 2 is the mass spectrum at polypeptide position of the present invention;
Fig. 3 is the liquid mass spectrum at polypeptide position of the present invention;
Fig. 4 is the Candida albicans activity figure that polypeptide of the present invention resists;
Fig. 5 is polypeptide anti-Staphylococcus aureus Activity Results figure of the present invention;
Fig. 6 is polypeptide of the present invention anti-colon bacillus Activity Results figure;
Fig. 7 is SDS-PAGE electrophoretogram of the present invention;
Fig. 8 is SDS-PAGE electrophoretogram of the present invention.
Embodiment
Embodiment 1
The preparation of garbanzo bean cotyledon polypeptide powder:
A, get garbanzo and germinate except the bean cotyledon after tooth, 3 times are rinsed to completely clean with water, use filtered through gauze respectively, merge the garbanzo bean cotyledon after cleaning, cryodrying, control temperature, at 40 DEG C, by dried garbanzo bean cotyledon, is pulverized through pulverizer, grinding particle size is 200 orders, be add sherwood oil at 1: 5 by feed liquid weight ratio, at room temperature stir 1 hour, repeatedly extract 3 times to not having grease to extract, suction filtration, merge precipitation, seasoning, to not having petroleum ether solvent residual, obtains the olecranon bean powder of degreasing;
B, be 50% edible ethanol by adding concentration in the olecranon bean powder of degreasing abundant in step a, solid-liquid ratio is 1: 5, at room temperature stirs extraction 2 hours, extracts 3 times at every turn, decompress filter, united extraction liquid, and separating sundries, obtains polypeptide and slightly extract solution for standby;
C, the polypeptide obtained in step b is slightly extracted solution, distilled water diluting to alcohol concn is utilized to be 10%, by the extraction solution of dilution, on used 10% ethanol, flow velocity is 3 milliliters/MIN, under UV-280NM, carry out the anti-phase C18 silicagel column that stripping equilibria is good, then be 50% ethanol elution by concentration, obtain thick polypeptide position:
D, the thick polypeptide position that step c is obtained utilize Rotary Evaporators be concentrated into residual without ethanolic soln after, adopt molecular weight cut-off to be the regenerated cellulose flat sheet membrane of 1KDa, pressure 0.15MPa in concentration process, temperature 25 DEG C, flow velocity 35m/s, cycles of concentration is 5-10 times, obtains garbanzo polypeptide enriched material, successively through centrifugal, spraying dry, sieves, ultraviolet sterilization, packaging, can obtain garbanzo polypeptide powder.
Embodiment 2
A, get the olecranon bean dregs after dry organic solvent extraction micromolecular compound, after pulverizing, grinding particle size is 200 orders, be add sherwood oil at 1: 5 by feed liquid weight ratio, at room temperature stir 2 hours, repeatedly extract 4 times to not having grease to extract, suction filtration, merge precipitation, seasoning, to not having petroleum ether solvent residual, obtains the olecranon bean powder of degreasing;
B, be 50% edible ethanol by adding concentration in the olecranon bean powder of degreasing abundant in step a, solid-liquid ratio is 1: 5, at room temperature stirs extraction 3 hours, extracts 4 times at every turn, decompress filter, united extraction liquid, and separating sundries, obtains polypeptide and slightly extract solution for standby;
C, the polypeptide obtained in step b is slightly extracted solution, distilled water diluting to alcohol concn is utilized to be 20%, by the extraction solution of dilution, on used 20% ethanol, flow velocity is 3 milliliters/MIN, under UV-280NM, carry out the anti-phase C18 silicagel column that stripping equilibria is good, then be 80% ethanol elution by concentration, obtain thick polypeptide position:
D, the thick polypeptide position that step c is obtained utilize Rotary Evaporators be concentrated into residual without ethanolic soln after, adopt molecular weight cut-off to be the regenerated cellulose flat sheet membrane of 1KDa, pressure 0.15MPa in concentration process, temperature 25 DEG C, flow velocity 35m/s, cycles of concentration is 5-10 times, obtains garbanzo polypeptide enriched material, successively through centrifugal, lyophilize, sieves, ultraviolet sterilization, packaging, can obtain garbanzo polypeptide powder.
Embodiment 3
The preparation of garbanzo bean cotyledon polypeptide powder:
A, get garbanzo and germinate except the bean cotyledon after tooth, 3 times are rinsed to completely clean with water, use filtered through gauze respectively, merge the garbanzo bean cotyledon after cleaning, cryodrying, control temperature, at 40 DEG C, by dried garbanzo bean cotyledon, is pulverized through pulverizer, grinding particle size is 200 orders, be add sherwood oil at 1: 5 by feed liquid weight ratio, at room temperature stir 2 hours, repeatedly extract 4 times to not having grease to extract, suction filtration, merge precipitation, seasoning, to not having petroleum ether solvent residual, obtains the olecranon bean powder of degreasing;
B, be 50% edible ethanol by adding concentration in the olecranon bean powder of degreasing abundant in step a, solid-liquid ratio is 1: 5, at room temperature stirs extraction 3 hours, extracts 4 times at every turn, decompress filter, united extraction liquid, and separating sundries, obtains polypeptide and slightly extract solution for standby;
C, the polypeptide obtained in step b is slightly extracted solution, distilled water diluting to alcohol concn is utilized to be 20%, the extraction solution of dilution, on used 20% ethanol, flow velocity is 3 milliliters/MIN, under UV-280NM, carry out the anti-phase C18 silicagel column that stripping equilibria is good, and be 60% ethanol elution by concentration, obtain thick polypeptide position:
D, the thick polypeptide position that step c is obtained utilize Rotary Evaporators be concentrated into residual without ethanolic soln after, adopt molecular weight cut-off to be the regenerated cellulose flat sheet membrane of 1KDa, pressure 0.15MPa in concentration process, temperature 25 DEG C, flow velocity 35m/s, cycles of concentration is 5-10 times, obtains garbanzo polypeptide enriched material, successively through centrifugal, lyophilize, sieves, ultraviolet sterilization, packaging, can obtain garbanzo polypeptide powder.
Embodiment 4
The preparation of garbanzo bean dregs polypeptide powder:
A, get the olecranon bean dregs after dry organic solvent extraction micromolecular compound, pulverize through pulverizer, grinding particle size is 200 orders, be that 1:5 adds sherwood oil by solid-liquid ratio, at room temperature stir 1 hour, repeatedly extract 3 times to not having grease to extract, suction filtration, merge precipitation, seasoning, to not having petroleum ether solvent residual, obtains the olecranon bean powder of degreasing;
B, be 50% edible ethanol by adding concentration in the olecranon bean powder of degreasing abundant in step a, solid-liquid ratio is 1: 5, at room temperature stirs extraction 2 hours, extracts 3 times at every turn, decompress filter, united extraction liquid, and separating sundries, obtains polypeptide and slightly extract solution for standby;
C, the polypeptide obtained in step b is slightly extracted solution, distilled water diluting to alcohol concn is utilized to be 10%-20%, the extraction solution of dilution, on used 10% ethanol, flow velocity is 3 milliliters/MIN, under UV-280NM, carry out the anti-phase C18 silicagel column that stripping equilibria is good, and be 50% ethanol elution by concentration, obtain thick polypeptide position:
D, the thick polypeptide position that step c is obtained utilize Rotary Evaporators be concentrated into residual without ethanolic soln after, adopt molecular weight cut-off to be the regenerated cellulose flat sheet membrane of 1KDa, pressure 0.15MPa in concentration process, temperature 25 DEG C, flow velocity 35m/s, cycles of concentration is 5-10 times, obtains garbanzo polypeptide enriched material, successively through centrifugal, spraying dry again, sieves, ultraviolet sterilization, packaging, can obtain garbanzo polypeptide powder.
Embodiment 5
The preparation of garbanzo bean cotyledon polypeptide powder:
A, get garbanzo and germinate except the bean cotyledon after tooth, 3 times are rinsed to completely clean with water, use filtered through gauze respectively, merge the garbanzo bean cotyledon after cleaning, cryodrying, control temperature, at 40 DEG C, by dried garbanzo bean cotyledon, is pulverized through pulverizer, grinding particle size is 200 orders, be add sherwood oil at 1: 5 by feed liquid weight ratio, at room temperature stir 2 hours, repeatedly extract 4 times to not having grease to extract, suction filtration, merge precipitation, seasoning, to not having petroleum ether solvent residual, obtains the olecranon bean powder of degreasing;
B, add 50% ethanol by the olecranon bean powder of degreasing abundant in step a, solid-liquid ratio is 1: 5, at room temperature stirs extraction 2 hours, extracts 3 times at every turn, decompress filter, united extraction liquid, and separating sundries, obtains polypeptide and slightly extract solution for standby;
C, the polypeptide obtained in step b is slightly extracted solution, distilled water diluting to alcohol concn is utilized to be 15%, by the extraction solution of dilution, on be the ethanol of 15% by concentration, flow velocity is 3 milliliters/MIN, under UV-280NM, carry out the anti-phase C18 silicagel column that stripping equilibria is good, and be 80% ethanol elution by concentration, obtain thick polypeptide position:
D, the thick polypeptide position that step c is obtained utilize Rotary Evaporators be concentrated into residual without ethanolic soln after, adopt molecular weight cut-off to be the regenerated cellulose flat sheet membrane of 1KDa, pressure 0.15MPa in concentration process, temperature 25 DEG C, flow velocity 35m/s, cycles of concentration is 5-10 times, obtains garbanzo polypeptide enriched material, successively through centrifugal, lyophilize, sieves, ultraviolet sterilization, packaging, can obtain garbanzo polypeptide powder.
Embodiment 6
The preparation of garbanzo bean dregs polypeptide powder:
A, get the olecranon bean dregs after dry organic solvent extraction micromolecular compound, pulverize through pulverizer, grinding particle size is 200 orders, be add sherwood oil at 1: 5 by solid-liquid ratio, at room temperature stir 1.5 hours, repeatedly extract 3 times to not having grease to extract, suction filtration, merge precipitation, seasoning, to not having petroleum ether solvent residual, obtains the olecranon bean powder of degreasing;
B, be 50% edible ethanol by adding concentration in the olecranon bean powder of degreasing abundant in step a, solid-liquid ratio is 1: 5, at room temperature stirs extraction 3 hours, extracts 3 times at every turn, decompress filter, united extraction liquid, and separating sundries, obtains polypeptide and slightly extract solution for standby;
C, the polypeptide obtained in step b is slightly extracted solution, distilled water diluting to alcohol concn is utilized to be 15%, by the extraction solution of dilution, on be the ethanol of 15% by concentration, flow velocity is 3 milliliters/MIN, under UV-280NM, carry out the anti-phase C18 silicagel column that stripping equilibria is good, and be 70% ethanol elution by concentration, obtain thick polypeptide position:
D, the thick polypeptide position that step c is obtained utilize Rotary Evaporators be concentrated into residual without ethanolic soln after, adopt molecular weight cut-off to be the regenerated cellulose flat sheet membrane of 1KDa, pressure 0.15MPa in concentration process, temperature 25 DEG C, flow velocity 35m/s, cycles of concentration is 5-10 times, obtains garbanzo polypeptide enriched material, successively through centrifugal, lyophilize, sieves, ultraviolet sterilization, packaging, can obtain garbanzo polypeptide powder.
Embodiment 7
The preparation of garbanzo bean cotyledon polypeptide powder:
A, the bean cotyledon water got after garbanzo is germinateed except tooth rinse 3 times to completely clean, use filtered through gauze respectively, merge the garbanzo bean cotyledon after cleaning, cryodrying, control temperature is at 40 DEG C, by dried garbanzo bean cotyledon, pulverize through pulverizer, grinding particle size is 200 orders, is add sherwood oil at 1: 5 by feed liquid weight ratio, at room temperature stir 2 hours, repeatedly extract 4 times to not having grease to extract, suction filtration, merges precipitation, seasoning, to not having petroleum ether solvent residual, obtains the olecranon bean powder of degreasing;
B, be 50% edible ethanol by adding concentration in the olecranon bean powder of degreasing abundant in step a, solid-liquid ratio is 1: 5, at room temperature stirs extraction 2.5 hours, extracts 4 times at every turn, decompress filter, united extraction liquid, and separating sundries, obtains polypeptide and slightly extract solution for standby;
C, the polypeptide obtained in step b is slightly extracted solution, distilled water diluting to alcohol concn is utilized to be 18%, by the extraction solution of dilution, on used 18% ethanol, flow velocity is 3 milliliters/MIN, under UV-280NM, carry out the anti-phase C18 silicagel column that stripping equilibria is good, and be 70% ethanol elution by concentration, obtain thick polypeptide position:
D, the thick polypeptide position that step c is obtained utilize Rotary Evaporators be concentrated into residual without ethanolic soln after, adopt molecular weight cut-off to be the regenerated cellulose flat sheet membrane of 1KDa, pressure 0.15MPa in concentration process, temperature 25 DEG C, flow velocity 35m/s, cycles of concentration is 5-10 times, obtains garbanzo polypeptide enriched material, successively through centrifugal, spraying dry again, sieves, ultraviolet sterilization, packaging, can obtain garbanzo polypeptide powder.
Embodiment 8
The preparation of garbanzo bean dregs polypeptide powder:
A, get garbanzo and to germinate the olecranon bean dregs after except the organic solvent extraction micromolecular compound of the bean cotyledon after tooth or drying, pulverize through pulverizer, grinding particle size is 200 orders, be that 1:5 adds sherwood oil by solid-liquid ratio, at room temperature stir 1.5 hours, repeatedly extract 4 times to not having grease to extract, suction filtration, merge precipitation, seasoning, to not having petroleum ether solvent residual, obtains the olecranon bean powder of degreasing;
B, add 50% ethanol by the olecranon bean powder of degreasing abundant in step a, solid-liquid ratio is 1: 5, at room temperature stirs extraction 2 hours, extracts 4 times at every turn, decompress filter, united extraction liquid, and separating sundries, obtains polypeptide and slightly extract solution for standby;
C, the polypeptide obtained in step b is slightly extracted solution, distilled water diluting to alcohol concn is utilized to be 10%%, the extraction solution of dilution, on used 20% ethanol, flow velocity is 3 milliliters/MIN, under UV-280NM, carry out the anti-phase C18 silicagel column that stripping equilibria is good, and be 60% ethanol elution by concentration, obtain thick polypeptide position:
D, the thick polypeptide position that step c is obtained utilize Rotary Evaporators be concentrated into residual without ethanolic soln after, adopt molecular weight cut-off to be the regenerated cellulose flat sheet membrane of 10KDa, pressure 0.15MPa in concentration process, temperature 25 DEG C, flow velocity 35m/s, cycles of concentration is 5-10 times, obtains garbanzo polypeptide enriched material, successively through centrifugal, spraying dry again, sieves, ultraviolet sterilization, packaging, can obtain garbanzo polypeptide powder.
Embodiment 9
By any one garbanzo polypeptide powder of embodiment 1-8, utilize SDS-PAGE electrophoresis, LC/MS commercial measurement peptide molecule quality, its result shows: confirm from SDS-PAGE electrophoretic analysis result, the polypeptide at this position is mainly distributed in the region between component 4.1KDA-20Kda; LC/MS technology confirms that this polypeptide position is by component further: 4002.5,4151.3,4037.43Da, 4191.8Da, 4276.8Da, 4249.63Da, 4346.2Da, 4426.8Da, 4459.3Da, 4531.55.8Da, 4614.15D4a, 5100.29Da, 5168.98Da, 5383.8.2Da, 5485.2Da, 5876.8Da, 6267.6Da, the intensive position coexisted of the multiple polypeptides of 6403.1Da, 7023.8Da, 7923.5.57Da, 7934.21Da and 7604.3Da etc. 20.
Embodiment 10
Any one garbanzo polypeptide powder of embodiment 1-8 is carried out screening active ingredients according to a conventional method: the antibacterial sieve method of cavities, cavities antimicrobial operation is as follows:
1. melt nutrient agar, treat that its temperature is down to 46 ± 0.5 DEG C, add cultured bacterium liquid, make test organisms suspension concentration be 5 × 105cfu/ml-5 × 106cfu/ml, be down flat ware, 15-20ml/ ware, place 20mine and make it solidify;
2. with Gelose punch punching, diameter is 5-6mm, 4-5 hole/ware, is uniformly distributed, at a distance of more than 25mm between each print center, with periphery more than the 15mm apart of flat board;
3. sample concentration is generally 100mg/ml (100mM); Every hole adds sample solution 20 μ l, builds plate, is placed in temperature 37 DEG C of incubator 30-60min, solution is absorbed completely, is inverted and cultivates 16h-18h, with the diameter of vernier caliper measurement bacterial restrain and record;
4. evaluate
The judgement of bacteriostatic action: inhibition zone diameter is greater than 7mm person, has been judged to bacteriostatic action; Inhibition zone diameter is less than or equal to 7mm person, is judged to without bacteriostatic action (table 1):
Table polypeptide antimicrobial acivity result
From table, Antimicrobial Screening result confirms, the garbanzo polypeptide position that the present invention obtains is all to CA (Candida albicans); EC (colon bacillus); SA (streptococcus aureus) has fine inhibit activities.
Claims (3)
1. the preparation method at garbanzo polypeptide position, the raw material that it is characterized in that in the method is the olecranon bean dregs after the organic solvent extraction micromolecular compound of bean cotyledon after garbanzo is germinateed except tooth or drying, and concrete operations follow these steps to carry out:
A, get garbanzo and to germinate the olecranon bean dregs after except the organic solvent extraction micromolecular compound of the bean cotyledon after tooth or drying, after pulverizing, be add sherwood oil at 1: 5 by feed liquid weight ratio, at room temperature stir 1-2 hour, repeatedly extract 3-4 time to not having grease to extract, suction filtration, merges precipitation, seasoning, to not having petroleum ether solvent residual, obtains the olecranon bean powder of degreasing;
B, add 50% ethanol by the olecranon bean powder of degreasing abundant in step a, feed liquid weight ratio is 1: 5, at room temperature stirs and extracts 2-3 hour, extracts 3-4 time at every turn, decompress filter, united extraction liquid, and separating sundries, obtains polypeptide and slightly extract solution for standby;
C, the polypeptide obtained in step b is slightly extracted solution, distilled water diluting to alcohol concn is utilized to be 10%-20%, the extraction solution of dilution, on used the ethanol of 10%-20%, flow velocity is 3 milliliters/MIN, under UV-280NM, carry out the anti-phase C18 silicagel column that stripping equilibria is good, and be 50%-80% ethanol elution by concentration, obtain thick polypeptide position:
D, the thick polypeptide position that step c is obtained utilize Rotary Evaporators be concentrated into residual without ethanolic soln after, adopt molecular weight cut-off to be the regenerated cellulose flat sheet membrane of 1KDa, pressure 0.15MPa in concentration process, temperature 25 DEG C, flow velocity 35m/s, cycles of concentration is 5-10 times, obtains garbanzo polypeptide enriched material, successively through centrifugal, spraying dry or lyophilize, sieve, ultraviolet sterilization, packaging, can obtain garbanzo polypeptide powder.
2. the garbanzo polypeptide powder that method obtains as claimed in claim 1 is preparing the purposes in antibacterium, antimycotic bioactive medicine.
3. the garbanzo polypeptide powder that method obtains as claimed in claim 1 is preparing the purposes in foodstuff additive.
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| CN105622715A (en) * | 2016-04-05 | 2016-06-01 | 中国科学院新疆理化技术研究所 | Quick preparation method and application of chickpea polypeptide powder |
| CN106432445A (en) * | 2016-10-14 | 2017-02-22 | 中国科学院新疆理化技术研究所 | Preparation method and applications of chickpea defensins |
| CN107365349A (en) * | 2017-08-11 | 2017-11-21 | 中国科学院新疆理化技术研究所 | A kind of preparation method and applications at cumin seed polypeptide position |
| CN107594249A (en) * | 2017-09-06 | 2018-01-19 | 陈漫霞 | A kind of preparation method of chick-pea beverage |
| CN111961118A (en) * | 2020-08-05 | 2020-11-20 | 合肥瑞克生物科技有限公司 | Antibacterial active peptide |
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