Biochemistry
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In vitro Digestion of Emulsions in a Single Droplet via Multi Subphase Exchange of Simulated Gastrointestinal Fluids
Chapters
Summary November 18th, 2022
A pendant drop surface film balance implemented with a multi-subphase exchange, nicknamed the OCTOPUS, allows for mimicking digestive conditions by the sequential subphase exchange of the original bulk solution with simulated gastrointestinal fluids. The simulated in vitro digestion is monitored by recording in situ the interfacial tension of the digested interfacial layer.
Transcript
Our method provides model to investigate how emotions are digested by simulating the process of digestion in a single aquas droplet, which is covered by emulsifier and is immersed in oil phase. We can evaluate the impact of different emulsifiers, and the action of different digestive components, and design interfaces that can resist or retard digestion at different locations within the gastrointestinal tract. We can measure interfacial tension in C2 throughout the whole simulated digestion process, and the elasticity and viscosity of the interfacial layer at the end of it digesting step.
The concentrations and the conditions of the digestion can be adjusted to the requirements of the experiment, and this can be applied sequentially, or simultaneously allowing to evaluate synergism and cumulative effects. And we work with a single droplet, so we need a very low amount of sample, and we have high precision control of experimental variables. We have applied this technique to design a strategy to take over this.
One of those approaches is to design in the fossil layer that retires or receipts the process of lipolysis. Another approach is the use of lipase innovators, and we have investigated the interfacial mechanism of action of lipase. This device was originally developed to study the digestion in vitro.
Nevertheless, it can be used for study mono and interaction with lipid, and also can be applied to study automatically the critical micro concentration. The experimental technique is accessible. A manual researcher has been trained to use it in one week.
You need to clean up the equipment thoroughly, and purify the samples and oil in order to prevent the presence of surface active contaminants. You need to get familiar with the software computer, and learn to control the lights and position of capability, and get rid of tiny bubbles. To begin, formal water droplet to check the surface tension of water at room temperature.
Set the differential density to air water in the left dialogue, and measure the surface tension in real time. for five minutes. Fill the clean cuvet with at least 0.002 liters of clean vegetable oil, and place it in the cuvet holder in the thermostatic cell.
Set the differential density to vegetable oil water and inject 40 microliters at a rate of 0.5 microliters per second. Measure the tension in real time every second until the end of the injection. This is a simple dynamic process.
Next, save the data and plot the interfacial tension as a function of droplet volume and a data sheet. Check that the droplet volume range provides a value for the interfacial tension independent of the droplet volume for clean water. Plot the interfacial area as a function of the droplet volume.
This curve will be used later to match the volume with the corresponding interfacial area. With the left syringe, inject the volume within the range of constant interfacial tension and record the interfacial tension for five minutes. This is to check the absence of surface active components in the system.
To perform the initial control, inject around 10 microliters of the emulsifier solution into the capillary for drop formation, and record the absorption at a constant interfacial area of around 20 square millimeter for one hour. The exact values of volume and area can be obtained from the calibration curve plotted previously. Program the dilutional reology by setting the amplitude of oscillation to 1.25 microliters in the period to 10 seconds.
Then program the absorption at the selected interfacial area for 10 seconds. Next, to record the gastric digestion program the absorption at the selected interfacial area for 10 seconds. Fill the left syringe with liquid from valve two.
Inject 125 microliters from valve two at five microliters per second with the left syringe, and simultaneously extract the same volume at the same rate with the right syringe. This is a visualization of the process of sub phase exchange of water with methylene blue. Unload the right syringe to exit valve eight and load again the left syringe with liquid from valve two.
Repeat these two steps 10 times to assure complete sub phase exchange with liquid and valve two and gastric enzymes. Then, record the absorption at the selected interfacial area for one hour and record the dilutional reology as shown earlier. To record the intestinal digestion after recording the absorption at the selected interfacial area, fill the left syringe with liquid from valve three and follow the same steps shown earlier for recording the gastric digestion.
Similarly, to record the desorption, after recording the absorption at the selected interfacial area, fill the left syringe with liquid from valve five and repeat the rest of the steps used to record the gastric digestion. Fill the micro centrifuge tubes with the artificial digestion media, and connect each of them to the respective valve by the corresponding tubing. Fill the tubing in valves two to eight by cleaning from valve two, valve three, valve four, and valve five to the external exit that is valve eight.
Fill the tubing in valve one by cleaning from valve one to valve six capillary five times. Place the capillary into the oil phase, and load the left syringe with valve one. Start sequentially processing the initial control gastric digestion, intestinal digestion, and desorption, saving the data at the end of each process.
Experimental results obtained for the gastric digestion of emulsifiers are shown in these figures. Gastric proteolysis of human serum albumin is presented here. Digestive media are applied by sub phases exchange with solutions at 37 degrees Celsius.
Here blue represents the initial buffer with protein and red represents simplified SGF with pepsin. During sub phase exchange with simplified SGF and pepsin, the interfacial tension increases owing to the hydrolysis of the protein, which dilutes the initial protein layer. The graphical image represents the gastric lipolysis of citrus pectin.
Here, blue represents the initial buffer with citrus pectin, yellow represents simplified SGF with gastric lipase, and gray represents simplified SGF. Sub phases exchange with gastric lipase decreases the surface tension while sub phases exchange with simplified SGF, provides a null response of the interfacial tension. Shown here is an example of the intestinal digestion profiles.
Absorption, desorption, profiles of bio salts, lipase, and lipase plus bile salts in simplified SIF at 37 degrees Celsius are presented here. The graphical image shows the evolution of the interfacial tension upon lipolysis of two variants of plueronic F127 and F68. A steep decrease can be seen in interfacial tension due to the absorption of lipase and bile salts, and the production of free fatty acids onto previously formed interfacial films of F68 and F127 at the oil water interface.
The desorption step shows the sub phase exchange with simplified SIF from bile salts, F68, and F127. In vitro digestion profile of AS48 absorbed film at the air water interface in human and bovine serum albumin absorbed films at the olive oil water interface. The representative images show the interfacial tension, the dilation elasticity, and the dilation viscosity of in vitro digestion of beta lactoglobulin absorbed film at the olive oil water interface.
The dilation parameters were measured at 1 hertz, 0.1 hertz, and 0.01 hertz after the digested interface was equilibrated in each step. It is important to match the balls when programming the process with the corresponding centrifuge tubes, capillary, or axis, the evolution of the drop site distribution, then, electrophoretic mobility set a potential of droplets, and the amino free fatty acid produces in lipolysis offer complementary information to corresponding with findings from interfacial tension. This technique provides interfacial layers with different digestibility profiles to deliver nutrients and drugs at different locations within gastrointestinal tract, and also to develop new encapsulation systems.
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