CN111466447B - Method for preparing Pickering emulsion stabilizer by ultrasonic-enzymatic method - Google Patents
Method for preparing Pickering emulsion stabilizer by ultrasonic-enzymatic method Download PDFInfo
- Publication number
- CN111466447B CN111466447B CN202010267104.4A CN202010267104A CN111466447B CN 111466447 B CN111466447 B CN 111466447B CN 202010267104 A CN202010267104 A CN 202010267104A CN 111466447 B CN111466447 B CN 111466447B
- Authority
- CN
- China
- Prior art keywords
- pickering emulsion
- rice protein
- emulsion stabilizer
- pickering
- protein peptide
- 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.)
- Active
Links
- 239000000839 emulsion Substances 0.000 title claims abstract description 131
- 239000003381 stabilizer Substances 0.000 title claims abstract description 66
- 238000000034 method Methods 0.000 title claims abstract description 36
- 238000006911 enzymatic reaction Methods 0.000 title description 2
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 55
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 55
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 52
- 241000209094 Oryza Species 0.000 claims abstract description 44
- 235000007164 Oryza sativa Nutrition 0.000 claims abstract description 44
- 235000009566 rice Nutrition 0.000 claims abstract description 44
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 43
- 239000000725 suspension Substances 0.000 claims abstract description 30
- 239000002105 nanoparticle Substances 0.000 claims abstract description 27
- 238000002360 preparation method Methods 0.000 claims abstract description 27
- 230000008569 process Effects 0.000 claims abstract description 17
- 239000011259 mixed solution Substances 0.000 claims abstract description 4
- 230000001804 emulsifying effect Effects 0.000 claims abstract description 3
- VFLDPWHFBUODDF-FCXRPNKRSA-N curcumin Chemical compound C1=C(O)C(OC)=CC(\C=C\C(=O)CC(=O)\C=C\C=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-FCXRPNKRSA-N 0.000 claims description 38
- 239000012071 phase Substances 0.000 claims description 31
- 239000000523 sample Substances 0.000 claims description 23
- 229940109262 curcumin Drugs 0.000 claims description 19
- 235000012754 curcumin Nutrition 0.000 claims description 19
- 239000004148 curcumin Substances 0.000 claims description 19
- VFLDPWHFBUODDF-UHFFFAOYSA-N diferuloylmethane Natural products C1=C(O)C(OC)=CC(C=CC(=O)CC(=O)C=CC=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-UHFFFAOYSA-N 0.000 claims description 19
- 239000003921 oil Substances 0.000 claims description 17
- 235000019198 oils Nutrition 0.000 claims description 17
- 238000010008 shearing Methods 0.000 claims description 14
- 238000003756 stirring Methods 0.000 claims description 14
- 235000019483 Peanut oil Nutrition 0.000 claims description 13
- 239000000312 peanut oil Substances 0.000 claims description 13
- 239000007788 liquid Substances 0.000 claims description 12
- 239000002244 precipitate Substances 0.000 claims description 11
- 102000004190 Enzymes Human genes 0.000 claims description 10
- 108090000790 Enzymes Proteins 0.000 claims description 10
- 230000036571 hydration Effects 0.000 claims description 9
- 238000006703 hydration reaction Methods 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 6
- 108091005658 Basic proteases Proteins 0.000 claims description 5
- 239000000796 flavoring agent Substances 0.000 claims description 5
- 235000019634 flavors Nutrition 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 5
- 239000013543 active substance Substances 0.000 claims description 4
- 239000008346 aqueous phase Substances 0.000 claims description 4
- 238000004108 freeze drying Methods 0.000 claims description 4
- 238000007710 freezing Methods 0.000 claims description 4
- 230000008014 freezing Effects 0.000 claims description 4
- 238000011068 loading method Methods 0.000 claims description 4
- 230000000415 inactivating effect Effects 0.000 claims description 2
- 238000012545 processing Methods 0.000 abstract description 8
- 108091005804 Peptidases Proteins 0.000 abstract description 7
- 239000004365 Protease Substances 0.000 abstract description 7
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 abstract description 7
- 239000003995 emulsifying agent Substances 0.000 abstract description 6
- 239000006227 byproduct Substances 0.000 abstract description 5
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 5
- 230000003078 antioxidant effect Effects 0.000 abstract description 4
- 238000009210 therapy by ultrasound Methods 0.000 abstract description 4
- 230000003301 hydrolyzing effect Effects 0.000 abstract description 3
- 235000013339 cereals Nutrition 0.000 abstract description 2
- 235000013305 food Nutrition 0.000 abstract description 2
- 231100000086 high toxicity Toxicity 0.000 abstract description 2
- 235000016709 nutrition Nutrition 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 238000009775 high-speed stirring Methods 0.000 abstract 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 33
- 239000002245 particle Substances 0.000 description 29
- 239000000203 mixture Substances 0.000 description 21
- 238000002604 ultrasonography Methods 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 230000029087 digestion Effects 0.000 description 9
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 7
- 230000002496 gastric effect Effects 0.000 description 7
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 description 6
- 230000003647 oxidation Effects 0.000 description 6
- 238000007254 oxidation reaction Methods 0.000 description 6
- 229910052719 titanium Inorganic materials 0.000 description 6
- 239000010936 titanium Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 5
- 238000000527 sonication Methods 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 3
- 230000009849 deactivation Effects 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 230000007071 enzymatic hydrolysis Effects 0.000 description 3
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 108090000145 Bacillolysin Proteins 0.000 description 2
- 102000035092 Neutral proteases Human genes 0.000 description 2
- 108091005507 Neutral proteases Proteins 0.000 description 2
- 230000003064 anti-oxidating effect Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000002000 scavenging effect Effects 0.000 description 2
- 230000000087 stabilizing effect Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 238000011481 absorbance measurement Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000010775 animal oil Substances 0.000 description 1
- 239000012296 anti-solvent Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 235000014121 butter Nutrition 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000002285 corn oil Substances 0.000 description 1
- 235000005687 corn oil Nutrition 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 235000019197 fats Nutrition 0.000 description 1
- 108010007119 flavourzyme Proteins 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 210000004051 gastric juice Anatomy 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000000887 hydrating effect Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
- A23D7/00—Edible oil or fat compositions containing an aqueous phase, e.g. margarines
- A23D7/02—Edible oil or fat compositions containing an aqueous phase, e.g. margarines characterised by the production or working-up
- A23D7/04—Working-up
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
- A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
- A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
- A23J3/346—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of vegetable proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/10—Foods or foodstuffs containing additives; Preparation or treatment thereof containing emulsifiers
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/30—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation
- A23L5/32—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation using phonon wave energy, e.g. sound or ultrasonic waves
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Colloid Chemistry (AREA)
Abstract
The invention discloses a method for preparing a Pickering emulsion stabilizer by ultrasonic-enzymatic combination, belonging to the technical field of food processing and grain byproduct deep processing. Taking rice protein as a raw material, hydrolyzing by using protease, and then carrying out ultrasonic treatment to obtain rice protein peptide nanoparticles, namely a Pickering emulsion stabilizer; then taking the rice protein peptide nano particle suspension as a water phase, adding oil phases with different volumes, and emulsifying the mixed solution under high-speed stirring to obtain the stable oil-in-water Pickering emulsion. The Pickering emulsifier which is nutritional, healthy, green, environment-friendly and simple in process is prepared by utilizing the rice protein peptide processing by-products, the prepared emulsifier has obvious antioxidant activity, the problems of high toxicity, complex preparation process, high cost and the like of the conventional emulsifier are effectively solved, and the comprehensive utilization of insoluble peptides in the rice protein peptide processing process is realized.
Description
Technical Field
The invention relates to a method for preparing a Pickering emulsion stabilizer by ultrasonic-enzymatic combination, belonging to the technical field of food processing and grain byproduct deep processing.
Background
The food-grade Pickering emulsion realizes the stability of an emulsion system by replacing a traditional emulsifier with edible solid particles, and compared with the traditional emulsion stabilized by a small molecular surfactant, the food-grade Pickering emulsion has higher safety of the solid particles which play a role in emulsification and have partial wettability, and can be irreversibly adsorbed and fixed on an oil-water interface, so that a compact adsorption layer is formed around emulsion droplets, a physical barrier on space is provided for the contact between the droplets, and the Pickering emulsion is endowed with stronger stability. In recent years, researches and developments on food-grade solid particle Pickering stabilizers are more and more favored by researchers.
The protein has incomparable nutritive value, unique conformational advantages and characteristics of changing with pH, so that the protein becomes a preferred material for preparing food-grade nanoparticles. At present, the preparation methods of protein nanoparticles mainly comprise chemical methods, such as an anti-solvent precipitation method, an acid heat treatment method, a pH adjustment method and the like, and the preparation methods have the defects of organic solvent residue, damage to the nutritional value and physiological activity of proteins and the like. In addition, the Pickering emulsion rich in oil and fat prepared by taking protein as a stabilizing agent is easy to oxidize, so that the flavor of the emulsion is deteriorated and the stability is lost. Although proteins have certain antioxidation, most active groups are embedded inside, the antioxidation effect is limited, and the oxidation stability of stable Pickering emulsion is required to be improved.
Disclosure of Invention
Aiming at the problems, the invention provides a method for preparing a Pickering emulsion stabilizer by ultrasonic-enzymatic combination.
The first purpose of the invention is to provide a preparation method of a Pickering emulsion stabilizer, which comprises the following steps:
taking rice protein as a raw material, hydrolyzing the rice protein by using protease, collecting insoluble rice protein peptide, and carrying out ultrasonic treatment on the insoluble rice protein peptide to obtain rice protein peptide nanoparticles, namely the Pickering emulsion stabilizer.
In one embodiment of the present invention, the method for hydrolyzing rice protein by protease comprises: preparing rice protein into suspension with the mass fraction of 3-5%, adjusting pH to 6.5-8.5, adding protease into the suspension for enzymolysis at 45-55 deg.C for 2-3h, inactivating enzyme after enzymolysis, centrifuging, washing precipitate with water, and freeze drying to obtain insoluble rice protein peptide.
In one embodiment of the present invention, the protease is any one of alkaline protease, neutral protease, trypsin, complex protease, and flavourzyme.
In one embodiment of the present invention, the amount of the protease added is 4000-8000U/g.
In one embodiment of the invention, the enzyme deactivation method is high-temperature enzyme deactivation, the temperature of the high-temperature enzyme deactivation is 85-90 ℃, and the water bath is 15-20min, so that the enzyme is inactivated.
In one embodiment of the present invention, the number of washing with water is 2 to 5.
In one embodiment of the invention, the freeze drying method comprises the steps of freeze drying the precipitate after centrifugation and water washing, placing the freeze-dried sample into a refrigerator at-60 ℃ for pre-freezing for 12 hours, and then placing the sample into a freeze dryer for drying, wherein the degree of vacuum of the freeze dryer is 0.1-0.15mBar, and the temperature is-55-60 ℃.
In one embodiment of the present invention, the ultrasonic treatment method is: preparing insoluble rice protein peptide into a solution with the mass fraction of 1-5%, adjusting the pH value to 7.0, stirring for 2-2.5h at 20-25 ℃, putting into a refrigerator with the temperature of 2-5 ℃ for hydration for 12-15h, taking out, treating for 10-30min by using a probe type ultrasonic processor, performing ultrasonic power of 120 plus 480W, and maintaining the temperature to be below 25 ℃ by using an ice bath in the ultrasonic process to obtain the rice protein peptide nanoparticle suspension.
In an embodiment of the invention, the sonication is: processing with probe type ultrasonic processor for 15min, with ultrasonic power of 360W.
In one embodiment of the invention, the pH is adjusted to 7.0 with HCl at a concentration of 0.1-0.5M or NaOH at a concentration of 0.1-0.5M.
The second purpose of the invention is to provide the Pickering emulsion stabilizer obtained by the preparation method.
The third purpose of the invention is to provide a Pickering emulsion, wherein the Pickering emulsion comprises the Pickering emulsion stabilizer.
The fourth purpose of the invention is to provide a preparation method of the Pickering emulsion, which comprises the following steps: taking the rice protein peptide nano particle suspension as a water phase, adding oil phases with different volumes, and emulsifying the mixed solution under high-speed shearing to obtain the stable oil-in-water Pickering emulsion.
In one embodiment of the invention, the different volumes of oil phase comprise from 10 to 75% by volume of the total volume.
In one embodiment of the invention, the oil phase species include vegetable oils (e.g., corn oil, peanut oil, etc.) and animal oils (e.g., lard, butter, etc.).
In one embodiment of the present invention, the conditions of the high speed shearing are: the shear rate is 7000-15000rpm, and the time is 2-20 min.
A fifth object of the present invention is to provide the use of the Pickering emulsion described above for loading active substances and flavor substances.
The invention has the beneficial effects that:
(1) due to the compact structural characteristics of rice protein and the release of hydrophobic polypeptide in the hydrolysis process, the degree of proteolysis is limited, and about 40% of insoluble aggregates, namely insoluble protein peptides, can be generated. The partial protein peptide retains the biological activity, but has poor solubility, is centrifugally removed in the production process of the active peptide, and is finally used as animal feed, thereby greatly reducing the industrial value of the partial protein peptide. Therefore, the Pickering emulsion stabilizer prepared by using the insoluble protein peptide can realize the comprehensive utilization of byproducts.
(2) The Pickering emulsion stabilizer is prepared from the rice protein peptide processing by-products, is nutrient and healthy, green and environment-friendly, has a simple preparation process, and effectively solves the problems of high toxicity, complex preparation process, high cost and the like of the conventional emulsifier.
(3) The Pickering emulsifier prepared by the invention has obvious antioxidant activity, and can be used for improving the oxidation stability of emulsion and preparing a high-quality emulsion system.
(4) The Pickering emulsion prepared by the invention can be used for loading active substances and flavor substances and realizing targeted transportation and directional release of the active substances and the flavor substances.
Drawings
FIG. 1 is a graph of the average particle size of Pickering emulsion stabilizers.
FIG. 2 is a graph of the average particle size of Pickering emulsions stabilized by Pickering emulsion stabilizers.
FIG. 3 is a graph of the oxidation resistance of the Pickering emulsion stabilizer.
Figure 4 is a graph of the stability of curcumin loaded Pickering emulsions during gastrointestinal digestion.
Detailed Description
The present invention will be described in detail with reference to examples.
Example 1:
preparation of Pickering emulsion stabilizer
(1) Dispersing rice protein in water at a feed-liquid ratio of 1:20(g/mL), uniformly stirring, adjusting the pH value of the rice protein dispersion to 8.0 by using 1mol/L NaOH solution, adding alkaline protease according to a ratio of 8000U/g, controlling the enzymolysis temperature to be 50 ℃, carrying out enzymolysis for 2h, carrying out enzyme inactivation in water bath at 90 ℃ for 20min, cooling to room temperature, centrifuging, discarding supernatant, washing precipitate with water for 3 times, then placing the precipitate into a refrigerator at-60 ℃ for pre-freezing for 12h, then placing the precipitate into a freeze dryer for drying, wherein the vacuum degree of the freeze dryer is 0.1mBar, and the temperature is 0 ℃ to obtain insoluble rice protein peptide;
(2) and (2) dispersing the rice protein peptide prepared in the step (1) in water, wherein the material-liquid ratio is 1:100(g/mL), adjusting the pH value to 7.0 by using 0.1M HCl or 0.1M NaOH, stirring for 2h at 25 ℃, putting the mixture into a refrigerator at 4 ℃ for hydration for 12h, taking the mixture out, treating the mixture for 15min by using a probe type ultrasonic processor (a titanium probe with the diameter of 10mm and the length of 13.65cm), performing ultrasonic power of 360W, and maintaining the temperature to be below 25 ℃ by using an ice bath in the ultrasonic process to obtain the rice protein peptide nanoparticle suspension, namely the Pickering emulsion stabilizer suspension.
Example 2
Preparation of Pickering emulsion containing Pickering emulsion stabilizer
(1) Taking 70mL of the Pickering emulsion stabilizer suspension prepared in example 1 as the aqueous phase;
(2) adding 30mL of peanut oil into the water phase as an oil phase, and shearing at 10000rpm for 10min at high speed to obtain Pickering emulsion.
Example 3:
preparation of Pickering emulsion stabilizer
(1) Dispersing rice protein in water at a feed-liquid ratio of 1:20(g/mL), uniformly stirring, adjusting the pH value of the rice protein dispersion to 8.0 by using 1mol/L NaOH solution, adding neutral protease according to a ratio of 8000U/g, controlling the enzymolysis temperature to be 50 ℃, carrying out enzymolysis for 2h, carrying out enzyme inactivation in water bath at 90 ℃ for 20min, cooling to room temperature, centrifuging, discarding supernatant, washing precipitate with water for 3 times, then placing the precipitate into a refrigerator at-60 ℃ for pre-freezing for 12h, then placing the precipitate into a freeze dryer for drying, wherein the vacuum degree of the freeze dryer is 0.1mBar, and the temperature is 0 ℃ to obtain insoluble rice protein peptide;
(2) and (2) dispersing the rice protein peptide prepared in the step (1) in water, wherein the material-liquid ratio is 1:100(g/mL), adjusting the pH value to 7.0 by using 0.1M HCl or 0.1M NaOH, stirring for 2h at 25 ℃, putting the mixture into a refrigerator at 4 ℃, hydrating for 12h, taking out the mixture, treating the mixture for 15min by using a probe type ultrasonic processor (a titanium probe, the diameter of which is 10mm and the length of which is 13.65cm), and keeping the temperature stable at below 25 ℃ by using an ice bath in the ultrasonic process, wherein the obtained rice protein peptide nanoparticle suspension is a Pickering emulsion stabilizer suspension.
Example 4
Preparation of Pickering emulsion containing Pickering emulsion stabilizer
(1) Taking 70mL of the Pickering emulsion stabilizer suspension prepared in example 3 as the aqueous phase;
(2) adding 30mL of peanut oil into the water phase as an oil phase, and shearing at 10000rpm for 10min at high speed to obtain Pickering emulsion.
Example 5:
preparation of Pickering emulsion stabilizer
(1) Same as in step (1) in example 1;
(2) and (2) dispersing the rice protein peptide prepared in the step (1) in water, wherein the material-liquid ratio is 1:50(g/mL), adjusting the pH value to 7.0 by using 0.1M HCl or 0.1M NaOH, stirring for 2h at 25 ℃, putting the mixture into a refrigerator at 4 ℃ for hydration for 12h, taking out the mixture, treating the mixture for 15min by using a probe type ultrasonic processor, wherein the ultrasonic power is 360W, and the temperature is kept to be stable below 25 ℃ by using an ice bath in the ultrasonic process, so that the obtained rice protein peptide nano particle suspension is Pickering emulsion stabilizer suspension.
Example 6
Preparation of Pickering emulsion containing Pickering emulsion stabilizer
(1) Taking 70mL of the Pickering emulsion stabilizer prepared in example 5 as a water phase;
(2) adding 30mL of peanut oil into the water phase as an oil phase, and shearing at 10000rpm for 10min at high speed to obtain Pickering emulsion.
Example 7:
preparation of Pickering emulsion stabilizer
(1) Same as in step (1) in example 1;
(2) and (2) dispersing the rice protein peptide prepared in the step (1) in water, wherein the material-liquid ratio is 1:100(g/mL), adjusting the pH value to 7.0 by using 0.1M HCl or 0.1M NaOH, stirring for 2h at 25 ℃, putting the mixture into a refrigerator at 4 ℃ for hydration for 12h, taking the mixture out, treating the mixture for 20min by using a probe type ultrasonic processor (a titanium probe with the diameter of 10mm and the length of 13.65cm), performing ultrasonic power of 360W, and maintaining the temperature to be below 25 ℃ by using an ice bath in the ultrasonic process to obtain a rice protein peptide nanoparticle suspension, namely a Pickering emulsion stabilizer suspension.
Example 8
Preparation of Pickering emulsion containing Pickering emulsion stabilizer
(1) Taking 70mL of the Pickering emulsion stabilizer prepared in example 7 as a water phase;
(2) adding 30mL of peanut oil into the water phase as an oil phase, and shearing at 10000rpm for 10min at high speed to obtain Pickering emulsion.
Example 9
Preparation of Pickering emulsion stabilizer
(1) Same as in step (1) in example 1;
(2) and (2) dispersing the rice protein peptide prepared in the step (1) in water, wherein the material-liquid ratio is 1:100(g/mL), adjusting the pH value to 7.0 by using 0.1M HCl or 0.1M NaOH, stirring for 2h at 25 ℃, putting the mixture into a refrigerator at 4 ℃ for hydration for 12h, taking the mixture out, treating the mixture for 10min by using a probe type ultrasonic processor (a titanium probe with the diameter of 10mm and the length of 13.65cm), performing ultrasonic power of 360W, and maintaining the temperature to be below 25 ℃ by using an ice bath in the ultrasonic process to obtain the rice protein peptide nanoparticle suspension, namely the Pickering emulsion stabilizer suspension.
Example 10
Preparation of Pickering emulsion containing Pickering emulsion stabilizer
(1) Taking 70mL of the Pickering emulsion stabilizer prepared in example 9 as the water phase;
(2) adding 30mL of peanut oil into the water phase as an oil phase, and shearing at 10000rpm for 10min at high speed to obtain Pickering emulsion.
Example 11
Preparation of Pickering emulsion stabilizer
(1) Same as in step (1) in example 1;
(2) and (2) dispersing the rice protein peptide prepared in the step (1) in water, wherein the material-liquid ratio is 1:100(g/mL), adjusting the pH value to 7.0 by using 0.1M HCl or 0.1M NaOH, stirring for 2h at 25 ℃, putting the mixture into a refrigerator at 4 ℃ for hydration for 12h, taking the mixture out, treating the mixture for 15min by using a probe type ultrasonic processor (a titanium probe with the diameter of 10mm and the length of 13.65cm), performing ultrasonic power of 120W, and maintaining the temperature to be below 25 ℃ by using an ice bath in the ultrasonic process to obtain a rice protein peptide nanoparticle suspension, namely a Pickering emulsion stabilizer suspension.
Example 12
Preparation of Pickering emulsion containing Pickering emulsion stabilizer
(1) Taking 70mL of the Pickering emulsion stabilizer prepared in example 11 as the water phase;
(2) adding 30mL of peanut oil into the water phase as an oil phase, and shearing at 10000rpm for 10min at high speed to obtain Pickering emulsion.
Example 13
Preparation of Pickering emulsion stabilizer
(1) Same as in step (1) in example 1;
(2) and (2) dispersing the rice protein peptide prepared in the step (1) in water, wherein the material-liquid ratio is 1:100(g/mL), adjusting the pH value to 7.0 by using 0.1M HCl or 0.1M NaOH, stirring for 2h at 25 ℃, putting the mixture into a refrigerator at 4 ℃ for hydration for 12h, taking the mixture out, treating the mixture for 15min by using a probe type ultrasonic processor (a titanium probe with the diameter of 10mm and the length of 13.65cm), and keeping the temperature stable below 25 ℃ by using an ice bath in the ultrasonic process, wherein the obtained rice protein peptide nanoparticle suspension is Pickering emulsion stabilizer suspension.
Example 14
Preparation of Pickering emulsion containing Pickering emulsion stabilizer
(1) Taking 70mL of the Pickering emulsion stabilizer prepared in example 13 as an aqueous phase;
(2) adding 30mL of peanut oil into the water phase as an oil phase, and shearing at 10000rpm for 10min at high speed to obtain Pickering emulsion.
Comparative example 1 (No enzymatic hydrolysis)
Preparation of Pickering emulsion stabilizer
Dispersing rice protein in water at a material-liquid ratio of 1:100(g/mL), adjusting pH to 7.0 with 0.1M HCl or 0.1M NaOH, stirring at 25 ℃ for 2h, placing in a refrigerator at 4 ℃ for hydration for 12h, taking out, treating for 15min with a probe type ultrasonic processor with ultrasonic power of 360W, and maintaining the stability at below 25 ℃ by using an ice bath during ultrasonic treatment to obtain a rice protein nanoparticle suspension, namely a Pickering emulsion stabilizer suspension.
Preparation of Pickering emulsion containing Pickering emulsion stabilizer
(1) Taking 70mL of the prepared Pickering stabilizer suspension as a water phase;
(2) adding 30mL of peanut oil into the water phase as an oil phase, and shearing at 10000rpm for 10min at high speed to obtain Pickering emulsion.
Example 15
The Pickering emulsion stabilizers and Pickering emulsions prepared in examples 1-14 and comparative example 1 were tested, and the particle sizes of the Pickering emulsion stabilizers and Pickering emulsions, and the antioxidant properties of the Pickering emulsion stabilizers were respectively tested.
And (3) particle size testing:
the Pickering emulsion stabilizer and the emulsion particle size testing method comprise the following steps: the particle size of the Pickering emulsion stabilizer was measured using a nanometer particle size and Zeta potentiometer, and the results are shown in FIG. 1; the emulsion particle size (fresh emulsion and emulsion after standing) was determined using a laser particle sizer, and the determination was repeated three times for each sample, and the results are shown in fig. 2.
As can be seen from FIG. 1, the particle size of the Pickering emulsion stabilizer obtained in the examples was influenced by the kind of enzyme, the concentration of insoluble peptide upon sonication, the sonication time and the sonication power. Compared with the comparative example 1, the nano particles obtained through double steps of enzymolysis and ultrasound have lower particle size, can be more quickly diffused to the surface of oil drops in the process of stabilizing the emulsion, and play a role in stabilizing the emulsion. Compared with example 1, insoluble peptide obtained by enzymolysis with alkaline protease is more susceptible to ultrasonic wave and is decomposed into small particles; increasing or decreasing the ultrasound time or ultrasound power both increased the particle size of the nanoparticles, indicating that ultrasound was unable to infinitely decrease the particle size of the insoluble peptides, and there was optimal ultrasound time and ultrasound power. In summary, the effect of preparing nanoparticles from insoluble peptides obtained by enzymolysis with alkaline protease is the best, the ultrasound time is 15min, and the ultrasound power is 360W, i.e. the conditions used in example 1.
Generally, the smaller the particle size of the emulsion, the greater its stability. As can be seen from fig. 2, compared with comparative example 1, the particle size of the fresh emulsion with stable nanoparticles obtained through the double steps of enzymolysis and ultrasound is smaller. However, the average particle size of the stable emulsion is greatly changed with different preparation processes of the nano particles. When the enzyme type was changed (example 4), the emulsion showed particle size fluctuations during storage and the emulsion began to delaminate after 7 days of storage. When the concentration of insoluble peptide was increased upon sonication (example 6), although the particle size of the nanoparticles increased, the emulsion was relatively stable due to the increased concentration of the stabilizer, and no significant change in particle size occurred. The ultrasonic time and the ultrasonic power have great influence on the particle size of the emulsion (examples 8 to 14), the ultrasonic time and the ultrasonic power are increased or reduced to remarkably increase the particle size of the fresh emulsion, the emulsion is layered quickly, and the emulsion is extremely unstable and the particle size of the emulsion is increased continuously in the storage process.
And (3) testing the oxidation resistance:
the Pickering emulsion stabilizers in examples 1-14 and comparative example 1 were tested for antioxidant performance by DPPH clearance. Taking 2mL of Pickering emulsion stabilizer solution with the mass fraction of 1%, adding 2mL of DPPH solution with the concentration of 0.1mmol/L (DPPH is dissolved by pure methanol), mixing uniformly, standing in a dark place for 30min, and measuring the light absorption value of a sample by using an ultraviolet spectrophotometer, wherein the wavelength is 517 nm. 2mL of sample +2mL of absolute methanol was used as a blank, and 2mL of deionized water +2mL of DPPH working solution was used as a control.
DPPH clearance is calculated according to equation (1-1):
DPPH clearance (%) - (1- (As-Ab)/Ac) × 100% (1-1)
In the formula:
as-light absorption value of sample
Ab-Absorbance value of blank
Absorbance of Ac control
The resulting DPPH clearance is shown in fig. 3.
As can be seen from fig. 3, the DPPH scavenging ability of the nanoparticles obtained by the double steps of enzymatic hydrolysis and ultrasound was significantly increased compared to that of comparative example 1, because active groups inside the protein were exposed after the enzymatic hydrolysis, contact sites with DPPH radicals were increased, radicals were more easily trapped, and the oxidation resistance was increased. In comparative examples 1 to 14, DPPH scavenging ability of nanoparticles is closely related to their particle size, also because the smaller the particle size of nanoparticles, the larger the specific surface area, the greater the probability of contact with radicals, and the stronger the oxidation resistance.
Example 16 use of Pickering emulsion
Preparation of Pickering emulsion loaded with curcumin
(1) A Pickering emulsion stabilizer suspension was prepared as in example 1;
(2) curcumin with the mass fraction of 0.1 percent is added into the peanut oil and stirred magnetically overnight, then centrifuged (12000rpm, 10min), and the precipitate is discarded.
(3) Taking 70mL of the prepared Pickering emulsion stabilizer suspension as a water phase, adding the curcumin-containing peanut oil as an oil phase, and shearing at 10000rpm for 10min at a high speed to obtain the Pickering emulsion loaded with curcumin.
Simulating gastrointestinal digestion
(1) Adjusting pH of 20mL of the Pickering emulsion to 1.5 with 1M HCl, incubating at 37 deg.C for 10min, adding 4% (w/w, based on the amount of nanoparticles in the granule) of simulated gastric juice, incubating for 1h, adjusting pH to 7.0 to complete simulated gastric digestion, and collecting part of the gastric digestion product for determination of curcumin content.
(2) After completion of gastric digestion according to the above procedure, 4% (w/w based on the amount of nanoparticles in the particles) of simulated intestinal fluid was added and incubated for 2 h. After the reaction is finished, taking a part of gastrointestinal tract digestion products for measuring the content of the curcumin.
Curcumin content determination
A standard curve was constructed by measuring the absorbance of known concentrations of curcumin in methanol at a wavelength of 425nm, and then the absorbance measurements were converted to curcumin concentrations by the standard curve.
Curcumin is unstable chemically, is very easy to hydrolyze or degrade, and greatly reduces the in vivo biological activity. Therefore, the digestion stability of curcumin loaded in Pickering emulsion is a main index for evaluating the biological function of curcumin. As shown in fig. 4, curcumin alone was mostly destroyed and less stable in the simulated gastrointestinal digestion process. After loading into the Pickering emulsion prepared in the embodiment, 79% of curcumin is retained in the process of simulating gastrointestinal digestion and is transported to the intestinal tract in a targeted mode.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (7)
1. A method for preparing a Pickering emulsion stabilizer is characterized by comprising the following steps:
(1) dispersing rice protein in water at a feed-liquid ratio of 1:20g/mL, uniformly stirring and adjusting the pH value to 8.0 to obtain a suspension; adding alkaline protease into the suspension according to a ratio of 8000U/g for enzymolysis at 50 ℃ for 2h, inactivating enzyme after enzymolysis, centrifuging, washing precipitate with water, pre-freezing in a refrigerator at-60 ℃ for 12h, and freeze-drying to obtain insoluble rice protein peptide;
(2) and (2) dispersing the insoluble rice protein peptide prepared in the step (1) in water, wherein the material-liquid ratio is 1:100g/mL, adjusting the pH value to 7.0, stirring for 2-2.5h at 20-25 ℃, putting into a refrigerator at 2-5 ℃ for hydration for 12-15h, taking out, treating for 15min by using a probe type ultrasonic processor, performing ultrasonic power of 360W, and maintaining the temperature to be below 25 ℃ by using an ice bath in the ultrasonic process to obtain the rice protein peptide nanoparticle suspension.
2. The Pickering emulsion stabilizer prepared according to the preparation method of claim 1.
3. A Pickering emulsion, characterized in that the Pickering emulsion comprises the Pickering emulsion stabilizer of claim 2.
4. A method of preparing the Pickering emulsion of claim 3, wherein the Pickering emulsion is prepared by: taking the suspension of the Pickering emulsion stabilizer of claim 2 as an aqueous phase, adding an oil phase, wherein the volume of the oil phase accounts for 30% of the total volume, and emulsifying the mixed solution through shearing treatment to obtain the oil-in-water Pickering emulsion.
5. The method according to claim 4, wherein the conditions of the shearing process are: the shear rate is 7000-15000rpm, and the time is 2-20 min.
6. Use of a Pickering emulsion according to claim 3 for loading active substances and flavor substances.
7. A method for preparing Pickering emulsion loaded with curcumin is characterized by comprising the following steps:
(1) preparing a Pickering emulsion stabilizer according to the method of claim 1;
(2) adding 0.1% of curcumin by mass into peanut oil, magnetically stirring overnight, centrifuging, and removing precipitate;
(3) taking the Pickering emulsion stabilizer prepared in the step (1) as a water phase, adding peanut oil containing curcumin as an oil phase, and shearing the mixed solution at a high speed of 10000rpm for 10min to prepare the Pickering emulsion loaded with curcumin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010267104.4A CN111466447B (en) | 2020-04-08 | 2020-04-08 | Method for preparing Pickering emulsion stabilizer by ultrasonic-enzymatic method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010267104.4A CN111466447B (en) | 2020-04-08 | 2020-04-08 | Method for preparing Pickering emulsion stabilizer by ultrasonic-enzymatic method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111466447A CN111466447A (en) | 2020-07-31 |
| CN111466447B true CN111466447B (en) | 2022-08-02 |
Family
ID=71750146
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010267104.4A Active CN111466447B (en) | 2020-04-08 | 2020-04-08 | Method for preparing Pickering emulsion stabilizer by ultrasonic-enzymatic method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111466447B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112617200A (en) * | 2020-12-09 | 2021-04-09 | 集美大学 | Preparation method of fish oil gel |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101253925A (en) * | 2008-04-01 | 2008-09-03 | 江南大学 | Preparation of rice protein antioxidation active peptide |
| CN109122906A (en) * | 2018-09-14 | 2019-01-04 | 南京财经大学 | A kind of rice protein lotion and preparation method thereof |
| CN110810617A (en) * | 2019-11-06 | 2020-02-21 | 江西省科学院应用化学研究所 | Preparation method of rice gluten with high emulsifying activity and high emulsifying stability |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105661552A (en) * | 2016-02-29 | 2016-06-15 | 华南理工大学 | Vitamin D nano-emulsion prepared by utilizing fragmented corn protein-tannic acid compound emulsifier and preparation method of vitamin D nano-emulsion |
| CN106753747A (en) * | 2016-12-05 | 2017-05-31 | 东北农业大学 | A kind of method that enzyme process prepares soybean grease and nanoemulsions |
| CN108208834B (en) * | 2018-01-05 | 2021-03-30 | 华南理工大学 | Curcumin-loaded soybean protein-based nanoparticles and preparation method and application thereof |
| CN108157585B (en) * | 2018-01-05 | 2021-05-14 | 华南理工大学 | Resveratrol-loaded soybean protein-based nano-particles and preparation method and application thereof |
| CN109170122A (en) * | 2018-07-27 | 2019-01-11 | 咸阳师范学院 | A kind of extracting soybean albumen simultaneously purifies and the method for recycling surfactant |
| CN110101084B (en) * | 2019-04-04 | 2023-02-10 | 华南理工大学 | Algae oil nano pickering emulsion and oil powder stabilized by yolk protein peptide particles as well as preparation method and application thereof |
| CN110786517A (en) * | 2019-11-12 | 2020-02-14 | 上海海洋大学 | Euphausia superba protein Pickering emulsion and preparation method thereof |
-
2020
- 2020-04-08 CN CN202010267104.4A patent/CN111466447B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101253925A (en) * | 2008-04-01 | 2008-09-03 | 江南大学 | Preparation of rice protein antioxidation active peptide |
| CN109122906A (en) * | 2018-09-14 | 2019-01-04 | 南京财经大学 | A kind of rice protein lotion and preparation method thereof |
| CN110810617A (en) * | 2019-11-06 | 2020-02-21 | 江西省科学院应用化学研究所 | Preparation method of rice gluten with high emulsifying activity and high emulsifying stability |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111466447A (en) | 2020-07-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | Modification of structural and functional characteristics of brewer's spent grain protein by ultrasound assisted extraction | |
| Ke et al. | Influence mechanism of polysaccharides induced Maillard reaction on plant proteins structure and functional properties: A review | |
| Jantakoson et al. | Effect of high pressure and heat treatments on black tiger shrimp (Penaeus monodon Fabricius) muscle protein | |
| Zheng et al. | Functional and structural properties of spirulina phycocyanin modified by ultra-high-pressure composite glycation | |
| CN109588721B (en) | Carotenoid-protein particle and preparation method and application thereof | |
| Roy et al. | Preparation of bio-functional surimi gel incorporation of fish oil and green tea extracts: Physico-chemical activities, in-vitro digestibility, and bacteriostatic properties | |
| CN111466447B (en) | Method for preparing Pickering emulsion stabilizer by ultrasonic-enzymatic method | |
| Skendi et al. | Recovery of high added-value compounds from brewing and distillate processing by-products | |
| Jhan et al. | Nanoreduction of millet proteins: effect on structural and functional properties | |
| CN1274361C (en) | Soybean peptide, its preparation and application | |
| CN100497652C (en) | Method for preparing water soluble wheat protein by composite modification | |
| CN111296852A (en) | Protein microcapsule for targeted inhibition of small intestine amylase and preparation method thereof | |
| CN114246326A (en) | Preparation method and application of modified egg white protein | |
| CN113925157A (en) | Beta-carotene emulsion and preparation method thereof | |
| Dabbour et al. | Ultrasonic-aided extraction and degossypolization of cottonseed meal protein: Optimization and characterization of functional traits and molecular structure | |
| CN112042937A (en) | Water-soluble lutein emulsion gel and preparation method thereof | |
| Li et al. | The changed structures of Cyperus esculentus protein decide its modified physicochemical characters: Effects of ball-milling, high pressure homogenization and cold plasma treatments on structural and functional properties of the protein | |
| CN108531290A (en) | Inhibit the method for lipid oxidation in fish oil | |
| CN104543635B (en) | A kind of astacin collagen jelly | |
| CN115644453A (en) | Method for stabilizing DHA emulsion by soybean polypeptide-polyphenol antioxidant particles | |
| CN104059956B (en) | A kind of preparation method for hiding antioxidation polypeptide in sheep blood | |
| CN114410723A (en) | Pea antioxidant peptide with good emulsibility and preparation method thereof | |
| CN1552892A (en) | Physiological active buck peptide by compound proteinase catalysis and use of products thereby | |
| CN113604527A (en) | Polypeptide nanofiber prepared by restriction enzymolysis and preparation method and application thereof | |
| Gao et al. | Physicochemical and rheological properties of interacted protein hydrolysates derived from tuna processing by‐products with sodium alginate |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |