Materials
Casein micelle concentrate (CMC) powder MC 80 was kindly provided by Milei GmbH Germany and citrus pectin (classic CU 201) was from Herbstreith & Fox (Herbstreith & Fox GmbH & Co. KG, Neuenbürg, Germany). Pectinase from Aspergillus niger, Tri-Sodium citrate, hydrochloric acid (1M), Sodium hydroxide (1M), and Sodium azide was from Merck (Merk, Darmstadt, Germany). Especially pure sodium dodecyl sulfate (SDS), ultra-pure BisTris, calcium chloride, and all salts (purity >99% or analytical grade) for SMUF preparation were obtained from VWR, Radnor, USA. Milli-Q water was obtained from our lab.
Preparation of working solutions
BisTris buffer solution (50 mM BisTris, 10 mM CaCl2) was prepared by adding 10.462 g BisTris in a volumetric flask containing about 980 ml milli-Q water under continuous stirring. After complete dissolution, 1.11 g CaCl2 was added to the solution and allowed to dissolve completely. The pH of the solution was adjusted to 6.8 with 1M HCl and NaOH and finally filled the volume up to 1L.
Simulated milk ultra-filtrate (SMUF) solution was prepared according to a protocol described by Dumpler et al 2017. All salts were dissolved step by step, allowing the complete dissolution of the previous salt then the next salt was added. Finally, pH was adjusted at 6.8 with 1M HCl and NaOH.
Pectin solution (2%) was prepared by dissolving 1 g Pectin in 49 g BisTris buffer solution with vigorous stirring at 80°C for 3 h then cooled down to room temperature. Finally, pH was adjusted to 6.8 with 1M HCl and NaOH.
Casein dispersion (7.36%) was prepared by dissolving 2 g of the CMC powder (protein >80% of which >92% casein) in 18 g of SMUF. The CMC powder was allowed to dissolve under continuous stirring at room temperature for 1 followed by 4 h at 4°C and finally 1 h at 37°C. To avoid microbial contamination, 0.5 g.L-1 sodium azide was added to the dispersion. For the preparation of citrate-treated CMP, the required amount of Tri-sodium citrate solution (200 mM in water) was added during the final 1 h stirring steps of casein dispersion.
Pectinase solution (activity ≈ 36 units/ml) was prepared by adding 0.47 ml pectinase (activity ≈ 800 units per ml) from Aspergillus niger to 10 g BisTris buffer and mixed properly. The solution was prepared immediately before adding to the film.
Preparation of Casein microparticle (CMPs)
The principle of the CMP production process was based on the depletion flocculation interaction between casein micelles (CMs) and pectin (Tuinier et al. 1999). The individual preparation steps are summarized in Fig. 1. The preparation of the CMPs was according to a protocol described by Schulte et al 2021. Briefly, the casein solution, pectin solution, and BisTris buffer solution mentioned above were mixed properly in a proportion of 4.1:1.5:4.4 (w/w) to obtain 3.0% casein and 0.3% pectin in the final solution. Then 3.9 g of this mixed solution was transferred to a glass petri dish (Ø 70 mm) and dried at room temperature for 16 h to produce a film. To hydrolyze this film 10 g pectinase solution was added to the petri dish. The enzymatic hydrolysis was performed in a ThermoMixture (Eppendorf, Eppendorf AG, Hamburg, Germany) at 47°C for 2 h with 160 rpm. After hydrolysis, the supernatant solution was collected in a falcon tube and centrifuged at 22°C and 1500 RFC for 10 min. The clear solution was poured out from the top and the resulting stabilized pellet of CMPs was then suspended again in BisTris buffer and stored at 4°C for further use.
Swelling experiments
The swelling behavior of CMP was studied according to the protocol developed by Schulte et al 2020a. Briefly, the swelling chamber was filled with CMP dispersion (in BisTris buffer, pH 6.8) and placed under Leica DMIL LED inverted microscope (Leica Microsystems, GmbH, Wetzlar, Germany) connected with Basler camera (Basler AG, Ahrensburg, Germany). The dispersion was allowed to stand for approx. 10 min to sediment the CMP into the sieve holes. A PHD ULTRA™ syringe pump (Harvard Apparatus, MA, USA) was connected with the swelling chamber by polyethylene tubes (Ø 0.55 mm). The pump flow rate was set at 0.05 ml per min for the exchange medium (ultrapure water, pH, 3 or 8). The swelling process of the CMP at different pH was started by replacing the buffer solution with an exchange medium. With the activation of the syringe pump, an image of a single CMP trapped in the sieve holes was started to record (at the rate of 2 frames per second for 2 h) using the Basler video recording software. Image frames were extracted using PyCharm (version 2021.1.3, JetBrains, Czech) and the area of the CMPs was calculated by a freehand selection of particles outer lines using ImageJ software (NIH, USA). All samples were measured in duplicate.
Dynamic swelling model and data analysis
For a more detailed analysis of the swelling behavior, the cross sections of the CMPs in the micrographs were first evaluated and plotted as a function of swelling time. The influence of citrate on the typical two-stage swelling behavior of the CMPs can be simulated with a dynamic model whose basic structure has already been described (Schulte et al., 2020b). Volumes are first calculated from the determined cross-sectional areas for the simulation, assuming the spherical approximation that is satisfied for the swelling process (Schulte et al., 2021). Within the model, the volume changes by two inflows and two outflows, respectively, which are controlled by valves. All incoming and outgoing volume flows are proportional to the current volume of the CMPs at each time point. The rate coefficients for the inflows and outflows assume a time-invariant value after a characteristic time through a step function (or through a transition function as exemplified in Fig. 3). The model is able to describe all measured swelling kinetics in a good approximation by a maximum of 9 parameters (4 characteristic times and 4 rate coefficients as well as an initial value for volume or particle area). The numerical integration of the underlying differential equations was performed with the program Stella, isee systems.
Stability experiments
For stability analysis, turbidity of CMPs solution was monitored using a Lambda 365 UV/VIS spectrometer (PerkinElmer, USA) according to the method described by Schulte et al 2021 with slight modification. Briefly, 1.5 ml CMP dispersion was filled in semi-micro cuvette (Eppendorf AG, Germany) and BisTirs buffer was used as reference. The turbidity was measured at a wavelength of 600 nm, slit width of 1 nm and absorbance was recorded for 900 s. Immediately after measurement, 40 µl SDS solution (520 mM in water) was added to CMP dispersion resulting in a 13.5 mM final concentration of SDS. The cuvette was then gently up and down 3 times to properly mix the SDS, placed back in the spectrometer, and measured again. All samples were measured in duplicate.