CN112106988A - Method for embedding and stabilizing carotene by taking egg lysozyme amyloid fiber as material - Google Patents
Method for embedding and stabilizing carotene by taking egg lysozyme amyloid fiber as material Download PDFInfo
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- carotene
- egg white
- amyloid fiber
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- white lysozyme
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- UPYKUZBSLRQECL-UKMVMLAPSA-N Lycopene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1C(=C)CCCC1(C)C)C=CC=C(/C)C=CC2C(=C)CCCC2(C)C UPYKUZBSLRQECL-UKMVMLAPSA-N 0.000 title claims abstract description 56
- 102000016943 Muramidase Human genes 0.000 title claims abstract description 56
- 108010014251 Muramidase Proteins 0.000 title claims abstract description 56
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 title claims abstract description 56
- 150000001746 carotenes Chemical class 0.000 title claims abstract description 56
- 235000005473 carotenes Nutrition 0.000 title claims abstract description 56
- 239000004325 lysozyme Substances 0.000 title claims abstract description 56
- 229960000274 lysozyme Drugs 0.000 title claims abstract description 56
- 235000010335 lysozyme Nutrition 0.000 title claims abstract description 56
- NCYCYZXNIZJOKI-UHFFFAOYSA-N vitamin A aldehyde Natural products O=CC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C NCYCYZXNIZJOKI-UHFFFAOYSA-N 0.000 title claims abstract description 56
- 239000000835 fiber Substances 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 9
- 239000000463 material Substances 0.000 title claims abstract description 6
- 230000000087 stabilizing effect Effects 0.000 title claims description 4
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims abstract description 42
- 102000002322 Egg Proteins Human genes 0.000 claims abstract description 42
- 108010000912 Egg Proteins Proteins 0.000 claims abstract description 42
- 235000014103 egg white Nutrition 0.000 claims abstract description 42
- 210000000969 egg white Anatomy 0.000 claims abstract description 42
- 239000000839 emulsion Substances 0.000 claims description 26
- 239000000243 solution Substances 0.000 claims description 22
- 230000014759 maintenance of location Effects 0.000 claims description 13
- 239000012224 working solution Substances 0.000 claims description 12
- OWMVSZAMULFTJU-UHFFFAOYSA-N bis-tris Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 claims description 9
- 238000005303 weighing Methods 0.000 claims description 8
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 claims description 7
- 229910001447 ferric ion Inorganic materials 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 6
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 6
- 239000012498 ultrapure water Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 230000031700 light absorption Effects 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 238000000502 dialysis Methods 0.000 claims description 3
- 239000006185 dispersion Substances 0.000 claims description 3
- 230000007613 environmental effect Effects 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 239000012452 mother liquor Substances 0.000 claims description 3
- 239000010413 mother solution Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 2
- 102000001049 Amyloid Human genes 0.000 claims 2
- 108010094108 Amyloid Proteins 0.000 claims 2
- 239000003814 drug Substances 0.000 abstract description 2
- 238000010438 heat treatment Methods 0.000 abstract description 2
- 230000002378 acidificating effect Effects 0.000 abstract 1
- 230000001804 emulsifying effect Effects 0.000 abstract 1
- 235000013402 health food Nutrition 0.000 abstract 1
- 238000005286 illumination Methods 0.000 abstract 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 abstract 1
- 229910052742 iron Inorganic materials 0.000 abstract 1
- 231100000956 nontoxicity Toxicity 0.000 abstract 1
- 238000004321 preservation Methods 0.000 abstract 1
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 4
- 235000013305 food Nutrition 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 102000006395 Globulins Human genes 0.000 description 2
- 108010044091 Globulins Proteins 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 208000002177 Cataract Diseases 0.000 description 1
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 102000034240 fibrous proteins Human genes 0.000 description 1
- 108091005899 fibrous proteins Proteins 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- 150000002634 lipophilic molecules Chemical class 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 229910052723 transition metal Inorganic materials 0.000 description 1
- 150000003624 transition metals Chemical class 0.000 description 1
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- 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
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- 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
- A23L29/03—Organic compounds
- A23L29/045—Organic compounds containing nitrogen as heteroatom
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
- A23P10/00—Shaping or working of foodstuffs characterised by the products
- A23P10/30—Encapsulation of particles, e.g. foodstuff additives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/01—Hydrocarbons
- A61K31/015—Hydrocarbons carbocyclic
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
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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 relates to a method for embedding carotene and improving the stability of carotene by using egg white lysozyme amyloid fibers as a material. Heating at high temperature under acidic condition to obtain egg white lysozyme amyloid fiber, and emulsifying and embedding carotene in the amyloid fiber. The preservation rate of carotene in ultraviolet illumination, high temperature and iron ion environment is mainly detected by a spectrophotometer method, and the stability of the carotene emulsified and embedded by the amyloid fiber is obviously improved. The material for embedding carotene in the invention is easy to produce, has low cost, no toxicity and small using amount, obviously improves the stability of the embedded carotene and has wide application prospect in the health food and medicine industries.
Description
Technical Field
The invention relates to the technical field of preparation of egg white lysozyme amyloid fiber solution, embedding of carotene and detection of carotene stability.
Background
Carotene is a substance with physiological activity, and has important effects in resisting oxidation, resisting cancer, enhancing immunity, preventing cardiovascular diseases, preventing and treating cataract, and protecting liver. However, as lipophilic compounds, carotene is very susceptible to degradation caused by heat, ultraviolet rays, transition metals, oxygen and free radicals, which greatly limits the application range in the food and pharmaceutical industries. In recent years, embedding carotene in emulsion to improve the stability, water solubility and bioavailability of fat-soluble carotene has become one of the research hotspots in the field of food technology. The high internal phase emulsion has great application prospect in the embedding load of functional substances due to the high internal phase characteristic. A large number of low-molecular or high-molecular surfactants are commonly used for stabilizing emulsions, but the surfactants have certain biotoxicity and are not suitable for being applied to the food and medicine industries. Accordingly, there has been increasing interest in the study of the stabilization of emulsions with macromolecular food-grade materials of the protein type, and much research has been done on globulin and globulin aggregates in the high internal phase emulsion field, but fibrous protein aggregates have not been reported. The egg white lysozyme used in the invention is derived from egg white, and has good biocompatibility and safety after being converted into amyloid fiber.
Disclosure of Invention
The invention aims to provide a method for embedding carotene and improving the stability of carotene after egg white lysozyme is converted into amyloid fiber.
The technical scheme of the method is as follows:
(1) the preparation of lysozyme amyloid fiber is characterized in that: the egg white lysozyme is dialyzed and purified by ultrapure water at 4 ℃, and then the lysozyme solution after dialysis is subjected to freeze-drying treatment. Weighing a certain mass of egg white lysozyme freeze-dried powder, dissolving the egg white lysozyme freeze-dried powder in ultrapure water to prepare a 2 wt% egg white lysozyme solution, adjusting the pH to 2 by using 2M HCl, and magnetically stirring and incubating for 8 hours at 90 ℃ to prepare an egg white lysozyme amyloid fiber mother solution. Egg white lysozyme amyloid fiber mother liquor (2 wt%, pH2) and Bis-Tris buffer solution (10mM, pH7.2) are diluted according to the volume ratio of 1: 3 to obtain egg white lysozyme amyloid fibers with the pH of 6. Under the condition that the volume ratio of egg white lysozyme amyloid fiber solution (2 wt%, pH2) to Bis-Tris buffer solution is the same, pH2 deionized water without egg white lysozyme amyloid fiber is mixed with Bis-Tris buffer solution to prepare corresponding blank Working Solution (WS). Diluting the egg white lysozyme amyloid fiber working solution with a corresponding blank working solution to prepare samples with the same pH value and different concentrations.
(2) The preparation of the embedding carotene lysozyme amyloid fiber emulsion is characterized in that: weighing a certain mass of carotene, dissolving the carotene in oil, mixing the egg white lysozyme amyloid fibers obtained in the step (1) with the oil solution according to a certain volume ratio, and dispersing for 2 minutes at 10000rpm by using a dispersion machine to form emulsion;
(3) the retention rate of the lysozyme amyloid fiber emulsion embedded with carotene is measured under the conditions of ultraviolet light, high temperature and ferric ion, and the method is characterized in that: and (3) standing the emulsion for half an hour, and treating the emulsion for 4-6 hours under different environmental conditions (ultraviolet light, high temperature and ferric ions). Extracting carotene in different samples, measuring the light absorption value by an ultraviolet spectrophotometer under the condition of 450nm, comparing carotene standard curves, and calculating the retention rate of the carotene. After 4-6 hours of treatment, the retention rate of carotene in the emulsion with stable egg white lysozyme amyloid fiber is obviously higher than that of carotene directly dissolved in oil.
The invention has the following advantages;
the amyloid fiber is applied to the high internal phase emulsion for the first time, and the loading capacity of the carotene is greatly improved.
The retention rate of carotene in the emulsion stabilized by egg white lysozyme amyloid fiber is obviously higher than that of carotene directly dissolved in oil.
Drawings
FIG. 1 is a graph showing the change of the carotene retention rate under UV light for 4 hours
FIG. 2 is a graph showing the change in the retention of carotene under heating at 90 ℃ for 6 hours
FIG. 3 is a graph showing the change in the retention of carotene in the presence of ferric chloride for 6 hours
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are further described below with reference to the embodiments of the present invention. The described embodiments are some, but not all embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The first embodiment is as follows:
(1) the egg white lysozyme is dialyzed and purified by ultrapure water at 4 ℃, and then the lysozyme solution after dialysis is subjected to freeze-drying treatment. Weighing a certain mass of egg white lysozyme freeze-dried powder, dissolving the egg white lysozyme freeze-dried powder in ultrapure water to prepare a 2 wt% egg white lysozyme solution, adjusting the pH to 2 by using 2M HCl, and magnetically stirring and incubating for 8 hours at 90 ℃ to prepare an egg white lysozyme amyloid fiber mother solution. The egg white lysozyme amyloid fiber mother liquor (2 wt%, pH2) and Bis-Tris buffer solution (10mM, pH7.2) are diluted according to the volume ratio of 1: 3 to obtain the egg white lysozyme amyloid fiber with the pH of 6. Under the condition that the volume ratio of egg white lysozyme amyloid fiber solution (2 wt%, pH2) to Bis-Tris buffer solution is the same, pH2 deionized water without egg white lysozyme amyloid fiber is mixed with Bis-Tris buffer solution to prepare corresponding blank Working Solution (WS). The egg white lysozyme amyloid fiber working solution is diluted by one time with a blank working solution to prepare 0.25 wt% lysozyme amyloid fiber with the pH value of 6.
(2) Weighing a certain mass of carotene, dissolving the carotene in oil, mixing the egg white lysozyme amyloid fibers obtained in the step (1) with the oil solution according to the volume ratio of 1: 3, and dispersing for 2 minutes at 10000rpm by using a dispersion machine to form a high internal phase emulsion with the carotene content of 0.01 wt% and the oil phase volume ratio of 75%;
(3) and (3) standing the emulsion for half an hour, and treating the emulsion for 4-6 hours under different environmental conditions (ultraviolet light, high temperature and ferric ions). Extracting carotene in different samples, measuring the light absorption value by an ultraviolet spectrophotometer under the condition of 450nm, comparing carotene standard curves, and calculating the retention rate of the carotene. After 4-6 hours of treatment, the retention rate of carotene in the emulsion with stable egg white lysozyme amyloid fiber is obviously higher than that of carotene directly dissolved in oil.
Example two:
(1) determination of Carotene marker
Weighing a certain amount of carotene, dissolving the carotene in oil, preparing into different concentrations with concentration gradients of 0.1 mu g/mL, 1 mu g/mL, 2.5 mu g/mL, 5 mu g/mL and 10 mu g/mL, measuring corresponding absorbance values by using an ultraviolet spectrophotometer under the wavelength of 450nm, drawing a curve, and fitting an equation.
(2) Carotene extraction
Weighing a certain mass of emulsion, adding 0.5mL of an extracting agent (ethanol: n-hexane is 2: 3), uniformly mixing by vortex, centrifuging at 12000rpm for 1 minute, taking supernate, adding n-hexane with proper volume for dilution, and detecting the light absorption value under the condition of 450nm by using a spectrophotometer.
As can be seen from figures 1 to 3, the emulsion-embedded carotene using egg white lysozyme amyloid fiber as an emulsifier has significantly improved stability under the conditions of ultraviolet light, high temperature of 90 ℃ and ferric ions.
Claims (4)
1. A method for embedding and stabilizing carotene by taking egg white lysozyme amyloid fiber as a material is characterized by comprising the following operation steps: preparing lysozyme amyloid fibers; preparing an embedding carotene lysozyme amyloid fiber emulsion; and (3) determining the retention rate of the lysozyme amyloid fiber emulsion embedding the carotene under the conditions of ultraviolet light, high temperature and ferric ions.
2. Preparation of lysozyme amyloid fiber according to claim 1, characterized in that: the egg white lysozyme is dialyzed and purified by ultrapure water at 4 ℃, and then the lysozyme solution after dialysis is subjected to freeze-drying treatment. Weighing a certain mass of egg white lysozyme freeze-dried powder, dissolving the egg white lysozyme freeze-dried powder in ultrapure water to prepare a 2 wt% egg white lysozyme solution, adjusting the pH to 2 by using 2M HCl, and magnetically stirring and incubating for 8 hours at 90 ℃ to prepare an egg white lysozyme amyloid fiber mother solution. Egg white lysozyme amyloid fiber mother liquor (2 wt%, pH2) and Bis-Tris buffer solution (10mM, pH7.2) are diluted according to the volume ratio of 1: 3 to obtain egg white lysozyme amyloid fibers with the pH of 6. The corresponding blank Working Solution (WS) was prepared by mixing pH2 deionized water without HEWL amyloid fibrils with Bis-Tris buffer at the same volume ratio of HEWL amyloid fibril solution (2 wt%, pH2) to Bis-Tris buffer. And (3) diluting the HEWL amyloid fiber working solution and the corresponding blank working solution to prepare samples with the same pH value and different concentrations.
3. The preparation of the emulsion of embedded carotene lysozyme amyloid fiber according to claim 1, characterized in that: weighing a certain mass of carotene, dissolving the carotene in oil, mixing the egg white lysozyme amyloid fibers obtained in the step (1) with the oil solution according to a certain volume ratio, and dispersing for 2min at 10000rpm by using a dispersion machine to form emulsion.
4. The method for measuring the retention rate of the carotene-embedded lysozyme amyloid fiber emulsion in the conditions of ultraviolet light, high temperature and ferric ions according to claim 1 is characterized in that: and (3) standing the emulsion for half an hour, and treating the emulsion for 4-6 hours under different environmental conditions (ultraviolet light, high temperature and ferric ions). Extracting carotene in different samples, measuring the light absorption value by an ultraviolet spectrophotometer under the condition of 450nm, comparing carotene standard curves, and calculating the retention rate of the carotene. After 4-6 h of treatment, the retention rate of carotene contained in the emulsion with stable egg white lysozyme amyloid fiber is obviously higher than that of carotene directly dissolved in oil.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108641103A (en) * | 2018-04-10 | 2018-10-12 | 华南理工大学 | Phase emulsion oil-in-water and preparation method in a kind of protein stabilized height |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108641103A (en) * | 2018-04-10 | 2018-10-12 | 华南理工大学 | Phase emulsion oil-in-water and preparation method in a kind of protein stabilized height |
Non-Patent Citations (3)
| Title |
|---|
| YAN-TENG XU，等: "Ovalbumin as an Outstanding Pickering Nano-stabilizer for High Internal Phase Emulsions", 《J. AGRIC. FOOD CHEM.》 * |
| 朱连昌等: "大豆伴球蛋白自组装纤维的乳化性质", 《现代食品科技》 * |
| 胡青莲，等: "卵清溶菌酶淀粉样纤维形成的研究", 《生物学杂志》 * |
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