This research was funded by projects RTI2018-101309-B-C21 and PID2020-631-116615RAI00, funded by MCIN/AEI/10.13039/501100011033 and by &#34;ERDF A way of making Europe&#34;. This work was (partially) supported by the Biocolloid and Fluid Physics Group (ref. PAI-FQM115) of the University of Granada (Spain).

1. Cleaning sequence for all glassware used in surface science experimentation
<ol>
	<li>Scrub the glassware with a concentrated cleaning solution (see Table of Materials) diluted in water (10%).</li>
	<li>Rinse thoroughly with a sequence of tap water, propanol, distilled water, and ultrapure water. Dry in a cabin and store in a closed cabinet until use.</li>
</ol>
2. Sample preparation
<ol>
	<li>Prepare artificial digestive media according to INFOGEST standardized protocols11,20 (see Table of Materials). For details, see Table 1 and include small adaptations to the requirements of the interfacial work to prevent surface active contamination and the dilution of samples (1:10)10.</li>
	<li>Prepare the emulsifier solution following the steps below.
	<ol>
		<li>Prepare 0.01&#160;L of a concentrated solution of (1 kg&#183;L&#8722;1) emulsifier or a mixture of emulsifiers (see Table of Materials) in an initial buffer (Table 1) and keep under mild agitation overnight.</li>
		<li>Dilute to 0.1 kg&#183;L&#8722;1 (or as required) to saturate the interface; reach a pseudo plateau in the interfacial tension after 1 h of adsorption at a constant interfacial area following the previously published report21.</li>
		<li>Keep under mild agitation for 15 min before use.</li>
	</ol>
	</li>
	<li>Purify the oil phase.
	<ol>
		<li>Prepare a mixture of vegetable oil (sunflower, olive, triolein, etc. )and magnesium metasilicate resins (see Table of Materials) in a proportion of 2:1 w/w in a large beaker. Keep under mild mechanical agitation for at least 3 h.</li>
		<li>Centrifuge the mixture at 8,000 x g for 30 min at room temperature in a commercial centrifuge (see Table of Materials).</li>
		<li>Filter the oil mixture under vacuum with a syringe filter (0.2 &#181;m pore size) (see Table of Materials). Store in clean amber bottles sealed and bubbled with nitrogen until use.</li>
	</ol>
	</li>
</ol>
3. Calibration and cleaning of the OCTOPUS
<ol>
	<li>Rinse all the tubing with ultrapure water by setting a sequence of cleaning both syringes and all valves through a capillary (valves 6/4) and to the external exit (valve 8-blue color). Perform this by pressing the clean button in the left dialog (Supplementary Figure 1A).</li>
	<li>Check the surface tension7 of water at room temperature by forming a water droplet and measuring in real time for 5 min (Supplementary Figure 1B, C).
	<ol>
		<li>Set the differential density to air-water (0.9982 kg&#183;L&#8722;1) in the left dialog, Supplementary Figure 1B.</li>
	</ol>
	</li>
	<li>Fill the clean cuvette (optical glass) with 0.002 L of clean vegetable oil and place it in the cuvette holder in the thermostatic cell (Figure 3).</li>
	<li>Set the thermostat and allow for temperature equilibration at 37 &#176;C.</li>
	<li>Check the interfacial tension of water-oil at room temperature7.
	<ol>
		<li>Set the differential density to vegetable oil-water (olive oil: 0.800 kg&#183;L&#8722;1) (Supplementary Figure 1C).</li>
		<li>Inject 40 &#181;L at a rate of 0.5 &#181;L&#183;s&#8722;1 and measure in real time every second until the end of the injection. This is a simple dynamic process (Supplementary Figure 1B, D).</li>
		<li>Plot the interfacial tension as a function of droplet volume in a data sheet.</li>
		<li>Check that the droplet volume range provides a value for the interfacial tension independent of the droplet volume. Plot the interfacial area as a function of droplet volume.</li>
		<li>Program a process containing two steps (Supplementary Figure 1B and Supplementary Figure 2A) following the steps below.
		<ol>
			<li>With an inner syringe, inject a volume contained within this range of constant interfacial tension.</li>
			<li>Maintain the interfacial area constant at the value selected in step 3.5.4 and record the interfacial tension for 5 min7.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Programming one experimental process in DINATEN for each digestive step
NOTE: For the process parameters, see Supplementary Figure 1B.
<ol>
	<li>Perform the initial control.
	<ol>
		<li>For drop formation, inject 10 &#181;L (&#177;5 &#181;L) of emulsifier solution into the capillary (valve 6) (Supplementary Figure 2A).</li>
		<li>Record the adsorption at a constant interfacial area21 of 20 mm2 (&#177;10 mm2) for 1 h (Supplementary Figure 2B).</li>
		<li>Record the dilatational rheology8 (Supplementary Figure 2C).
		<ol>
			<li>Set the amplitude of oscillation to 1.25 &#181;L, period 10 s.</li>
			<li>Record the adsorption at the selected interfacial area (step 4.1.2) for 10 s.</li>
			<li>Repeat step 4.1.3 at different periods: 5 s, 20 s, 50 s, and 100 s.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Record gastric digestion.
	<ol>
		<li>Record the adsorption21 at the selected interfacial area for 10 s.</li>
		<li>Subphase exchange7 with liquid in valve 2 (sSGF) and gastric enzymes (Table 1) (Supplementary Figure 2D).
		<ol>
			<li>Fill the left syringe from valve 2. Inject 125 &#181;L into valve 6-capillary with the left syringe at 5 &#181;L&#183;s&#8722;1.</li>
			<li>Extract 125 &#181;L from the capillary with the right syringe at 5 &#181;L&#183;s&#8722;1. Unload the right syringe to exit valve 8. Repeat steps 4.2.2.1-4.2.2.2 10 times to assure complete exchange.</li>
		</ol>
		</li>
		<li>Record the adsorption21 at the selected interfacial area in step 4.1.2 for 1 h (Supplementary&#160;Figure 2B).</li>
		<li>Record the dilatational rheology8 (Supplementary Figure 2C).
		<ol>
			<li>Set the amplitude of oscillation to 1.25 &#181;L, period 10 s.</li>
			<li>Record the adsorption of the selected interfacial area in step 4.1.2 for 10 s. Repeat at different periods: 5 s, 20 s, 50 s, 100 s.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Record intestinal digestion.
	<ol>
		<li>Record the adsorption21 at the selected interfacial area in step 4.1.2 for 10 s (Supplementary Figure 2B).</li>
		<li>Subphase exchange7 with liquid in valve 3 (sSIF) and intestinal enzymes/bile salts/phospholipids (Table 1) (Supplementary Figure 2D).
		<ol>
			<li>Fill the left syringe from valve 2. Inject 125 &#181;L into valve 6-capillary with the left syringe at 5 &#181;L&#183;s&#8722;1. Extract 125 &#181;L from the capillary with the right syringe at 5 &#181;L&#183;s&#8722;1.</li>
			<li>Unload the right syringe to exit valve 8. Repeat steps 4.3.2.1-4.3.2.2 10 times to assure complete exchange.</li>
		</ol>
		</li>
		<li>Record the adsorption21 at the selected interfacial area in step 4.1.2 for 1 h.</li>
		<li>Record the dilatational rheology8 (Supplementary Figure 2C).
		<ol>
			<li>Set the amplitude of oscillation to 1.25 &#181;L, period 10 s.</li>
			<li>Record the adsorption at the selected interfacial area in step 4.1.2 for 10 s.</li>
			<li>Repeat at different periods: 5 s, 20 s, 50 s, 100 s.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Record the desorption following the steps below.
	<ol>
		<li>Record the adsorption21 at the selected interfacial area in step 4.1.2 for 10 s (Supplementary Figure 2B).</li>
		<li>Subphase exchange7 with liquid in valve 5 (sSIF) (Table 1, Supplementary Figure 2D).
		<ol>
			<li>Fill the left syringe from valve 5. Inject 125 &#181;L into valve 5-capillary with the left syringe at 5 &#181;L&#183;s&#8722;1.</li>
			<li>Extract 125 &#181;L from the capillary with the right syringe at 5 &#181;L&#183;s&#8722;1. Unload the right syringe to exit valve 8. Repeat steps 4.4.2.1-4.4.2.2 10 times to assure complete exchange.</li>
		</ol>
		</li>
		<li>Record the adsorption21 at the selected interfacial area in step 4.1.2 for 1 h (Supplementary Figure 2B).</li>
		<li>Record the dilatational rheology8 (Supplementary Figure 2C).
		<ol>
			<li>Maintain the amplitude of 1.25 &#181;L, period 10 s.</li>
			<li>Record the adsorption at the selected interfacial area in step 4.1.2 for 10 s.</li>
			<li>Repeat step 4.4.4 at different periods: 5 s, 20 s, 50 s, 100 s.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. Setting up the experiment
<ol>
	<li>Fill the microcentrifuge tubes with the artificial digestion media and connect each of them to the respective valve by the corresponding tubing.</li>
	<li>Fill the tubing in valves 2-5 by cleaning from valve 2, valve 3, valve 4, and valve 5 to the external exit (valve 8) (Supplementary Figure 1A).</li>
	<li>Fill the tubing in valve 1 by cleaning from valve 1 to valve 6-capillary 5 times.</li>
	<li>Place the capillary into the oil phase. Load the left syringe with valve 1 (initial solution, Table 1).</li>
	<li>Start sequentially processing step 4.1-initial, step 4.2-gastric, step 4.3-intestines, and step 4.4-desorption, saving the data at the end of each process.</li>
</ol>
6. Calculation of the dilatational rheological parameters with the image processing software CONTACTO8
NOTE: For details, see Maldonado-Valderrama et al.8.
<ol>
	<li>Load the images corresponding to the area oscillation at a given frequency and amplitude (Supplementary Figure 3A).</li>
	<li>Press Rheology (Supplementary Figure 3B) and obtain the dilatational parameters (Supplementary Figure 3C).</li>
	<li>Copy-paste the results into the data spread sheet.</li>
</ol>
7. Plotting the experimental results
<ol>
	<li>Recalculate the time column in each of the steps of the digestion process by adding the last data of the time of the previous step.</li>
	<li>Plot the interfacial tension versus additive time for each of the steps of the digestion process used.</li>
	<li>Plot the final interfacial tension/dilatational elasticity and viscosity obtained at the end of each step versus the digestion phase: initial, gastric digestion, duodenal digestion, and desorption.</li>
</ol>

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The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.

This article describes a generalized protocol to measure in vitro digestion of interfacial layers by using pendant drop equipment. The protocol can be adjusted to the specific requirements of the experiment by tuning the composition of the digestive buffers, which are based on the INFOGEST11,20 harmonized protocol to facilitate comparison with literature. The digestive enzymes and biosurfactants can be added individually, sequentially, or together. This ...

Understanding how fat is digested, which involves emulsion digestion, is important to rationally design products with tailored functionality1. The substrate for fat digestion is an emulsion since fat is emulsified upon consumption by mechanical action and mixing with biosurfactants in the mouth and stomach. Also, most of the fat consumed by humans is already emulsified (such as milk products), and in the case of infants or some elderly people, this is the only form of consumption. Hence, the design of emulsion-based products with specific digestion profiles is very important in nutrition1. Moreover, emulsions can encapsulate and deliver nutrients, drugs, or lipophilic bioactives2 to tackle different gastrointestinal conditions such as obesity3, nutrient fortification, food allergies, and digestive diseases. In oil-in-water emulsions, lipid droplets are surrounded by interfacial layers of emulsifiers such as proteins, surfactants, polymers, particles, and mixtures4. The role of emulsifiers is twofold: stabilize the emulsion5 and protect/transport the encapsulated compound to a specific site. Achieving a tailored digestibility of emulsions depends on their initial composition but also requires monitoring the continuous evolution of this interface during the transit through the gastrointestinal tract (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64158/64158fig01.jpg" /> 
Figure 1: Applying interfacial engineering of emulsions to tackle some of the main gastrointestinal conditions. <a href="https://www.jove.com/files/ftp_upload/64158/64158fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Lipid digestion is ultimately an interfacial process because it requires the adsorption of lipases (gastric or pancreatic) onto the oil-water interface of emulsified lipid droplets through the interfacial layer to reach and hydrolyze the triglycerides contained in the oil into free fatty acids and monoacylglycerides6. This is schematized in Figure 2. Gastric lipase competes with pepsin and phospholipids in the stomach for the oil-water interface (Figure 2, gastric digestion). Then, pancreatic lipase/colipase compete with trypsin/chymotrypsin, phospholipids, bile salts, and digestive products in the small intestine. Proteases can alter the interfacial coverage, preventing or favoring lipase adsorption, while bile salts are highly surface active and displace most of the remaining emulsifier to promote lipase adsorption (Figure 2, intestinal digestion). Eventually, the rate and extent of lipolysis depend on the interfacial properties of the initial/gastric digested emulsion, such as the thickness, inter/intramolecular connections, and electrostatic and steric interactions. Accordingly, monitoring the evolution of the interfacial layer as it is digested offers an experimental platform to identify interfacial mechanisms and events affecting lipase adsorption and, hence, lipid digestion.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64158/64158fig02.jpg" /> 
Figure 2: Schematic diagram illustrating the role of interfaces in gastrointestinal lipid digestion. Pepsin hydrolysis alters interfacial composition at the gastric phase, while gastric lipase hydrolyzes triglycerides. In the small intestine, trypsin/chymotrypsin further hydrolyze the interfacial film, while lipolysis proceeds by the adsorption of BS/lipases, the hydrolysis of triglycerides, and the desorption of lipolytic products by solubilization in the BS micelles/complex. <a href="https://www.jove.com/files/ftp_upload/64158/64158fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The pendant drop equipment at the University of Granada (UGR) is implemented with a patented technology, the coaxial double capillary, that enables subphase exchange of the bulk solution7. The capillary, which holds the pendant drop, consists of an arrangement of two coaxial capillaries that are independently connected to each channel of a double microinjector. Each microinjector can operate independently, allowing the exchange of the dropped content by through-flow7. Accordingly, the subphase exchange consists of the simultaneous injection of the new solution with the inner capillary and the extraction of the bulk solution with the outer capillary using the same flow rate. This process allows the replacement of the bulk solution with no disturbance of the interfacial area or the volume of the droplet. This procedure was later upgraded to a multi-subphase exchange, which allows up to eight sequential subphase exchanges of the droplet bulk solution8. This enables the simulation of the digestive process in a single aqueous droplet suspended in lipidic media by sequentially exchanging the bulk solution with artificial media mimicking the different compartments (mouth, stomach, small intestine). The whole setup is represented in Figure 3, including the details of the components. The syringes in the microinjector are connected to the eight vias valves, each connecting to a microcentrifuge tube containing the artificial digestive fluid with components described in Figure 2.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64158/64158fig03.jpg" /> 
Figure 3: General view of the OCTOPUS with all components. The CCD camera, microscope, micro-positioner, thermostabilized cell, and double capillary connected independently to a double microinjector with two syringes connected to eight vias valves. Each syringe connects with capillary, four&#160;microcentrifuge tubes with sample and one discharge. <a href="https://www.jove.com/files/ftp_upload/64158/64158fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 4A shows how each of the artificial digestive fluids is injected into the pendant drop by subphase exchange through the double capillary. Each digestive compound detailed in Figure 2 can be applied simultaneously/sequentially, simulating the passage through the gastrointestinal tract. The artificial digestive fluids contain different enzymes and biosurfactants, which alter the interfacial tension of the initial emulsifier, as schematized in Figure 4B. The software DINATEN (see Table of Materials), also developed at the UGR, records the evolution of the interfacial tension in real time as the initial interfacial layer is digested in vitro. Also, after each digestive phase, the dilatational elasticity of the interfacial layer is computed by imposing periodic oscillations of volume/interfacial area onto the stabilized interfacial layer and recording the response of the interfacial tension. The period/frequency and the amplitude of the oscillation can be varied, and image processing with the software CONTACTO provides the dilatational rheological parameters8.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64158/64158fig04.jpg" /> 
Figure 4: Examples of digestion profiles. (A) The initial emulsifier layer is subjected to artificial digestive media placed in the microcentrifuge by sequential subphase exchange of the different solutions into the pendant drop. (B) The general evolution of the interfacial tension (y-axis) of the initial emulsifier as a function of time (x-axis) as it is digested in vitro by the various enzymes/biosurfactants in the artificial media. A final subphase exchange with plain intestinal fluid measures the desorption of digested lipid by solubilization in mixed micelles. <a href="https://www.jove.com/files/ftp_upload/64158/64158fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This study presents the general protocol designed to measure in vitro digestion of interfacial layers with pendant drop equipment9. The initial interfacial layer is subjected sequentially to conditions mimicking the passage through the gastrointestinal tract, as depicted in Figure 2. These different digestive media are injected into the pendant drop by subphase exchange of the different solutions contained in the microcentrifuge tubes (Figure 4A). The composition of these media can be customized depending on the gastrointestinal conditions that will be evaluated, namely, gastric/intestinal proteolysis/lipolysis, allowing for measuring cumulative effects and sinergies10. The experimental conditions used to mimic the digestion process in each compartment follow the international consensus protocol published by INFOGEST detailing the pH and amounts of electrolytes and enzymes11. The experimental device based on pendant drop allows recording of the interfacial tension in situ throughout the simulated digestion process. The dilatational rheology of the interfacial layer is computed at the end of each digestive step. In this way, each emulsifier offers a digestion profile illustrating the properties of the digested interfaces, as depicted in Figure 4B. This allows the extraction of conclusions regarding its susceptibility or resistance to the different stages of the digestive process. In general, the artificial digestive media contains acid/basic pH, electrolytes, proteases (gastric and intestinal), lipases (gastric and intestinal), bile salts, and phospholipids, which are dissolved in their respective digestive fluids (gastric or intestinal). Figure 4B shows a generic profile of the evolution of an emulsifier&#39;s interfacial tension, first subjected to protease action, followed by lipases. In general, proteolysis of the interfacial layer promotes an increase in the interfacial tension owing to the desorption of hydrolyzed peptides9,12, while lipolysis results in a very steep reduction in the interfacial tension due to the adsorption of bile salts and lipases13. A final subphase exchange with intestinal fluid depletes the bulk solution of unadsorbed/digested material and promotes the desorption of soluble compounds and the solubilization of digested lipids in mixed micelles. This is quantified by the increased interfacial tension recorded (Figure 4B).
In summary, the experimental design implemented in the pendant drop to simulate in vitro digestion in a single droplet allows for measuring cumulative effects and synergies as the digestion process is applied sequentially to the initial interfacial layer10. The composition of each digestive media can be easily tuned to account for the particularities of the digestive conditions, including gastrointestinal pathologies or infant digestive media14. Then, identification of the interfacial mechanisms affecting proteolysis and lipolysis can be used to modulate digestion by the interfacial engineering of emulsions. The obtained results can be applied in designing novel food matrices with tailored functionalities such as low allergenicity, controlled energy intake, and decreased digestibility15,16,17,18,19.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alpha-chymotrypsin from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>C4129</td>
			<td>Enzyme</td>
		</tr>
		<tr>
			<td>Beta-lactoglobulin</td>
			<td>Sigma-Aldrich</td>
			<td>L0130</td>
			<td>Emulsfier</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>9048-46-8</td>
			<td>Emulsfier</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>10043-52-4</td>
			<td>Electrolyte</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Kronton instruments</td>
			<td>Centrikon T-124</td>
			<td>For separating oil and resins</td>
		</tr>
		<tr>
			<td>Citrus pectin</td>
			<td>Sigma-Aldrich</td>
			<td>P9135</td>
			<td>Emulsfier</td>
		</tr>
		<tr>
			<td>co-lipase FROM PORCINE PANCREAS</td>
			<td>Sigma</td>
			<td>C3028</td>
			<td>Enzyme</td>
		</tr>
		<tr>
			<td>CONTACTO</td>
			<td>University of Granada (UGR)</td>
			<td></td>
			<td>https://core.ugr.es/dinaten/, last access: 07/18/2022</td>
		</tr>
		<tr>
			<td>DINATEN</td>
			<td>University of Granada (UGR)</td>
			<td></td>
			<td>https://core.ugr.es/dinaten/, last access: 07/18/2022</td>
		</tr>
		<tr>
			<td>Gastric lipase</td>
			<td>Lipolytech</td>
			<td>RGE15-1G</td>
			<td>Enzyme</td>
		</tr>
		<tr>
			<td>Human Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>70024-90-7</td>
			<td>Emulsifier</td>
		</tr>
		<tr>
			<td>INFOGEST</td>
			<td></td>
			<td></td>
			<td>http://www.proteomics.ch/IVD/</td>
		</tr>
		<tr>
			<td>Lipase from porcine pancreas, type II</td>
			<td>Sigma-Aldrich</td>
			<td>L33126</td>
			<td>Enzyme</td>
		</tr>
		<tr>
			<td>Magnesium metasilicate resins</td>
			<td>Fluka</td>
			<td>1343-88-0</td>
			<td>Resins to purify oil</td>
		</tr>
		<tr>
			<td>Micro 90</td>
			<td>International products</td>
			<td>M-9051-04</td>
			<td>Cleaner</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>7647-14-5</td>
			<td>Electrolyte</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Scharlau</td>
			<td>10049-21-5</td>
			<td>To prepare buffer</td>
		</tr>
		<tr>
			<td>OCTOPUS</td>
			<td>Producciones Cient&#237;ficas y T&#233;cnicas S.L. (G&#243;jar, Spain)</td>
			<td></td>
			<td>Pendandt Drop Equipment implemented with multi subphase exchange</td>
		</tr>
		<tr>
			<td>Olive oil</td>
			<td>Sigma-Aldrich</td>
			<td>1514</td>
			<td>oil</td>
		</tr>
		<tr>
			<td>Pancreatic from porcine pancreas</td>
			<td>Sigma</td>
			<td>P7545-25 g</td>
			<td>Enzyme</td>
		</tr>
		<tr>
			<td>Pepsin</td>
			<td>Sigma-Aldrich</td>
			<td>P6887</td>
			<td>Enzyme</td>
		</tr>
		<tr>
			<td>Pluronic F127</td>
			<td>Sigma</td>
			<td>P2443</td>
			<td>Emulsifier</td>
		</tr>
		<tr>
			<td>Pluronic F68</td>
			<td>Sigma</td>
			<td>P1300</td>
			<td>Emulsfier</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma</td>
			<td></td>
			<td>Bile salts</td>
		</tr>
		<tr>
			<td>Sodium glycodeoxycholate</td>
			<td>Sigma</td>
			<td>C9910</td>
			<td>Bile salts</td>
		</tr>
		<tr>
			<td>Sodium taurocholate</td>
			<td>Sigma</td>
			<td>86339</td>
			<td>Bile salts</td>
		</tr>
		<tr>
			<td>Syringe Filter</td>
			<td>Millex-DP</td>
			<td>SLGP033R</td>
			<td>&#160;Syringe Filter 0.22 &#181;m pore size polyethersulfone</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T1426</td>
			<td>Enzyme</td>
		</tr>
	</tbody>
</table>

in vitro digestion of emulsions in a single droplet via multi subphase exchange of simulated gastrointestinal fluids

Emulsions are currently being used to encapsulate and deliver nutrients and drugs to tackle different gastrointestinal conditions such as obesity, nutrient fortification, food allergies, and digestive diseases. The ability of an emulsion to provide the desired functionality, namely, reaching a specific site within the gastrointestinal tract, inhibiting/retarding lipolysis, or facilitating digestibility, ultimately depends on its susceptibility to enzymatic degradation in the gastrointestinal tract. In oil-in-water emulsions, lipid droplets are surrounded by interfacial layers, where the emulsifiers stabilize the emulsion and protect the encapsulated compound. Achieving a tailored digestibility of emulsions depends on their initial composition but also requires monitoring the evolution of those interfacial layers as they are subjected to different phases of gastrointestinal digestion. A pendant drop surface film balance implemented with a multi-subphase exchange allows for simulating the in vitro digestion of emulsions in a single aqueous droplet immersed in oil by applying a customized static digestion model. The transit through the gastrointestinal tract is mimicked by the subphase exchange of the original droplet bulk solution with artificial media, mimicking the physiological conditions of each compartment/step of the gastrointestinal tract. The dynamic evolution of the interfacial tension is recorded in situ throughout the whole simulated gastrointestinal digestion. The mechanical properties of digested interfaces, such as interfacial dilatational elasticity and viscosity, are measured after each digestion phase (oral, gastric, small intestine). The composition of each digestive media can be tuned to account for the particularities of the digestive conditions, including gastrointestinal pathologies and infant digestive media. The specific interfacial mechanisms affecting proteolysis and lipolysis are identified, providing tools to modulate digestion by the interfacial engineering of emulsions. The obtained results can be manipulated for designing novel food matrices with tailored functionalities such as low allergenicity, controlled energy intake, and decreased digestibility.

This section shows different examples of digestion profiles measured with the OCTOPUS. The general appearance of the simulated digestion profile matches is shown in Figure 4B. The interfacial tension is usually represented against time in the digestion profile. The different phases/digestion steps considered are represented in different colors. The first phase forms the initial layer and corresponds to the adsorption phase of the emulsifier or protein/surfactant/polymer, depending on each ca...

Watch this Scientific Journal Video about In vitro Digestion of Emulsions in a Single Droplet via Multi Subphase Exchange of Simulated Gastrointestinal Fluids at JoVE.com

In vitro Digestion of Emulsions in a Single Droplet via Multi Subphase Exchange of Simulated Gastrointestinal Fluids

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.

In vitro Digestion of Emulsions in a Single Droplet via Multi Subphase Exchange of Simulated Gastrointestinal Fluids

Emulsions are currently being used to encapsulate and deliver nutrients and drugs to tackle different gastrointestinal conditions such as obesity, ...

in-vitro-digestion-emulsions-single-droplet-via-multi-subphase

Research

JoVE Journal

Biochemistry

2.3K Views.  University of Granada. 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. 

Video: In vitro Digestion of Emulsions in a Single Droplet via Multi Subphase Exchange of Simulated Gastrointestinal Fluids

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for skin-care cosmetics. The goals of the protocol are: (1) to process chicken feathers into KH by alkaline-enzymatic hydrolysis and purify it by dialysis, and (2) to test if adding KH into an ointment base (OB) increases hydration of the skin and improves skin barrier function by diminishing transepidermal water loss (TEWL). During alkaline-enzymatic hydrolysis feathers are first incubated at a higher temperature in an alkaline environment and then, under mild conditions, hydrolyzed with proteolytic enzyme. The solution of KH is dialyzed, vacuum dried, and milled to a fine powder. Cosmetic formulations comprising from oil in water emulsion (O/W) containing 2, 4, and 6 weight% of KH (based on the weight of the OB) are prepared. Testing the moisturizing properties of KH is carried out on 10 men and 10 women at time intervals of 1, 2, 3, 4, 24, and 48 h. Tested formulations are spread at degreased volar forearm sites. The skin hydration of stratum corneum (SC) is assessed by measuring capacitance of the skin, which is one of the most world-wide used and simple methods. TEWL is based on measuring the quantity of water transported per a defined area and period of time from the skin. Both methods are fully non-invasive. KH makes for an excellent occlusive; depending on the addition of KH into OB, it brings about a 30% reduction in TEWL after application. KH also functions as a humectant, as it binds water from the lower layers of the epidermis to the SC; at the optimum KH addition in the OB, up to 19% rise in hydration in men and 22% rise in women occurs.

The goal of the protocol is to prepare keratin hydrolysate from chicken feathers by alkaline-enzymatic hydrolysis and to test whether adding keratin hydrolysate into a cosmetics ointment base improves skin barrier function (heightening hydration and diminishing transepidermal water loss). Tests are conducted on men and woman volunteers.

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

Ion Exchange Chromatography (IEX) Coupled to Multi-angle Light Scattering (MALS) for Protein Separation and Characterization

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination of the high-specificity separation technique IEX with the accurate molar mass analysis achieved by MALS allows the characterization of heterogeneous protein samples, including mixtures of oligomeric forms or protein populations, even with very similar molar masses. Therefore, IEX-MALS provides an additional level of protein characterization and is complementary to the standard size-exclusion chromatography with multi-angle light scattering (SEC-MALS) technique.
Here we describe a protocol for a basic IEX-MALS experiment and demonstrate this method on bovine serum albumin (BSA). IEX separates BSA to its oligomeric forms allowing a molar mass analysis by MALS of each individual form. Optimization of an IEX-MALS experiment is also presented and demonstrated on BSA, achieving excellent separation between BSA monomers and larger oligomers. IEX-MALS is a valuable technique for protein quality assessment since it provides both fine separation and molar mass determination of multiple protein species that exist in a sample.

Ion Exchange Chromatography IEX Coupled to Multi-angle Light Scattering MALS for Protein Separation and Characterization

This protocol describes the use of high-specificity ion-exchange chromatography with multi-angle light scattering for an accurate molar mass determination of proteins, protein complexes, and peptides in a heterogeneous sample. This method is valuable for quality assessment, as well as for the characterization of native oligomers, charge variants, and mixed-protein samples.

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination ...

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide&#8211;ribosome&#8211;mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked&#160;bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and ...

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have more realistic biological responses that resemble the in vivo environment. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more predictive study models with a broad range of biotechnological applicability. However, reproducible platforms that can achieve large-scale production of tissue spheroids have become an unmet need in fully exploring and boosting their potential. Herein, the large-scale production of homogeneous tissue spheroids is reported using a low-cost and time-effective methodology. A 3D printed stamp-like device is developed to generate up to 4,716 spheroids per 6-well plate. The device is fabricated by the stereolithography method using a photocurable resin. The final device is composed of cylindrical micropins, with a height of 1.3 mm and a width of 650 &#181;m. This approach allows the fast generation of homogeneous spheroids and co-cultured spheroids with uniform shape and size and &gt;95% cell viability. Moreover, the stamp-like device is tunable for different sizes of well plates and Petri dishes. It is easily sterilized and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to leverage their translation for multiple areas of industry, such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have ...

Smartphone as an Analytical Device to Quantify Protein Using Bradford Assay

RGBradford: Protein Quantitation with a Smartphone Camera

Protein quantitation is an essential procedure in life sciences research. Amongst several other methods, the Bradford assay is one of the most used. Because of its widespread, the limitations and advantages of the Bradford assay have been exhaustively reported, including several modifications of the original method to improve its performance. One of the alterations of the original method is the use of a smartphone camera as an analytical instrument. Taking advantage of the three forms of the Coomassie Brilliant Blue dye that exist in the conditions of the Bradford assay, this paper describes how to accurately quantify protein in samples using color data extracted from a single picture of a microplate. After performing the assay in a microplate, a picture is taken using a smartphone camera, and RGB color data is extracted from the picture using a free and open-source image analysis software application. Then, the ratio of blue to green intensity (in the RGB scale) of samples with unknown concentrations of protein is used to calculate the protein content based on a standard curve. No significant difference is observed between values calculated using RGB color data and those calculated using conventional absorbance data.

Author Spotlight: An Alternative Approach to Protein Quantification by Bradford Assay Using a Smartphone 

This paper provides a protocol for protein quantification using the Bradford assay and a smartphone as an analytical device. Protein levels in samples can be quantified using color data extracted from a picture of a microplate taken with a smartphone.

Protein quantitation is an essential procedure in life sciences research. Amongst several other methods, the Bradford assay is one of the most used. ...