CN109965282B - Functional enhancement type Pickering emulsion constructed by utilizing black tea nano aggregate and nano selenium as well as preparation method and application thereof - Google Patents
Functional enhancement type Pickering emulsion constructed by utilizing black tea nano aggregate and nano selenium as well as preparation method and application thereof Download PDFInfo
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- CN109965282B CN109965282B CN201910100190.7A CN201910100190A CN109965282B CN 109965282 B CN109965282 B CN 109965282B CN 201910100190 A CN201910100190 A CN 201910100190A CN 109965282 B CN109965282 B CN 109965282B
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- 244000269722 Thea sinensis Species 0.000 title claims abstract description 97
- 239000000839 emulsion Substances 0.000 title claims abstract description 74
- 235000006468 Thea sinensis Nutrition 0.000 title claims abstract description 70
- 235000020279 black tea Nutrition 0.000 title claims abstract description 70
- 239000011669 selenium Substances 0.000 title claims abstract description 66
- 229910052711 selenium Inorganic materials 0.000 title claims abstract description 65
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 title claims abstract description 48
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 9
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- 229910021641 deionized water Inorganic materials 0.000 description 1
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 description 1
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- 125000002924 primary amino group Chemical group [H]N([H])* 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
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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Abstract
The invention belongs to the technical field of materials, and discloses a functional enhancement type Pickering emulsion constructed by black tea nano aggregate combined with nano selenium, and a preparation method and application thereof. The method comprises the following steps: (1) And separating black tea nanometer aggregates by ultrafiltration and centrifugation, wherein the average diameter is more than 10nm and the average diameter is less than or equal to 10 nm. (2) Constructing Pickering emulsion by using black tea nano aggregate and nano selenium: the black tea nano aggregate and nano selenium are taken as Pickering particles, the pH of a water dispersed phase is regulated, and the mixture is mixed with an oil phase, and stable Pickering emulsion is obtained after shearing homogenization and high-pressure homogenization. The invention adopts a black tea nano aggregate combined nano selenium composite system as a Pickering stabilizer to jointly construct the function-enhanced Pickering emulsion, widens the application of nano selenium and enhances the health efficacy of the Pickering emulsion.
Description
Technical Field
The invention belongs to the technical field of materials, and particularly relates to a functional enhancement type Pickering emulsion constructed by utilizing black tea nano aggregate and nano selenium, and a preparation method and application thereof.
Background
Compared with the traditional emulsion, the Pickering emulsion is a novel emulsion which uses solid particles with certain affinity/hydrophobicity to replace the traditional surfactant to be stable, and the solid particles are irreversibly adsorbed at an oil-water interface, so that the emulsion has high thermodynamic stability, and the Pickering emulsion has the advantages of less emulsifier consumption, low cost and low toxicity and has wide application in the fields of foods, cosmetics and the like.
The black tea water extract contains active components such as theabrownin, tea polyphenol, tea polysaccharide, protein, alkaloid and the like, and all the substances can spontaneously aggregate to form aggregates with the particle size of about 1-100 nm, and can be obtained through ultrafiltration and centrifugal separation. The black tea nano aggregate contains hydrophilic groups such as hydroxyl, carboxyl, amino and the like, and a hydrophobic region, and has certain hydrophilicity/hydrophobicity.
Selenium (Se) is one of 8 essential trace elements for the human body. The nano selenium is amorphous zero-valent element selenium with the average diameter within the range of 1-100 nm, and has the advantages of high oxidation resistance, anticancer, low toxicity and the like compared with other forms of selenium. At present, the tea water extract (CN 201710770329) and the tea nano aggregate (CN 201710771056) are successfully used for preparing nano selenium, and the tea water extract and the tea nano aggregate contain substances such as tea polyphenol, alkaloid, theanine and the like, and each substance is a substance basis of the tea for playing a health care effect, so that the functional characteristics of the nano selenium can be further enhanced. In application, nano-selenium is generally used as a selenium supplement, and in recent years, the prior patent (CN 201110215019) utilizes the combination of nano-selenium, a template and a medicament to prepare a multifunctional carrier, thereby promoting the application of nano-selenium in medicines such as cancer treatment and the like, and the researches provide a new idea for constructing the multifunctional carrier of food active components.
Disclosure of Invention
In order to overcome the defects and shortcomings in the prior art, the primary purpose of the invention is to provide a preparation method for constructing a function-enhanced Pickering emulsion by utilizing black tea nano aggregate and nano selenium; the black tea nano aggregate and nano selenium are used as Pickering stabilizers to prepare the function-enhanced Pickering emulsion.
The invention further aims at providing Pickering emulsion prepared by the preparation method.
It is a further object of the present invention to provide the use of a Pickering emulsion as described above.
The aim of the invention is achieved by the following technical scheme:
a preparation method for constructing a function-enhanced Pickering emulsion by using black tea nano aggregate and nano selenium comprises the following steps:
(1) Separating black tea nano aggregate;
(2) And preparing Pickering emulsion by using the black tea nano aggregate and nano selenium.
The black tea nano aggregate separation in the step (1) specifically comprises the following steps: adding a solvent with the temperature of 25-100 ℃ and the volume percentage concentration of 70-100% into the black tea raw material, wherein the dosage of the solvent is calculated by adding 10-250 mL of solvent into each g of black tea raw material; and leaching for 2-120 min under the water bath condition of 25-100 ℃ to obtain black tea extract, centrifuging for 10-40 min under the conditions of the temperature of 4 ℃ and the centrifugal force of 1000-4000 g by using a centrifugal ultrafiltration tube with the molecular weight cut-off of 3-100 kDa, collecting inner tubes and outer tubes to obtain black tea nano aggregate solutions with different particle size ranges, freeze-drying, and storing at-20 ℃ to obtain the black tea nano aggregate.
The raw material of said black tea is tea treeCamellia sinensis(L.)O.Kuntze]Fresh leaves and tender tips are used as raw materials, and products are prepared by the processes of enzyme deactivation, rolling, piling and drying, preferably Fuzhuan tea, qing brick tea, liupu tea, kang Zhuancha, pu' er tea or Qianliang tea.
The solvent is water, methanol or ethanol.
The dosage of the solvent is calculated by adding 50mL of solvent into each g of black tea raw material; the temperature of the water bath is 100 ℃; the leaching time is 30 min; the molecular weight cut-off of the centrifugal ultrafiltration tube is 100 kD; the centrifugal force is 4000g; the centrifugation time is 20 min; the obtained black tea nanometer aggregate is two colloidal particles with average diameter more than 10nm and average diameter less than or equal to 10 nm.
The preparation of Pickering emulsion by using black tea nano aggregate and nano selenium in the step (2) specifically comprises the following steps: preparing 5-20 times of nano selenium diluent, and dissolving the black tea nano aggregate by using the nano selenium diluent to enable the mass percentage of the black tea nano aggregate to be 0.5% -6.0%; adding sodium azide with the mass fraction of 0.02%, adjusting the pH of a black tea nano aggregate disperse phase to 2-11, adding an oil phase according to the volume ratio of oil to water of 1:9-6:4, shearing and homogenizing at a high speed of 10000-20000 r/min for 1-2 min, and homogenizing at a high pressure of 40MPa for 1-3 times to obtain Pickering emulsion.
The nano-selenium is liquid phase nano-selenium prepared by a soft template method; the oil phase is water-insoluble.
The dilution multiple of the nano selenium diluent is 20 times; the mass percentage of the black tea nano aggregate is 2%; the pH value of the black tea nano aggregate disperse phase is 7; the volume ratio of the oil to the water is 4:6; the rotation speed of the high-speed shearing homogenization is 20000r/min, and the time is 2 min; the pressure of the high-pressure homogenization is 40MPa, and the homogenization times are 3 times.
Pickering emulsion prepared by the preparation method.
The Pickering emulsion is applied to the fields of foods, cosmetics and biological medicines.
Measuring the particle size of Pickering emulsion liquid drops by a laser light scattering technology, and evaluating the size of the liquid drops of the Pickering emulsion; the Zeta potential of the Pickering emulsion system is measured by a laser Doppler velocimetry technology, the electrostatic stability of the Pickering emulsion obtained by the invention is evaluated, and the microstructure of emulsion liquid drops is observed by an optical microscope.
The tumor cell survival rate is detected by MTT method, and the anticancer activity of the nano-selenium obtained by the invention is evaluated.
The tumor cell model comprises: HCT116 cell line, hepa1c1c7 cell line, MDA-MB-231 cell line, hepG2 cell line, hela cell line, etc., preferably HCT116 cell line.
Compared with the prior art, the invention has the following advantages and beneficial effects: the invention adopts black tea nano aggregate and nano selenium as Pickering stabilizer to jointly construct the function-enhanced Pickering emulsion, widens the application of nano selenium and enhances the health efficacy of the Pickering emulsion.
Drawings
Fig. 1 is a particle size distribution of black tea nano-aggregates (average diameter >10 nm) in combination with nano-selenium Pickering emulsion droplets.
FIG. 2 is a particle size distribution of black tea nano-aggregates (average diameter. Ltoreq.10 nm) combined with nano-selenium Pickering emulsion droplets.
FIG. 3 shows the Zeta potential of dark tea nanoclusters (average diameter >10 nm and average diameter. Ltoreq.10 nm) combined with a nano-selenium Pickering emulsion system.
FIG. 4 shows the microstructure of a black tea nano-aggregate (average diameter >10 nm and average diameter. Ltoreq.10 nm) combined with nano-selenium Pickering emulsion.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
In order to make the purpose and the technical scheme of the invention clearer and more clear, the invention is further described in detail by taking black tea nano aggregate combined tea-nano selenium as a stabilizer for example. The equipment and reagents used in the present invention are all commonly used in the art. It should be understood that the examples described herein are for illustrative purposes only and are not intended to limit the present invention.
Example 1: pickering emulsion prepared from black tea nano aggregate and nano selenium
(1) Accurately weighing 4.5 g ground tea leaves, adding 225mL of ultra-pure water with the temperature of 100 ℃, leaching in a water bath with the temperature of 100 ℃ for 30min, shaking once every 10 min, and carrying out suction filtration with quantitative filter paper and constant volume after leaching to obtain tea extract; and (3) freeze-drying the tea extract to obtain freeze-dried powder, namely the tea extract (black tea extract takes black tea as a leaching raw material).
(2) Transferring the tea extract obtained in the step (1) of 4 mL into an inner tube of a 100 kDa ultrafiltration tube, and centrifuging at 4 ℃ and 4000g for 20 min; collecting inner/outer tube solution to obtain tea nanometer aggregate solution with different particle size ranges (average diameter >10 nm and average diameter less than or equal to 10 nm), and lyophilizing to obtain lyophilized powder, which is tea nanometer aggregate, to be used (black tea nanometer aggregate uses black tea as raw material).
(3) Respectively preparing 100 mM sodium selenite solution and 500 mM vitamin C solution. Fixing Na in reaction system 2 SeO 3 Concentration of 20 mmol/L, vc and Na 2 SeO 3 The ratio is 8:1 (20 mM:160 mM), the tea water extract prepared by the same method in the step (1) or the tea nano aggregate prepared by the same method in the step (2) is used as a template, the addition amount is 500 mg/L, the mixture is kept stand and reacts for 1 h under the water bath condition of 40 ℃, the mixture is centrifuged for 30min at the temperature of 4 ℃ and the speed of 11000 r/min, the mixture is repeated for 3 times, and the mixture is resuspended in the ultra-pure water with the same volume to obtain the nano selenium colloid solution.
(4) Fixing the dilution multiple of the nano selenium colloid solution to be 20 times of the volume, wherein the addition quantity of the black tea nano aggregate is 2% by mass, adding sodium azide with the mass fraction of 0.02%, respectively adjusting the pH of a water phase to be 7 by using 1 mol/L HCl or 1 mol/L NaOH, keeping away from light, standing overnight (12-14 h), adding soybean oil (20 mL:30 mL) according to the volume ratio of oil to water of 4:6, emulsifying for 2 min at a high speed of 20000r/min, homogenizing for 3 times under a high pressure of 40MPa, measuring the droplet size of the Pickering emulsion, and the Zeta potential value of the system, observing the microstructure of the droplet, wherein the result is shown in the figure 1-4, and the stability of the nano aggregate (average diameter of more than 10 nm) combined nano selenium Pickering emulsion of the black tea nano aggregate is higher than that of the nano aggregate (average diameter of less than or equal to 10 nm).
Example 2: emulsion droplet size and particle size distribution determination
The particle size and particle size distribution of the emulsion after the novel preparation or storage of the invention are measured by using a Markov 3000 laser particle sizer, and deionized water and 1% SDS are respectively used as dispersing agents. The relative refractive index of the emulsion was set to 1.095, which is the ratio of the refractive index of soybean oil (1.456) to the refractive index of water (1.33). The particle size of the emulsion is indicated as d4,3 (volume weighted average particle size). The results show that: the particle size of the black tea nano aggregate (average diameter is more than 10 nm) combined nano selenium Pickering emulsion liquid drops is 2.93+/-0.02 mu m, and the particle distribution is concentrated and mainly comprises small particle liquid drops; the particle size of the nano-selenium Pickering emulsion liquid drops used for the black tea nano-aggregate (the average diameter is less than or equal to 10 nm) is 8.56+/-0.06 mu m, the particle distribution is wide, and the large particle liquid drops are the main.
Example 3: determination of the Electrostatic stability of emulsion systems
The emulsion Zeta potential is measured by adopting an LDV technology, and the electrostatic stability of the function-enhanced Pickering emulsion is built by utilizing black tea nano aggregate and nano selenium. The results show that: the Zeta potential value of the black tea nano aggregate (the average diameter is more than 10nm and the average diameter is less than or equal to 10 nm) combined nano selenium Pickering emulsion system is more than 40 mV, and the electrostatic stability of the system is high. Wherein, the Zeta potential value of the black tea nano aggregate (average diameter is more than 10 nm) combined nano selenium Pickering emulsion system is 48.1+/-1.3 mV, and the Zeta potential value of the black tea nano aggregate (average diameter is less than or equal to 10 nm) combined nano selenium Pickering emulsion system is 57.5+/-0.6 mV.
Example 4: observation of microstructure of emulsion droplets
The invention uses black tea nano aggregate and nano selenium to construct function-enhanced Pickering emulsion, respectively uses ultrapure water and 1% SDS to dilute the emulsion by 10 times, and uses an optical microscope to observe the size and the shape of emulsion liquid drops (adjusting the multiple of an objective lens, collecting data by a computer and taking a picture). The results show that: the black tea nano aggregate (average diameter is more than 10nm and average diameter is less than or equal to 10 nm) is spherical in combination with nano selenium Pickering emulsion liquid drops. Wherein, the black tea nanometer aggregate (average diameter is more than 10 nm) is combined with the nanometer selenium Pickering emulsion, the liquid drops are similar in size, small in liquid drops and more in number; the size of droplets of the nano aggregate (average diameter is less than or equal to 10 nm) combined nano selenium Pickering emulsion is large, and the droplets are large and the number is small. Consistent with the laser particle sizer results.
Example 5: determination of emulsion anticancer Activity
Single cell suspension prepared by trypsinization of adherent HCT116 cells/MDA-MB-231 at 5X 10 4 Inoculating 96-well culture plate into each cell/well, placing at 37deg.C, and 5% CO 2 The black tea nano aggregate and nano selenium are used for constructing the function-enhanced Pickering emulsion, the function-enhanced Pickering emulsion is diluted by 10 times, 20 times, 40 times, 80 times, 160 times and 320 times by a culture medium, and then the culture medium is added according to 100 mu L/hole for continuous culture of 48 h. 100. Mu.L/well of MTT diluent 0.25/mg/mL was added to the plates and placed at 37℃in 5% CO 2 Continuously culturing 2 h in an incubator, discarding supernatant, adding 200 mu L of DMSO solution into each well, placing in a shaking table of 100 r/min, shaking for 15-20 min, and measuring OD of each well 550 Value of OD in control group 550 The value was 100%, and the half Inhibitory Concentration (IC) of each group of nano-selenium for inhibiting cell proliferation was calculated 50 ) As shown in Table 1, wherein the dark tea nano-aggregate (average diameter is less than or equal to 10 nm) is combined with the nano-selenium Pickering emulsion to inhibit HCT116 cell proliferation more than the dark tea nano-aggregate (average diameter>10 nm) is combined with nano-selenium Pickering emulsion.
TABLE 1 dark tea nano-aggregate combination nano-selenium Pickering emulsion for inhibiting HCT116 cell proliferation
semi-Inhibitory Concentration (IC) 50 ）
| Dilution factor | |
| Emulsion (average diameter)>10 nm） | 63.40±5.49 |
| Emulsion (average diameter is less than or equal to 10 nm) | 46.85±4.93 |
(dark tea nanometer aggregate combined nanometer selenium Pickering emulsion in the concentration range, MDA-MB-231 cell survival rate is greater than 0.5.)
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (7)
1. The preparation method for constructing the function-enhanced Pickering emulsion by utilizing the black tea nano aggregate and the nano selenium is characterized by comprising the following steps of:
(1) Separating black tea nano aggregate:
adding water with the temperature of 25-100 ℃ and the volume percentage concentration of 70-100% into the black tea raw material, wherein the dosage of the solvent is calculated by adding 50mL of solvent into each g of black tea raw material; leaching for 30min under water bath condition of 100deg.C to obtain black tea extractive solution, centrifuging for 20min at 4deg.C with centrifugal force 4000g using centrifugal ultrafiltration tube with molecular weight cut-off of 100 kDa, collecting inner tube and outer tube to obtain black tea nanometer aggregate solution with different particle size ranges, lyophilizing, and storing at-20deg.C to obtain black tea nanometer aggregate;
(2) Preparing Pickering emulsion by using black tea nano aggregate and nano selenium:
preparing 20 times of nano selenium diluent, and dissolving black tea nano aggregate by using the nano selenium diluent to enable the mass percentage of the black tea nano aggregate to be 2%; adding sodium azide with the mass fraction of 0.02%, adjusting the pH of a black tea nano aggregate disperse phase to 7, adding an oil phase according to the volume ratio of oil to water of 4:6, shearing and homogenizing at a high speed of 20000r/min for 2 min, and homogenizing at a high pressure of 40MPa for 3 times to obtain Pickering emulsion.
2. The preparation method for constructing the function-enhanced Pickering emulsion by utilizing black tea nano aggregate and nano selenium according to claim 1, which is characterized by comprising the following steps of: the black tea raw material is a product prepared by taking fresh tea leaves and tender tips of tea trees as raw materials and processing the raw materials through the processes of deactivation of enzymes, rolling, piling and drying.
3. The preparation method for constructing the function-enhanced Pickering emulsion by utilizing black tea nano aggregate and nano selenium according to claim 1, which is characterized by comprising the following steps of: the black tea raw materials comprise Fuzhuan tea, qingdao tea, liupu tea, kang Zhuancha, pu' er tea or Qianliang tea.
4. The preparation method for constructing the function-enhanced Pickering emulsion by utilizing black tea nano aggregate and nano selenium according to claim 1, which is characterized by comprising the following steps of: the obtained black tea nanometer aggregate is two colloidal particles with average diameter more than 10nm and average diameter less than or equal to 10 nm.
5. The preparation method for constructing the function-enhanced Pickering emulsion by utilizing black tea nano aggregate and nano selenium according to claim 1, which is characterized by comprising the following steps of: the nano-selenium is liquid phase nano-selenium prepared by a soft template method; the oil phase is water-insoluble.
6. A Pickering emulsion prepared according to the method of any one of claims 1-5.
7. Use of a Pickering emulsion according to claim 6 in the cosmetic and biomedical fields.
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