Real-Time Force Measurement Between Emulsion Droplets During Enzymatic Breakdown

Summary

This protocol employs dual-trap optical tweezers to measure inter-droplet forces in real time during the enzymatic digestion of emulsions. It facilitates the study of emulsion stability and droplet dynamics, with applications in food science and pharmaceuticals.

Abstract

This protocol introduces a unique methodology for investigating the stability of emulsions and the dynamic disintegration of emulsion droplets during enzymatic digestion using dual-trap optical tweezers. The process involves preparing the emulsion, introducing the enzyme, and configuring a force measurement system to monitor inter-droplet forces in real time. By employing dual-trap optical tweezers, the method enables precise manipulation and observation of emulsion droplets, facilitating the measurement of forces between droplets during enzymatic disintegration. This experimental technique offers significant insights into the interactions and stability of emulsions during digestion, with particular relevance to research in food science, pharmaceuticals, and other industries involving organic emulsions. The method is adaptable and applicable to various optical tweezer setups and different types of emulsions or enzymes, serving as a versatile tool for exploring mechanical properties and disintegration processes across a range of fields. Furthermore, this visual approach provides the advantage of real-time monitoring of force dynamics, opening new avenues for studying emulsion behavior in diverse applications.

Introduction

Emulsions are heterogeneous systems consisting of two immiscible liquids, where one liquid is dispersed as droplets within the other. These systems are ubiquitous in various industries, including food, pharmaceuticals¹, cosmetics, and petrochemicals. The stability and functionality of emulsions are critically dependent on the interactions between droplets, which can be influenced by various factors such as surface chemistry, environmental conditions, and the presence of enzymes².

In many applications, particularly in food science and drug delivery, emulsions are exposed to enzymatic environments^3,4. For instance, in the human digestive system, ingested emulsion-based foods and drugs encounter various enzymes that can alter their structure and function. Understanding the dynamics of droplet interactions during enzymatic digestion is crucial for designing stable and effective emulsion-based products^5,6.

Traditional methods for studying emulsion stability^2,7, such as light scattering or rheological measurements, provide valuable bulk information but cannot directly observe and quantify interactions between individual droplets⁸. This limitation has hindered understanding of the microscale processes that occur during the enzymatic digestion of emulsions.

Optical tweezers have emerged as a powerful tool for manipulating microscopic objects and measuring forces at the micro- and nano-scale⁹. By harnessing the momentum of light, optical tweezers can trap and move individual droplets with exceptional precision. This capability opens new possibilities for studying droplet-droplet interactions in complex environments^10,11.

This protocol presents a unique method that combines dual optical tweezers with real-time force measurements to investigate the interactions between emulsion droplets during enzymatic digestion. As a representative example, the method is demonstrated using an emulsion system composed of rapeseed oil or one fat droplet stabilized by whey protein, undergoing digestion by trypsin. This model system is particularly relevant to food science and nutrition, as it mimics common food emulsions and their behavior in the digestive tract¹².

The approach allows for direct observation and quantification of forces between individual droplets as they undergo enzymatic breakdown, providing unprecedented insights into the dynamics of emulsion stability under digestive conditions. The method uses the optical tweezers system, a state-of-the-art dual-trap optical tweezers setup, to manipulate emulsion droplets and measure inter-droplet forces with high precision¹³.

This experimental technique bridges the gap between bulk emulsion studies and molecular-level investigations, offering a unique perspective on the behavior of emulsions during enzymatic digestion. The insights gained from this method have far-reaching implications for various fields, including food engineering, pharmaceutical formulations, and fundamental colloid science. While a specific oil-protein-enzyme system is used as an example, the methodology presented here can be adapted to study a wide range of organic emulsion systems and digestive enzymes in various industries.

Protocol

Figure 1 illustrates a schematic of dual-trap optical tweezers droplet trapping. The reagents and the equipment used in this study are listed in the Table of Materials.

1. Preparation of whey protein dispersion

1. Prepare a 5% (w/v) whey protein dispersion in deionized water.
   1. Using an electronic balance, accurately weigh 2.5 g of whey protein powder.
   2. Using a graduated cylinder, measure 47.5 mL of deionized water.
2. Combine the ingredients.
   1. Transfer the weighed protein powder and measured water into a beaker.
   2. Add a magnetic stir bar to the beaker.
3. Seal the beaker.
   1. Cover the beaker with sealing film to prevent evaporation and contamination.
4. Mix the dispersion.
   1. Place the beaker on a magnetic stirrer. Set the stirring speed to 1500 rpm (Figure 2A) until the protein powder is completely dissolved and the solution is evenly dispersed.
5. Hydrate the protein
   1. To ensure complete hydration of the whey protein and minimize foaming, prepare the dispersion one day prior to emulsion preparation.
   2. Allow the 5% whey protein dispersion to mix overnight at room temperature.
      CAUTION: Ensure that all glassware and utensils are clean and free from contaminants to avoid affecting the protein dispersion.
      NOTE: The overnight hydration step is crucial for optimal protein functionality and emulsion stability. Skipping this step may result in reduced emulsification efficiency.This protocol section outlines the initial steps for preparing the whey protein dispersion, which will be used to stabilize the rapeseed oil droplets in the subsequent emulsion preparation. The following steps typically include the preparation of the oil-in-water emulsion, setup of the optical tweezers experiment, and introduction of trypsin for digestion studies.

2. Preparation of oil-in-water emulsions

1. Optimize the formulation.
   1. Prepare the emulsion by mixing 15% oil with 85% protein solution by volume to achieve a protein-to-lipid mass ratio of 0.3.
2. Prepare the lipid phase.
   1. Rapeseed oil is liquid at room temperature. Add 30 mL of rapeseed oil into a centrifuge tube for later use.
   2. Milk fat is solid at room temperature. Take approximately five scoops of milk fat and place them into a centrifuge tube.
   3. Place the tube into a beaker and add approximately 40 mL of deionized water to the beaker.
   4. Use a digital heating plate to heat the beaker to 42 °C and maintain this temperature for 30 min to ensure complete melting (Figure 2B).
      NOTE: The melting point of milk fat is approximately 30 °C to 40 °C. The laboratory temperature is maintained at 26 °C. Melted milk fat can remain liquid for a certain period, but it will gradually solidify at room temperature if left standing.
3. Combine ingredients.
   1. In a 5 mL centrifuge tube, add 0.6 mL of oil (rapeseed oil or melted milk fat) and 3.4 mL of the prepared whey protein solution.
4. Homogenize the emulsion.
   1. Use a high-shear mixer (HSM). Set the speed to 25,000 rpm and mix for 5 min (Figure 2C).
   2. Optionally, pass the coarse emulsion through a high-pressure homogenizer. Typical conditions: 2-3 passes at 500-1000 bar pressure.
5. Dilute the emulsion.
   1. Prepare a 1:1000 dilution of the emulsion. Measure 4 µL of the original emulsion using a pipette. Add this to 3996 µL of deionized water. Mix thoroughly to ensure uniform dilution.

3. Initial setup of the optical tweezers

1. Prepare and clean the microfluidic flow cells (see Table of Materials).
   NOTE: The device is equipped with six microfluidic channels. This experiment uses a single channel-only the leftmost channel in Figure 3B is used.
   1. To prepare the microfluidic channel, clean all necessary equipment and flow channels. Thoroughly rinse all equipment with deionized water to ensure no residues remain.
   2. To clean, flush the required channel with ethanol. Set the flow rate to 1.0 bar and flush with ethanol. 1 h before the experiment, flush bovine serum albumin (BSA) into the flow cell. BSA forms a "blocking" layer on surfaces to reduce non-specific adsorption of emulsion droplets or other biomolecules.
2. Flow the prepared emulsion into the experimental channel.
3. Prepare the instrument before the experiment.
   1. Open the optical tweezers instrument.
   2. Add approximately 70 µL of deionized water to the lower objective lens.
   3. Insert the sample chamber until a "click" is heard.
   4. Place a drop of immersion oil on top of the sample chamber (Figure 3C).
   5. Gently lower the condenser lens until it lightly touches the top surface of the sample chamber.
   6. Close the optical tweezers instrument before turning on the laser. The laser operates at 1064 nm.
4. Locate the Z-plane and adjust the optical path.
   1. Turn on the laser and set the power to 100%.
   2. Adjust the lower knob (Figure 3A) on the optical tweezers system to locate the laser spot. Moving from bottom to top (clockwise adjustment), observe the laser spots three times. Set the Z-plane between the second and third sets of laser spots.
      NOTE: The three sets of laser spots appear when the laser is focused on the lower surface of the flow cell, the upper surface of the lower coverslip, and the lower surface of the upper glass slide. The laser should be focused between the upper surface of the coverslip and the lower surface of the glass slide.
   3. Reduce laser power to 30%.
   4. Verify that 7 to 11 interference fringes appear on the screen (Figure 3E). If the number is too high or too low, adjust the lower knob.
      NOTE: The number of interference fringes reflects the optimization of the spacing between the condenser and the flow cell. Within the range of 7 to 11 fringes, the condenser effectively captures sufficient light to ensure a good signal-to-noise ratio for measured signals, such as mechanical response. This spacing also maintains a safe distance to avoid lens contact with the flow cell and reduce optical path scattering or reflection interference.
5. Adjust the flow channels. Close unnecessary channels and ensure that only the required experimental channel is open.
6. Set the laser power. Usually, set the laser to 100% to capture droplets, but adjust to the required intensity for specific experimental conditions.
7. Set up data storage. Configure the file-saving directory to ensure immediate acquisition of experimental data.

4. Measurement of the force-distance curve for the emulsion

1. Set the flow rate to 0.5 bar. Droplets should be visible on the screen for approximately 5 s. Then, stop the flow by selecting Vacuum and close the channel valve.
2. Capture the droplets.
   1. Use the joystick to capture two droplets of similar size within the channel. A schematic of successfully captured droplets is shown in Figure 4.
   2. Adjust the distance between the two droplets to approximately 10 µm. If a tether forms, a noticeable change in force will be observed.
   3. Fix the x and y positions of the right optical trap and the y position of the left optical trap. Mark the two droplets with magenta and green boxes, respectively.
3. Measure the Power Spectral Density (PSD).
   1. Navigate to the Calibration menu.
   2. On the left screen, select Measure. If noise levels are excessive, repeat the measurement until satisfactory results are obtained.
   3. Select Apply to confirm the calibration.
4. Clear the original data and measure the force-distance.
   1. At this stage, the joystick should control only the left optical trap. Slowly move the left trap toward the right droplet at a constant speed (1.0 µm/s) until the distance reaches zero. This procedure yields the required force-distance curve.
5. Select the data and save.
   1. Use the left mouse button to select the data interval of interest, starting from a 10 µm droplet separation down to 0 µm. At 0 µm distance, the measured force typically reaches its maximum value.
6. Save the selected data.
   1. Right-click to save the selected data segment to the initial folder directory.

5. Preparation of the Tris buffer solution and trypsin solution

1. Prepare the Tris buffer solution with the following composition: 11.860 g/L Tris base, 0.735 g/L calcium chloride dihydrate (CaCl₂·2H₂O), and 8.766 g/L sodium chloride (NaCl). Adjust the final volume to 100 mL. Adjust the pH initially to approximately 8.4 using hydrochloric acid (HCl), and confirm the final pH using pH test strips.
2. Prepare the trypsin solution by dissolving 100 mg of Trypsin 1:250 (derived from porcine pancreas) in 1000 mL of Tris buffer. Stir the mixture with a magnetic stirrer at 4 °C and 500 rpm for 15 min. Subsequently, centrifuge the solution at 4 °C for 15 min.

6. Trypsin action and dynamic detection scheme

1. Add trypsin solution to the emulsion.
   1. Using a pipette with a range of 0.5 µL to 10 µL, draw 4 µL of the trypsin solution and carefully add it to the prepared 4 mL emulsion.
2. Perform real-time dynamic monitoring.
   1. Following the addition of the trypsin solution, dynamically monitor the emulsions using the optical tweezer technology. Measurements are taken every 10 min, with the first measurement conducted 10 min after the addition of the trypsin solution.

7. Data processing

1. Obtain the experimental data in .h5 format and perform the required format conversion using a data processing environment to produce tabular data files. The converted files consist of two sections, recording the Force and Distance data, respectively.
2. Fit the data from these two sections using curve fitting techniques, and visually plot the resulting fitted curves for further analysis.

Results

In this study, emulsions containing rapeseed oil and whey protein, and milk fat and whey protein, were analyzed using optical tweezers. First, the force-distance relationship between the two emulsions was measured to establish baseline data (Figure 5A-F). Trypsin was then added to each emulsion, and the force-distance relationship was re-measured every 10 min.

Figure 5 shows the force-distance (FD) cu...

Discussion

The core innovation of this methodology lies in the synchronized operation of dual optical traps and real-time mechanical monitoring technology (Figure 3A), enabling high-precision dynamic analysis at the single-droplet level. By independently trapping two droplets using dual optical tweezers and precisely regulating their separation to an initial 10 µm distance (see step 4.2.2), the method overcomes the traditional limitations of manipulating a single droplet. Figu...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bluelake software	LUMICKS, Netherland		optical tweezer control software
CaCl2.H4O2
Dome LED digital heating plate agitator	SCILOGEX	SCI280-Pro
Electronic balance	Shanghai Hengping Instrument Co., LTD	FA1204
HCl
High speed homogenizer	Ningbo Kemai Instrument Co., LTD	KM-S10
Immersion oil
Light-induced vortex mixing apparatus	Shanghai HUXI Industrial Co., LTD	Vortex-5S
Microsoft Office Excel	Microsoft
Milk fat	SM Mlekovita	Maslo Klarowane
NaCl
Optical tweezers platform	LUMICKS,Netherland	m-trap
Origin software	Origin Lab	2024	To draw a force and distance fitting curve
Pipette gun	Eppendorf	10 μL,100 μL,1000 μL
Rapeseed oil	Bunge Polska Sp.z o.o.	Kujawski
Sealing film	Parafilm	4IN*125FT
Tris
Trypsin	Sangon Biotech	A100458-0010	Trypsin 1:250, from Porcine pancreas
Whey protein concentrate	SM Mlekovita	WPC