## 3 Results and Discussion  
### Disintegration of CM: Defining PEF Parameters  
As already described, there are reasonable assumptions that PEF application can change properties of CM to disintegrate the structure and, therefore, prepare them for being loaded with \(\beta\)-carotene. These assumptions are based on the fact that CM are stabilized from the outside by an electrical double layer and from the inside by calcium phosphate clusters, which causes a natural polarization of the CM (cf. Figure 1). The electric double layer includes a layer of carboxyl groups of caseinacropeptide and a relatively mobile layer of calcium ions [38]. If the negative charge is reduced by changing the external conditions, such as an electric field, conditions comparable to the isoelectric state are created, leading to a coagulation of the micelles. This is in agreement with [PERSON] [39], who found that the size of CM at pH 8 can be influenced by PEF. Additionally, changes in charge distribution in the electric double layer can be caused by electrical pulses [40]. Hence, different PEF treatment conditions should lead to disintegration and restructuring of CM. The possible effect of PEF on CM can be studied by comparing the results of the CM analysis before and after the treatment. Figure 3 shows a schematic diagram of CM structure in native (a) and in disintegrated state (b). As can be estimated from these structures, relevant parameters for identifying the respective CM state are specific surface area (SSA), charge and charge density, as well as the surface potential. Properties pointing to CM disintegration are larger SSA due to volume changes and higher charge density. A higher charge density results from charge carriers being more available (e.g., for the titrants used for determination). Furthermore, surface potential is also relevant as a lower negative value is found for treated CM compared to native (untreated) CM (data not shown). Hence, PEF parameters for CM disintegration have to be identified so that CM properties are adapted in the way of a higher SSA and charge density, as well as a lower negative surface potential. Figure 3: Schematic diagram of potential CM structure in native (a) and in disintegrated state (b). Previous studies (data not shown) have shown that the biggest differences in the effect of disintegration were found at different electric field strengths and pulse widths. Only a limited effect on CM disintegration was detected for specific energy intake. Therefore, the lowest energy level was used for further investigations in order to allow a higher capacity for industrial application, and also lower energy costs. The results concerning SSA are summarized in Figure 4 and correlated with CLSM images. Here, the difference in SSA before and after PEF treatment (ASSA) is correlated with electric field strength and pulse width. Since CMR's isolation is very complex and cost-intensive, only limited amounts were available, so the industrial semi-finished product MCC 80 was used for these basic preliminary tests. The application of low electric field strength and short pulse widths result in the most negative difference, meaning an increase in SSA, which is correlated with the disintegration of agglomerates/CM. High electric field strength and long pulse width lead to the most positive difference, which is correlated with a decrease in SSA and the formation of agglomerates/CM, respectively. These aspects are verified and visualized using CLSM. As a control sample, a CLSM image of rehydrated, spray-dried CM is also shown for comparison. PEF also modifies the charge density of CM (cf. Figure 5). As already described, an increase in charge density means that the availability of free charge carriers has also increased, which implies disintegration of CM. Consequently, a decrease in charge density stands for a decrease in the availability of free charge carriers, implying an agglomeration, respectively.