## 4 Utilizing the Marx Generator in Biomass Processing
### Application of the Marx Generator for PEF Treatment of Milk Casein
#### 4.2.1 Materials and Methods of CSM Treatment  
The generator produced was also used to process milk casein in a pulsed electric field. Casein micelles (CSM, 92% protein) were purchased from VWR Chemicals (Leuven,  
Figure 9: Visualization of PEF-extracted proteins by SDS-PAGE analysis. The numbers above the gels indicate the strength of the applied electric field (in kV/cm) in Range 1 (**a**) and Range 2 (**b**). ‘L’ denotes the ladder, and ‘C’ indicates the control.  
Belgium). Sodium hydroxide and sodium chloride were obtained from ApplChem GmbH (Darmstadt, Germany).  
CSM protein suspension (1%, \(w/v\)) was prepared in potassium phosphate buffer (0.1 M, pH = 7) and stirred overnight. The protein suspension with an electrical conductivity of about 1.4 mS/cm was then treated using both ranges of the Marx generator when the capacitors were charged to 500-1500 V. For this treatment, a modular serial flow-through co-linear chamber was designed with two active treatment areas and the meshes in contact with the electrodes. The design of this chamber was the same as the chamber described in ref. [41]. The stainless steel meshes in this chamber act as electrodes and provide a homogeneous distribution of the electric field in the active treatment areas. The distance between these meshes was 5 mm when the diameter of the treatment region was about 15 mm. The average resistance of the chamber with this suspension was about 400 \(\Omega\).  
During the experiment, the suspension was pumped into the tubes using a peristaltic pump (Masterflex, Cole-Parmer, Vernon Hills, IL, USA). The flow rate was adapted to the repetition frequency of the generator pulses so that each \"portion\" of the suspension (located between the electrodes and affected by the electric field) was treated with only one pulse. This made it possible to obtain more correct results. The characteristic repetition of electrical impulses in this case was 1 Hz. Such a time interval between pulses was sufficient to fully load the capacitors. The repetition rate as well as the shape of the pulses, and the maximum voltage were stable. To avoid turbulence processes in the liquid in the cuvette caused by the electrode grid and the change in the shape of the channel in the cuvette, it was also necessary to limit the flow rate of this liquid. Therefore, this limited the maximal repetition rate of the pulse. The electroporation chamber was set up in a vertical position. The voltage pulses at the electrodes, and the current was recorded with an oscilloscope. In these experiments, a protein suspension of about 50 mL was treated.  
The particle size and \(\zeta\)-potential were measured using the dynamic laser light scattering (DLS) of protein samples before and after electroporation using a Malvern Zetasizer analyzer (Nano ZS, Malvern Instrument Co., Ltd., Worcestershire, UK). The diluted samples (1:20) were measured at refractive indexes of 1.45 and 1.33 for the protein and dispersant, respectively [42].  
The protein concentration was adjusted to 0.5 mg/mL with 0.1 M phosphate buffer (pH 7.0). In total, 3 mL of each sample was added to a quartz cuvette with a 10 mm path length. The intrinsic fluorescence spectra were recorded with an LS-50B luminescence spectrometer (PerkinElmer Inc., UK). The excitation and emission slit were 10 nm. The fluorescence emission spectra were recorded from 300 to 450 nm at an excitation wavelength of 290 nm [43].  
All experiments were performed in triplicate. Mean values and standard deviations were calculated. The results were analyzed by one-way analysis of variance (ANOVA) using Excel software and SPSS (IBM SPSS Statistics, Version 25, SPSS Inc., Chicago, IL, USA) with a statistical significance of \(p<0.05\).