# Influence of Processing Steps on Structural, Functional, and Quality Properties of Beef Hamburgers
## 3 Results and Discussion
### Structural and Functional Properties  
To check the influence of each process step on the _ANIC_, the meat samples were histologically analyzed for their degree of non-intact muscle fibers. During the first grinding step, the _ANIC_ was approximately doubled (factor 2.4) from 3.79 \(\pm\) 2.61 Vol.% in raw material to 9.21 \(\pm\) 4.81 Vol.% after the first grinding, thereby reducing the volume of the particle by a factor of ca. 100 (Figure 2A). During the second grinding step, the volume of the particles was further reduced by a factor ca. 81, and the _ANIC_ significantly increased about threefold (factor 2.7, Figure 2A) from 8.87 \(\pm\) 3.91 Vol.% after mixing to 23.86 \(\pm\) 14.16 Vol.% after the second grinding. Both the mixing and the forming steps did not significantly change the _ANIC_ (\(p\) > 0.05). Hence, the grinding steps have the biggest influence on muscle fiber disintegration, whereby the increase is higher in the second grinding compared to the first grinding. The higher standard deviations in Figure 2A might be caused by variations in the raw material meat, as the displayed mean value is composed out of three biological replicates made from different meat cut at different days. It is known that parameters, such as physiological parameters of the animal [23], sex, age, or slaughtering conditions [24] influence the meat quality, wherefore changes between different batches are reasonable. Additionally, the histological  
This outcome was expected, as grinding decreases the particle size through cutting and shearing the meat through end-hole plates, which disintegrates muscle structures [2]. The findings of [PERSON] [2] that mechanical treatment, such as mixing or forming, under pressure are key parameters in muscle fiber disintegration could not be verified in this study. This might be explained by quite gentle mixing and forming conditions in the present study. Increasing mixing time, speed, or higher formation pressure might cause stronger disintegration of meat muscle fibers [25; 26]. To check the influence of each process step on the water-holding capacity, the samples were analyzed for their drip loss (\(DL\)). As muscle fiber disintegration is associated with increased \(DL\)[6], it is considered as a quality determining product parameter in this study. The centrifugation method was chosen over the sedimentation method, i.e., without applying an external force other than gravity (e.g., 24 h, 1 g) [14], as preliminary experiments revealed no \(DL\) on hamburgers over several days (data not shown). [PERSON] [27] described that all methods determining the free-water content can be used to determine the water-holding capacity of meat. Both traditional sedimentation, as well as accelerated methods applying centrifugal forces, are applicable. It is assumed that the sedimentation method might be more suitable for meat samples releasing free water easily, e.g., due to meat defects [21]. The \(DL\) increased slightly but not significantly from \(6.61\pm 2.83\%\) in the meat piece to \(10.50\pm 3.45\%\) after the first grinding (Figure 2B). For all other process steps, the \(DL\) remains nearly constant at 10.38 to 11.15%. As the volume reduction of the meat particles in the first grinding is stronger compared to the reduction in the second grinding, more muscle fibers are cut, and more intramuscular fibrillar substances might leak out. Thus, it is reasonable that the \(DL\) slightly increased during the first grinding step. In contrast to the present study, [PERSON] et al. [12] found a more pronounced positive correlation between muscle fiber disintegration caused by rupture of myofibrils and water release from mechanically stressed pork meat. Additionally, they found that meat treated with a meat grinder lost more water compared to meat treated gentler with a meat activator or tenderizer. This underlines the hypothesis of a higher \(DL\) with increasing \(ANIC\)[12] but could not be fully confirmed in this study. Following the idea of [PERSON] et al.