CN112781958A - Method for representing fluidity of specific components of emulsion molecules - Google Patents
Method for representing fluidity of specific components of emulsion molecules Download PDFInfo
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- CN112781958A CN112781958A CN202110140491.XA CN202110140491A CN112781958A CN 112781958 A CN112781958 A CN 112781958A CN 202110140491 A CN202110140491 A CN 202110140491A CN 112781958 A CN112781958 A CN 112781958A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N11/00—Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
Abstract
The invention discloses a method for representing the fluidity of a specific component of an emulsion, which comprises the steps of firstly preparing a latticed lysine slide, mixing a single emulsion molecule with a fluorescent probe, fixing, and then collecting an image and data of the fluorescent recovery of the specific component of the emulsion to be detected after bleaching by using a laser confocal microscope; the time value (t) at which the fluorescence recovered to 1/2 which is the final stable value was calculated1‑half) The fluidity of different specific components was analyzed by comparison of the diffusion coefficient (K) and the ratio of dynamic molecules (F1 mobile fraction). The invention adopts the combination of the laser confocal microscope and the fluorescent probe, compared with the traditional method, the invention is convenient for counting and counting the emulsion molecules in the visual field; the fluidity of specific components of the emulsion can be more intuitively characterized; adding the evaluation index of the accurate fluidity of the emulsion molecules,provides a new method for exploring and researching the emulsification property on an oil-water interface.
Description
Technical Field
The invention belongs to the technical field of food analysis, and particularly relates to a method for representing the fluidity of specific components of an emulsion.
Background
An emulsion is a dispersion of one liquid in the form of droplets dispersed in another liquid immiscible with it. The emulsion is generally opaque and milky white. The diameter of the droplets is usually between 100nm and 10 μm, and they can be observed by a general optical microscope. Emulsions can be of both the oil-in-water and water-in-oil types. The stability of the food emulsion determines the structure, taste and stability of food, and the generation rule and the stabilizing mechanism of an emulsifying system are key difficulties in the research of the emulsion stability. In the food field, common emulsion systems include natural milk, pickering emulsion, microemulsion, nano emulsion and other nano particle conveying systems, which belong to the dispersion systems. In the field of food analysis and detection, the fluidity of the emulsion is mainly determined by physical methods, including the use of a viscometer, a fluorescence gradiometer and a rheometer, and the rheological property of the emulsion is characterized by measuring the change of viscosity along with the change of shear rate, but the physical methods are not enough to intuitively characterize the fluidity of specific components of single molecules of the emulsion.
Fluorescence light bleaching recovery (FRAP), also known as photobleaching fluorescence recovery technique or photobleaching fluorescence recovery technique, is a technique that can be used to monitor differential fusion mobility of proteins and lipids in cell membranes or artificial bilayers. The principle of the method is that high-energy laser is used for irradiating a certain specific area of a cell to irreversibly quench a fluorescent molecule marked in the area, the area is called a fluorescence bleaching area, and then, due to the movement of lipid molecules or protein molecules in cytoplasm, fluorescent molecules in a surrounding non-bleaching area continuously migrate to the photobleaching area, so that the fluorescence intensity of the fluorescence bleaching area gradually returns to the original level. Fluorescent photobleaching recovery enables direct characterization of the mobility of the labeled components, while also enabling measurement of the rate of membrane protein diffusion.
In addition, no relevant published reports are found on a characterization method for the fluidity of specific components of the emulsion, so that the blank area needs to be filled.
Disclosure of Invention
In view of the problems existing in the existing methods for characterizing the fluidity of the specific components in the emulsion, the invention aims to provide a method for visually characterizing the fluidity of the specific components in the emulsion, which has the characteristics of visual reflection, simple operation, rapid comparison and the like.
In order to solve the technical problems, the invention provides the following technical scheme:
a method for representing the fluidity of a specific component of an emulsion comprises the steps of marking molecules of the specific component on an emulsion film by a fluorescent probe technology, and measuring the migration condition of a single molecule on the emulsion film by using a fluorescent photobleaching recovery technology of a laser confocal microscope to represent the fluidity; the specific components are components capable of being labeled by fluorescent probes, including lipids and proteins.
Further, the emulsion is a common emulsion dispersion system, and comprises natural milk, milk produced by a food processing technology, pickering emulsion, microemulsion, nano emulsion and the like.
Further, the single molecules of the emulsion mainly comprise proteins and lipids, wherein the lipids comprise triglyceride, sphingomyelin, polyethylene glycol phosphate and the like, and the proteins comprise water-soluble proteins, enzymes and the like.
As a preferred embodiment of the method for characterizing the fluidity of the specific ingredients of the emulsion, according to the present invention, wherein: the sample is pretreated and fixed, before use, the slide is soaked in ultrapure water for 2h, soaked in acid overnight, washed clean with ultrapure water, soaked in 95% ethanol for 12h, baked at 70 ℃, treated under high pressure, and then a 5mm by 5mm grid containing a plurality of 0.5mm by 0.5mm single grids is placed at the center of the slide, polylysine (0.025mg/ml) is dripped, and the slide is coated overnight at 4 ℃. Before use, the mixture is washed by ultrapure water, washed by absolute ethyl alcohol and air-dried by an ultra-clean bench. When preparing the sample, 2 μ L of the sample was dropped uniformly into the grid area for fixation.
Further, the fluorescence photobleaching and recovery curve is characterized by three parameters, therefore, the migration condition of a single molecule on the emulsion film is mainly characterized by the following three parameters:
(1) time value t at which the fluorescence recovered to 1/2 of the final stable value1-halfThe time value corresponding to the time obtained by subtracting the minimum value from the time obtained by adding the half of the difference between the maximum stable value after the fluorescence recovery and the minimum value reached by bleaching represents the fluorescence recovery rateSpeed is high and low;
(2) a dynamic molecular ratio (F1, mobile fraction) which is the ratio of the difference between the maximum stable value and the minimum bleaching value after the fluorescence is recovered to the difference between the bleaching starting value and the minimum value and represents the dynamic molecular ratio of a specific component in the tested emulsion;
(3) the diffusion coefficient K (1/s), which is the reciprocal of the difference between the time corresponding to the maximum stable value and the time corresponding to the minimum bleached value, characterizes a measure of the mean square displacement per unit time and represents a physical quantity of the degree of molecular diffusion in the emulsion measured.
As a preferred embodiment of the method for characterizing the fluidity of the specific ingredients of the emulsion, according to the present invention, wherein: the comparative analysis of the fluidity of a particular ingredient in an emulsion comprises the steps of: preparing observation sample, adjusting working parameters of laser confocal microscope, setting bleaching parameters of corresponding probes, bleaching for a period of time, outputting fluorescence bleaching recovery data of emulsion, and calculating time value (t) when fluorescence is recovered to 1/2 of final stable value1-half) The diffusion coefficient (K) and the ratio of dynamic molecules (F1, mobile fraction) in turn characterize the flowability.
As a preferred embodiment of the method for characterizing the fluidity of the specific ingredients of the emulsion, according to the present invention, wherein: the preparation and observation sample comprises the steps of taking 10 mu L of emulsion, correspondingly mixing the 10 mu L of emulsion with 0.1mg/mLNile Red, 1mg/mL Rh-DOPE, 0.1mg/mLNile Blue, 1mg/mLNBD-PC and 0.5mg/mLNBD-SM according to a ratio of 5:1, 40:1, 50:1, 40:1 and 5:1(v/v), dyeing the mixture in a dark place for more than 30min, taking 10 mu L of sample, placing the sample on a glass slide with gridded lysine as a substrate, and covering the glass slide.
The method for preparing the latticed lysine slide is used for fixing single emulsion molecules, and comprises the following specific steps:
(1) firstly, soaking a slide in ultrapure water for 2 hours, soaking in nitric acid overnight, washing with the ultrapure water, soaking in 95% ethanol for 12 hours, baking at 70 ℃, and carrying out high-pressure sterilization treatment at 121 ℃ and 100 kPa;
(2) placing a 5mm by 5mm grid on a slide according to the size of a field of view in a microscope, wherein the grid is preferably placed at the center of the slide for the convenience of observation; adding polylysine (0.025mg/ml) dropwise, and coating at 4 deg.C overnight;
(3) before use, the grid-shaped lysine slide is obtained by washing with ultrapure water, washing with absolute ethyl alcohol and air-drying on an ultra-clean bench.
As a preferred embodiment of the method for characterizing the fluidity of the specific ingredients of the emulsion, according to the present invention, wherein: the working parameters of the laser confocal microscope are adjusted to include that the Nile Red, the NBD-SM and the NBD-PC fluorescent probes are excited by an Ar + laser, the excitation wavelength of the Nile Red fluorescent probes is 514nm, and the excitation wavelengths of the NBD-SM and the NBD-PC fluorescent probes are 488 nm; the Nile Blue and Rh-DOPE fluorescent probes are excited by a He-Ne laser, the excitation wavelengths are 633nm and 543nm respectively, the Nile Blue and Rh-DOPE fluorescent probes are observed by a multiplied 63 oil mirror, the scanning range Frame Size (nm) is 512 x 512, and the specific parameters are shown in Table 1.
TABLE 1 Instrument parameters
As a preferred embodiment of the method for characterizing the fluidity of the specific ingredients of the emulsion, according to the present invention, wherein: the setting of the bleaching parameters of the corresponding probes comprises setting of different sizes of bleaching regions and bleaching time, and specific parameters are shown in table 2.
TABLE 2 bleaching conditions for five different fluorescent probes
As a preferred embodiment of the method for characterizing the fluidity of the specific ingredients of the emulsion, according to the present invention, wherein: the raw material system is an emulsion system with the particle size of 0.5-100 mu m.
As a preferable proposal of the method for representing the fluidity of the specific components of the emulsion, the size of the bleaching area determines the speed of fluorescence recovery in experimental research, namely, the larger the bleaching area is, the longer the time required for fluorescence recovery is; the size of the relative bleaching zone (bleaching zone/particle size) determines the recovery rate of fluorescence: the larger the relative bleaching zone, the longer the time required for fluorescence recovery. Therefore, it is preferable that the bleached area size of the corresponding probe is set to 10 pixels, which corresponds to about 2.5 μm in practice, and the size of the emulsion is uniform in the same set of parallel data.
As a preferred embodiment of the method for characterizing the fluidity of the specific ingredients of the emulsion, according to the present invention, wherein: the results of the measurements were averaged over 3 emulsion samples in the specified range.
The invention has the beneficial effects that:
the invention uses different fluorescent probes to mark specific components of the emulsion, places a sample on a gridding processed lysine-based glass slide for immobilization pretreatment, observes the marked emulsion by using a laser confocal microscope, provides an image of the recovery of the component from fluorescent bleaching in the emulsion, and calculates the time value (t) when the fluorescence is recovered to 1/2 which is the final stable value1-half) The diffusion coefficient (K) and the ratio of dynamic molecules (F1, mobile fraction) reflect the flowability of different components, and a fluorescence bleaching recovery (FRAP) kinetic method is established, so that the method for representing the flowability of specific components of the emulsion provided by the invention has higher intuitiveness and accuracy. In the traditional method, researchers judge the fluidity of membrane components by measuring the content of fatty acids aiming at phospholipids, firstly extract the total lipid of an emulsion by using a chloroform-methanol mixed solvent, then separate the obtained lipid substances in a developing solution by using a silica gel sheet, and finally analyze the structural fluidity by using Gas Chromatography (GC) after multiple times of extraction, concentration, centrifugation and drying. Compared with the traditional method, the method has the advantages that the sample is pre-treated by the gridding lysine slide, so that the emulsion molecule is better fixed; all complex steps of extracting the milk fat and analyzing the fatty acid composition are saved; the detection time taken is short.
Drawings
FIG. 1 is a graph showing the recovery of emulsion fluorescence bleaching (emulsion triglyceride labeling) in the aqueous peanut oil extraction process of example 1;
FIG. 2 is a graph of the fluorescent bleach recovery of breast milk of example 2 (probability of presence of sphingomyelin marking the emulsion and crescent moon); FIG. 3 is a fluorescent bleach recovery plot for breast milk of example 2; (labeling emulsion sphingomyelin);
FIG. 4 is a graph of the recovery of fluorescence bleaching for the nanoemulsion prepared in example 3; (marking the emulsion water soluble protein);
FIG. 5 is a graph showing the recovery of emulsion fluorescence bleaching (emulsion triglyceride labeling) in the aqueous peanut oil extraction process of a comparative example;
fig. 6 shows a grid-treated lysine-based glass slide, wherein the slide length a is 75mm, the slide width b is 25mm, the slide thickness h is 1mm, the grid size d is 5mm, and the size of a single grid in the grid p is 0.5 mm.
Detailed Description
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention will be described in detail with reference to the following embodiments in order to make the aforementioned objects, features and advantages of the invention more comprehensible.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Examples
Firstly, preparing a grid-shaped lysine slide for fixing a single emulsion molecule, wherein the specific method for pretreating the slide comprises the following steps:
(1) before use, the slide is soaked in ultrapure water for 2h, soaked in nitric acid overnight, washed clean with ultrapure water, soaked in 95% ethanol for 12h, baked at 70 ℃, and sterilized at 121 ℃ under 100 kPa;
(2) according to the size of the visual field in the microscope, a 5mm by 5mm grid containing a plurality of 0.5mm by 0.5mm single grids is placed at the center of the slide, polylysine (0.025mg/ml) is dripped, and the slide is coated overnight at 4 ℃; the basis of the grid size setting is as follows: the loading amount of the emulsion is about no more than 5mm in diameter covered on a glass slide, and considering that the size of the emulsion single-molecule liquid drop is small, 40X and 63X oil mirrors can be used for observation; the length and width of the single field of view for this imaging were no more than 500 μm, so the grid size was set to 5mm by 5mm, and the size of the single cells in the grid was 0.5mm by 0.5mm, as shown in fig. 6.
(3) Before use, the grid-shaped lysine slide is obtained by washing with ultrapure water, washing with absolute ethyl alcohol and air-drying on an ultra-clean bench.
Example 1 emulsion in a milk-peanut aqueous phase oil extraction Process produced by a food processing Process
(1) Preparing an observation sample: the peeled peanuts are crushed into pulp by a traditional Chinese medicine crusher, and emulsion and free oil are generated by primary alkali extraction and secondary alkali extraction.
And (3) taking 10 mu L of emulsion sample, mixing the emulsion sample with 0.1mg/mLNile Red according to a ratio of 5:1(v/v), dyeing the mixture for more than 30min in a dark place, taking 2 mu L of sample, placing the sample on a glass slide with the gridded lysine as a substrate, and quickly and gently covering the glass slide with a cover slip.
(2) Adjusting the working parameters of the laser confocal microscope:
excited by an Ar + laser, the excitation wavelength is 514nm, the scanning range Frame Size (nm) is 512 x 512, the other parameters are the same as the parameters in the table 1, namely the emission light receiving range is 538-740nm, the pinhole is 128.1AU, and the gain value is 700.
(3) Setting bleaching parameters of the probe:
selecting 10pixel pairs of bleaching area sizeThe single emulsion molecule was bleached with the bleaching settings as in table 2, i.e. the laser line attenuator transmission ratio was 0.01% and the number of scans was 30 scans. The figure of the fluorescent bleaching recovery of triglyceride in the inner core of the emulsion marked by Nile Red is shown in figure 1, and the single triglyceride molecule in the emulsion is obviously marked and detected, which indicates that the single triglyceride molecule in the emulsion is fully fixed. The characterization data are shown in Table 3, the time value (t) at which 1/2 is restored to the final stable value by fluorescence1-half) The fluidity of the labeled components was compared with the diffusion coefficient (K) and the ratio of dynamic molecules (F1 mobile fraction). For single emulsion molecules with the particle size of about 5 mu m, the average dynamic molecular proportion is about 65 percent; t is t1-halfThe average value is about 45s, which shows that the fluorescence recovery time is longer and the recovery rate is slower; the larger the value of K, the larger the diffusion coefficient.
TABLE 3 fluorescent bleaching recovery index table for emulsion marked by Nile Red
Example 2: natural milk-mother milk
Natural milk includes all natural milk, such as human milk (also called breast milk) and other non-human milk, and the present embodiment takes breast milk as an example. The fat in the breast milk exists in the form of milk fat globules with different sizes, the Milk Fat Globule Membrane (MFGM) accounts for about 2-6% of the weight of the fat globules, is a loose net structure composed of phospholipids, glycolipids, proteins, lipid raft domains rich in cholesterol and sphingomyelin, and the like, and has a complex structure. The crescent area refers to a convex substance attached to human milk fat globule HMFGs, and the relevant literature explains the fact that the cytoplasmic residues of the lactating cells connected with the HMFGs during secretion are named as crescent areas due to the shapes of the cytoplasmic residues, so that the cause of cytoplasmic crescent on the fat globule is accurately determined, and the secretion mechanism of the milk fat globule can be further clarified. The method can characterize the fluidity of sphingolipid, namely sphingomyelin, and accurately and conveniently calculate the existence probability of crescent areas.
(1) Preparing an observation sample:
mixing 10 μ L breast milk with 0.5mg/mLNBD-SM at a ratio of 5:1(v/v), staining in dark for more than 30min, placing 2 μ L breast milk on a gridded lysine-based glass slide, and rapidly and gently covering with a cover glass.
(2) Adjusting the working parameters of the laser confocal microscope:
excited by an Ar + laser, the excitation wavelength is 488nm, the scanning range Frame Size (nm) is 512 x 512, the other parameters are the same as the parameters in the table 1, namely the emission light receiving range is 521-635nm, the pinhole is 155.4AU, and the gain value is 909.
(3) Setting bleaching parameters of the probe:
after NBD-SM labeling, the probability of existence of crescent moon areas was 1.2. + -. 0.39% by the mean counts per field of view for the grid, and the representation is shown in FIG. 2.
Fig. 2 shows images of the crescent area marked by NBD-SM under the laser scanning confocal microscope CLSM, wherein a is a 2D image, b is a 3D image, and c is a crescent area count image).
The bleaching area size of 10 pixels is selected to bleach the crescent area of the single milk fat globule, the bleaching conditions of the probe are shown in table 2, namely the transmission ratio of the laser line attenuator is 0.2%, and the scanning times are 3 scans. The recovery of fluorescence bleaching of the emulsion after NBD-SM labeling is shown in FIG. 3, where individual sphingomyelin molecules in the emulsion are visibly labeled and detected, indicating that the individual sphingomyelin molecules in the emulsion are sufficiently immobilized. The characterization data are shown in Table 4, by dynamic molecular ratio (F1 mobile fraction) and t1-halfValues to compare the flowability of the labeled components. As can be seen from FIG. 3, the fluorescence recovery after bleaching of NBD-SM labeled sphingomyelin is low, and as can be seen from Table 4, the F1 value is low, so both the image and the value reflect the low dynamic ratio of sphingomyelin molecules; t of sphingomyelin1-halfThe average value is about 1.8s, the fluorescence recovery time is short, and although the recovery degree is low, the fluorescence recovery rate is high; the larger the value of K, the larger the diffusion coefficient.
TABLE 4 NBD-SM tagged indicator Table for fluorescent bleaching recovery of emulsions
Therefore, the invention applies different fluorescent probes and laser confocal microscopes to a breast milk natural system, and can evaluate the fluidity of specific components in the emulsion by calculating and comparing the characterization indexes.
Example 3: nanoemulsion-nanoemulsion was prepared by dissolving catechin and powdered zein in ethanol.
Dissolving catechin 2g and zein powder in ethanol 20g, and stirring to mix well. And (3) mixing 10 mu L of sample with 0.1mg/mLNile Blue according to a ratio of 5:1(v/v), dyeing for more than 30min in a dark place, placing 2 mu L of sample on a glass slide with a gridded lysine as a substrate, and quickly and gently covering a cover glass.
(2) Adjusting the working parameters of the laser confocal microscope:
excited by He-Ne laser, the excitation wavelength is 633nm, observed by a X63 oil mirror, the scanning range Frame Size (nm) is 512 x 512, and the other parameters are the same as those in Table 1, namely the emission light receiving range is 538-740nm, the pinhole is 128.1AU, and the gain value is 700.
(3) Setting bleaching parameters of the probe:
the bleaching area size of 10 pixels is selected to bleach the single emulsion molecule, the bleaching adjustment is shown in table 2, namely the transmission ratio of the laser line attenuator is 0.01%, and the scanning times are 30 scans. The fluorescence bleaching recovery diagram of the water-soluble protein of the emulsion marked by Nile Blue is shown in FIG. 4, and the single water-soluble protein molecule in the emulsion is obviously marked and detected, which indicates that the single water-soluble protein molecule in the emulsion is fully fixed. The characterization data are shown in Table 5. Time value (t) at 1/2 at which the fluorescence returns to the final stable value1-half) The fluidity of the labeled components was compared with the diffusion coefficient (K) and the ratio of dynamic molecules (F1 mobile fraction). For single emulsion molecules with the particle size of about 6 mu m, the dynamic molecular proportion is about 30 percent on average and can be reduced along with the increase of the particle size; t is t1-halfThe average value is about 7.5s, and the fluorescence is recoveredThe time is shorter, and the recovery rate is higher; the K value did not change much with increasing particle size, and was 0.0142 on average, indicating that the diffusion coefficient was relatively stable.
TABLE 5 fluorescent bleaching recovery index table for emulsion marked by Nile Blue
Comparative example 4 emulsion in a milk-peanut aqueous phase oil extraction Process produced by a food processing Process
This comparative example employed the same treatment method as in example 1, except that treatment with a grid-like lysine slide was not used.
The result of the treatment without grid-shaped lysine slide is shown in fig. 6, on one hand, the emulsion in the visual field has high molecular weight and is overlapped, and the image of a single molecule cannot be clearly obtained; on the other hand, the emulsion molecules move fast, the scanning speed of the instrument cannot capture a real-time result, and the imaging result shows that the emulsion flows too fast and the molecules deform, so that the representation of the experiment cannot be carried out. The invention provides a method for fixing a special component in milk, which is characterized in that the mobility of milk has great influence on the measurement and analysis process in the measurement process of milk molecules, particularly single molecules, and how to fix the special component in milk in order to more conveniently and accurately represent the mobility of the special component in the milk.
The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.
Claims (10)
1. A method for representing the fluidity of a specific component of an emulsion is characterized in that a fluorescent probe is used for marking molecules of the specific component on an emulsion film, and the migration condition of a single molecule on the emulsion film is measured by using the fluorescent photobleaching recovery technology of a laser confocal microscope to represent the fluidity; the specific components are components capable of being labeled by a fluorescent probe and comprise lipid and protein, wherein the lipid comprises triglyceride, sphingomyelin and polyethylene glycol phosphate, and the protein comprises water-soluble protein and enzyme.
2. The method for characterizing the fluidity of specific components of emulsion according to claim 1, wherein the emulsion is an emulsion dispersion system comprising natural milk, milk produced by food processing technology, pickering emulsion, microemulsion and nano-emulsion, and the particle size of the emulsion is 0.5-100 μm.
3. The method for characterizing the fluidity of a specific component of an emulsion according to claim 1, wherein the fluidity includes the time value t at which the fluorescence returns to 1/2, which is the last stable value1-halfA dynamic molecular ratio F1 and a diffusion coefficient K, where t1-halfRepresenting the speed of the fluorescence recovery rate, F1 representing the dynamic molecular ratio of the specific components in the emulsion tested, and K representing the physical quantity of the degree of molecular diffusion in the emulsion tested.
4. The method for visually characterizing the flow dynamics of different components in an emulsion according to claim 1, wherein the specific method for labeling specific components in the emulsion by using the fluorescent probe technology comprises the following steps: mixing the emulsion with the fluorescent probe, dyeing in dark for more than 30min, placing the sample on a glass slide, and covering with a cover glass.
5. The method for characterizing the fluidity of specific components of emulsion according to claim 4, wherein the glass slide is pre-treated before the sample is placed, and is made into a grid-shaped lysine glass slide with a grid-shaped treated lysine base for immobilizing single emulsion molecules.
6. The method for characterizing the fluidity of the specific components of the emulsion according to claim 5, wherein the grid-shaped lysine glass slides are prepared by: placing 5mm by 5mm grid containing several 0.5mm by 0.5mm single grids on the glass slide after autoclaving, dripping polylysine, and coating at 4 deg.C overnight; and washing and drying to obtain the latticed lysine slide.
7. The method for characterizing the fluidity of specific components of emulsion according to claim 1, wherein the fluorescent probe comprises 0.1mg/mL Nile Red, 1mg/mL Rh-DOPE, 0.1mg/mL Nile Blue, 1mg/mL NBD-PC, 0.5mg/mL LNBD-SM, and the emulsion is mixed with the fluorescent probe at 5:1, 40:1, 50:1, 40:1, 5:1 (v/v).
8. The method for characterizing the fluidity of specific components of emulsion according to claim 5, wherein the Nile Red, NBD-SM and NBD-PC fluorescent probes are all Ar+Excitation by a laser, wherein the excitation wavelength of the Nile Red fluorescent probe is 514nm, and the excitation wavelengths of the NBD-SM fluorescent probe and the NBD-PC fluorescent probe are 488 nm; the Nile Blue and Rh-DOPE fluorescent probes are excited by a He-Ne laser, and the excitation wavelengths are 633nm and 543nm respectively.
9. The method for characterizing the fluidity of specific components of emulsion according to claim 1, wherein the confocal laser microscopy is performed by means of a x 63 oil microscope, the scanning range is 512 x 512nm, the emission light receiving range is 490-800 nm, the pinhole is 90-160 AU, and the gain value is 700-950.
10. The method for characterizing the fluidity of a specific component of an emulsion according to claim 1, wherein the diameter of the bleaching region is 5/1.25 or 10/2.5 or 15/3.75pixel/μm, the bleaching time is 3 to 800s, the number of scanning is 3 to 500, and the transmission ratio of the laser line attenuator is 0.01 to 2%.
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| CN112781958B (en) | 2022-07-08 |
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