CN116158535B - Mulberry leaf protein-mulberry anthocyanin composite emulsion and preparation method thereof - Google Patents
Mulberry leaf protein-mulberry anthocyanin composite emulsion and preparation method thereof Download PDFInfo
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- 240000000249 Morus alba Species 0.000 title claims abstract description 119
- 235000008708 Morus alba Nutrition 0.000 title claims abstract description 119
- 239000004410 anthocyanin Substances 0.000 title claims abstract description 57
- 239000000839 emulsion Substances 0.000 title claims abstract description 50
- 235000010208 anthocyanin Nutrition 0.000 title claims abstract description 45
- 229930002877 anthocyanin Natural products 0.000 title claims abstract description 45
- 150000004636 anthocyanins Chemical class 0.000 title claims abstract description 45
- 239000002131 composite material Substances 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 238000004945 emulsification Methods 0.000 title description 2
- 150000004676 glycans Chemical class 0.000 claims abstract description 37
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 37
- 239000005017 polysaccharide Substances 0.000 claims abstract description 37
- 101710138460 Leaf protein Proteins 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 95
- 239000000243 solution Substances 0.000 claims description 33
- 235000019441 ethanol Nutrition 0.000 claims description 30
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 27
- 239000002244 precipitate Substances 0.000 claims description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 26
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 21
- 239000012153 distilled water Substances 0.000 claims description 19
- 239000006228 supernatant Substances 0.000 claims description 17
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 17
- 238000000605 extraction Methods 0.000 claims description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 12
- 102000004169 proteins and genes Human genes 0.000 claims description 12
- 238000005119 centrifugation Methods 0.000 claims description 11
- 238000002137 ultrasound extraction Methods 0.000 claims description 11
- 238000011068 loading method Methods 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 9
- 230000001105 regulatory effect Effects 0.000 claims description 7
- 239000011347 resin Substances 0.000 claims description 7
- 229920005989 resin Polymers 0.000 claims description 7
- 235000012424 soybean oil Nutrition 0.000 claims description 7
- 239000003549 soybean oil Substances 0.000 claims description 7
- 229910001868 water Inorganic materials 0.000 claims description 7
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 229920005654 Sephadex Polymers 0.000 claims description 5
- 230000002378 acidificating effect Effects 0.000 claims description 5
- 229920002678 cellulose Polymers 0.000 claims description 5
- 239000001913 cellulose Substances 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 238000010438 heat treatment Methods 0.000 claims description 5
- 238000000265 homogenisation Methods 0.000 claims description 5
- 239000012535 impurity Substances 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 230000007935 neutral effect Effects 0.000 claims description 5
- 239000003960 organic solvent Substances 0.000 claims description 5
- 239000008363 phosphate buffer Substances 0.000 claims description 5
- 238000004537 pulping Methods 0.000 claims description 5
- 238000010298 pulverizing process Methods 0.000 claims description 5
- 238000007873 sieving Methods 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 239000008055 phosphate buffer solution Substances 0.000 claims description 3
- 239000012507 Sephadex™ Substances 0.000 claims description 2
- 239000003480 eluent Substances 0.000 claims description 2
- 239000012460 protein solution Substances 0.000 claims description 2
- 238000002390 rotary evaporation Methods 0.000 claims description 2
- 239000012527 feed solution Substances 0.000 claims 1
- 235000013305 food Nutrition 0.000 abstract description 7
- 238000012545 processing Methods 0.000 abstract description 3
- 241000196324 Embryophyta Species 0.000 abstract description 2
- 235000018102 proteins Nutrition 0.000 description 10
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- 230000009286 beneficial effect Effects 0.000 description 6
- 238000003760 magnetic stirring Methods 0.000 description 6
- 238000009210 therapy by ultrasound Methods 0.000 description 4
- 108010073771 Soybean Proteins Proteins 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000001804 emulsifying effect Effects 0.000 description 3
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 235000019710 soybean protein Nutrition 0.000 description 3
- 238000010025 steaming Methods 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- 239000013543 active substance Substances 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 235000006708 antioxidants Nutrition 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- VEVZSMAEJFVWIL-UHFFFAOYSA-O cyanidin cation Chemical compound [O+]=1C2=CC(O)=CC(O)=C2C=C(O)C=1C1=CC=C(O)C(O)=C1 VEVZSMAEJFVWIL-UHFFFAOYSA-O 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000003995 emulsifying agent Substances 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- GCPYCNBGGPHOBD-UHFFFAOYSA-N Delphinidin Natural products OC1=Cc2c(O)cc(O)cc2OC1=C3C=C(O)C(=O)C(=C3)O GCPYCNBGGPHOBD-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 240000001549 Ipomoea eriocarpa Species 0.000 description 1
- 235000005146 Ipomoea eriocarpa Nutrition 0.000 description 1
- YKRGDOXKVOZESV-WRJNSLSBSA-N Paeoniflorin Chemical compound C([C@]12[C@H]3O[C@]4(O)C[C@](O3)([C@]1(C[C@@H]42)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)C)OC(=O)C1=CC=CC=C1 YKRGDOXKVOZESV-WRJNSLSBSA-N 0.000 description 1
- 108010064851 Plant Proteins Proteins 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 235000007336 cyanidin Nutrition 0.000 description 1
- 235000007242 delphinidin Nutrition 0.000 description 1
- JKHRCGUTYDNCLE-UHFFFAOYSA-O delphinidin Chemical compound [O+]=1C2=CC(O)=CC(O)=C2C=C(O)C=1C1=CC(O)=C(O)C(O)=C1 JKHRCGUTYDNCLE-UHFFFAOYSA-O 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 235000018927 edible plant Nutrition 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- YKRGDOXKVOZESV-UHFFFAOYSA-N paeoniflorin Natural products O1C(C)(C2(CC34)OC5C(C(O)C(O)C(CO)O5)O)CC3(O)OC1C24COC(=O)C1=CC=CC=C1 YKRGDOXKVOZESV-UHFFFAOYSA-N 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000000518 rheometry Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000000733 zeta-potential measurement Methods 0.000 description 1
Classifications
-
- 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
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/185—Vegetable proteins
-
- 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
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/006—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from vegetable materials
- A23J1/007—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from vegetable materials from leafy vegetables, e.g. alfalfa, clover, grass
-
- 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
- A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
-
- 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
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- 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
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
- C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
- C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B61/00—Dyes of natural origin prepared from natural sources, e.g. vegetable sources
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B67/00—Influencing the physical, e.g. the dyeing or printing properties of dyestuffs without chemical reactions, e.g. by treating with solvents grinding or grinding assistants, coating of pigments or dyes; Process features in the making of dyestuff preparations; Dyestuff preparations of a special physical nature, e.g. tablets, films
- C09B67/0096—Purification; Precipitation; Filtration
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- 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
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Abstract
The invention discloses a mulberry leaf protein-mulberry anthocyanin composite emulsion and a preparation method thereof, and relates to the technical field of food processing. The method specifically comprises the following steps: (1) preparing mulberry leaf protein; (2) preparing mulberry leaf polysaccharide; (3) preparing a mulberry anthocyanin extract; (4) homogenizing to prepare emulsion. The invention utilizes the mulberry leaf protein, the mulberry leaf polysaccharide and the mulberry anthocyanin which belong to the same plant system source to prepare the emulsion, thereby improving the stability of the whole emulsion system.
Description
Technical Field
The invention relates to the technical field of food processing, in particular to a mulberry leaf protein-mulberry anthocyanin composite emulsion and a preparation method thereof.
Background
Anthocyanin is a small molecular active substance, and mainly comprises cyanidin, delphinidin, paeoniflorin, morning glory pigment, etc. Anthocyanin has antioxidant, antibacterial, antitumor, and antiinflammatory effects. However, anthocyanins have low stability to environmental conditions (e.g., temperature, pH, light, water, oxygen, metal ions, etc.) during processing and storage, making their use in food products a significant challenge.
Emulsion is one of the most complex and important systems in food systems, protein is often utilized as an emulsifier, and researches show that anthocyanin has stronger protein affinity, and the oxidation stability of the composite emulsion can be effectively improved by adding anthocyanin into soybean protein isolate. The anthocyanin and the protein are combined to form a complex, so that not only can the stability of the anthocyanin be improved, but also the antioxidant capacity and other functional characteristics of the protein can be enhanced. However, it is difficult to prepare stable composite systems by simple mixing. Polysaccharides are also commonly used as emulsion stabilizers to improve system rheology, and polysaccharides also play an important role in system architecture and stability.
The mulberry leaves are a medicine and food dual-purpose resource, the protein content in the mulberry leaves is high, and the protein content of part of mulberry leaves can account for 20-30% of the dry basis weight. The mulberry leaf protein has complete amino acid variety and higher essential amino acid content, and is an edible plant protein raw material with great development value. In recent years, research on mulberry leaf proteins is mainly focused on the extraction process, and no report on the application of the mulberry leaf proteins in a food system is yet made. The mulberry leaf protein has good foamability, emulsifying property and emulsifying stability and has the potential of becoming a food-grade emulsifier. However, in the processes of heat treatment, enzyme treatment and the like in the extraction process, the physicochemical properties of the mulberry leaf protein are easily changed, so that the development and the utilization of the mulberry leaf protein are limited to a certain extent.
The mulberry leaf polysaccharide is an active substance extracted from mulberry leaves, has various pharmacological effects of reducing blood sugar, reducing blood fat, resisting oxidation, resisting aging, enhancing immunity and the like, and has higher development and utilization values in the industries of health care products, medicines and the like. The application of mulberry leaf polysaccharide in a stable food system is also reported recently.
Disclosure of Invention
In view of this, the present invention provides a method of
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a preparation method of mulberry leaf protein-anthocyanin composite emulsion specifically comprises the following steps:
(1) Vacuum freeze drying folium Mori, pulverizing, and sieving with 40 mesh sieve to obtain folium Mori powder; adding 0.1mol/L NaOH solution into mulberry leaf powder, performing ultrasonic extraction, centrifuging for the first time, and collecting supernatant; regulating the pH of the supernatant to 4.8, standing at 4 ℃ after flocculent precipitate is generated on protein, centrifuging for the second time, and collecting the precipitate and supernatant for later use; washing the precipitate with 95% ethanol, redissolving with PBS, dialyzing in distilled water for 1d, and vacuum freeze drying to obtain mulberry leaf protein;
(2) Collecting the supernatant of the second centrifugation in the step (1), regulating the pH to be neutral, concentrating in vacuum, adding absolute ethyl alcohol, standing at 4 ℃ to separate out a precipitate, and centrifugally collecting the precipitate; dissolving the precipitate with distilled water, slowly dripping onto a treated DEAE-52 cellulose chromatographic column, performing gradient elution with NaCL solution, collecting the component with highest polysaccharide content, loading onto a SephadexG-100 sephadex column, eluting with distilled water, collecting and mixing eluates according to peak value, and vacuum freeze-drying to obtain mulberry leaf polysaccharide;
(3) Pulping fresh mulberry with a high-speed refiner, adding acidified ethanol, performing ultrasonic extraction, repeating the extraction for 2 times, and combining the extracting solutions; removing ethanol from the extractive solution by rotary evaporation, suction filtering with Buchner funnel, loading onto chromatographic column filled with AB-8 macroporous resin, eluting with distilled water and ethyl acetate to remove impurities, eluting anthocyanin with 60% acidic ethanol, collecting eluate, concentrating under reduced pressure to remove organic solvent, and vacuum freeze drying to obtain Mori fructus anthocyanin extract;
(4) Dissolving mulberry leaf protein in phosphate buffer solution, magnetically stirring for 2 hours after heat treatment, then adjusting the pH to 7.0, adding mulberry anthocyanin, magnetically stirring for 1 hour at room temperature, adding soybean oil and mulberry leaf polysaccharide, stirring for 1 hour, and finally homogenizing to prepare emulsion.
Further, in the step (1), the feed liquid ratio of the mulberry leaf to the NaOH solution is g: mL = 1:30-50; the ultrasonic power is 300W, the extraction temperature is 40 ℃, and the extraction time is 20-60min.
Further, in the step (1), the ratio of mulberry leaf to NaOH solution is preferably g: mL = 1:40; the ultrasonic extraction time is preferably 45min.
The adoption of the further technical scheme has the beneficial effects that the mulberry leaf protein has high extraction rate and stable protein structure.
Further, in the step (2), the volume of the vacuum concentration is 1/10-20 of the initial volume.
Further, in the step (2), the gradient concentration of the NaCL solution in the gradient elution is 0mol/L, 0.1mol/L, 0.3mol/L and 0.5mol/L;
the component with the highest polysaccharide content is eluent eluted by 0.3mol/L NaCL solution.
The adoption of the further technical scheme has the beneficial effect that the mulberry leaf polysaccharide with higher purity and smaller molecular weight is obtained.
Further, in the step (3), the acidified ethanol is an ethanol solution containing 0.1% citric acid;
the ratio of mulberry pulp to acidified ethanol feed liquid is g: ml=1:2-5, preferably 1:3; the ultrasonic power is 300W, the extraction temperature is 40 ℃, and the extraction time is 20-60min, preferably 30min.
Further, in the step (3), the ratio of the mulberry pulp to the acidified ethanol solution is preferably g: mL = 1:3; the ultrasonic extraction time is preferably 30min.
The adoption of the further technical scheme has the beneficial effect that the mulberry anthocyanin is fully extracted.
Further, in the step (3), the volume of distilled water used for macroporous resin elution is 1 time of the volume of the chromatographic column, the volume of ethyl acetate is 1 time of the volume of the chromatographic column, and the volume of 60% acidified ethanol is 5 times of the volume of the chromatographic column.
The adoption of the further technical scheme has the beneficial effect that the high-purity mulberry anthocyanin is obtained.
Further, in the step (4), the concentration of the phosphate buffer solution is 0.02mol/L; the mass concentration of the mulberry leaf protein is 30mg/mL, the volume ratio of the mulberry leaf protein solution to the soybean oil is 5:1, the mass ratio of the mulberry leaf protein to the anthocyanin is 40:1, and the mass ratio of the mulberry leaf protein to the mulberry leaf polysaccharide is 5:1; the heat treatment is carried out in a water bath at 80 ℃ for 10min, the homogenization speed is 12000r/min, and the time is 3min.
The further technical scheme has the beneficial effects that the mulberry leaf polysaccharide is added according to a proper proportion, so that the stability of the mulberry leaf protein-anthocyanin composite emulsion is improved.
The invention also provides the mulberry leaf protein-anthocyanin composite emulsion obtained by the preparation method.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention utilizes the mulberry leaf protein, the mulberry leaf polysaccharide and the mulberry anthocyanin which belong to the same plant system source to prepare the emulsion, thereby improving the stability of the whole emulsion system.
2. The invention combines the mulberry protein and the mulberry polysaccharide extraction, thereby improving the comprehensive utilization rate of mulberry resources.
3. The emulsion prepared by the invention can improve the thermal stability of anthocyanin.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The preparation method of the mulberry leaf protein-mulberry anthocyanin composite emulsion specifically comprises the following steps:
(1) Vacuum freeze drying folium Mori, pulverizing, and sieving with 40 mesh sieve to obtain folium Mori powder. Weighing 20g of mulberry leaf powder, adding 800mL of NaOH solution (0.1 mol/L), carrying out ultrasonic treatment for 45min, carrying out ultrasonic power of 300W, carrying out first centrifugation at 40 ℃, collecting supernatant, adjusting the pH of the supernatant to 4.8, standing at 4 ℃ after flocculent precipitate is generated on protein, carrying out second centrifugation, collecting precipitate, washing the precipitate with 95% ethanol, carrying out redissolution with PBS, dialyzing for 1d in distilled water, and carrying out vacuum freeze-drying to obtain mulberry leaf protein.
(2) Collecting the supernatant of the second centrifugation in the step (1), regulating the pH to be neutral, concentrating to 100mL in vacuum, adding absolute ethyl alcohol, and standing at 4 ℃ to separate out the precipitate. Centrifuging to collect precipitate, dissolving with distilled water, slowly dripping onto treated DEAE-52 cellulose chromatographic column, eluting with gradient (0, 0.1, 0.3, 0.5 mol/L) NaCL solution at flow rate of 5mL/min, collecting one tube of 5mL, measuring polysaccharide content by phenol-sulfuric acid method, collecting eluate with high polysaccharide content eluting with 0.3mol/L NaCL solution according to peak value, loading onto SephadexG-100 column of dextran gel, eluting with flow rate of 5mL/min, collecting eluate, eluting with distilled water each tube of 5mL, collecting and combining eluate according to peak value, and vacuum freeze drying to obtain mulberry leaf polysaccharide.
(3) Pulping fresh mulberry by a high-speed refiner, taking 200g of mulberry pulp, adding 600mL of acidified ethanol, repeating ultrasonic extraction for 2 times, and extracting for 30min each time at the extraction temperature of 40 ℃ under the ultrasonic power of 300W. Mixing the extractive solutions, steaming to remove ethanol, vacuum filtering with Buchner funnel, loading onto chromatography column (diameter of 2.6cm and height of 60 cm) filled with AB-8 macroporous resin, eluting with 300mL and 300mL ethyl acetate sequentially to remove impurities after adsorption, eluting anthocyanin with 1.5L acidic ethanol (60%), collecting eluate, concentrating under reduced pressure to remove organic solvent, and vacuum lyophilizing to obtain Mori fructus anthocyanin extract.
(4) 1.5g of mulberry leaf protein is taken and dissolved in 50mL of phosphate buffer (0.02 mol/L), water bath is carried out for 10min at 80 ℃, magnetic stirring is carried out for 2h, 37.5mg of mulberry anthocyanin is added, magnetic stirring is carried out for 1h at room temperature, 10mL of soybean oil and 0.3g of mulberry leaf polysaccharide are added, stirring is carried out for 1h, and then homogenization is carried out for 3min at 12000r/min, thus obtaining emulsion.
Example 2
The preparation method of the mulberry leaf protein-mulberry anthocyanin composite emulsion specifically comprises the following steps:
(1) Vacuum freeze drying folium Mori, pulverizing, and sieving with 40 mesh sieve to obtain folium Mori powder. Weighing 20g of mulberry leaf powder, adding 600mL of NaOH solution (0.1 mol/L), carrying out ultrasonic treatment for 20min, carrying out ultrasonic treatment with power of 300W and temperature of 40 ℃, carrying out first centrifugation, collecting supernatant, adjusting pH of the supernatant to 4.8, standing at 4 ℃ after flocculent precipitate is generated on protein, carrying out second centrifugation, collecting precipitate, washing the precipitate with 95% ethanol, carrying out redissolution with PBS, dialyzing for 1d in distilled water, and carrying out vacuum freeze-drying to obtain mulberry leaf protein.
(2) Collecting the supernatant of the second centrifugation in the step (1), regulating the pH to be neutral, concentrating to 100mL in vacuum, adding absolute ethyl alcohol, and standing at 4 ℃ to separate out the precipitate. Centrifuging to collect precipitate, dissolving with distilled water, slowly dripping onto treated DEAE-52 cellulose chromatographic column, eluting with gradient (0, 0.1, 0.3, 0.5 mol/L) NaCL solution at flow rate of 5mL/min, collecting one tube of 5mL, measuring polysaccharide content by phenol-sulfuric acid method, collecting eluate with high polysaccharide content eluting with 0.3mol/L NaCL solution according to peak value, loading onto SephadexG-100 column of dextran gel, eluting with flow rate of 5mL/min, collecting eluate, eluting with distilled water each tube of 5mL, collecting and combining eluate according to peak value, and vacuum freeze drying to obtain mulberry leaf polysaccharide.
(3) Pulping fresh mulberry by a high-speed refiner, taking 200g of mulberry pulp, adding 400mL of acidified ethanol, repeating ultrasonic extraction for 2 times, and extracting for 20min each time at the extraction temperature of 40 ℃ under the ultrasonic power of 300W. Mixing the extractive solutions, steaming to remove ethanol, vacuum filtering with Buchner funnel, loading onto chromatography column (diameter of 2.6cm and height of 60 cm) filled with AB-8 macroporous resin, eluting with 300mL and 300mL ethyl acetate sequentially to remove impurities after adsorption, eluting anthocyanin with 1.5L acidic ethanol (60%), collecting eluate, concentrating under reduced pressure to remove organic solvent, and vacuum lyophilizing to obtain Mori fructus anthocyanin extract.
(4) 1.5g of mulberry leaf protein is taken and dissolved in 50mL of phosphate buffer (0.02 mol/L), water bath is carried out for 10min at 80 ℃, magnetic stirring is carried out for 2h, 37.5mg of mulberry anthocyanin is added, magnetic stirring is carried out for 1h at room temperature, 10mL of soybean oil and 0.3g of mulberry leaf polysaccharide are added, stirring is carried out for 1h, and then homogenization is carried out for 3min at 12000r/min, thus obtaining emulsion.
Example 3
The preparation method of the mulberry leaf protein-mulberry anthocyanin composite emulsion specifically comprises the following steps:
(1) Vacuum freeze drying folium Mori, pulverizing, and sieving with 40 mesh sieve to obtain folium Mori powder. Weighing 20g of mulberry leaf powder, adding 1000mL of NaOH solution (0.1 mol/L), carrying out ultrasonic treatment for 60min, carrying out ultrasonic power of 300W, carrying out first centrifugation at 40 ℃, collecting supernatant, adjusting the pH of the supernatant to 4.8, standing at 4 ℃ after flocculent precipitate is generated on protein, carrying out second centrifugation, collecting precipitate, washing the precipitate with 95% ethanol, carrying out redissolution with PBS, dialyzing for 1d in distilled water, and carrying out vacuum freeze-drying to obtain mulberry leaf protein.
(2) Collecting the supernatant of the second centrifugation in the step (1), regulating the pH to be neutral, concentrating to 100mL in vacuum, adding absolute ethyl alcohol, and standing at 4 ℃ to separate out the precipitate. Centrifuging to collect precipitate, dissolving with distilled water, slowly dripping onto treated DEAE-52 cellulose chromatographic column, eluting with gradient (0, 0.1, 0.3, 0.5 mol/L) NaCL solution at flow rate of 5mL/min, collecting one tube of 5mL, measuring polysaccharide content by phenol-sulfuric acid method, collecting eluate with high polysaccharide content eluting with 0.3mol/L NaCL solution according to peak value, loading onto SephadexG-100 column of dextran gel, eluting with flow rate of 5mL/min, collecting eluate, eluting with distilled water each tube of 5mL, collecting and combining eluate according to peak value, and vacuum freeze drying to obtain mulberry leaf polysaccharide.
(3) Pulping fresh mulberry by a high-speed refiner, taking 200g of mulberry pulp, adding 1000mL of acidified ethanol, repeating ultrasonic extraction for 2 times, and extracting at 40 ℃ for 60min each time under the ultrasonic power of 300W. Mixing the extractive solutions, steaming to remove ethanol, vacuum filtering with Buchner funnel, loading onto chromatography column (diameter of 2.6cm and height of 60 cm) filled with AB-8 macroporous resin, eluting with 300mL and 300mL ethyl acetate sequentially to remove impurities after adsorption, eluting anthocyanin with 1.5L acidic ethanol (60%), collecting eluate, concentrating under reduced pressure to remove organic solvent, and vacuum lyophilizing to obtain Mori fructus anthocyanin extract.
(4) 1.5g of mulberry leaf protein is taken and dissolved in 50mL of phosphate buffer (0.02 mol/L), water bath is carried out for 10min at 80 ℃, magnetic stirring is carried out for 2h, 37.5mg of mulberry anthocyanin is added, magnetic stirring is carried out for 1h at room temperature, 10mL of soybean oil and 0.3g of mulberry leaf polysaccharide are added, stirring is carried out for 1h, and then homogenization is carried out for 3min at 12000r/min, thus obtaining emulsion.
Comparative example 1
Step (2) does not prepare mulberry leaf polysaccharide, step (4) does not add mulberry leaf polysaccharide, and the other steps are the same as in example 1.
Comparative example 2
The mulberry leaf protein of step (4) was replaced with a commercially available soybean protein, and the other steps were the same as in example 1.
Comparative example 3
Step (2) does not prepare mulberry leaf polysaccharide, step (4) does not add mulberry leaf polysaccharide, and the mulberry leaf protein in step (4) is replaced by commercial soybean protein, and other steps are the same as in example 1.
Performance testing
The stability index and the heat stability of the mulberry anthocyanin were measured for the emulsions of example 1 and comparative examples 1-3, and the specific steps were as follows:
(1) Zeta potential measurement: samples were diluted 100-fold with phosphate buffer (10 mmol/L, ph=7). The Zeta potential of the emulsion was measured using a Zeta potentiometer at 25℃for 2min and 3 times for each sample, and the average value was taken.
(2) Determination of emulsion Activity and emulsion stability: the emulsion sample was diluted 100-fold with SDS solution (0.1%), absorbance at the initial state and absorbance after 10min of standing were measured with an ultraviolet spectrophotometer, colorimetry was performed at 500nm, and the emulsion activity index (m 2/g) and emulsion stability index (min) were calculated according to the formula.
(3) Anthocyanin thermostability: the mulberry juice emulsion (50% of mulberry juice and 50% of emulsion sample) added with 0.05mol/L of organic acid is respectively placed in a constant-temperature water bath at 80 ℃ for 7 hours, the maximum absorbance is measured every 1 hour, a-ln (ct/c 0) -time curve is drawn, and the thermal degradation half-life period t1/2 of anthocyanin is calculated.
The measurement results are shown in Table 1:
TABLE 1 stability index and Mulberry anthocyanin thermal stability of emulsions of example 1 and comparative examples 1-3
| Project | Example 1 | Comparative example 1 | Comparative example 2 | Comparative example 3 |
| Zeta potential | -36.67 | -31.10 | -34.00 | -30.33 |
| Emulsifying Activity index (m) 2 /g) | 74.14 | 63.43 | 74.26 | 65.52 |
| Emulsion stability index (min) | 91.25 | 80.12 | 84.15 | 75.25 |
| Half-life of thermal degradation t 1/2 (h) | 3.44 | 2.92 | 3.28 | 2.87 |
As is clear from the above table, the emulsion prepared in example 1 has the best emulsion stability and anthocyanin thermal stability, and the emulsion activity index is similar to that of comparative example 2. Namely, the mulberry protein, the mulberry polysaccharide and the mulberry anthocyanin are prepared into the emulsion, so that the stability of the whole emulsion system is effectively improved, and meanwhile, the heat stability of the mulberry anthocyanin is effectively improved.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. The preparation method of the mulberry leaf protein-anthocyanin composite emulsion is characterized by comprising the following steps of:
(1) Vacuum freeze drying folium Mori, pulverizing, and sieving with 40 mesh sieve to obtain folium Mori powder; adding 0.1mol/L NaOH solution into mulberry leaf powder, performing ultrasonic extraction, centrifuging for the first time, and collecting supernatant; regulating the pH of the supernatant to 4.8, standing at 4 ℃ after flocculent precipitate is generated on protein, centrifuging for the second time, and collecting the precipitate and supernatant for later use; washing the precipitate with 95% ethanol, redissolving with PBS, dialyzing in distilled water for 1d, and vacuum freeze drying to obtain mulberry leaf protein;
(2) Collecting the supernatant of the second centrifugation in the step (1), regulating the pH to be neutral, concentrating in vacuum, adding absolute ethyl alcohol, standing at 4 ℃ to separate out a precipitate, and centrifugally collecting the precipitate; dissolving the precipitate with distilled water, slowly dripping onto a treated DEAE-52 cellulose chromatographic column, performing gradient elution with NaCL solution, collecting the component with highest polysaccharide content, loading onto a SephadexG-100 sephadex column, eluting with distilled water, collecting and mixing eluates according to peak value, and vacuum freeze-drying to obtain mulberry leaf polysaccharide;
(3) Pulping fresh mulberry with a high-speed refiner, adding acidified ethanol, performing ultrasonic extraction, repeating the extraction for 2 times, and combining the extracting solutions; removing ethanol from the extractive solution by rotary evaporation, suction filtering with Buchner funnel, loading onto chromatographic column filled with AB-8 macroporous resin, eluting with distilled water and ethyl acetate to remove impurities, eluting anthocyanin with 60% acidic ethanol, collecting eluate, concentrating under reduced pressure to remove organic solvent, and vacuum freeze drying to obtain Mori fructus anthocyanin extract;
(4) Dissolving mulberry leaf protein in phosphate buffer solution, magnetically stirring for 2 hours after heat treatment, then adjusting the pH to 7.0, adding the mulberry anthocyanin extract, magnetically stirring for 1 hour at room temperature, adding soybean oil and mulberry leaf polysaccharide, stirring for 1 hour, and finally homogenizing to prepare emulsion.
2. The method for preparing a mulberry leaf protein-anthocyanin composite emulsion according to claim 1, wherein in the step (1), the feed liquid ratio of mulberry leaf to NaOH solution is g: mL = 1:30-50; the ultrasonic power is 300W, the extraction temperature is 40 ℃, and the extraction time is 20-60min.
3. The method for preparing a mulberry leaf protein-anthocyanin composite emulsion according to claim 2, wherein in the step (1), the feed liquid ratio of mulberry leaf to NaOH solution is g: mL = 1:40; the ultrasonic extraction time is 45min.
4. The method for preparing a mulberry leaf protein anthocyanin composite emulsion of claim 1 wherein in step (2) the volume of vacuum concentration is 1/10 to 20 of the initial volume.
5. The method for preparing a mulberry leaf protein-anthocyanin composite emulsion of claim 4, wherein in the step (2), the gradient concentration of the NaCL solution in the gradient elution is 0mol/L, 0.1mol/L, 0.3mol/L and 0.5mol/L;
the component with the highest polysaccharide content is eluent eluted by 0.3mol/L NaCL solution.
6. The method for preparing a mulberry leaf protein-anthocyanin composite emulsion of claim 1 wherein in step (3), the acidified ethanol is an ethanol solution containing 0.1% citric acid;
the ratio of mulberry pulp to acidified ethanol feed liquid is g: mL = 1:2-5; the ultrasonic power is 300W, the extraction temperature is 40 ℃, and the extraction time is 20-60min.
7. The method for preparing a mulberry leaf protein-anthocyanin composite emulsion of claim 6, wherein in the step (3), the ratio of mulberry pulp to acidified ethanol feed solution is g: mL = 1:3; the ultrasonic extraction time is 30min.
8. The method according to claim 7, wherein in the step (3), the distilled water used for the macroporous resin elution is 1 time of the column volume, the ethyl acetate is 1 time of the column volume, and the 60% acidified ethanol is 5 times of the column volume.
9. The method for preparing a mulberry leaf protein-anthocyanin composite emulsion of claim 1 wherein in step (4), the concentration of the phosphate buffer is 0.02mol/L; the mass concentration of the mulberry leaf protein is 30mg/mL, the volume ratio of the mulberry leaf protein solution to the soybean oil is 5:1, the mass ratio of the mulberry leaf protein to the anthocyanin is 40:1, and the mass ratio of the mulberry leaf protein to the mulberry leaf polysaccharide is 5:1; the heat treatment is carried out in a water bath at 80 ℃ for 10min, the homogenization speed is 12000r/min, and the time is 3min.
10. The mulberry leaf protein-anthocyanin composite emulsion prepared by the preparation method of the mulberry leaf protein-anthocyanin composite emulsion of claim 1.
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