# Studying the Process of Enzyme Treatment on Beef Meat-Bone Paste Quality
## 3 Results and Discussion
### Particle Size and Microstructural Analysis  
The research investigated the effect of enzyme treatment on the size distribution of bone particles from cow bones after fine grinding. Two samples were studied: a control sample without enzyme treatment and the other after enzyme treatment in a solution of pepsin and ascorbic acid. In experiment 1, without enzyme treatment, the majority of bone particles (86.4%) were between 0.25 and 0.75 mm in size (Figure 4). In contrast, after enzyme treatment in experiment 2, the majority of bone particles (86.33%) were between 0.05 and 0.2 mm in size. This result indicates the efficacy of the enzyme treatment process in breaking down larger bone particles into smaller ones. The presence of particles below 0.05 mm, albeit in a small percentage (1.20%), further highlights the ability of enzyme treatment to generate finer particles.  
This shift in particle size distribution underscores the role of enzyme treatment in promoting the breakdown of bone particles into smaller dimensions. The enzymatic action of pepsin, coupled with the influence of ascorbic acid, appears to facilitate the disintegration of larger bone particles, leading to a more refined particle size distribution. Pepsin is a protease enzyme that breaks down proteins, while ascorbic acid (vitamin C) acts as a reducing agent [45]. This process has the potential to improve the bioavailability and utilization of bone-derived nutrients and enhance the handling properties of bone powder.  
In further studies, we determined the sizes of bone particles after grinding by microscopy. After fine grinding without enzyme treatment, the average size of bone particles  
Figure 4: Distribution of mass fraction of bone particles depending on particle size.  
was 0.21 mm based on the results of bone particle measurements. The largest size was 0.37 mm; the smallest was 0.14 mm (Figure 5).  
After the enzyme treatment process of meat-bone paste, the average size of bone particles was 0.052 mm according to the measurement results. The largest size was 0.095 mm; the smallest was 0.025 mm (Figure 6). From the bone particle size results, the bone particles after enzyme treatment were on average four times smaller than the bone particles of the meat-bone paste obtained without enzyme treatment.  
Enzymatic treatment with repsin and ascorbic acid of meat-bone paste from the rib bones of cattle showed the splitting of bone particles. The dependence of the reaction on  
Figure 5: Bone particle size of meat and bone paste without enzyme treatment.  
Figure 6: Bone particle size of meat-bone paste after enzyme treatment.  
temperature was established: with an increase in temperature, the activity of the enzyme increased, and popsin showed the greatest activity at a temperature of 30 \({}^{\circ}\)C.  
The processing of meat-bone paste with popsin and ascorbic acid at a temperature of 30 \({}^{\circ}\)C showed the greatest enzyme activity after undergoing the reaction for a duration of more than 4 h. Another parameter affecting the reaction rate is the enzyme amount, which affected the activity of the enzyme to a small extent, whereas the amount of meat-bone paste had a significant effect: the more meat-bone paste, the lower the popsin activity. A reduction in chicken bone particle size was observed in [46]. The research revealed a notable reduction in the particle size of chicken bone powder, with a 44.21% decrease in the median particle size following steam explosion. Notably, the steam explosion treatment yielded smaller particle sizes of chicken bone powder, demonstrating advantages in terms of both time and energy efficiency when compared to alternative treatments [46]. [PERSON] (2021) used enzymatic acid hydrolysis of bone with popsin and hydrochloric acid (1M) for the production of gelatin [47].  
The application of popsin and ascorbic acid-treated meat-bone paste in food production eliminates bone particles, ensuring its safe integration into meat product technologies.