Study on the Combined Effects of Bromelain (Ananas comosus) Enzyme Treatment and Bacteria Cultures on the Physicochemical Properties and Oxidative Stability of Horse Meat

Abstract

This study investigates the impact of bromelain, a plant enzyme, on the physicochemical and sensory properties of horse meat, as well as the effects of different bacterial cultures (Lactococcus lactis, Lactococcus lactis subsp. lactis biovar diacetylactis, Lactobacillus acidophilus, and Bifidobacterium longum) on the inhibition of lipid oxidation and control of pH during chilled storage. Horse meat (longissimus dorsi) samples (n = 14) were treated with bromelain in two forms (powder and aqueous solution) and with three methods: immersion in enzyme solution, spreading enzyme powder on meat, and syringing enzyme solution into the meat. After fermentation, a part of the meat samples (n = 6) was treated with different bacteria compositions at a 5% weight ratio and stored at 0–2 °C for 6 days. Injecting 3–5% bromelain solutions was most effective at tenderizing the meat, reducing shear force by up to 56% after 8 h. This injection also maximized the water-holding capacity (78–81%) and minimized cooking losses (21–26%), compared to 38% for the control meat sample without treatment. Syringing with 3% bromelain yielded the highest sensory scores across the tenderness, flavor, and overall palatability parameters. The combination of L. acidophilus, Lc. lactis, and B. longum at a ratio of 1.5:1.5:2 was highly effective in reducing oxidative spoilage and optimizing pH levels, thereby ensuring extended meat storability. This study demonstrates that bromelain treatment is an effective method for improving the tenderness, WHC, and sensory properties of horse meat. The LAB combination showed efficient acid formation, crucial for enhancing meat preservation.

1. Introduction

The tenderness of meat is one of the important indicators that can determine the consumer properties of the product (juiciness, softness, flavor, aroma, consistency, and appearance), and is considered one of the most important properties of cooked meat [1,2]. The level of tenderness is directly related to the composition and structure of the muscle fibers, particularly the amount and type of connective tissue present in the meat. Meat with high hardness containing a high amount of connective tissue remains just as hard after cooking, its yield decreases, losses increase, and it becomes less juicy. Further use of such meat leads to deterioration in the quality of meat products [3]. Thus, increasing the tenderness of meat and increasing the storability of meat products are important problems in meat processing. Various enzymes are used to improve the tenderness of meat [4,5,6]. Of particular interest are studies related to the application of effective enzyme preparations from cheap and readily available raw materials that do not have extraneous odors or flavors and are sanitarily safe. The use of enzymes is widespread in the food industry, which mainly utilizes three sources: microorganisms, plants, or animal tissues. Such enzymes include hyaluronidase, papain, trypsin, pepsin, ficin, protosubtilin, testipan, and others [7]. These enzymes work by breaking down tough muscle fibers and connective tissues, resulting in improved texture, increased juiciness, and enhanced overall palatability of the meat. One of the primary challenges associated with enzyme preparations in the meat industry is guaranteeing their uniform spread throughout the meat’s thickness. However, with the use of jet injection and electrophysical processing methods (electromassaging, mechanical treatment, etc.) this problem has been mostly solved [8,9].

Plant enzymes are receiving considerable attention from researchers in meat processing for their multiple advantages. These include their low cost, ability to reduce meat toughness, the presence of biologically active compounds, and the potential antioxidant and antimicrobial properties [10,11]. The application of plant enzymes allows for more efficient use of meat with high levels of connective tissue, and the improvement of consistency and other quality indicators in ready meat products [12]. Researchers have proposed various methods for treating meat with plant enzymes, such as spraying enzyme solutions onto the meat surface, immersing meat in enzyme solutions, coating meat with enzyme powder, and injecting meat with enzyme solutions [13]. Papain, bromelain, ficin, actinidin, zingibain, and cucumin are plant proteases that are used to tenderize meat [14].

Bromelain, a proteolytic enzyme extracted from pineapples, particularly from the stem and fruit, is widely used in meat tenderization. As a cysteine protease, bromelain exhibits broad-spectrum activity, breaking down a variety of protein bonds. The mechanism of action involves the degradation of collagen and elastin, primary contributors to meat toughness [15]. It enhances the biological value of meat by increasing the content of essential amino acids such as lysine, glutamic acid, glycine, methionine sulfoxide and alanine [16,17]. Studies have demonstrated bromelain’s superior performance in enhancing meat tenderness and flavor compared to other plant proteases like papain. Kim and Taub (1991) found that bromelain-treated freeze-dried beef round had a superior flavor compared to papain-treated beef round [18]. Additionally, bromelain has been found to completely hydrolyze beef steaks from round muscles [19], and its application to pork loin resulted in better tenderization [20]. The efficiency of bromelain as a meat tenderizer depends on factors such as meat type, enzyme concentration, activity, temperature, and processing time [21]. Optimal enzyme concentrations range from 3–20%, with maximum activity at 50 °C [22,23].

In the production of meat products, a significant problem is increasing the shelf life of finished products. This can be achieved in various ways. One method is the use of agents with antimicrobial activity against bacteria causing food spoilage. In this case, the use of lactic acid bacteria is implied [24,25]. Lactic acid bacteria have great potential for use in biopreservation because they are safe for human consumption [26]. Some strains of Lc. lactis produce a bacteriocin called nisin. Nisin inhibits the growth of heat-resistant gram-positive spore-forming bacteria of the genera Bacillus, Clostridium, Mycobacterium tuberculosis, Lactobacillus, Corynebacterium, a few species of Streptomyces, and Micrococcus pyogenes, and prevents possible food poisoning, as well as extending the shelf life of products. However, nisin has no antimicrobial effect on Escherichia coli, Salmonella typhi, Shigella, or some Neisseria species. [27,28].

Lactobacillus acidophilus is also widely used for the fermentation of meat products [29]. It is a homofermentative, microaerophilic, short-chain Gram-positive microorganism with bacilliform morphology. It is thermally stable and remains active over a wide pH range, and has a strong inhibitory effect against food spoilage and pathogenic bacteria, which makes it an important class of biopreservatives [30]. Lactococcus lactis subsp. lactis bv. diacetylactis strains are often used in the dairy industry as starter cultures because of the production of acetoin and diacetyl, important compounds that provide an oily flavor to dairy products [31]. Bifidobacteria exhibit high antagonistic activity, and have the ability to destroy toxic metabolites, grow under anaerobic conditions, accumulate aromatic compounds, and act as reducing agents. These characteristics make bifidobacteria attractive for use in sausage production. Additionally, bifidobacteria have the ability to bind to air oxygen and significantly reduce the redox potential, which likely helps protect lipids from oxidation [32,33].

The high antagonistic ability and positive properties of lactic acid and bifidobacteria allow their use as a biological preservative in meat products. The novelty of this research lies in the application of bromelain fermentation, combined with lactic acid and bifidobacteria, to enhance the tenderization and physicochemical properties of horse meat. This research contributes to the understanding of meat processing techniques and offers potential solutions for improving the quality of horse meat. The study provides a comprehensive analysis of how this combined treatment impacts the physicochemical properties and microstructure of horse meat, offering new insights into meat tenderization techniques.

The aim of this work is to investigate the effects of the plant enzyme bromelain, lactic acid, and bifidobacteria on the physicochemical properties, tenderization, and microstructure of horse meat.

2. Materials and Methods

2.1. Samples

The object of the research was horse meat with a pronounced amount of connective tissue from the peasant farm “Mukinov” of Ernazar village, Beskaragai district, Abai region (Kazakhstan). The amount of connective tissue was determined visually. Five female horses from 1.5–2 years old of the local breed “Jabe” were slaughtered in the slaughterhouse of the farm “Mukinov” (Abay region, Kazakhstan). The meat (longissimus dorsi) obtained after slaughter was cooled in refrigerated chambers at (−4)–(−2) °C for 24 h. The longissimus dorsi is characterized by a higher protein content, and a lower content of connective and bone tissue compared to other cuts of horse meat. The chilled meat (50 kg) was transported in special refrigerated chambers to the laboratory. The meat was deboned manually using special deboning knives. In the process of deboning, large pieces of meat were cut into smaller pieces weighing no more than 200 g.

2.2. Technology of Meat Processing with Enzyme

Bromelain was purchased from Sinoway Industrial Co., Ltd., Xiamen, China, with the following characteristics: color—light yellow powder, proteolytic activity—2040 GDU/g, pH 7, and thermal stability at 50 °C. The bromelain powder was obtained from the central part of a pineapple (Ananas comosus).

The enzyme was used in two forms: in the form of a powder and in the form of an aqueous solution with a bromelain concentration from 0.5% to 2.5%. The aqueous solution of bromelain was prepared as follows: 0.5 g, 1.0 g, 1.5 g, 1.5 g, 2.0 g, and 2.5 g of bromelain powder were dissolved in a volume of 100 mL of water heated to 50 °C.

Treatment (3 replicates per treatment) was carried out by three methods: treatment with bromelain powder, immersion in enzyme solution, injecting enzyme solution into the raw material of the meat (

Table 1. Marking of meat samples during enzymatic processing.

#	Sample	Treatment
1	Control	Horse meat without treatment
2	BS 0.5	Horse meat in 0.5% bromelain solution
3	BS 1.0	Horse meat in 1.0% bromelain solution
4	BS 1.5	Horse meat in 1.5% bromelain solution
5	BS 2.0	Horse meat in 2.0% bromelain solution
6	BS 2.5	Horse meat in 2.5% bromelain solution
7	BP 1	Horse meat treated with bromelain powder (1% by weight of raw material)
8	BP 2	Horse meat treated with bromelain powder (2% by weight of raw material)
9	BP 3	Horse meat treated with bromelain powder (3% by weight of raw material)
10	BP 4	Horse meat treated with bromelain powder (4% by weight of raw material)
11	BP 5	Horse meat treated with bromelain powder (5% by weight of raw material)
12	BI 1	Horsemeat treated by injection with 1% bromelain solution
13	BI 2	Horse meat treated by injection with 2% bromelain solution
14	BI 3	Horse meat treated by injection with 3% bromelain solution
15	BI 4	Horse meat treated by injection with 4% bromelain solution
16	BI 5	Horse meat treated by injection with 5% bromelain solution

).

In the treatment with bromelain powder, pieces of meat were rubbed with bromelain powder, placed in a stainless steel food container and held for 4 h at (+2)–(+4) °C.

In the immersion in enzyme solution treatment, the meat was immersed into the container with the enzyme solution and left to ferment for 4 h at (+2)–(+4) °C.

In the injection of the enzyme treatment, the solution was injected into the muscle using a hand syringe injector (volume 30 mL) with a needle length of 7.5 cm (Ningbo Guangmu Hardware Co., Ltd., Ningbo, China). The depth of needle insertion into the 200 g pieces of meat was 2.0–2.5 cm. A marinade injector was used to syringe approximately 10 mL of the enzyme solution. The amount of enzyme solution injected into the meat samples amounted to 5% of the meat weight. After injection, the meat samples were placed on stainless steel food containers and held for 4 h at (+2)–(+4) °C.

2.3. Treatment of Meat with Lactic Acid Bacteria

In this experimental work, the following starter cultures were chosen: Lc. lactis, L. lactis ssp. lactis bv. diacetylactis, B. longum, and L. acidophilus.

For the study, the mother starter of these cultures was prepared beforehand. To prepare the mother starter, skim milk was pasteurized at 100 °C for 20 min, then cooled to the optimum temperature for bacterial fermentation. For Lc. Lactis and L. lactis ssp. lactis bv. diacetylactis this was 28 °C, for B. longum this was 37 °C, and for L. acidophilus this was 42 °C. Next, a dry bacteria concentrate was introduced, and the milk was fermented at these temperatures, then cooled to (+2)–(+4) °C.

To prepare the production starter, skim milk was pasteurized at 90 °C for 20 min, then cooled to the fermentation temperature (28 °C, 37 °C, and 42 °C, respectively), then the mother starter was added in the amount of 5% of the weight of skim milk and fermented for a certain period of time for each culture individually (

Table 2. Characteristics of bacteria starter.

Type of Bacteria	Clotting Time, h	Titratable Acidity, ⁰T	Total Viable Count CFU/g × 109	Welling Temperature, ⁰T
Lc. lactis	10–12	80	2.8	30
L. lactis ssp. lactis bv. diacetylactis	7–8	80	2.4	28
B. longum	8–10	85	2.4	37
L. acidophilus	9–12	100	2.4	42

). The finished starter was cooled to a temperature of (+2)–(+4) °C.

Meat samples (n = 6) were treated with different bacteria compositions at a 5% weight ratio. The studies were conducted within 6 days during the meat storage at a temperature of 0–2 °C.

2.4. Determination of Fat Content

Fat was determined according to GOST 23042-2015 [34]. This method is based on multiple extraction of fat by a solvent from the dried analyzed sample in a Soxhlet extraction apparatus, with subsequent removal of the solvent and drying of the extracted fat to a constant weight. Petroleum ether was used as a solvent.

The mass fraction of fat X as a % is calculated according to the Formula (1):

$$\mathrm{X}={\displaystyle \frac{\left(m_2-m_1\right)\ast 100}{m}}$$

(1)

where m₂ is the mass of the extraction flask with fat, g;

m₁ is mass of the extraction flask, g;

100 is the percentage conversion factor;

m is the mass of the analyzed sample, g.

2.5. Determination of Protein Content

Protein was determined according to GOST 25011-2017 [35]. The Kjeldahl method is based on mineralization of the product sample with concentrated sulfuric acid in the presence of oxidant, inert salt—potassium sulfate and catalyst—copper sulfate. The method is based on mineralization of organic substances of the sample with subsequent determination of nitrogen by the amount of ammonia formed. In this case amino groups of protein are converted into ammonium sulfate dissolved in sulfuric acid. The method is based on mineralization of organic matter of the sample with subsequent determination of nitrogen by the amount of ammonia formed.

Mass fraction of protein is calculated by the Formula (2):

$$X={\displaystyle \frac{0.0014\ast (V_1-V_2)\ast K\ast 100}{m}}·6.25$$

(2)

where 0.0014 is the amount of nitrogen equivalent to 0.1 mol/dm³ of hydrochloric acid solution, g;

V₁ is the volume of 0.1 mol/dm³ of hydrochloric acid solution or the volume of 0.05 mol/dm³ used for titration of the test sample, cm³;

V₂ is the volume of 0.1 mol/dm³ of hydrochloric acid solution or the volume of 0.05 mol/dm³ used for titration of the control sample, cm³;

K is the correction factor to the nominal concentration of hydrochloric acid solution;

100 is the percentage conversion factor;

m is the mass of the sample, g;

6.25 is the protein conversion factor.

2.6. Determination of Water Content

Moisture was determined according to GOST 33319-2015 [36]. The method is based on drying the analyzed sample with sand to a constant mass at a temperature of (103 ± 2) °C.

2.7. Determination of Ash Content

Ash was determined according to GOST 31727-2012 [37]. The method is based on the drying, charring, and ashing of the test sample at 550 °C and the subsequent determination of the mass fraction of total ash. The cup (crucible) with the sample was placed in a cold muffle furnace and the temperature of the furnace was gradually increased for 5–6 h up to 550 °C. The ashing continued at a temperature of 550 °C. The cup (crucible) was removed from the furnace and cooled in the desiccator to room temperature. The mass fraction of total ash, X %, is calculated with Formula (3):

$$\mathrm{X}={\displaystyle \frac{(m_2-m_0)\ast 100}{(m_1-m_0)}}$$

(3)

where m₂ is the mass of the cup with ash, g;

m₀ is the cup weight, g;

m₁ is the mass of the cup with the test sample, g.

2.8. Determination of Organoleptic Properties

The organoleptic evaluation was carried out according to GOST 9959-2015 [38]. Organoleptic evaluation was carried out to determine the indicators, such as appearance, color, taste, smell (aroma), consistency, etc, by the sensory organs. The quality indicators of the whole product were determined in the following sequence: (a) appearance, color and surface condition—visually, by external inspection; (b) smell (aroma) on the surface of the product, and (c) consistency, by pressing with a spatula or fingers. The tasters (21 people) were trained members of the laboratory of food products and biotechnology of Shakarim University. In the process of tasting, the participants filled out a questionnaire and assigned each sample of the product an appropriate score.

Each meat sample was evaluated three times. Water was provided to clean the palates of the tasters between samples. The horse meat samples were cut with a sharp knife into thin slices to preserve the characteristic appearance and pattern on the cut. In the first stage of tasting, the color, appearance, and pattern on the transverse and longitudinal slices were determined, followed by odor, aroma, flavor, and juiciness. Lastly, the consistency of the product was determined by pressing, cutting, and chewing. Scoring of the quality of the meat products used a 5-point scale; 1 point—very bad (technical defect); 2 points—bad (nutritionally inferior product); 3 points—satisfactory; 4 points—good; 5 points—excellent quality.

The samples were tasted in a certain order, and the tasters had no information about the number under which a certain sample was coded. As a result, the information contained in the tasting sheets was statistically processed.

2.9. Determination of Cooking Loss

The meat samples were vacuum-packed in polyethylene bags. Then, the meat was immersed in water heated to 80 °C and cooked until the temperature in the center of the meat was 72–75 °C. After cooking, the meat was cooled with running cold water for 30 min and transferred to a storage chamber for 12 h at +2 °C [39].

The weight loss of the meat during heat treatment was calculated according to Formula (4):

$$C_l={\displaystyle \frac{m_1-m_2}{m_1}}100$$

(4)

where C_l is the losses during the heat treatment and cooling of the product, kg;

m₁ is the mass of meat before heat treatment, kg;

m₂ is the mass of meat after heat treatment, kg.

2.10. Determination of Shear Stress

Meat samples were cut on a special device by lightly pressing a slice of the product onto a rotating tubular knife. The resulting smooth cylindrical sample with a diameter of 10 mm was pulled out using an ejector. In the absence of a specialized device for cutting the samples, the samples were cut manually in the form of a square with sides measuring 0.02 × 0.02 m.

The prepared product sample was carefully placed on the table. The force required to cut the sample was recorded on the scoreboard of the structurometer (Radius Company, Russia). After that, all measurement data were processed on the computer. In this case, the value of shearing stress was determined by dividing the force acting on the product by the area of the string passing over the surface of the product according to the Formula (5) [40]:

$${\theta }_m={\displaystyle \frac{P}{F}}$$

(5)

where P is the shearing force, N;

F is the cutting surface area, m².

2.11. Determination of Water-Holding Capacity

To determine the water-holding capacity (WHC), a sample of minced meat weighing 4–6 g was evenly spread with a glass rod on the inner surface of the wide part of the gyrometer. The gyrometer was tightly sealed with a cap and placed in a water bath with the narrow side down for 15 min at the boiling point of water. After that, the mass of the released moisture was determined by the number of readings on the scale of the gyrometer [41].

The water-holding capacity of the meat (WHC, %) was calculated according to Formula (6):

WHC = W − WRC,

(6)

The water release capacity (WRC, %) was calculated according to Formula (7):

WRC = a n m⁻¹ 100,

(7)

where W is the total mass fraction of moisture in the sample, %;

a is the gyrometer graduation rate, a = 0.01 cm³;

n is the number of graduations;

m is the mass of the sample, g.

2.12. Determination of pH

The active acidity of the medium (pH) was determined by potentiometric method on a pH meter 150MI device (Measuring Technology Ltd., Moscow, Russia) by immersing two electrodes in the solution and fixing the pH value on the scale of the device. The solution (aqueous extract) was prepared from ground meat and water (1:10 ratio). The pH was measured after infusion for 30 min at 20 °C [42].

2.13. Determination of Peroxide Number

The peroxide number was determined with the method of oxidation of hydroiodic acid by peroxides contained in the fat according to GOST 8285-91 [43]. A sample of 1 g of fat was weighed into a conical flask, then 10 mL of chloroform and 10 mL of glacial acetic acid were added, and 0.5 mL of potassium iodide was quickly added. The flask was closed and the contents were mixed by rotating, then kept in a dark place for 10 min. Then, 100 mL of distilled water containing 1 mL of 1% starch solution was added to the flask. The released iodine was titrated with 0.01 n sodium thiosulfate solution until the blue color disappeared.

The fat peroxide number y (% iodine) was calculated according to Formula (8):

$$y={\displaystyle \frac{(V-V_1)\times K\times 0.00127\times 100}{a}}$$

(8)

where V is the amount of 0.01 n sodium thiosulfate solution consumed for titration of the test solution, mL;

V₁ is the amount of 0.01 n sodium thiosulfate solution consumed during titration of the control solution, mL;

K is the correction factor to the titer of 0.01 n sodium thiosulfate solution;

a is the weight of the fat, g;

0.00127 is the amount of iodine equivalent to 1 mL of 0.01 n sodium thiosulfate solution, g.

In order to express the peroxide number (mmol) of active oxygen per 1 kg of fat (mmol (½ O₂) /kg), the conversion factor given in GOST 26593-85 [44] was used. The resulting peroxide number expressed in percentage of iodine (grams of iodine per 100 g of fat) was multiplied by 78.

2.14. Determination of Water Activity (a_w)

The measurement of water activity was conducted using an Aqualab 4TE water activity meter (Addium Inc., Pullman, Washington, DC, USA). Prior to the measurement, samples were ground and weighed, followed by an even distribution on the device’s cup [45].

2.15. Determination of Titratable Acidity, ⁰T

The acidity of the starter was determined according to the method of the national standard GOST 3624-92 [46]. The method was based on the neutralization of acids with sodium hydroxide solution in the presence of the indicator phenolphthalein. In a flask with a capacity of 100 to 250 cm³, we measured 20 cm³ distilled water and analyzed the product in a volume of 10 cm³ with three drops of phenolphthalein. The mixture was thoroughly mixed and titrated with sodium hydroxide solution until the a weak pink coloration appeared, corresponding to the control color standard, which does not disappear within 1 min.

The acidity, in degrees Turner (⁰T), was found by multiplying the volume of sodium hydroxide solution used to neutralize the acids contained in a given volume of product by a factor of 10.

2.16. Clotting Time, h

Clotting time was determined by the formation of a stable clot and titratable acidity characteristic of each type of starter according to GOST 34372-2017 [47].

2.17. Microstructure

Observations of the microstructure of meat–bone paste were made using a low vacuum scanning electron microscope JSM-6390LV JEOL (Tokyo, Japan). The accelerating voltage applied to the electron beam was 15 KV, at a magnification of X100 [48].

2.18. Statistical Analysis

The experiments were performed in triplicate. Standard deviation values were indicated for all measurements. Differences in the measurements of the experimental and control groups were calculated using analysis of variation (one-way ANOVA) using the Tukey test. A p-value of ˂0.05 was considered significant.

3. Results

3.1. Determination of Chemical Composition

The protein, fat, ash, and moisture content were not significantly changed (p > 0.05) depending on the method of treatment and the concentration of the enzyme solution. The chemical compositions of the meat samples treated with bromelain are presented in the Supplementary File (Table S1).

3.2. Shear Force of Meat before and after Enzyme Treatment

The tenderness of meat is a crucial attribute affecting consumer satisfaction and product quality. In this study, the effect of bromelain enzyme treatment on the shear force, a measure of meat tenderness, was investigated in horse meat samples with a high amount of connective tissue. The shear force is inversely related to meat tenderness, with lower values indicating softer meat [49,50]. The results demonstrate that bromelain treatment effectively reduced the shear force of horse meat, thereby improving its tenderness (Figure 1). The shear force in the control sample was not significantly changed (p > 0.05). Across all methods of treatment, a clear trend of decreasing shear force was observed with an increasing duration of treatment and concentration of bromelain. This indicates that longer treatment durations and higher bromelain concentrations lead to greater tenderization of meat.

Regardless of the method of treatment with bromelain, the results of the shear stress tests in all experimental samples were statistically significantly (p < 0.05) different from the control sample in terms of both the treatment method and treatment time. The injection method (Figure 1C) proved to be the most effective in reducing the shear force, with the 5% bromelain solution injection (sample 15) resulting in the lowest shear force of 118 N (p < 0.05) after 8 h of treatment, compared to the initial shear force of 270 N.

Among the immersion treatments (Figure 1A), the 2.5% bromelain solution (sample 5) yielded the lowest shear force of 140 N after 8 h, followed by the 2% solution (sample 4) at 143 N.

For the powder treatment method (Figure 1B), the 3% bromelain powder (sample 8) showed the best performance, with a shear force of 163 N after 6 h and 170 N after 8 h.

The data also suggest that the injection method was more effective than the immersion method, which in turn was more effective than the powder treatment method in reducing the shear force and improving tenderness.

The results of our study align with previous research on the effect of bromelain enzyme treatment on the shear force of various types of meat. Our findings demonstrate that bromelain treatment effectively reduces the shear force of horse meat, thereby improving its tenderness. This outcome is consistent with the results of Ketnawa and Rawdkuen (2011), who found that bromelain extract (BE) significantly decreased the shear force values in all BE-treated samples compared to the control [51]. Similarly, Naveena et al. (2004) observed a decrease in shear force values in buffalo meat when ginger rhizome extract was added, suggesting that enzymatic treatment can effectively tenderize meat with extensive muscle fiber and connective tissue [52]. This finding supports our results, indicating that enzymatic treatment can be an effective method for reducing the shear force of meat. Saengsuk et al. (2021) reported that bromelain-treated restructured pork steak had 64–80% lower shear force values compared to non-treated samples, due to degradation of the myofibrillar and collagen structures [53]. Jun-hui et al. (2020) [54] found that bromelain proteases reduced hardness and shear force in squid and meat, respectively.

The tenderizing mechanism of bromelain is generally attributed to its proteolytic activity, which breaks down tough myofibrillar and connective tissue proteins, resulting in more tender meat texture.

The research findings demonstrate the potential of bromelain enzyme treatment in improving the tenderness of horse meat, as evidenced by the significant reduction in the shear force values. The injection method with higher bromelain concentrations and longer treatment times emerged as the most effective approach for achieving optimal tenderness. These results offer valuable insights for the development of enzymatic tenderization processes in the meat industry, particularly for products with a high amount of connective tissue.

3.3. Sensory Analysis

In the next step, we investigated the effect of bromelain enzyme treatment on the sensory properties of horse meat with a high amount of connective tissue. Figure 2 shows the average score of the organoleptic evaluation of the samples. The evaluation was carried out according to a 5-point system.

In the first method, where meat samples were immersed in bromelain solution, the total sensory score ranged from 23.42 to 24.2 points. The highest total score (24.2 points) was observed in the meat sample treated with a 1.5% bromelain solution. However, the highest concentration (2.5%) did not necessarily yield the highest sensory scores, suggesting an optimal concentration range for maximum sensory enhancement. In the second method, where bromelain powder was rubbed into the meat, the total sensory score ranged from 23.58 to 24.32 points. The 2% bromelain powder treatment yielded the highest overall score of 24.32, with top marks for appearance (4.9), consistency (4.9), and aroma (4.87). Scores increased up to the 2%, powder but declined at higher concentrations. Similarly, syringing of the bromelain solution into the meat thickness also demonstrated improvements in sensory attributes with increasing bromelain concentration. Injection of a 3% bromelain solution resulted in the highest sensory scores across all treatments, with perfect 5.0 ratings for consistency and aroma.

When comparing the treated samples with the control (untreated) sample, it is evident that bromelain treatment significantly improved the sensory profile of horse meat. The treated samples consistently scored higher in all sensory attributes, reflecting enhanced tenderness, flavor, and overall palatability compared to the control. Notably, sample 13 (IBS 3), treated with 3% bromelain solution via syringing, exhibited the highest sensory score of 24.7 points, indicating superior sensory attributes across all evaluated parameters. This method likely facilitated deeper penetration and uniform distribution of the enzyme within the meat, leading to superior tenderization and flavor development.

3.4. Study of Water-Holding Capacity

When studying the dynamics of water-holding capacity, it was noted that when increasing the dosage of the enzyme, the maximum values of water-holding capacity were reached within 4 h. When increasing the dosage of the enzyme after a short-term increase in water-holding capacity, there is a decrease in the ability of meat raw material to retain moisture afterwards (Figure 3).

This is explained by the fact that with increasing dosage and longer treatment of meat with the enzyme preparation, deep proteolysis of protein macromolecules occurs with an increase in the amount of low molecular weight products of hydrolyzation, resulting in decreased hydration, which affects the content of adsorption-bound water in meat and worsens its functional and technological properties [55]. Bromelain hydrolyzes proteins by binding to specific peptide bonds, with its cysteine residue acting as a nucleophile to cleave these bonds. This process breaks down large proteins, particularly collagen and elastin in meat, into smaller peptides and amino acids, allowing the bromelain to continuously act on other protein molecules [56].

The results of the studies show that injection with enzyme solution increased the water-holding capacity of meat. When the meat was immersed in bromelain solution of different concentrations, the WHC values did not change significantly. Thus, at the 0.5% concentration of bromelain solution, the WHC value of horse meat was 69.5%, while at the concentration of 2.5% the WHC was 68.3% (p > 0.05). However, the values of the WHC of meat treated with bromelain solution are statistically significantly (p < 0.05) higher than the control sample of meat without treatment.

When sprinkling the meat with bromelain powder (mass of 2% of the meat weight), an increase in the WHC of meat up to 65.9% was observed, which is statistically significantly (p < 0.05) higher than in the control sample. However, when sprinkled with bromelain powder in the amount of 1%, 3%, 4%, and 5%, the WHC values were within the WHC value of the control sample of horse meat (p > 0.05).

Syringing meat with bromelain solution of different concentrations significantly increased the WHC values (p < 0.05) in comparison to the control. The data show that the method of syringing meat, in comparison with other methods, maximally increases the WHC from 71.1 to 75.1% depending on the concentration of the solution. In this case, it is most preferable to choose up to 3% bromelain solution for syringing, as with the 5% bromelain solution the WSS decreases to 71.1% compared to the 3% bromelain solution (75.1%) (p < 0.05).

Chaurasiya et al. (2015) noted a noteworthy rise in water-holding capacity (WHC) in meat treated with reverse micellar extracted fruit bromelain (RMEB), reaching 91.1%, compared to 56.6% in the control group [15]. The increase in the WHC of meat following enzymatic treatment can be attributed to the biochemical changes in protein molecules during hydrolysis, which result in the release and cleavage of actomyosin into actin and myosin. These proteins have a better ability to absorb water and transfer into a soluble state, leading to an improved WHC in meat. Therefore, enzymatic treatment can be an effective method for enhancing the quality and shelf life of meat products [57,58].

3.5. Cooking Losses

Cooking losses, or yield, are a critical parameter influencing the economic viability and consumer acceptability of meat products. Excessive moisture and weight loss during thermal processing can result in diminished juiciness, tenderness, and overall quality [59,60]. The results of the study showed that the cooking losses (yield) of the meat samples varied depending on the method of treatment and the concentration of the enzyme solution. Fermented meat samples were exposed to heat treatment—cooking for 2 h in order to assess losses during heat treatment. The data of the conducted studies are presented in the diagram in Figure 4.

Compared to the control group (38% loss), all bromelain-treated samples exhibited lower cooking losses, ranging from 21% to 32%. This suggests that bromelain treatment can effectively improve meat yield by minimizing the moisture loss during cooking.

The cooking loss of the control meat sample without bromelain treatment was 38%, which is significantly higher (p < 0.05) than in the case of bromelain treatment regardless of the method of treatment. However, among the methods of treatment with bromelain, the lowest mass loss during heat treatment of meat was recorded in the meat samples syringed with bromelain solution. It should be noted that syringing is better carried out at a 3% concentration of bromelain solution, because at this concentration the mass loss is low (21%). The obtained results of weight loss in the immersion in bromelain solution and sprinkling with bromelain powder methods of meat processing do not differ significantly (p > 0.05). The injection method emerged as the most promising approach, facilitating deep and uniform distribution of the enzyme within the meat tissue. The 3% bromelain solution injection yielded the lowest cooking loss of 21%, a significant reduction (p < 0.05) compared to the untreated control (38%). This can be attributed to the improved structural integrity and water-binding capacity resulting from the targeted breakdown of connective tissue fibers by the bromelain.

The conducted complex of research showed that positive indicators of meat quality were achieved by the processing of horse meat with an enzyme solution with the inoculation method, and the best sample was test 13 (horse meat processed by syringing with 3% bromelain solution). Chaurasiya et al. (2015) explored the effect of reverse micellar extracted fruit bromelain (RMEB) on meat tenderization but did not observe a significant change in cooking losses compared to the control group [15]. However, Botinestean et al. (2018) reported increased cooking losses in beef steak samples treated with 0.3% bromelain (38.6%) compared to non-treated samples (30.6%) [19]. Saengsuk et al. (2021) also observed higher cooking losses in restructured pork steak samples treated with 0.1% bromelain [53].

3.6. Microstructure of Meat Muscles before and after Treatment

In the softening of meat by enzymes, a major role is played by protein denaturation, leading to the destruction of the actomyosin complex, and limited proteolysis, contributing to the breakage of the long chains of myofibrillar proteins which also reduces its stiffness to a certain extent. Enzyme treatment reduces the amount of high molecular weight proteins and significantly increases the amount of low molecular weight proteins [61].

Photographs of the microstructure of meat samples (Figure 4) show that in horse meat treated with bromelain, sections of muscle fibers with different degrees of destruction were found. The microstructural studies of the control sample (untreated) show that in the horse meat samples, the muscle fibers are mostly rectilinear and tightly adhered to each other. In some groups of fibers in the form of bends, folds between such fibers are more often seen layers of connective tissue. These bends are explained by the different degrees of shrinkage in neighboring muscle fibers; corrugated fibers have a weak shrinkage or are generally dissolved, and straight fibers neighboring with them are strongly reduced [62].

When treated with bromelain, the transverse striation of horse meat muscle fibers is clearly visible. Each myofibril in the fiber has its own striation that does not coincide with the striation of a neighboring myofibril (see Figure 5, BP1). It can be seen that the structure of meat became grainy with the presence of homogeneous and pyknotic nuclei [63]. At some distance from the place of enzyme injection, some fragments of muscle fibers with poorly distinguishable transverse striation were observed. After injection with a solution of bromelain, muscle fibers were found to be straight, with wavy fibers occurring. Some groups of fibers were tightly adjacent to each other, while others were separated by large spaces and gaps created during syringing. The injected solution was distributed mainly in the connective tissue layers and between muscle fibers, pushing them apart. This reduced the mechanical strength of the horse meat muscle tissue. Thus, the most homogeneous and uniform meat structure was observed when the bromelain solution was syringed, compared to immersion in the solution and rubbing of the bromelain powder.

3.7. Treatment of Fermented Meat with Lactic Acid Bacteria

At the next stage, studies on the selection of lactic acid bacteria were carried out in order to use them in the biotechnological processing of meat to increase storability. Previous studies have shown that it is reasonable to use bacteria such as Lactobacillus bulgaricus, Lactobacillus lactis, Lactobacillus acidophilus, Lactiplantibacillus plantarum, Lactiplantibacillus brevis, and Lactiplantibacillus casei, etc. in the processing of raw meat materials. [64,65,66].

The peroxide number was analyzed as a controlling indicator, which allows the determination of the oxidative spoilage of the product. The data are shown in

Table 3. Changes in peroxide number during storage of meat.

Storage Time, Days	Different LAB Combinations and Ratios						Control Sample (Horse Meat)
	Lc. lactis, L. lactis ssp. lactis bv. diacetylactis, B. longum (1.5:1.5:2)	Lc. lactis, L. lactis ssp. lactis bv. diacetylactis, B. longum (1.5:2:1.5)	Lc. lactis, L. lactis ssp. lactis bv. diacetylactis, B. longum (2:1.5:1.5)	L. acidophilus, Lc. lactis, B. longum (1.5:1.5:2)	L. acidophilus, Lc. lactis, B. longum (1.5:2:1.5)	L. acidophilus, Lc. lactis, B. longum (2:1.5:1.5)
	Sample 1	Sample 2	Sample 3	Sample 4	Sample 5	Sample 6
1 day	0.015 ± 0.000 e	0.013 ± 0.000 c	0.012 ± 0.000 b	0.011 ± 0.000 a	0.014 ± 0.000 d	0.014 ± 0.000 d	0.016 ± 0.000 f
2 days	0.040 ± 0.001 b	0.037 ± 0.001 a	0.036 ± 0.001 a	0.035 ± 0.001 a	0.035 ± 0.001 a	0.036 ± 0.001 a	0.049 ± 0.001 c
3 days	0.044 ± 0.001 a	0.044 ± 0.001 ab	0.043 ± 0.001 a	0.042 ± 0.001 a	0.045 ± 0.001 b	0.044 ± 0.001 ab	0.051 ± 0.001 c
4 days	0.053 ± 0.001 b	0.051 ± 0.001 a	0.051 ± 0.001 a	0.050 ± 0.001 a	0.055 ± 0.001 b	0.053 ± 0.001 b	0.063 ± 0.001 c
5 days	0.069 ± 0.001 c	0.062 ± 0.001 a	0.065 ± 0.001 b	0.061 ± 0.001 a	0.070 ± 0.001 c	0.070 ± 0.001 c	0.076 ± 0.001 d
6 days	0.075 ± 0.001 c	0.067 ± 0.001 b	0.069 ± 0.001 b	0.062 ± 0.001 a	0.077 ± 0.001 c	0.077 ± 0.001 c	0.089 ± 0.001 d

The data are presented as the mean ± standard deviation. ^a–f Means within the same row with different letters mean there are significant differences among different samples (p < 0.05).

.

Lactic acid bacteria (LAB) are known for their beneficial effects in food preservation, particularly in improving storability and extending shelf life. In this study, the effect of different starter compositions of LAB on the peroxide number of horse meat samples during storage at refrigeration temperatures was investigated. The peroxide number serves as an indicator of oxidative deterioration, with lower values indicating better oxidative stability and increased shelf life of the meat product.

According to the results, all lactic acid bacteria starter culture treatments effectively reduced the peroxide number compared to the untreated control sample, indicating their ability to inhibit oxidative rancidity and extend the shelf life of the meat. In all experimental groups, the peroxide number increased with the storage time, indicating a gradual oxidation of the meat. However, the rate of increase varied among the different starter compositions. The lowest peroxide numbers were observed in experiment 4, where the starter composition consisted of 1.5 parts L. acidophilus, 1.5 parts Lc. lactis, and 2 parts B. longum. This suggests that this particular starter composition was most effective in delaying the oxidative deterioration of the meat. Comparatively, the control group (meat without treatment) showed the highest peroxide numbers throughout the storage period. This indicates that the LAB treatment significantly improved the storability of the meat by slowing down the oxidation process. LAB produce organic acids like lactic acid, which can lower the pH of meat, creating an unfavorable environment for spoilage microorganisms and inhibiting their growth. Additionally, LAB can scavenge free radicals, which are responsible for initiating lipid oxidation [67,68].

3.8. pH Changes during Storage of Meat Samples

It is known that microorganisms introduced into minced meat with starters change its structure through enzymes, forming new substances that improve the quality of the product [69]. Nitrate-reducing micrococci, homofermentative lactic acid bacteria and pediococci, and yeasts in the form of pure or mixed cultures are mainly used as starter cultures. During the salting and ripening of meat products, microflora play an active role in stabilizing coloration, improving organoleptic characteristics and increasing the shelf life [70,71]. Meat and meat products are a favorable environment for the development of lactic acid bacteria. They find all the necessary substances for normal life activity in meat—carbon sources, nitrogen, vitamins, and mineral salts. The pH and humidity of meat also promote their growth [72]. Further, the pH changes during the storage of meat samples at 0–2 °C were investigated (

Table 4. Changes in pH during meat storage at 0–2 °C.

Storage Time, Hours	Lc. lactis, L. lactis ssp. lactis bv. diacetylactis, B. longum (1.5:1.5:2)	Lc. lactis, L. lactis ssp. lactis bv. diacetylactis, B. longum (1.5:2:1.5)	Lc. lactis, L. lactis ssp. lactis bv. diacetylactis, B. longum (2:1.5:1.5)	L. acidophilus, Lc. lactis, B. longum (1.5:1.5:2)	L. acidophilus, Lc. lactis, B. longum (1.5:2:1.5)	L. acidophilus, Lc. lactis, B. longum (2:1.5:1.5)	Control Sample (Horse Meat)
	Sample 1	Sample 2	Sample 3	Sample 4	Sample 5	Sample 6
0	6.10 ± 0.11 a	6.10 ± 0.07 a	6.10 ± 0.09 a	6.10 ± 0.08 a	6.10 ± 0.06 a	6.10 ± 0.07 a	6.33 ± 0.10 a
2	6.0 ± 0.05 a	6.0 ± 0.06 a	6.0 ± 0.05 a	6.0 ± 0.07 a	6.03 ± 0.08 a	6.00 ± 0.05 a	6.27 ± 0.10 a
4	5.82 ± 0.08 ab	5.86 ± 0.09 ab	5.87 ± 0.08 ab	5.80 ± 0.05 a	5.81 ± 0.07 a	5.85 ± 0.08 ab	6.11 ± 0.09 b
6	5.70 ± 0.06 a	5.85 ± 0.09 ab	5.85 ± 0.07 ab	5.60 ± 0.05 a	5.76 ± 0.06 ab	5.75 ± 0.07 ab	6.01 ± 0.08 b
8	5.70 ± 0.08 b	5.70 ± 0.06 b	5.70 ± 0.07 b	5.30 ± 0.06 a	5.63 ± 0.05 b	5.60 ± 0.06 b	5.98 ± 0.08 c
10	5.67 ± 0.07 b	5.65 ± 0.06 b	5.65 ± 0.07 b	5.27 ± 0.09 a	5.59 ± 0.05 a	5.50 ± 0.05 a	5.92 ± 0.10 b
12	5.54 ± 0.08 b	5.57 ± 0.09 b	5.58 ± 0.06 b	5.21 ± 0.05 a	5.51 ± 0.07 b	5.30 ± 0.06 a	5.81 ± 0.08 b
14	5.53 ± 0.08 b	5.55 ± 0.09 b	5.56 ± 0.08 b	5.20 ± 0.07 a	5.45 ± 0.06 ab	5.28 ± 0.05 a	5.79 ± 0.09 b
16	5.50 ± 0.07 b	5.51 ± 0.08 b	5.50 ± 0.08 b	5.19 ± 0.06 a	5.31 ± 0.07 ab	5.26 ± 0.06 a	5.71 ± 0.08 b
18	5.30 ± 0.06 ab	5.44 ± 0.09 b	5.45 ± 0.08 b	5.17 ± 0.07 a	5.29 ± 0.07 a	5.24 ± 0.06 a	5.63 ± 0.09 b
20	5.29 ± 0.08 ab	5.30 ± 0.09 ab	5.40 ± 0.08 b	5.14 ± 0.06 a	5.26 ± 0.07 a	5.20 ± 0.08 a	5.60 ± 0.10 b
22	5.27 ± 0.09 a	5.30 ± 0.10 ab	5.30 ± 0.09 ab	5.05 ± 0.05 a	5.21 ± 0.07 a	5.15 ± 0.06 a	5.58 ± 0.08 b
24	5.21 ± 0.05 a	5.30 ± 0.09 ab	5.34 ± 0.05 ab	5.04 ± 0.07 a	5.17 ± 0.08 a	5.10 ± 0.06 a	5.55 ± 0.08 b

The data are presented as the mean ± standard deviation. ^a–c Means within the same row with different letters mean there are significant differences among different samples (p < 0.05).

).

It is known that in fresh meat, after slaughtering the muscle tissue is in a relaxed state, has a high water-binding capacity, and a pH value close to neutral. After slaughtering the animal, the oxygen supply is stopped and anaerobic hydrolytic breakdown of glycogen with the formation of lactic acid occurs [73]. In the muscle tissue of horse meat after slaughter, glycolytic changes proceed more slowly. The initial phase of autolysis (rigor mortis progress) is determined by the pH shift within 48 h, and the pH rise is observed after 96 h.

During slaughtering, the pH of the meat should be above 6 but not below 5.2 to prevent adverse effects on the juiciness of the meat. With each hour of storage, the toughness of the meat increases and its cutting resistance increases. As can be seen from the table, all samples show a decrease in active acidity with the use of the starter compared to the control sample. Different ratios of cultures give different pH values. The combination of Lc. lactis, L. lactis ssp. lactis bv. diacetylactis, and B. longum decreased the pH to 5.3 after 18 h in experiment 1, after 20 h in experiment 2, and after 22 h in experiment 3. This symbiosis of cultures has lower energy for acid formation.

A more intensive pH reduction is observed when using a symbiosis of cultures L. acidophilus, Lc. lactis, and B. longum. This is primarily due to the fact that this combination uses bifidobacteria, which actively develop at lower temperatures. L. acidophilus is considered a strong acid former.

At the same time, in experiment 4 we observed that at the ratio of L. acidophilus, Lc. lactis, and B. longum (1.5:1.5:2), the pH decrease was more intensive than in other samples, such as experiment 5, experiment 6. This is due to the fact that this symbiosis includes bifidobacteria, which more actively develop at lower temperature compared to acidophilus bacillus.

Acidophilus bacillus develops less at low temperatures, and at 15 °C its growth is slightly retarded [74]. In experiment 4, the pH reached 5.3 in 8 h, 16 h in experiment 5, and 12 h in experiment 6. This is due to the fact that bifidobacteria show more active energy in lactic acid formation at low temperatures.

The pH value of the medium is one of the conditions for the development of microflora, including putrefactive and sanitary-indicative microflora. If the medium has high acidity, the growth of putrefactive microorganisms is inhibited. Evaluating the conducted research allowed us to make a choice in favor of the combination of bacteria L. acidophilus, Lc. lactis, and B. longum, taken in the ratio (1.5:1.5:2), in sample 4.

During the ripening of horse meat, the processes caused by the vital activity of microorganisms and the activity of tissue enzymes are of great importance. At the same time, glycogen decomposition occurs with the formation of lactic acid, the rate of accumulation of which depends on the genus, species, and strain of the probiotic cultures. The pH value is important for the processes of meat structure formation. It is known that at pH values close to 5.2–5.3, collagen swelling, hydrolysis of intermolecular bonds, and increased activity of cellular enzymes, especially cathepsins, occurs. In addition, rapid and continuous reduction of the meat pH to 5.2–5.3 suppresses the growth of pathogenic microorganisms [75,76].

A decrease in the pH values of meat is associated with the formation of lactic acid with the participation of bifidobacteria, L. acidophilus, and Lc. lactis. It was found that the optimal value of pH = (5.3 ± 0.1) was achieved when the studied samples were kept at the ratio of L. acidophilus, Lc. lactis, and B. longum (1.5:1.5:2) in experiment 4, with a fermentation time of 8 h. As seen from the presented data, the maximum rate of lactic acid formation was achieved using experiment 4.

4. Conclusions

This research study comprehensively investigated the impact of plant enzyme (bromelain) treatment and bacteria cultures on improving the quality and storability of horse meat. The bromelain treatment demonstrated significant improvements in the tenderness, water-holding capacity, and sensory properties of horse meat, with the syringing method showing the most substantial enhancement. Therefore, while bromelain treatment may affect the tenderization of horse meat, its impact on the chemical composition of the meat is not significant. The use of various bacteria starter cultures effectively reduced oxidative spoilage and controlled pH changes during chilled storage, with the combination of L. acidophilus, Lc. lactis, and B. longum in a ratio of 1.5:1.5:2 proving to be the most effective in extending the shelf life of the meat. The findings from this study provide valuable insights into the potential applications of bromelain treatment and bacteria cultures in the meat industry, offering promising biotechnological strategies for enhancing meat quality and storability.