# Studying the Process of Enzyme Treatment on Beef Meat-Bone Paste Quality
## 3 Results and Discussion  
### Enzymatic Treatment of Meat-Bone Paste  
Enzymes play a very important role in the food industry; in some cases, carrying out or helping to carry out many technological processes while in others complicating their implementation. Enzyme preparations are actively used in meat production technology. The improvement of organoleptic, functional and technological properties largely depends on the enzymes contained in meat [37; 38]. In bone tissue, collagen molecules are indeed closely associated with the mineral phase, consisting of calcium phosphate mineral nanoparticles, predominantly hydroxyapatite. This interaction creates a complex and hierarchical structure in bone, contributing to its strength and resilience. The hierarchical structure of bone collagen is stabilized due to molecular entanglement, intermolecular interactions and the mechanical relationship between hydroxyapatite crystals and specific amino acid groups (e.g., polyaspartic acid sequences, carboxy glutamic acid residues and glutamic acid sequences) in bone collagen [39; 40]. For each enzyme, there is a pH interval of the medium that is optimal for the manifestation of its highest activity. For example, the optimal pH values for pepsin are 1.5-2.5; trypsin, 8.0-8.5; saliva amylase, 7.2; arginase, 9.7; acid phosphatase, 4.5-5.0; and succinate dehydrogenase, 9.0. The pH value at which the reaction proceeds at the maximum speed is considered optimal. At higher and lower pH, the activity of the enzyme decreases. With a decrease in pH, the acidity increases, and the concentration of H\({}^{+}\) ions increases. Consequently, the number of positive charges in the medium increases [41]. The dependence of enzyme activity on the duration of treatment was monitored (Figure 1). The enzyme treatment parameters were determined by at least five parallel experiments. The results show that the enzyme reaction rate varied with the treatment duration. At 60 min, the rate was 0.004 mmol/min, increasing to 0.014 mmol/min at 120 and 180 min, and then gradually decreasing to 0.006 mmol/min at 480 min. This decrease in enzyme reaction rate may be attributed to the depletion of substrate or the inhibition of enzyme activity due to the accumulation of reaction products. The sustained high enzyme activity at 180 min suggests an equilibrium point where the catalytic potential is optimized. The rate of the enzymatic reaction strongly depends on the temperature, increasing with increasing temperature. However, due to the protein nature of the enzyme, denaturation of the enzyme occurs with a further increase in temperature. The temperature at which the reaction rate is maximal is called the temperature optimum. The dependence of pepsin enzyme activity on temperature is described by a curve with a maximum velocity at temperatures from 25 to 35 \({}^{\circ}\)C (Figure 2). At a substrate temperature of 10 \({}^{\circ}\)C, the enzyme reaction rate was 0.0023 mmol/min, which increased significantly to 0.0107 mmol/min at a substrate temperature of 20 \({}^{\circ}\)C. The enzyme reaction rate continued to increase, reaching a maximum value of 0.0129 mmol/min at a substrate temperature of 30 \({}^{\circ}\)C. Beyond this temperature, the reaction rate starts to decrease, with values of V = 0.0119 mmol/min at 40 \({}^{\circ}\)C. At temperatures close to and above 50 \({}^{\circ}\)C, the enzyme reaction rate decreased significantly, indicating that the enzyme had lost its activity due to thermal denaturation of the protein enzyme. This decrease in enzyme activity is attributed to the unfolding of the enzyme's tertiary structure, leading to the disruption of the active site and the loss of enzyme function [42,43]. Overall, the results demonstrate that the enzyme reaction rate is highly sensitive to substrate temperature, with enzyme activity increasing significantly with increasing temperature up to a certain point, after which thermal denaturation of the enzyme occurs, leading to a decrease in enzyme activity.