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. 2020 Nov 22;58(7):2806–2814. doi: 10.1007/s13197-020-04889-2

The effects of curing agents on the proteolysis and lipid oxidation of pastırma produced by the traditional method

Emel Oz 1, Emre Kabil 2, Mükerrem Kaya 1,✉
PMCID: PMC8196147  PMID: 34194114

Abstract

In the study, the effects of nitrate and/or nitrite (150 mg/kg KNO3, 300 mg/kg KNO3, 150 mg/kg NaNO2, and 150 mg/kg KNO3 + 150 mg/kg NaNO2) on proteolytic changes (free amino acid composition, SDS-PAGE) in pastırma were investigated. Pastırma samples were also analyzed in terms of some qualitative (pH, aw, TBARS, residual nitrite, salt) properties. The lowest total free amino acid content (1818.3 mg/ 100 g DM) was observed in the combination of 150 mg/kg KNO3 + 150 mg/kg NaNO2, while the highest content (2847.49 mg/ 100 g DM) was observed in 150 mg/kg KNO3. Although the pastırma groups generally exhibited similar SDS-PAGE profiles, differences were detected at some band intensities. The lowest TBARS value (22.24 μmol MDA/kg) was observed in 150 mg/kg KNO3 + 150 mg/kg NaNO2. As a result, the use of 150 mg/kg KNO3 in the pastırma curing process causes more intense proteolysis.

Keywords: Pastırma, Curing agent, Free amino acid composition, SDS-PAGE profile, Residual nitrite

Introduction

Pastırma is a traditional Turkish dry-cured meat product. The production process lasts for approximately 1 month depending on the muscle size and process conditions (Kaban 2009; Oz and Kaya 2019). In pastırma, which is included in the intermediate moisture foods class, curing is an important stage in terms of product safety and quality. Nitrate and nitrite are important additives for the formation of the characteristic red color and prevention of the growth of foodborne pathogens, especially Clostridium botulinum. These compounds also play a role in flavor formation and delaying oxidative rancidity (Sebranek and Bacus 2007). Nitrite acts as an antioxidant in the curing process. In addition, a part of nitrite binds to myoglobin and forms NO-myoglobin, which is responsible for the color improvement of cured meat products, the other part of nitrite also binds to proteins or other substances. Nitrate, another curing agent, shows these effects in cured meat products after being reduced to nitrite by microorganisms with nitrate reductase activity (Honikel 2008). Nitrate is a curing agent commonly used in the production of pastırma, together with salt (Gökalp et al. 1999a; Kaban 2013). Furthermore, nitrite can also be used alone or in combination with nitrate in the production of pastırma (Gökalp et al. 1999a; Uğuz et al. 2011; Erdemir and Aksu 2017).

Lipid oxidation is an important factor on the development of the typical aroma of dry-cured meat products (Harkouss et al. 2015). This reaction strongly affects the quality (negatively or positively) and chemical safety of product (Jin et al. 2012). Processing conditions affect the kinetics of oxidation reaction to a large extend (Gandemer 2002). Curing agent is also one of the factors affecting lipid oxidation due to its antioxidant properties (Kaban 2013).

Proteolysis is another important phenomenon in the production of dry-cured meat products. In such products, endogenous muscle peptidases play an important role in proteolysis, and intense degradation occurs in the protein fraction (Larrea et al. 2006; Gallego et al. 2018). Low molecular weight peptides and free amino acids resulting from protein degradation that occurs depending on production conditions play a significant role in the texture, flavor, and final quality of the end product (Toldra 2006). Free amino acids and small peptides are the main non-volatile compounds especially affect the taste properties of dry-cured meat products (Garrido et al. 2012). Texture is another key factor for the acceptance of meat products by consumers. Protein/peptide/amino acid profiles may affect the quality of meat products due to the fact that proteins are main figures in texture (Mora et al. 2018). However, excessive proteolysis may cause excessive softness, and mushy texture (Virgili and Schivazappa 2002).

The many internal and external factors such as initial pH value, fiber type of muscles, water content, salt amount and temperature play important role at the level of proteolysis (Oz and Kaya 2019). In additon, Toldra et al. (1993) stated that the curing agent affects the activity of many proteolytic enzymes that is closely related to the levels of proteolysis. Also, Waade and Stahnke (1997) reported that nitrite affects the free amino acid formation mechanism in dry fermented sausages. The effects of muscle difference (Oz and Kaya 2019), salt content (Soyer et al. 2011), salt variety (Kızılkaya 2012), and the use of starter culture (Aktaş et al. 2005) on proteolytic changes in pastırma, has been investigated. In addition, the effect of different nitrite levels on free amino acid composition of pastırma was investigated in another study (Erdemir and Aksu 2017). However, the effects of nitrate and/or its combinations with nitrite on proteolytic changes in pastırma have not been investigated yet. Therefore, in the present study, the effects of different curing agents and their mixture (I. 150 mg/kg KNO3, II. 300 mg/kg KNO3, III. 150 mg/kg NaNO2 and IV. 150 mg/kg KNO3 + 150 mg/kg NaNO2) on proteolytic changes and lipid oxidation in pastırma were investigated. Furthermore, the effects of different curing mixtures on some physicochemical properties of pastırma were determined.

Materials and methods

Materials

In the study, M. longissimus thoracis et lumborum muscles obtained from two beef carcasses and acquired from Erzurum Meat and Milk Institution was used as a raw material.

Methods

Pastırma production

The production of pastırma was carried out by the traditional method under natural conditions in a local plant. Briefly, the two muscles obtained from each carcass were divided into two parts of equal weight, and therefore, a total of eight pieces of meat, including four pieces from one carcass, were obtained. For each group, 150 mg/kg KNO3, 300 mg/kg KNO3, 150 mg/kg NaNO2 and 150 mg/kg KNO3 + 150 mg/kg NaNO2 were used as curing agents, respectively. The meat pieces were covered with a curing mixture containing 8% salt, 0.3% sucrose and curing agent per 1 kg of meat and kept at 8–10 °C for 48 h. Then, meat pieces were rinsed to remove excess salt from the surface and hunged for drying under natural conditions (12–19 °C). After 6 days all samples were pressed (15 kg weight for per kg of meat) at 10–11 °C and dried again under natural conditions (12–25 °C) for 5 days. After drying process, samples were then pressed at 25 °C for 45 min dried at 12–23 °C for 5 days. Then, meat pieces were treated with seasoning mixture (Çemen: 500 g flour (Trigonella foenum graecum), 350 g smashed fresh garlic, 75 g red pepper, 75 g paprika, and 1200 mL water) and kept at 8–10 °C for 24 h. At the end of production, the surface of meat samples was trimmed to a 2–3 mm thickness çemen paste and dried under natural conditions (12–23 °C) for 10 days (Oz and Kaya 2019).

Moisture, crude protein and fat content

To determine the moisture content, 10 g of the assay sample was dried at 102–105 °C until constant weighing was achieved. The crude protein content of the raw material was determined by the Kjeldahl method, while the fat content was determined by Soxhlet’s ether extraction method (Gökalp et al. 1999b).

pH value

The pH value of the samples homogenized with distilled water (1:10 w/v) was determined using a pH meter (ATI ORION 420, MA 02129, USA) calibrated with buffer solution (Gökalp et al. 1999b).

Water activity

In order to determine aw values, a water activity device (Novasina, TH-500 aw Sprint) was used. Prior to its use, the device was calibrated with six different salt solutions at 25 °C.

Thiobarbituric acid reactive substances (TBARS) value

For TBARS analysis, 2 g of each sample was weighed, and 12 mL of TCA solution (7.5% TCA, 0.1% EDTA, 0.1% Propyl gallate dissolved in 3 mL ethanol) was added to it and homogenized for 15–20 s. The samples were then filtered through filter paper (Whatman 1). 3 mL of the filtrate was taken, and 3 mL of TBA (0.02 M) solution was added to it. The samples were kept in a boiling water bath for 40 min and then cooled in cold water for 5 min. Subsequently, after centrifugation at 2000 g for 5 min, the measurement was performed in the spectrophotometer at a wavelength of 530 nm, and the result was presented in μmol malondialdehyde (MDA)/kg. For drawing a standard curve, 1,1,3,3-tetraethoxypropane was used (Lemon 1975).

Residual nitrite and salt content

For residual nitrite analysis, 10 g of the sample was taken, and 10 mL of saturated borax solution and 50 mL of hot distilled water were added to it and homogenized using an Ultra-Turrax homogenizer. The samples were then kept in a boiling water bath for 15 min. 2 mL of Carrez I and Carrez II solutions were added to the samples cooled to room temperature, and the volume was completed to 200 mL with distilled water, and the samples were incubated at room temperature for 30 min. Afterward, the samples were filtered through Whatman 595 filter paper. 10 mL of the filtrate was mixed with an equal volume of Griess solution and incubated at room temperature for 30 min. The absorbance of the samples was measured at 540 nm, and the residual nitrite content was calculated as mg/kg by considering the sample amount, dilution factor, and standard curve. In order to determine the salt content of the samples, 20 mL of the above-mentioned filtrate was taken. 3–5 drops of 10% potassium chromate indicator were dropped on it, and it was titrated with 0.1 M silver nitrate solution until a weak red color was formed. The result was presented in % salt (Tauchmann 1987).

Free amino acid composition

The free amino acid composition of the samples was determined according to the method presented by Antoine et al. (1999) and modified by Oz and Kaya (2019). 0.5 g of the sample was homogenized with 40 mL of 0.1 N HCl solution, and then the homogenate was incubated at 4 °C overnight. It was centrifuged at 10,000 g at 4 °C for 50 min. The resulting supernatant was filtered through a 0.22 μm filter and then analyzed by HPLC (Thermo scientific). In the analysis of the samples, Zorbax Eclipse-AAA 4.6 × 150 mm, 3.5 μm was used as the column, and a fluorescence detector was used as the detector. A mix solution of 17 amino acids (Sigma-Aldrich, 79,248) was used as the amino acid standard. OPA (ortho-phthalaldehyde) and FMOC (9-fluorenylmethyl chloroformate) were used as amino acid derivatization reagents, while 0.4 N borate (pH 10.2) was used as a buffer solution. In the chromatography system, 40 mM NaH2PO4 (pH 7.8) was used as mobile phase A, and Acetonitrile (ACN): Methanol (MeOH): Water /45:45:10, v/v/v solutions were used as mobile phase B. The mobile phase flow rate was set at 2 mL/min, and the column temperature was set at 40 °C. The results were given in mg/100 g dry matter.

SDS-PAGE profile of sarcoplasmic and myofibrillar proteins

The extraction of sarcoplasmic and myofibrillar proteins was carried out by performing some modifications according to the method presented by Wu et al. (2014). Each sample was homogenized in 30 mM phosphate buffer (pH 7.4) at a ratio of 1:10 (w/v). The homogenate was centrifuged at 10,000 g at 4 °C for 20 min. The supernatant was collected by filtration through glass wool. This process was repeated two times for pellets. The supernatants were combined as a sarcoplasmic protein fraction and stored at 4 °C until use. The resulting pellet was homogenized with nine volumes of 100 mM phosphate buffer (pH 7.4, 0.7 M potassium iodide, and 0.02% sodium azide). The homogenate was centrifuged at 10,000 g at 4 °C for 20 min. The supernatant was collected as a myofibrillar protein fraction.

Myofibrillar and sarcoplasmic protein extracts were mixed with 50 mM Tris buffer (pH 6.8, 0.05% bromophenol blue, 3% SDS (w/v), 75 mM dithiothreitol, 2 M thiourea, 8 M urea) at a ratio of 1:1 (v/v). The mixture was incubated at 100 °C for 5 min and stored at −20 °C until its use in SDS-PAGE analysis. 5% gel (acrylamide/bisacrylamide 29:1 (w/w), 0.1% (w/v) SDS, 0.05% (v/v) TEMED, 0.075% (w/v) APS, 0.125 M Tris-HCl, pH 6.8) was used as the loading gel. For the separation of sarcoplasmic and myofibrillar proteins, 12% (acrylamide/bisacrylamide 29:1 (w/w), 0.1% (w/v) SDS, 0.05% (v/v) TEMED, 0.075% (w/v) APS, 0.375 M Tris-HCl, pH 8.8) separation gel was used. The protein concentration of the samples was set at 1.0 mg/mL, and 10 μL of the sample was loaded into each gel well. Sample execution in electrophoresis continued at 100 V until the stain trace reached the end of the gel. After the completion of electrophoresis, the gel was stained with a staining solution containing Coomassie Brilliant Blue R-250 (Coomassie Brilliant Blue R-250 (1 g/L), 50% (v/v) methanol, 10% (v/v) acetic acid). After staining, the stain was removed with washing solution (10% (v/v) acetic acid, 10% (v/v) methanol, 80% (v/v) distilled water) until the gel background became clear. The protein standard mixture (BIO-RAD, Broad Range, USA) was used to estimate the molecular mass of the samples.

Statistical analyses

The research was conducted according to the randomized complete block design with two replicates. The obtained data were subjected to the two-way analysis of variance (ANOVA) and the differences between means were evaluated by Duncan’s multiple range test (SPSS 20.0).

Results and discussion

Chemical composition and pH value of meats used for pastırma production

The moisture, crude protein, and fat contents of the muscles (M. longissimus thoracis et lumborum) used in the present study as a raw material were determined to be 75.39 ± 0.27–76.01 ± 0.47%, 21.56 ± 0.11–20.09 ± 0.03%, 1.93 ± 0.28–1.65 ± 0.27%, respectively. In the production of pastırma, the pH value of the raw material is a significant factor in terms of product quality, and it is not desirable that this value is higher than 5.8 (Gökalp et al. 1999a). The mean pH values of the raw materials in the current study were determined to be 5.56 ± 0.02 and 5.58 ± 0.01, respectively.

pH value

The pH values of the pastırma produced using different curing agents and mixtures are presented in Table 1. The highest pH value was determined in the group in which the curing mixture of 150 mg/kg KNO3 + 150 mg/kg NaNO2 was used. No statistical difference was observed between the other groups (p > 0.05). This result is estimated to be due to the slower growth of lactic acid bacteria in the group cured with 150 mg/kg KNO3 + 150 mg/kg NaNO2.

Table 1.

The effect of curing mix on some qualitative properties of pastırma (mean ± SD)

Curing mix pH aw TBARS (μmol MDA/kg) Residual nitrite (mg/kg) Salt (%)
150 ppm KNO3 5.73 ± 0.02b 0.89 ± 0.03a 34.31 ± 1.90a 3.49 ± 1.31a 9.61 ± 0.14a
300 ppm KNO3 5.72 ± 0.05b 0.88 ± 0.03a 27.56 ± 1.56b 3.14 ± 1.66a 9.81 ± 0.31a
150 ppm NaNO2 5.71 ± 0.01b 0.88 ± 0.01a 26.69 ± 3.89b 4.32 ± 0.61a 8.93 ± 0.20b
150 ppm KNO3 + 150 ppm NaNO2 5.80 ± 0.05a 0.89 ± 0.02a 22.24 ± 2.84c 3.78 ± 1.63a 9.66 ± 0.31a
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Different letters (a–c) in the same column are significantly different (P < 0.05)

SD Standard Deviation

The mean pH values determined to be 5.56–5.58 in the raw materials varied between 5.71–5.80 in the pastırma samples. The increase in pH in the pastırma groups is thought to be the result of proteolysis occurring during production. Thus, as a result of proteolysis, the increase was observed in the non-protein nitrogenous substance (NPN-S) content, and the compounds of basic character in NPN-S were reported to cause a partial increase in the pH value of pastırma (Aktaş et al. 2005; Kaban 2009). The pH values determined in the present study are consistent with other studies on pastırma (Aksu et al. 2016; Akkose et al. 2017). Furthermore, the pH value of each pastırma group was found to be below the maximum limit specified in the Turkish Food Codex Communiqué on Meat and Meat Products (≤6.0) (Anon. 2018).

Water activity (aw) value

It is stated that the aw value should be below 0.90 in terms of microbiological stability in pastırma, which is included in the intermediate moisture foods class. Moreover, it is emphasized that this value should not decrease below 0.85 in terms of sensory properties (Leistner 1988). In the present study, the aw values of the pastırma groups varied between 0.88 and 0.89. The use of different curing mixtures did not have a significant effect on the aw value, which constitutes an important obstacle in pastırma (p > 0.05) (Table 1). Similarly, Akkose et al. (2017) reported that different nitrate levels did not have a significant effect on the water activity of pastırma.

TBARS value

The TBARS value is a parameter commonly used in determining lipid oxidation that affects the quality and acceptability of meat and meat products (Beltran et al. 2003) and this value mainly reflects the secondary products of lipid oxidation such as malondialdehyde and hydroxyl-nonenal (Huang et al. 2014). As seen from Table 1, the TBARS value of the pastırma groups were ranged from 22.24 μmol MDA/kg to 34.31 μmol MDA/kg. It has been declared that the TBARS value of pastırma after ripening period is about 30 μmol MDA/kg (Kaban 2009; Hazar et al. 2017). The highest and the lowest TBARS values were determined in the groups in which 150 mg/kg KNO3 and the combination of 150 mg/kg KNO3 + 150 mg/kg NaNO2 were used, respectively. In processes in which nitrate is used as a curing agent, nitrate should be converted to nitrite for revealing its protective effects. Microorganisms with nitrate reductase activity play an important role in this transformation (Kaban 2009). The high TBARS value in the groups in which 150 mg/kg KNO3 and 300 mg/kg KNO3 were used is thought to be related to the low nitrate reductase activity of these groups. In other words, the differences of TBARS value between groups may be related to counts of catalase positive cocci with nitrate reductase activity in the groups. Similarly, Hazar et al. (2017) reported that the TBARS value of pastırma cured with nitrate was higher than that of with nitrite.

Aksu et al. (2016) reported that the lowest TBARS value was determined in the group using 150 mg/kg NaNO2 in pastırma produced by using NaNO2 at different levels (0, 50, 100, and 150 mg/kg). On the other hand, differences between TBARS values may also be related to the different antioxidant properties of curing agents. Aksu et al. (2016) reported that nitrite shows an antioxidant effect by converting to nitrate as a result of oxidation by free oxygen.

Residual nitrite

The results of the residual nitrite content of pastırma produced using different curing mixtures are presented in Table 1. Accordingly, the use of different curing mixtures did not have a significant effect on the residual nitrite content of pastırma (p > 0.05). Similarly, Akkose et al. (2017) reported that different levels of nitrate (150, 300, 450, and 600 mg/kg KNO3) did not have a significant effect on the residual nitrite content of pastırma. Although different curing agents and amounts were used in the present study, no statistical difference was observed between the residual nitrite contents of the pastırma groups, indicating that the spontaneous flora with nitrate reductase activity in the groups using nitrate converted nitrate to nitrite.

In the present study, even if 300 mg/kg KNO3 was used, it was determined that the residual nitrite content in the end product was below 10 mg/kg and the residual nitrite contents of the pastırma groups were in compliance with the maximum limit (50 mg/kg) reported in the TSE 1071 pastırma standard. In the production process of cured ripened meat products, the residual nitrite content of end products may decrease due to the conversion of nitrite to nitrite oxide to form compounds affecting the meat pigment, nitrosomyoglobin, and aroma (Sanchez Mainar and Leroy 2015; Bosse et al. 2016).

Salt content

Salt used in the production of pastırma is an important additive, which is effective in reducing the water activity, ensuring the microbiological safety of the product, and forming the desired texture and flavor (Uğuz et al. 2011). In this study, the use of different curing mixtures had a significant effect on the salt content of pastırma (p < 0.05). While the lowest salt content was determined in the group cured with 150 mg/kg NaNO2, no statistical difference was observed between the other groups.

Free amino acid composition

The free amino acid composition of pastırma produced using different curing mixtures is presented in Table 2. The use of different curing mixtures showed a significant effect (p < 0.05) on the aspartic acid, histidine, arginine, tyrosine, phenylalanine, and proline contents of the pastırma samples, and a very significant effect (p < 0.01) on the serine, glycine, cystine, and valine contents. Different curing mixtures did not have a significant effect on the glutamic acid, threonine, alanine, methionine, isoleucine, leucine, and lysine contents of the samples (p > 0.05). Alanine was determined to be the dominant amino acid in all groups. Alanine has also been reported to be among the dominant amino acids in pastırma and other cured ripened meat products (Cordoba et al. 1994; Jurado et al. 2007; Ceylan and Aksu 2011; Deniz et al. 2016; Erdemir and Aksu 2017; Oz and Kaya 2019). In this study, in addition to alanine, high amounts of leucine, lysine, glutamic acid, arginine, and isoleucine amino acids were also detected. These results overlap with other studies on the free amino acid composition of pastırma (Ceylan and Aksu 2011; Deniz et al. 2016; Erdemir and Aksu 2017, Oz and Kaya 2019).

Table 2.

The effect of curing mix on the free amino acid composition of pastırma (mg/100 g DM) (mean ± SD)

Amino acid 150 ppm KNO3 300 ppm KNO3 150 ppm NaNO2 150 ppm KNO3 + 150 ppm NaNO2
Aspartic acid 11.35 ± 3.25a 8.51 ± 4.14ab 8.16 ± 0.91ab 4.62 ± 2.16b
Glutamic acid 141.73 ± 10.88a 111.51 ± 33.38a 138.25 ± 12.41a 119.62 ± 50.19a
Serine 145.34 ± 32.12a 46.28 ± 29.81b 72.89 ± 13.18b 73.09 ± 41.90b
Histidine 22.87 ± 9.63a 6.97 ± 2.57b 17.86 ± 14.54ab 6.92 ± 3.10b
Glycine 65.41 ± 12.42a 64.98 ± 9.75a 42.33 ± 14.35b 28.92 ± 13.21b
Threonine 27.85 ± 6.02a 15.17 ± 3.69a 16.52 ± 6.47a 19.61 ± 12.30a
Arginine 185.58 ± 25.22a 88.78 ± 22.31b 139.37 ± 33.53ab 119.69 ± 57.43b
Alanine 1615.31 ± 98.45a 1363.25 ± 414.69a 1253.04 ± 430.12a 1052.01 ± 82.39a
Tyrosine 41.34 ± 25.95a 22.11 ± 5.75ab 22.39 ± 11.63ab 5.72 ± 2.51b
Cystine 12.46 ± 3.81a 4.76 ± 1.73b 5.87 ± 1.22b 6.10 ± 3.72b
Valine 73.66 ± 8.11a 41.27 ± 18.28b 36.14 ± 8.54b 38.18 ± 14.29b
Methionine 19.11 ± 9.66a 9.43 ± 3.52a 21.46 ± 11.53a 13.30 ± 3.39a
Phenylalanine 37.21 ± 1.72a 21.67 ± 8.59b 19.09 ± 10.63b 18.30 ± 4.07b
Isoleucine 90.02 ± 49.61a 62.80 ± 23.65a 71.10 ± 19.94a 65.27 ± 42.62a
Leucine 186.40 ± 30.45a 125.41 ± 13.93a 125.56 ± 41.84a 109.04 ± 61.92a
Lysine 161.76 ± 7.26a 99.40 ± 39.03a 136.73 ± 13.46a 125.14 ± 69.63a
Proline 10.09 ± 1.5b 14.36 ± 2.2a 15.23 ± 2.10a 12.77 ± 4.71ab
Total AA 2847.49 ± 338.06a 2106.66 ± 637.02b 2141.99 ± 646.4b 1818.3 ± 469.54c
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Different letters (a–c) in the same letters are significantly different (P < 0.05)

SD Standard Deviation

Taste which is derived from nonvolatile compounds is one of the main sensory impressions of meat and meat products (Khan et al. 2015). Free amino acids are very important due to their contribution to specific taste of dry-cured meat products (Zhou et al. 2017). Hughes et al. (2002) reported that lysine, tyrosine, aspartic acid, glutamic acid and alanine are amino acids that strongly affect the taste of dry cured meat products. In the present study, glutamic acid content which is associated with umami taste ranged between 111.52–141.73 mg/100 g DM. It has been reported that glutamic acid is an important amino acid affecting the flavor of the meat and the taste properties of dry cured meat products (Jurado et al. 2007). On the other hand, the lowest proline content belonged to the group cured with 150 mg/kg KNO3. It has been stated that proline a major contributor to bitter taste, since peptides containing proline support binding to the bitter taste receptor (Tamura et al. 1990; Zhou et al. 2017).

The nutritional value of a food is closely related to its essential amino acid content (Paleari et al. 2003). The high amounts of leucine, lysine, arginine, and isoleucine essential amino acids in all pastırma samples are an important result for the nutritional value of the product. Furthermore, arginine, valine, and phenylalanine essential amino acids were found in higher amounts in the group cured with 150 mg/kg KNO3.

Free amino acids may involved in degradation reactions that generate volatile compounds which are responsible for the flavor of dry-cured meat products. It has been reported that volatile compounds generate from free amino acids in dry-cured meat products by Maillard and Strecker reactions (Jurado et al. 2007). In the present study, high total free amino acid content is may positively affect the sensory properties of pastırma. Berardo et al. (2015) reported that lower amounts of free amino acids may negatively affect the sensorial qualities, as they are important for flavour development as a source of Strecker aldehydes.

The use of different curing mixtures had a very significant effect on the total free amino acid content of pastırma (p < 0.01). While the lowest total free amino acid content was determined in the group in which the combination of 150 mg/kg KNO3 + 150 mg/kg NaNO2 was used, the highest content was determined in the group using 150 mg/kg KNO3. These results show that the use of 150 mg/kg KNO3 in the curing process leads to more intense proteolysis. This is may be related to the effect of curing agents on enzymes responsible for the formation of free amino acids. The increase in the free amino acid content in ripened meat products is known to be closely related to aminopeptidase activity. Toldra et al. (1993) reported that the use of nitrate up to 200 mg/kg did not have a significant effect on aminopeptidase activity, whereas the use of nitrite had an inhibitory effect on the activity of many proteolytic enzymes.

As a result, the curing agent affected the level of proteolysis, but the chemical mechanism behind the effect of the curing agent on proteolysis remained unclear. Villaverde et al. (2014) stated that the complex redox properties of curing agents may have different consequences on proteins and most of the chemistry involved is waiting to be unveiled.

SDS-PAGE profile

Myofibrillar and sarcoplasmic proteins are very important muscle proteins in determining the functionality of meat proteins (Toldra 2010; Marino et al. 2014). Peptides and free amino acids released with ripening are also largely formed as a result of the enzymatic degradation of these proteins (Toldra and Etherington 1988; Toldra 2010). The SDS-PAGE profiles of sarcoplasmic and myofibrillar proteins of pastırma produced using different curing mixtures are presented in Figs. 1 and 2, respectively.

Fig. 1.

Fig. 1

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SDS–PAGE profile belonged to sarcoplasmic proteins of pastırma samples cured with the four types of curing mix. STD: Protein standard, treatment 1: 150 mg/kg KNO3, treatment 2: 300 mg/kg KNO3, treatment 3: 150 mg/kg NaNO2, treatment 4: 150 mg/kg KNO3 + 150 mg/kg NaNO2

Fig. 2.

Fig. 2

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SDS–PAGE profile belonged to myofibrillar proteins of pastırma samples cured with the four types of curing mix. STD: Protein standard, treatment 1: 150 mg/kg KNO3, treatment 2: 300 mg/kg KNO3, treatment 3: 150 mg/kg NaNO2, treatment 4: 150 mg/kg KNO3 + 150 mg/kg NaNO2

The band profiles of sarcoplasmic proteins were generally similar. A high band intensity was determined in all groups at 70, 64.2, 58.8, 47.6, 44.4, 40.5, 38.4, 36, 30.7, 29.9, 28.6, 19,4, and 6.5 kDa. However, the band intensity of some sarcoplasmic proteins (66.2, 64.2, 58.8, 47.6, 44.4 kDa) was found to be higher in the group using 150 mg/kg NaNO2. This is thought to be associated with the lower salt content in this group. Larrea et al. (2006) reported that the salt content was an important factor affecting the solubility of soluble sarcoplasmic proteins. Myoglobin (17 kDa) that is an important sarcoplasmic protein could not be detected in the present study. Myoglobin may become insoluble by reacting with nitric oxide (Cordoba et al. 1994). The high molecular weight bands identified in this study are thought to be formed as a result of the degradation of myofibrillar proteins. Marino et al. (2014) reported that some myofibrillar proteins disintegrated with ripening and were released in the soluble fraction. Moreover, the high molecular weight bands identified in the current study were also determined in other studies on pastırma (Uğuz 2007; Kızılkaya 2012).

The gel profile of myofibrillar proteins shows that the band profiles of treatment groups are generally similar. Furthermore, the protein band intensity of 200 kDa, estimated to be myosin, was found to be slightly lower in the pastırma group cured with 150 mg/kg KNO3. This is thought to be due to the fact that the degradation of myosin, an important member of myofibrillar proteins, is slightly more intense in this group. On the other hand, as seen in the electrophoretogram, the intensity of protein bands of 200, 138, 56, 42, 38, 36, 26, and 6,5 kDa was found to be high in all groups. Toldra et al. (1997) identified many fragments in the range of 50–100 kDa as a result of proteolysis in dry-cured meat products. Similarly, many fragments in the range of 50–100 kDa were identified in all pastırma groups in this study.

Conclusion

The curing agent had a significant effect on both lipid oxidation and proteolytic changes in pastırma. If nitrate, which is an important curing agent in pastırma, is used at a level of 150 mg/kg, more intense proteolysis occurs. Moreover, there is also a need for studies investigating the effects of different curing combinations on the enzyme activity of pastırma.

Acknowledgments

This research was supported by Atatürk University research center with Project No: 286 2015/405. This research presented in part at the International Congress on Engineering and Life Science, Kastamonu, Turkey, April 26–29, 2018.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Footnotes

This research was supported by Atatürk University research center with Project No: 286 2015/405.

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