## 3 Results and Discussion
### Amount of Non-Intact Cells  
When a mechanical force is applied to the muscle fibers, the well-ordered fibrillar structures and connective tissue are disrupted, making proteins and intramuscular substances open for extraction [31], and the amount of non-intact muscle fibers (ANIC) is significantly increased [32; 33]. The amount of ANIC negatively influences the quality and functionality of minced meat and is, therefore, officially restricted to 20% vol. in Germany [4; 34].  
The lowest amount of ANIC (30%) in our experiment was observed for the minced pork made exclusively from fresh meat chilled at 3 \({}^{\circ}\)C (Figure 5). This result is in concurrence with the investigation of [PERSON], [PERSON], [PERSON], [PERSON], [PERSON], [PERSON], [PERSON], [PERSON], [PERSON], [PERSON], [PERSON], and [PERSON] [4] where lower amounts of ANIC also could not be registered when the two step mincing process was utilized. We already know that the first step increases the amount of ANIC by a factor of 2.4, and that the second step raises it further by a factor of 2.7 [32]. In terms of the meat mixing and forming of minced meat under pressure, the results are contradicting. The results of [PERSON] [33] suggest that these processing steps  
Figure 4: Boxplots of sensory evaluation of (**a**) firmness, (**b**) juiciness, (**c**) inner cohesion, and (**d**) inner structure (n = 96) of cooked meatballs consisting of varying frozen to fresh meat ratios; 0 means soft, dry, loose cohesion, and fine structure, whereas 10 means firm, juicy, strong cohesion, and coarse inner structure. Dotted line marks mean, full line marks median, box marks lower and upper quartile, and whiskers mark distance until lower and upper extreme. Different letters represent significant differences (\(p<0.05\)) for each parameter.  
are crucial for the muscle fiber disintegration, while the reports of [PERSON], [PERSON], [PERSON], [PERSON], and [PERSON] [32] have not provided evidence that they have a significant influence on the ANIC formation. Presumably, the more gentile mixing and lower pressures applied during forming diminish the negative effect of these processing steps. This also explains why only 6% of artisanal-produced and almost 45% of industrial-produced minced meat had an ANIC over the regulatory limit in Germany [35].  
The amount of non-intact cells significantly (\(p<0.05\)) increased with an increased portion of frozen meat added to the meat batter. The highest amount of ANIC was measured in the 50/50 frozen to fresh meat ratio with 70% vol. (Figure 5). This phenomenon could be explained by the effect of freezing on the microstructure of meat. Depending on the freezing rate, larger (slow freezing) or smaller (fast freezing) ice crystals out of the immobilized and free water will be formed [13] and they are capable of disrupting muscle cells [36]. It seems that the freezing of the meat used in our experiments led to the increased amount of ANIC in the minced pork. Therefore, the more frozen meat we added to the batter, the more ANIC was detected in the final product (Figure 5).  
The higher standard deviations in Figure 5 might be caused by the differences in the unprocessed material (fresh and frozen meat), as the presented mean value is composed out of the four biological replicates made from different meat cuts, animals, and at different days. Additionally, meat samples may exhibit micro-regions or \"mushy\" regions where crystallization and/or solidification may take place in separated areas, rather than uniformly [37] and the fact that for the histological analyses of ANIC only one sample (meat dice of 2 \(\times\) 2 cm) per biological replicate is taken, only adds up to the observed variations in the final results. Finally, we would like to concur with the conclusion of [PERSON] and [PERSON] [38] who acknowledged that with the addition of frozen meat into the comminated meat products, an ANIC that is in compliance with German regulatory requirements is technologically (practically) impossible.