Studying the Process of Enzyme Treatment on Beef Meat-Bone Paste Quality

Abstract

Animal bones, particularly from cattle after slaughter, are commonly discarded, posing environmental challenges and highlighting the need for sustainable valorization. This study investigated the effect of enzyme and organic acid treatment on physicochemical properties, particle size, microstructure and safety of meat-bone paste (MBP). Two samples were prepared: a control (MBP-C) without enzyme treatment and an experimental sample (MBP-E) treated with pepsin and ascorbic acid. Results showed that the enzyme reaction rate increased from 0.004 mmol/min at 60 min to 0.014 mmol/min at 120–180 min before declining to 0.006 mmol/min at 480 min, suggesting substrate depletion or product inhibition. Temperature greatly influenced reaction rates, peaking at 0.0129 mmol/min at 30 °C, with significant declines at higher temperatures due to enzyme denaturation. The enzyme’s kinetic performance was proportional to the pepsin concentration, demonstrating enhanced catalytic efficiency at higher enzyme concentrations. Particle size analysis revealed that enzyme treatment significantly reduced bone particle size, with 86.33% of particles measuring between 0.05 and 0.2 mm, compared to 86.4% between 0.25 and 0.75 mm in the untreated sample. Microscopy confirmed these findings, showing an average particle size reduction from 0.21 mm to 0.052 mm after enzyme treatment. Physicochemical analysis revealed no significant differences in chemical composition between the two samples. However, enzyme-treated MBP-E exhibited a lower pH (5.9) compared to MBP-C (7.02), attributed to the addition of ascorbic acid. Water-binding capacity significantly increased in MBP-E (82.54% vs. 77.28%), indicating enhanced hydration and collagen loosening during enzymatic action. Enzyme treatment significantly reduced the total viable count and eliminated pathogenic bacteria (E. coli, Listeria, Salmonella), improving MBP safety. These findings highlight the potential of this approach for valorizing animal bones as a valuable food ingredient while promoting sustainable waste management practices.

1. Introduction

Animal bones, particularly those derived from cattle after the slaughtering process, are commonly treated as solid waste and often disposed of without adequate utilization. This prevalent practice poses significant challenges, primarily contributing to environmental pollution and underscoring the need for more sustainable and resourceful approaches to handle such biological byproducts [1,2]. Therefore, deep processing of animal bones after slaughter is a relevant topic for both industry and scientists. The main barrier to the use of bone raw material in the technology of meat products is its great hardness and elasticity. Significant hardness and elasticity of bone tissue are achieved due to a unique combination of organic base with mineral salts that are insoluble in water. The main organic part of bone is bone collagen (ossein), which provides strength to connective tissue. It accounts for 93% of the total amount of bone proteins, the rest being glycoproteins, lipids, and glucosaminoglycans (chondroitin sulfate, keratan sulfate, and other glucosamines and galactosamines) [3]. Connective tissue is formed by intertwining collagen fibers and their bundles. Collagen fibers stretch very well, which gives the skin elasticity. The collagen molecule is a helix formed from three chains [4,5]. Collagen fibrils consist of rod-shaped molecules (tropocollagen) that are 300 nm long and 1.5 nm thick. Tropocollagen consists of three polypeptide chains, so-called α-chains, forming a triple helix. In these fibrils, tropocollagen molecules are linked together using transverse covalent cross-links localized mainly at the ends of the molecules, the so-called telopeptides [6]. Due to its dense helical structure, native tropocollagen is highly resistant to alkaline, acid, and enzymatic hydrolysis. However, its heat peptides do not possess such resistance since they are not integrated into the helical structure but represent disordered globular areas [7].

To split the bone structure, the chemical hydrolysis method is used in combination with organic acids and enzymes. Chemical hydrolysis is a method in which acidic materials are used in the hydrolysis process, namely organic acids (acetic acid, citric acid, lactic acid) and inorganic acids (hydrochloric acid). Organic acids are capable of dissolving non-collagen cross-links and are also capable of destroying collagen cross-links, which produce collagen that is more soluble during extraction [8,9]

Disrupting the supramolecular structure of collagen to achieve dissolution or solubilization typically involves a combination of mechanical and chemical processes. Mechanical processes, such as fine grinding, are employed to break down collagen-containing raw materials into smaller particles (less than 100 microns), facilitating the subsequent chemical processes for solubilization [10,11]. There are two methods for destruction of the supramolecular structure of collagen with preservation of the structure and properties of tropocollagen—the enzymatic treatment and the alkaline salt treatment. Enzymatic treatment involves the use of enzymes, such as collagenase, which specifically target and break down collagen molecules. These enzymes hydrolyze peptide bonds within the tropocollagen structure, leading to the degradation of collagen fibrils and fibers. As a result, the strength of fibrils and fibers sharply decreases, and the subsequent action of the reagent provides collagen dissolution [12,13,14].

Enzymes are widely used in the food industry. Specialists and scientists have developed processes for separating meat from bones using proteases, as well as methods for splitting meat waste into fat, soluble protein, insoluble protein, and bone fractions using various enzymes [15]. In China, methods for processing meat-bone raw materials from the poultry processing industry for food purposes have been proposed [16,17]. There are known studies on the use of ultrasonic pre-treatment to promote enzymatic extraction of chicken bone protein [18]. Cao and others [19] proposed a method for acid treatment and extraction of collagen with pepsin from the waste of skin and bones of the golden pompano. Scientists from Indonesia studied the isolation of collagen from goat bone and determined its characteristics using enzymatic hydrolysis with pepsin at various concentrations [20].

The use of enzymes in meat processing plays a crucial role in enhancing both meat quality and processing efficiency. These enzymes, derived from microorganisms, plants, or animal tissues, are known for their ability to break down tough muscle fibers and connective tissues, thereby improving meat texture, juiciness, and overall palatability. Commonly used enzymes, such as papain, trypsin, pepsin, ficin, and microbial proteases, are well-recognized for their ability to tenderize meat and facilitate processing [21]. Pepsin, one of the key enzymes mentioned, is a globular protein with a molecular weight of about 34,500 Da (daltons). The pepsin molecule is a polypeptide chain consisting of 340 amino acids and contains 3 disulfide bonds and a phosphoric acid group. Pepsin is an endopeptidase, an enzyme that cleaves central peptide bonds in protein and peptide molecules [22]. Its optimal catalytic activity is observed at a low pH range of approximately 1.5 to 2.0. The low pH is necessary for the activation of pepsinogen, the inactive precursor of pepsin, into its active form [23,24]. Beyond its role in tenderizing meat, enzymatic processing holds promise in the bioconversion of animal and poultry byproducts into value-added products. This approach not only enhances the nutritional value of byproducts but also contributes to environmental sustainability by reducing waste [25,26].

The goal of this research was to study the effect of enzyme and organic acid treatment of meat-bone paste on the particle size, microstructure and physicochemical properties. Processing of meat-bone raw material into a fine paste allows the addition of this product to meat products (sausages, pâtés, cutlets, meatballs, etc.). Since the meat-bone paste is produced without heat treatment, it retains all the highly valuable vitamins, protein and mineral substances. The use of meat-bone paste for food purposes could solve the problem of insufficient supply of mineral and protein components to the human organism. Furthermore, the costs for producers of meat products may be significantly reduced.

2. Materials and Methods

2.1. Meat-Bone Paste Production

Fresh rib bones (moisture 33.89%, protein 24.30%, fat 12.70%, ash 29.11%) were obtained from meat processing enterprises and large meat pavilions of the city of Semey, Kazakhstan and cut to the size of 50–70 mm. To obtain a finely dispersed meat-bone paste, the rib bones of cattle were crushed in a bone crusher. Then, crushed bones (3–5 mm) were ground in a rotary ultrafine grinding machine, “Supermascolloider MKZA 10-15” (Masuko Sangyo Co., Ltd., Kawaguchi city, Japan), with a gap between rotary knives of 0.01 mm [27,28].

2.2. Enzymatic Treatment

The enzymatic treatment utilized ascorbic acid (99.8% purity, LLC Khimproduct, Kyiv, Ukraine) and pepsin (800 IMCU/g proteolytic activity, ≥85% active substance, Vernal LLP, Almaty, Kazakhstan). First, 100 g of meat-bone paste was weighed. A film or lid was placed over the bowl containing the paste to prevent moisture evaporation. Following this, a solution of pepsin and ascorbic acid was prepared. The working pH was adjusted to 2.0 by adding 2 mL of ascorbic acid (0.05 M), 5 mL of distilled water, and 10 g of pepsin. The working pH of 2.0 was selected based on the optimal pH range for pepsin activity, ensuring maximum enzyme efficiency during the reaction. This solution was then added to the meat-bone paste and thoroughly mixed to ensure even distribution. The temperature was regulated between 28 and 30 °C using a water or thermostatic bath, and the reaction duration was set to 4 h. The temperature range of 28–30 °C was chosen to facilitate pepsin activity without denaturing the enzyme or compromising the integrity of the meat-bone paste. This range reflects the optimal temperature for pepsin, allowing for effective collagen breakdown while minimizing thermal damage to the proteins and minerals present in the paste. The reaction was influenced by factors such as pH, temperature, enzyme concentration, and substrate concentration.

2.3. Determination of Chemical Composition

The moisture content was determined by drying the samples in a drying oven (temperature—150 °C; duration—60 min) and calculated according to the standard [29]. Fat determination was performed using a Soxhlet extraction device according to the method described in GOST 23042-86 [30]. To measure the ash content, the samples were calcined in a muffle furnace (500–600 °C) and then were calculated according to [31]. Finally, the protein content was analyzed by the Kjeldahl method according to the standard GOST 25011-81 [32].

2.4. Determination of pH

The active acidity of the medium (pH) was determined by the potentiometric method on the pH meter 340 by dipping two electrodes into a solution while fixing the pH value on the scale of the device. The solution (water extract) was prepared from the crushed product with water (in a ratio of 1:10). The pH was measured after infusing for 30 min at a temperature of 20 °C [33].

2.5. Microstructure of Bone Particles

To identify the size of bone particles, the microstructure of bone particles of meat-bone paste was studied. Measurement of the size of bone particles was performed using a scanning electron microscope “JSM-6390LV” (JEOL company, Akishima City, Japan). To prepare the sample for scanning on a microscope, meat-bone paste was treated with a 2% NaOH solution when heated in a boiling water bath for complete decomposition of meat cuts and tissues according to standard GOST 32224-2013 [34].

The remaining bone particles were dried at a temperature of 103 to 105 °C. The dried bone residue was passed through a sieve. The obtained samples were then analyzed under a microscope. In the microscope software, the bone particle sizes were observed with increasing resolution from 50 to 200 times. Using a special ruler in the program settings, each particle with a clearly defined contour was measured separately.

2.6. Water-Binding Capacity

The method used for determination of the water-binding capacity consisted of releasing water from the test sample through light pressing, sorption of the released water with filter paper (Whatman, qualitative grade 1, 150 mm), and determination of the amount of separated moisture based on the area of the wet spot left by the released water on the filtered paper.

2.7. Determination of Dynamic Viscosity

Dynamic viscosity was determined on a Brookfield RVT analog viscometer (Brookfield, MA, USA) by immersing the spindle and rotating it in the test medium. Using a circular scale, the scale readings were recorded and the viscosity of the medium was calculated.

2.8. ICP-MS Method for Elemental Composition Analysis

To determine the mineral composition, 1–2 g of meat-bone paste was turned to ash by drying at 400 °C for 4 h, then at 600 °C for 2 h. A representative 1 g (dry weight) of ashes was digested by adding 3 mL HNO₃ and 2 mL of HF (hydrofluoric acid). This was placed in a microwave at 200 °C for 20 min (Berghof Speed Wave microwave system, Bremen, Germany). After microwave digestion, the samples were diluted with 1% HNO₃ in a 10 mL vessel.

The content of elements in muscle samples was determined with an inductively coupled plasma mass spectrometer (ICP-MS, Varian-820 MS, Varian Company, Canberra, Australia). The method was validated with certified reference materials. Calibration standards Var-TS-MS, IV-ICPMS-71A (Inorganic Ventures Company, Christiansburg, VA, USA) were used for calibrating the mass spectrometer. The sensitivity of the mass spectrometer was tuned using a diluted calibration solution Var-TS-MS with a concentration of Ba, Be, Ce, Co, B, Pb, Mg, Tl, and Th of 10 µg/L. Three calibration solutions were used for the detector calibration. They were IV-ICPMS-71A of Cd, Pb, Cu, and Zn elements diluted to 10, 50, and 100 µg/L. Discrepancies between the certified values and concentrations quantified were below 10%. The operating parameters of the inductively coupled plasma mass spectrometer Varian ICP 820–MS were as follows: plasma flow, 17.5 L/min; auxiliary flow, 1.7 L/min; sheath gas, 0.2 L/min; nebulizer flow, 1.0 L/min; sampling depth, 6.5 mm; RF power, 1.4 kW; pump rate, 5.0 rpm; and stabilization delay, 10.0 s. The limits of quantification (LOQ) for the ICP-MS method used were in the range of 1–10 µg/L. All analyses were performed in triplicates, expressing the results as mg/100 g sample [35].

2.9. Determination of Microbiological Indicators

Microbiological evaluation of the product was carried out by methods for bacteriological analysis according to [36]. The following indicators were determined: the total number of microorganisms in 1 g of the product; the presence of bacteria of the Escherichia coli group of the genus Proteus; and the presence of pathogenic microorganisms.

The protocol begins with sample preparation, where 10 g of the sample is weighed to prepare the initial dilution. A series of tenfold dilutions is then prepared using saline solution. For total viable counts (TVC), 0.2 cm³ of the appropriate dilution is pipetted onto the Petritest™ substrate, evenly distributed, and incubated at 36 ± 1 °C for 12–24 h. Colonies are counted on Petritests with 15 to 300 colonies, and results are calculated to express counts per 1 cm³ of sample.

Total coliform count (TCC) follows a similar procedure using Petritest™ with an indicator that stains enterobacteria colonies red. For yeasts and molds analysis, the same inoculation process is used but incubation occurs at 24 ± 1 °C for 24 h (preliminary count) and 120 h (final count). Colonies are counted on Petritests with 15 to 150 yeast colonies or 5 to 50 mold colonies. In all cases, results are adjusted to represent counts per 1 cm³ of sample by multiplying the colony count by the dilution factor and then by 5.

2.10. Statistical Analysis

To ensure the accuracy and reliability of the data, all measurements were performed in triplicate. Statistical analysis was carried out using Excel 2016 (Microsoft Corporation, Redmond, Washington, DC, USA) and Statistica 12 PL (StatSoft, Inc., Tulsa, OK, USA) software packages. A one-way analysis of variance (ANOVA) was conducted to evaluate the significance of differences between the meat-bone paste control (MBP-C) and meat-bone paste enzyme-treated (MBP-E) samples. Following the ANOVA, Tukey’s Honest Significant Difference (HSD) test was applied to perform pairwise comparisons between the means of the control and enzyme-treated samples. Comparisons where the observed differences between means exceeded this critical value were considered significant at the p ≤ 0.05 level. This approach ensures that any significant differences identified are robust and not due to random variation, offering a detailed understanding of the effects of enzyme treatment on the physicochemical properties of the meat-bone paste.

3. Results and Discussion

3.1. 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 °C (Figure 2).

At a substrate temperature of 10 °C, the enzyme reaction rate was 0.0023 mmol/min, which increased significantly to 0.0107 mmol/min at a substrate temperature of 20 °C. The enzyme reaction rate continued to increase, reaching a maximum value of 0.0129 mmol/min at a substrate temperature of 30 °C. Beyond this temperature, the reaction rate starts to decrease, with values of V = 0.0119 mmol/min at 40 °C. At temperatures close to and above 50 °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. In enzymatic kinetics, the dependence of the reaction rate on the amount of substrate for a given amount of enzyme is very important (Figure 3).

With an increase in the number of enzyme molecules, the reaction rate increases continuously and in direct proportion to the amount of the enzyme, since a larger number of enzyme molecules produces a larger number of product molecules [44]. The observed trend underscores the direct relationship between pepsin concentration and the rate of the enzymatic reaction. Initially, as the concentration of pepsin increased, the reaction rate exhibited a proportional rise, highlighting the catalytic efficiency of higher enzyme concentrations.

3.2. 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 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 pepsin 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 temperature was established: with an increase in temperature, the activity of the enzyme increased, and pepsin showed the greatest activity at a temperature of 30 °C.

The processing of meat-bone paste with pepsin and ascorbic acid at a temperature of 30 °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 pepsin 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]. Voroshilin (2021) used enzymatic acid hydrolysis of bone with pepsin and hydrochloric acid (1M) for the production of gelatin [47].

The application of pepsin and ascorbic acid-treated meat-bone paste in food production eliminates bone particles, ensuring its safe integration into meat product technologies.

3.3. Physicochemical Analysis

The chemical composition of meat-bone paste before and after enzyme treatment showed no significant differences (

Table 1. Chemical composition of meat-bone paste.

Indicator in Percent	MBP-C	MBP-E
Protein %	13.70 ± 0.28	14.12 ± 0.23
Fat %	4.35 ± 0.05	4.23 ± 0.06
Ash %	15.98 ± 0.19	15.47 ± 0.28
Water %	65.97 ± 0.85	66.18 ± 1.19

MBP-C—meat-bone paste without enzyme treatment (control); MBP-E—meat-bone paste after enzyme treatment.

). The enzymatic action, in conjunction with other factors like the influence of ascorbic acid, can facilitate the loosening of collagen structures and enhance the hydration of proteins. Pepsin specifically targets collagen, a major protein component of bone. Breaking down collagen into smaller peptides could contribute to higher protein values in the analysis [48]. Additionally, the enzymatic treatment appears to induce changes in the fat component of the meat-bone paste. The splitting of fat cells during treatment may contribute to the observed slight decrease in fat content in MBP-E compared to MBP-C, although the difference is minimal. The slight decrease in ash content after enzymatic treatment might be due to solubilization and leaching of some minerals by the enzymes and acids.

The pH of the MBP-E sample was lower than that of the MBP-C sample, indicating a more acidic environment due to the enzyme treatment process. This decrease in pH could be attributed to the addition of ascorbic acid during enzyme treatment, which can influence the functional properties of the meat-bone paste (

Table 2. Physical properties of meat-bone paste.

Indicator	MBP-C	MBP-E
pH	7.02 ± 0.12 b	5.9 ± 0.09 a
Water-binding capacity (%)	77.28 ± 1.50 a	82.54 ± 1.05 b
Viscosity (Pa × s)	13.35 ± 0.16 b	11.27 ± 0.13 a

^a,b means within the same row with different uppercase letters differing significantly among different samples (p < 0.05). MBP-C—meat-bone paste without enzyme treatment (control); MBP-E—meat-bone paste after enzyme treatment.

).

Additionally, the water-binding capacity of MBP-E was higher than that of the control sample, suggesting that the enzyme treatment process led to an increased ability of the meat-bone paste to retain water. This could be due to the breakdown of protein structures during enzyme action, resulting in improved water-binding capacity. This is explained by the fact that in the treatment process, the proteins are hydrated, and the amount of immobilized moisture in the meat-bone paste increases due to the loosening of collagen structure and splitting of fat cells. The loosening of collagen promotes additional binding of water molecules [49]. Zinina et al. (2024) also noted an increase in the water-binding capacity of protein hydrolysates after enzymatic treatment with microbial culture of chicken gizzards [50].

The decrease in viscosity of MBP-E compared to the control sample indicates that the enzyme action process altered the rheological properties of the meat-bone paste, possibly due to the changes in protein and water interactions caused by the enzymatic activity during enzyme treatment.

The decline in viscosity observed in MBP-E can be attributed to two primary factors. Firstly, the increase in water-binding capacity in MBP-E contributes to a more hydrated matrix, leading to a reduction in the viscosity of the meat-bone paste. The enhanced water-binding capacity indicates that the enzyme action process promotes the incorporation of moisture into the paste, altering its rheological behavior. Secondly, the decrease in viscosity is linked to the structural changes induced by the enzymatic action of pepsin. The enzymatic action of pepsin, in conjunction with ascorbic acid, leads to destructive changes in collagen fibers within the meat-bone paste. The described changes in collagen fibers, including breaks between fibrils and fibers, swelling, and an increase in air interlayers, are consistent with the effects of enzymatic collagen breakdown. Pepsin’s action cleaves peptide bonds within collagen, leading to structural alterations. The disruption of collagen structure is known to impact the rheological properties of food matrices, resulting in reduced viscosity [51]. Thus, enzyme treatment of meat-bone paste improves consistency, strengthens moisture binding, and promotes protein loosening and fat breakdown in meat-bone paste, which leads to faster achievement of readiness during heat treatment.

The application of proteolytic enzymes in meat processing has been widely studied for its ability to improve meat tenderness. Studies show that dipping meat in enzyme solutions after osmotic dehydration effectively increases tenderness, with papain-treated samples achieving the highest scores for tenderness and reduced hardness [52]. These enzymes work by breaking down both myofibrillar and collagenous proteins, significantly improving the texture of various meat cuts, including those with high connective tissue content [53].

For low-grade beef, the use of proteolytic enzymes has been found to be particularly beneficial in softening connective tissue. This enzymatic treatment, when followed by the application of transglutaminase, enhances moisture binding and retention, further improving the functional properties of the meat [54]. Such processes are essential for adding value to lower-grade cuts, making them more suitable for consumption and minimizing waste.

Exogenous proteases, such as actinidin and zingibain, have shown potential in tenderizing meat from lower-quality sources. However, the effectiveness of these enzymes depends on optimizing conditions like temperature and pH for specific enzyme activity [55]. These findings support the growing role of enzymes in improving meat texture, functionality, and overall quality.

3.4. Mineral Composition of Meat-Bone Paste

Bone collagen, due to the specific structure and function of bone tissue, can facilitate the deposition of crystalline mineral substances, such as calcium phosphate. This process, known as mineralization, is critical for bone strength and rigidity. The mineral crystals that accumulate within the collagen fibers make bone collagen more stable and durable compared to other collagen types, which do not undergo such mineral deposition. This enhanced stability is essential for the mechanical strength of bones [56].

This study highlights the exceptional richness of MBP in calcium and phosphorus, with significantly higher levels compared to beef meat. The primary minerals found in this paste were calcium (Ca) and phosphorus (P), with concentrations of 5318.13 mg and 2342.78 mg per 100 g, respectively (

Table 3. The mineral content of meat-bone paste after enzyme treatment (MBP-E).

Name	Content, mg/100 g
Ca (calcium)	5318.13 ± 71.22
P (phosphorous)	2342.78 ± 46.35
Na (sodium)	1279.63 ± 18.17
S (sulfur)	1085.93 ± 20.53
Cl (chlorine)	847.49 ± 13.80
K (potassium)	377.92 ± 6.42
Mg (magnesium)	207.62 ± 1.69
Fe (iron)	8.35 ± 0.08
Zn (zinc)	7.20 ± 0.11
Cu (copper)	4.35 ± 0.04
Mn (manganese)	0.41 ± 0.00

). These levels are significantly higher than the recommended daily intake for adults, making this paste a valuable supplement for bone health and energy metabolism [57]. The paste also contains high levels of sodium (Na), sulfur (S), chlorine (Cl), and potassium (K), with concentrations of 1279.63 mg, 1085.93 mg, 847.49 mg, and 377.92 mg per 100 g, respectively. These minerals play crucial roles in various physiological processes, such as fluid balance, nerve function, and muscle contraction [58,59].

The MBP tested also contained high levels of magnesium (207.62 mg/100 g), crucial for bone health and enzyme function. Other trace elements like zinc (7.20 mg/100 g), copper (4.35 mg/100 g) and manganese (0.41 mg/100 g) were also present. These minerals are essential for various biological and metabolic processes, such as enzyme activation, oxygen transport, and antioxidant defense [60,61].

The daily recommended intake of the following minerals is: calcium, 1000 mg; phosphorus, 1500 mg; and magnesium, 500 mg) [62]. This is particularly relevant for populations prone to deficiencies in these vital minerals. According to the USA’s National Institute of Health, the average person does not obtain up to 500 mg of calcium in their daily diet [63]. Calcium contained in food products is a hard-to-digest element, so it is extremely difficult to ensure optimal intake of calcium into the human body through traditional food products [64]. Thus, incorporating MBP into meat products could significantly enhance mineral consumption.

The mineral composition of MBP, especially its high calcium and phosphorus content, makes it a promising source of essential minerals for dietary supplementation, with potential applications in food product development.

The analysis of the mineral composition of cattle meat-bone paste revealed a high calcium content—5318 mg/100 g—which is significantly higher in the offal of cattle (liver—5.00 mg/100 g; heart—8.00 mg/100 g; kidneys—13.00 mg/100 g; tongue—6.39 mg/100 g, brain—43.00 mg/100 g) and poultry (chicken liver—15 mg/100 g).

3.5. Microbiological Examination

The data showcased a significant reduction in the total viable count of mesophilic aerobic and facultative anaerobic microorganisms after enzyme treatment, measuring at 1 × 10⁵ CFU/g, compared to 2 × 10⁵ CFU/g in the untreated meat-bone paste. Importantly, both counts fall well below the regulatory threshold of 5 × 10⁵ CFU/g (

Table 4. Microbiological indicators of meat-bone paste.

Microbiological Indicators	Regulatory Threshold	MPB-C	MPB-E
Total viable count of mesophilic aerobic and facultative anaerobic microorganisms	Not be more than 5 × 105 CFU/g	2 × 105 CFU/g	1 × 105 CFU/g
Bacteria of the E. coli group (coliforms)	Not allowed in 0.0001 g	Not detected in 0.0001 g	Not detected in 0.0001 g
L. monocytogenes	Not allowed in 25.0 g	Not detected in 25.0 g	Not detected in 25.0 g
Pathogenic microorganisms including Salmonella	Not allowed in 25.0 g	Not detected in 25.0 g	Not detected in 25.0 g

). This reduction underscores the efficacy of enzyme treatment in minimizing microbial load. The absence of bacteria from the E. coli group (coliforms), Listeria monocytogenes, and Salmonella in the treated meat-bone paste further strengthens the safety profile. The elimination of these pathogenic microorganisms is crucial for ensuring the product’s suitability for consumption.

The safety analysis, centered on the total viable count and the absence of specific pathogens, sheds light on the effectiveness of enzyme treatment in enhancing the microbiological safety of meat-bone paste. The reduction in the total viable count post-treatment signifies a tangible improvement in hygienic conditions and the overall quality of the product. The analysis did not reveal any excess of the maximum permissible concentrations of pathogenic microflora content in meat-bone paste, which corresponds to the requirements of the Technical Regulation of the Customs Union 021/2011 “On the safety of food products”. This research highlights the potential of enzymatic treatment for sustainable valorization of animal bone waste, offering insights into improving both nutritional value and safety of meat-bone paste for various food applications.

3.6. Implications, Limitations and Recommendations for Future Studies

The development of innovative food production technologies is essential to address mineral deficiencies in human nutrition and optimize the use of non-traditional raw materials. In meat processing, bones are often underutilized, despite their high content of essential nutrients such as calcium, phosphorus, and magnesium. This study demonstrates that processing bones into MBP using enzyme and organic acid treatment creates a valuable, microbiologically safe food additive. The enzymatic treatment transforms high-molecular-weight proteins into more digestible, low-molecular-weight compounds, enhancing the nutritional profile of MBP. The application of MBP in food production offers a promising avenue for enriching meat products with essential macro- and micro-elements without compromising nutritional value. This approach addresses mineral deficiencies in human nutrition, particularly calcium, phosphorus, magnesium, and iron, while potentially reducing production costs and optimizing resource utilization in the meat industry.

However, several limitations and areas for future research should be addressed. More comprehensive microbiological testing is needed to ensure long-term safety and stability. Sensory evaluations are crucial to assess the impact on the flavor and texture of products incorporating MBP. Studies on the bioavailability and absorption of minerals in the human body are necessary to confirm the nutritional benefits. Enzyme-treated MBP offers a sustainable solution for enriching processed meat products while optimizing resource utilization in the meat industry. Future research should explore the full sensory and microbiological effects of this treatment and assess its industrial viability. This approach holds promise for improving the nutritional quality of food products and addressing mineral deficiencies in human diets.

4. Conclusions

This research aimed to investigate the impact of enzyme and organic acid treatment on the physicochemical properties of meat-bone paste. Enzyme reaction rates were influenced by treatment duration, substrate temperature, and enzyme concentration, highlighting the intricate relationship between these factors. Notably, optimal enzyme activity was observed at 180 min and a substrate temperature of 30 °C. Enzyme and acid treatment significantly reduced bone particle size, indicating the effectiveness of this process in enhancing the handling properties of bone powder. The chemical analysis revealed alterations in the composition of meat-bone paste following enzymatic treatment, including changes in protein content, pH, and water-binding capacity. These modifications suggest potential improvements in the functional properties of the paste. Importantly, microbiological examination demonstrated a substantial reduction in microbial load after enzyme treatment, ensuring the safety of the final product. Overall, this research contributes valuable insights into the utilization of animal bones and underscores the importance of sustainable approaches in addressing environmental challenges. The conducted research can be useful for meat-processing enterprises for the solving of problems on waste-free processing of bone raw materials and secondary products of cattle and poultry for food purposes by fine grinding and enzyme processing, increasing economic efficiency and expanding the range of functional meat products.