Meat Science 96 (2014) 1325–1331

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Proteolysis and sensory properties of dry-cured bacon as affected by the partial substitution of sodium chloride with potassium chloride Haizhou Wu, Yingyang Zhang, Men Long, Jing Tang, Xiang Yu, Jiamei Wang, Jianhao Zhang ⁎ National Center of Meat Quality and Safety Control, Synergetic Innovation Cener of Food Safety and Nutrition, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China

a r t i c l e

i n f o

Article history: Received 21 July 2013 Received in revised form 23 October 2013 Accepted 30 October 2013 Keywords: Dry-cured bacon Sodium chloride substitution Potassium chloride Proteolysis Sensory properties

a b s t r a c t Quadriceps femoris muscle samples (48) from 24 pigs were processed into dry-cured bacon. This study investigated the influence of partial substitution of sodium chloride (NaCl) with potassium chloride (KCl) on proteolysis and sensory properties of dry-cured bacon. Three salt treatments were considered, namely, I (100% NaCl), II (60% NaCl, 40% KCl), and III (30% NaCl, 70% KCl). No significant differences were observed among treatments in the proteolysis, which was reflected by SDS–PAGE, proteolysis index, amino acid nitrogen, and peptide nitrogen contents. Furthermore, there were no significant differences in the moisture content between control and treatment II, whereas the moisture content in treatment III was significantly higher (p b 0.05) in comparison with control (treatment I). The sensory analysis indicated that it was possible to reduce NaCl by 40% without adverse effects on sensory properties, but 70% replacement of NaCl with KCl resulted in bacon with less hardness and saltiness and higher (p b 0.05) juiciness and bitterness. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Sodium chloride (NaCl; salt) is one of the most familiar food ingredients known to people. Salt is also an essential ingredient in processed meats, owing to its contribution to the desirable salty taste, flavor, and the microbiological stability and the typical textural properties of meat products (Kilcast & Angus, 2007). Nevertheless, despite the importance of sodium chloride in producing food, the overconsumption of sodium has the propensity to develop hypertension and cardiovascular diseases (Doyle & Glass, 2010). For these reasons, consumer demand for a variety of low-salt products, especially meat products, has increased considerably (Ruusunen & Puolanne, 2005). However, sodium chloride must be decreased without sacrificing the food quality and food safety characteristics. From the technological point of view, the main methods are focused toward the reduction of the sodium chloride added or substitution with other salts like KCl, MgCl2, and CaCl2, but these actions may lead to a modification of the processing techniques (Aliño, Grau, Baigts, & Barat, 2009). The reduction of sodium chloride added has led to some problems like an excess of proteolysis because of the intense action of tissue proteases leading to defective textures like softness. The tissue proteases also could overact on proteins and polypeptides generating excessive concentration of low molecular weight nitrogen compounds like free amino acids and small peptides, which could lead to unpleasant flavor such as bitter taste (Martín, Córdoba, Antequera, Timón, & Ventanas, 1998; Toldra, 1998). ⁎ Corresponding author at: College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China. Tel./fax: +86 25 84399096. E-mail address: [email protected] (J. Zhang). 0309-1740/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.meatsci.2013.10.037

Potassium chloride has been the most widely used salt substitute, and its intake can reduce the development of hypertension and cardiovascular diseases (Aljuraiban et al., 2012), whereas its excessive addition to meat products results in a bitter and metallic taste (Gelabert, Gou, Guerrero, & Arnau, 2003). During the last decades, a number of attempts have been made to produce acceptable low sodium meat products using the replacement of NaCl by KCl. For example, curing salts with up to 50% of KCl in Italian salami (Zanardi, Ghidini, Conter, & Ianieri, 2010) or 60% in dry-cured pork loin (Gou, Guerrero, Gelabert, & Arnau, 1996) were used with no significant negative effect on their sensory properties. These results indicated that the replacement up to 50% of NaCl with KCl is adequate to acquire an acceptable meat product without influence on flavor, texture, color, and also microbiological stability. However, there is no available information on the effect of the replacement of NaCl by KCl on proteolysis during the whole dry-curing process. Therefore, the objective of this research was to analyze the influence of the partial replacement of NaCl by KCl on the proteolysis during the processing of dry-cured bacons and sensory properties of the final products. 2. Materials and methods 2.1. Bacon-making procedure and sampling Twenty-four Taihu × Duroc × York crossbreed pigs (100–120 kg live wt.) were used in this study. Forty-eight samples of the quadriceps femoris muscle (1.5 ± 0.5 kg per quadriceps femoris) were taken from these postmortem porcine carcasses in a local slaughterhouse. After 24 h of chilling at 4 °C in a cold chamber, 3 of them were used to analyze

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the raw material. The remaining 45 muscle samples were randomly divided into 3 groups of 15 for the production of bacon. Bacon was produced as treatment I: control, 100% NaCl; treatment II: 60% NaCl, 40% KCl; treatment III: 30% NaCl, 70% KCl (wt/wt). The amount of the salt mixture was 3% of the total weight of muscle samples. Moreover, the amount of sodium chloride and the percent of replacement by potassium chloride were chosen according to the results published by Jin et al. (2012) and our preliminary experiments. The same method was used for all treatments. The salting stage was carried out at 4 °C and 90% relative humidity (RH) for 3 days. After salting, the bacon joints were taken to the last processing stage (drying-ripening) for 12 days according to the following temperature and humidity procedures: temperature progressively increased from 13 °C to 31 °C at 1.5 °C per day, and RH progressively reduced from 85% to 79% at 0.5% per day (Jin et al., 2010). Total processing time was 15 days. The central part of the quadriceps femoris muscle from the bacon joints was sampled at different points from the beginning of the process: 0 (raw meat), 3 (the end of salting), and 7, 11, and 15 days (the drying–ripening period). After sampling, samples were vacuum-packed and stored at −40 °C for further analysis. Three replicated samples were tested at every process point and treatment. 2.2. Moisture determination The moisture content was measured by air-drying the sample at 100 °C to constant weight according to the methodology of the International Organization for Standardization (ISO Norm R-1442, 1979).

Table 1 Sensory attributes, definitions, and extremes.a Sensory trait

Definition

Redness Yellowness Hardness

Intensity of red color in the lean (pale pink to dark red) Level of yellow color of the fat (white to intense yellow) Effort required to bite thorough lean and to convert the sample to a swallowable state (very tender to very firm) Impression of lubricated food during chewing (not to very juicy) Level of salt taste (not to very salty) Level of bitter taste (not to very bitter)

Juiciness Saltiness Bitterness a

Each attribute scored in an unstructured line of 10 cm.

buffer, pH 7.4, using a polytron (3 × 15 s at 15,000 rpm) homogenizer model T18 (IKA-Werke GmbH and Co., Staufen, KG, Germany) with cooling in ice and then the homogenate was centrifuged at 10,000g for 20 min at 4 °C. The supernatant was collected by filtering through glass wool and kept at 4 °C. This procedure was repeated with the pellets up to three times. Supernatants were combined as the sarcoplasmic protein fraction and stored at 4 °C until use. The resulting pellet was finally homogenized with 9 volumes of 100 mM phosphate buffer, pH 7.4, containing 0.7 M potassium iodide and 0.02% sodium azide using a polytron (3 × 15 s at 10,000 rpm) homogenizer model T18 (IKA-Werke GmbH and Co.) and cooled in ice, and then the homogenate was centrifuged at 10,000g for 20 min at 4 °C. The supernatant was collected as the myofibrillar protein fraction and stored at 4 °C until use. The protein concentration of both sarcoplasmic and myofibrillar protein extracts was determined using a standard assay with Bradford reagent (Sigma, St. Louis, MO) and bovine serum albumin as the standard (Sigma).

2.3. Sodium and potassium analysis At the end of the production of dry-cured bacon, 3 bacon joints per treatment were used to determine the contents of sodium and potassium, and each measuring was performed in triplicate basing on the specifications of the Association of Official Analytical Chemists (AOAC, 2005). 2.4. Sensory analysis At the end of ripening stage, bacon joints from the three types of salt were subjected to sensory analysis to determine the influence of the partial substitution of NaCl by KCl on sensory qualities. The sensory analysis was carried out according to the general guidance of the International Organization for Standardization (ISO 6658, 2005). Ten trained specialists were selected as assessors (ISO 8586, 2012). The sensory evaluation took place in a separate test room in separate booths and in normal white light (ISO 8589, 2007). Dry-cured bacons were warmed using a microwave oven for 2 min and cut in about 5-mm-thick slices. The samples were presented to the panelists with three-digit codes and in random order, and water was provided for rinsing of the mouth between samples (Ruusunen et al., 2003). The choice of the sensory trait was carried out by open discussion in two preliminary sessions. The retained six traits of the dry-cured bacons were the following: redness, yellowness, hardness, juiciness, saltiness, and bitterness. Each attribute was scored in an unstructured line of 10 cm. The sensory traits, their definitions, and extremes are explained in Table 1 according to the work published by Ruiz, Garcıa, Muriel, Andrés, and Ventanas (2002). Sensory evaluation was repeated in three sessions carried out in three different days. The final scores were averaged over all assessors. 2.5. Extraction of sarcoplasmic and myofibrillar muscle proteins The sarcoplasmic and myofibrillar proteins were extracted according to the procedure as described previously (Molina & Toldra, 1992) with minor modifications, as noted in the following description. The minced muscle was homogenized 1:10 (w/v) with 30 mM phosphate

2.6. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) The proteins of sarcoplasmic and myofibrillar fractions were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) according to the operation as reported previously (Huang, Huang, Ma, Xu, & Zhou, 2012). The extracts of the sarcoplasmic and myofibrillar proteins were mixed in a ratio 1:1 (v/v) with 50 mM Tris buffer, pH 6.8, containing 0.05% bromophenol blue, 3% (w/v) SDS, 75 mM dithiothreitol, 2 M thiourea, and 8 M urea. The mixture was heated at 100 °C for 5 min and stored at − 20 °C until use for SDS–PAGE. Gels were made according to the method of Laemmli (1970). A 5% stacking gel (acrylamide: bisacrylamide = 29:1 (w/w), 0.1% (w/v) SDS, 0.5‰ (v/v) TEMED, 0.075% (w/v) APS, and 0.125 M Tris–HCl, pH 6.8) was used to determine the protein concentrations of sample. A 12% separating gel (acrylamide: bisacrylamide = 29:1 [w/w], 0.1% SDS, 0.05% TEMED, 0.075% (w/v) APS, and 0.375 M Tris–HCl, pH 8.8) were used for separating sarcoplasmic proteins and myofibrillar proteins. The protein concentration of each sample was adjusted to 1.0 mg/mL, and then 10 μL of these samples were injected in each lane into the gels, respectively. Electrophoresis was performed in Bio-Rad Protean II xi cell (Bio-Rad Laboratories, Hercules, CA) and run at 200 V at 4 °C, until the dye track reached the end of the gels. The gel was stained with Coomassie Brilliant Blue R-250 (1 g/L) containing 50% (v/v) methanol and 10% (v/v) acetic acid and destained with the solution containing 10% (v/v) acetic acid and 10% (v/v) methanol in distilled water, until the background was clear. Standard proteins (PageRuler ™ Unstained Low Range Protein Ladder; Thermo Fisher Scientific Inc., Shanghai, China.) with MW ranging from 10 to 200 kDa were simultaneously run for molecular mass estimation. Gels were scanned (GT-800F; Epson, Japan) at a resolution of 800 dpi, and then the densities of bands were quantified by Quantity One (Bio-Rad Laboratories Inc., Benicia, CA). The relative band density was calculated as a percentage ratio between density of each band and

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total band density (sum of all band densities) of each lane. All electrophoresis analyses were performed in duplicate. 2.7. Total nitrogen, non-protein nitrogen, and proteolysis index analysis Total nitrogen (TN) content was determined by the Kjeldahl method, using a Kjeltec™ 2300 Auto Distillation Unit (FOSS, Hillerød, Denmark) and expressed as mg/g muscle dry matter of the sample. Non-protein nitrogen (NPN) was quantified according to the method as described previously (Careri et al., 1993) with small modifications. Five grams of minced sample were homogenized 1:5 (w/v) with 10% trichloroacetic acid, using a polytron (3 × 20 s at 5000 rpm) homogenizer model T18 (IKA-Werke GmbH and Co.), cooled in ice, and then stored at 4 °C to react overnight. The homogenate was centrifuged at 5000 rpm for 10 min at 4 °C and filtered through Whatman No. 4 filter paper. The non-protein nitrogen (NPN) was measured from the filtrate by the Kjeldahl method, using a Kjeltec™ 2300 Auto Distillation Unit (FOSS) and expressed as mg/g muscle dry matter of the sample. The proteolysis index (PI) was calculated as the percentage ratio between NPN and TN (Careri et al., 1993). 2.8. Amino acid nitrogen and peptide nitrogen analysis To analyze amino acid nitrogen (AN) and peptide nitrogen (PN), an extract of muscle was prepared according to the method as reported previously (Ketelaere, Demeyer, Vandekerckhove, & Vervaeke, 1974). Five grams of minced sample were homogenized with 50 mL of perchloric acid 0.6 M, using a polytron (3 × 15 s at 10,000 rpm) homogenizer model T18 (IKA-Werke GmbH and Co.) with cooling in ice and then the homogenate was centrifuged at 5000 rpm for 10 min at 4 °C and filtered through Whatman No. 54 filter paper. The resulting pellet was rehomogenized with 10 mL of perchloric acid 0.6 M, using a polytron (3 × 15 s at 10,000 rpm) homogenizer model T18 (IKAWerke GmbH and Co.) with cooling in ice and then the homogenate was centrifuged at 5000 rpm for 10 min at 4 °C and filtered through Whatman No. 54 filter paper. The two filtrates were pooled and adjusted to pH 6 with sodium hydroxide, chilled, filtered, and diluted up to 100 mL with distilled water. Amino acid nitrogen (AN) and peptide nitrogen (PN) were determined by the ninhydrin reaction basing on the operation as described previously (Moore & Stein, 1954). Amino acid nitrogen (AN) from the muscle extract as acquired above was measured after peptide precipitation with sulfosalicylic acid l0%. Peptide nitrogen (PN) content was quantified by the difference between the amino acid nitrogen content, after hydrolysis of peptides with 6 M chloride acid of the muscle extraction as acquired above and the previously determined AN. The contents of amino acid nitrogen (AN) and peptide nitrogen (PN) were expressed as milligrams per gram of muscle dry matter of the sample. 2.9. Statistical analysis Three bacon joints were tested for each treatment. For each of bacon joint, three replicates of every parameter were analyzed. The data were analyzed using one-way ANOVA. The averages were compared using Fisher's least significant difference (LSD) procedure, and differences were considered significant at p b 0.05. Statistical processing was performed using the SPSS® software package (Release 16.0 for Windows, 2004; SPSS Inc., Chicago, IL). 3. Results and discussion 3.1. Moisture content and salt content The results of moisture content (%) change during dry-cured bacons processing using three types of salt are shown in Fig. 1. No significant differences (p ≥ 0.05) were observed throughout their processing

Fig. 1. Moisture content (%) change during dry-cured bacon processing using three types of salt. Treatment I: control, 100% NaCl; treatment II: 60% NaCl, 40% KCl; treatment III: 30% NaCl, 70% KCl. Labeled error bars indicate the standard deviation for each treatment. Different bar letters indicate significant differences among treatments (p b 0.05).

between the moisture content of the dry-cured bacons salted with treatment II and treatment I. However, treatment III showed a significantly higher (p b 0.05) moisture content at 3 days (end of salting) compared with treatment I and treatment II. This could be due to the quicker penetration of the salt mixtures containing KCl that would hinder the exit of water from the inside of the meat (Aliño, Grau, Baigts, & Barat, 2009). In addition, the differences between the moisture content of treatment III and treatments I and II were reduced at the end of the ripening stage (15 days). This phenomenon can be attributed to the severe dehydration of the meat during the drying stage. These results are in line with those of other workers (Aliño et al., 2009) for dry-cured loin. The content of sodium and potassium in the dry-cured bacons subjected to three types of salt and the dietary reference intakes (DRIs) of the sodium and potassium are presented in Table 2. The control sample (with treatment I) contained the highest amount of sodium (2837.82 mg/100 g), which was similar to a normal level for bacons with these characteristics (Jin et al., 2010). The partial substitution of sodium chloride by potassium chloride significantly (p b 0.05) reduced the sodium content and increased potassium content. The trends in sodium and potassium contents can be attributed to the types of the salt employed during their production. For a supposed 50 g serving, sodium contents for treatments I, II, and III cover 61%, 39%, and 26% of the DRIs value (IOM, 2004) for the tolerable upper intake level (UL) of sodium, respectively. The reduction of sodium content for meat products could result in some health benefits because excessive sodium intake is related to the development of several health complications such as stomach cancer, kidney stones, and hypertension (Doyle & Glass, 2010). Furthermore, epidemiological studies have indicated that potassium intake is inversely associated with the level of blood pressure and hypertension development (Aljuraiban et al., 2012). 3.2. Sensory analysis The results of the sensory analysis of the dry-cured bacons salted with three types of salt are presented in Table 3. There were no significant differences (p N 0.05) between the control bacons (with treatment I) and those salted with treatment II in terms of redness, yellowness, hardness, juiciness, and bitterness. However, a slight but significant decrease (p b 0.05) in saltiness scores was observed in treatment II compared with control (treatment I). This could be due to the fact that the 40% replacement of NaCl with KCl results in a less salty taste in the dry-cured bacon, and a less salty taste is sometimes positively valuated by the assessors (Gimeno, Astiasarán, & Bello, 1998).

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Table 2 Sodium and potassium content (mean ± standard deviation) in the dry-cured bacons subjected to three types of salt: I: control, 100% NaCl; II: 60% NaCl, 40% KCl; III: 30% NaCl, 70% KCl and dietary reference intakes (DRIs) of the sodium and potassium. Ion

I

II

III

DRIsd

Na+(mg/100 g) K+(mg/100 g)

2837.82 ± 14.26a 634.63 ± 17.34c

1828.07 ± 35.56b 1731.29 ± 23.72b

1215.22 ± 8.64c 2691.53 ± 48.46a

1500–2300 4700

a–c d

Means in the same row with different superscripts differ significantly (p b 0.05). From the United States National Academy of Sciences.

In the present study, mean sensory scores revealed significant differences (p b 0.05) in the hardness, juiciness, saltiness, and bitterness between control and treatment III. Lower hardness score and higher juiciness score of treatment III were observed as compared with control. Soft texture of treatment III could have contributed to lower hardness score for products with treatment III. This could be due to the higher moisture content of treatment III. And this finding also agrees with the aforementioned results carried out in 3.1. This finding in the change of hardness was similar to results obtained by Gimeno et al. (1999) and Gou et al. (1996), who also observed a significant reduction (p b 0.01) of hardness measured by texture profile analysis for Spanish dry fermented sausages manufactured with some NaCl substitutes. We also found that bacons with treatment III had lower saltiness score and higher bitterness score as compared with control (treatment I). This result could be due to K+ bitter taste markedly detected by assessors, when 70% NaCl was substituted by KCl. These results are in line with previous studies in fermented sausages (Gelabert et al., 2003) and Italian salami (Zanardi et al., 2010) and suggested that replacements up to 50% of NaCl by KCl did not influence the color and texture and also supplied an acceptable flavor without a bitterness.

densitometry. Relative band density as a percentage of total band density (sum of all band densities) of myofibrillar protein bands is presented in Table 5. Generally, all the bands were very similar in the samples of treatment I and treatment II. It was also observed that a majority of the bands were very similar between the samples of treatment III and treatments I and II. Nevertheless, a higher intensity of bands at 86.2, 44.0, and 12.9 kDa was observed in treatment III compared with the treatments I and II. On the contrary, it was also found that a lower intensity of bands at 37.1 and 34.4 kDa was shown in treatment III compared with the treatments I and II. These results are in agreement with the results obtained by Armenteros, Aristoy, Barat, and Toldrá (2009), Armenteros, Aristoy, and Toldrá (2009), who also found partial substitution (35% and 50%) of NaCl by KCl in the dry-cured loin had very similar patterns of myofibrillar proteins. 3.4. Assessment of proteolysis During the processing of dry-cured meat products, the protein degradation results in an increase in non-protein nitrogen concentration. These changes in proteins could be an important contribution of flavor compounds such as small peptides and amino acids (Toldra, 1998).

3.3. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) The electrophoretic profiles of sarcoplasmic proteins extracted from the dry-cured bacons salted with the three types of salt are shown in Fig. 2. The SDS–PAGE patterns of all bacons were similar, indicating that the sarcoplasmic protein breakdown was not affected by KCl levels in salt. To quantify the proteolysis of sarcoplasmic proteins in our study, the SDS–PAGE was measured by densitometry. Relative band density as a percentage of total band density (sum of all band densities) of sarcoplasmic protein bands are presented in Table 4. In general, all the bands were very similar in the samples of treatment II and treatment I. It was also found that most of all bands were very similar in the samples of treatment III and treatments I and II. However, a higher intensity of bands at 102.5, 41.3, and 37.6 kDa was apparent in treatment III compared with the treatments I and II. On the other hand, the intensity of bands at 33.8 kDa and below 26.6 kDa was lower for treatment III as compared with the treatments I and II. These results indicated that the high-proportion substitution (70%) of NaCl by KCl may inhibit some large fragments degradation into small fragments. Fig. 3. shows the electropherogram (SDS–PAGE) obtained for myofibrillar proteins extracted from the dry-cured bacons salted with the three types of salt. For the sake of quantifying the proteolysis of myofibrillar proteins in this study, the SDS–PAGE was measured by Table 3 Sensory analysis (mean ± standard deviation) in the dry-cured bacons subjected to three types of salt: I: control, 100% NaCl; II: 60% NaCl, 40% KCl; III: 30% NaCl, 70% KCl. Parameters

I

Redness Yellowness Hardness Juiciness Saltiness Bitterness

6.29 1.83 5.54 4.62 5.76 1.14

a–c

II ± ± ± ± ± ±

0.32a 0.61a 0.29a 0.37b 0.46a 0.24b

5.86 2.01 5.82 4.25 4.46 1.28

III ± ± ± ± ± ±

0.58a 0.46a 0.46a 0.22b 0.35b 0.45b

6.13 2.35 4.63 5.58 2.15 4.61

± ± ± ± ± ±

0.24a 0.39a 0.31b 0.34a 0.28c 0.53a

Means in the same row with different superscripts differ significantly (p b 0.05).

Fig. 2. SDS–polyacrylamide gel of sarcoplasmic proteins in the dry-cured bacons salted with the three types of salt. aStd: PageRuler molecular weight standards. bTreatment I: control, 100% NaCl; treatment II: 60% NaCl, 40% KCl; treatment III: 30% NaCl, 70% KCl.

H. Wu et al. / Meat Science 96 (2014) 1325–1331 Table 4 Relative band density as a percentage of total band density (sum of all band densities) of sarcoplasmic protein bands. aTreatment I: control, 100% NaCl; treatment II: 60% NaCl, 40% KCl; treatment III: 30% NaCl, 70% KCl. MW (kDa) N200 172.1 152.1 132.5 117.4 102.5 84.2 80.5 74.5 69.3 64.9 59.8 46.1 43.2 41.3 37.6 35.7 33.8 28.3 26.6 18.1 15.8 a–b

Ia

II a

0.22 0.51a 0.22a 0.17a — 0.77b 3.20a 0.39a 0.64a 4.20a 0.95a 20.42a 20.79a 2.97a 2.72b 8.95b 3.81a 10.25a 11.07a 5.59a 1.41 0.75

0.14 0.64a 0.25a 0.14a 0.25 0.94b 3.01a 0.36a 0.66a 4.02a 1.03a 20.09a 21.13a 3.05a 3.09b 7.85b 4.64a 9.96a 11.61a 4.86a 1.36 0.92

Table 5 Relative band density as a percentage of total band density (sum of all band densities) of myofibrillar protein bands. Treatment I: control, 100% NaCl; treatment II: 60% NaCl, 40% KCl; treatment III: 30% NaCl, 70% KCl. MW (kDa)

III a

a

0.12 0.48a 0.25a 0.22a — 4.01a 2.54a 0.37a 0.58a 4.52a 1.23a 19.35 a 18.51 a 2.01a 6.77a 14.62a 4.25a 4.80b 11.45a 3.92b — —

Means in the same row with different superscripts differ significantly (p b 0.05).

We further studied whether the extent of proteolysis in dry-cured bacons was affected by salting treatment during processing. The extent of proteolysis was evaluated by measuring proteolysis index (PI), PN, and AN in the dry-cured bacons subjected to three types of salt.

Fig. 3. SDS–polyacrylamide gel of myofibrillar proteins in the dry-cured bacons salted with the three types of salt. aStd: PageRuler molecular weight standards. bTreatment I: control, 100% NaCl; treatment II: 60% NaCl, 40% KCl; treatment III: 30% NaCl, 70% KCl.

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N200 152.8 133.5 103.2 94.4 86.2 81.1 57.7 43.8 37.0 34.4 30.4 22.0 18.4 16.1 12.9 a–b

I

II a

4.83 0.81a 0.87a 1.57a 1.35a — 0.83a 13.63a 22.91b 14.13a 18.82a 1.79a 5.28a 5.16a 4.21a 3.83b

III a

6.70 1.16a 0.75a 1.78a 0.99a — 0.47a 11.32a 24.25b 13.75a 18.09a 1.82a 4.83a 5.98a 3.78a 4.33b

5.20a 0.77a 0.48a 2.12a 1.57a 0.56 0.66a 12.55a 28.68a 9.35b 14.56b 2.38a 5.23a 5.52a 3.08a 7.31a

Means in the same row with different superscripts differ significantly (p b 0.05).

The proteolysis index (PI) of dry-cured bacons throughout the process is shown in Fig. 4. The proteolysis index (PI) increased progressively during their processing, reaching maximum levels at the end of the ripening stage, indicating the occurrence of proteolysis. These trends agree with previous studies in Jinhua ham (Zhao et al., 2008) and in dry-fermented sausage (Ikonić et al., 2013). However, the results found in this study are not totally in line with the results obtained by Bedia, Méndez, and Bañón (2011), who did not find statistically significant differences (p b 0.05) in the proteolysis index (PI) during the processing of semi-ripened Salami. This phenomenon could be attributed to raw material quality characteristics and process conditions (Morales, Serra, Guerrero, & Gou, 2007). Furthermore, the proteolysis index (PI) in the control (treatment I), treatment II, and treatment III was similar (p N 0.05) throughout the process in Fig. 4, which indicated that the extent as well as the level of proteolysis was similar among all salting treatments. These also suggest that the partial replacement of NaCl by KCl could not affect the protease activity. Fig. 5 shows the changes in the peptide nitrogen (PN) during drycured bacon processing using three types of salt. The content of peptide nitrogen (PN) did not change during the first stages of salting. It mainly increased during the drying-ripening (3–15 days), reaching the

Fig. 4. Proteolysis index (PI) change during dry-cured bacon processing using three types of salt. Treatment I: control, 100% NaCl; treatment II: 60% NaCl, 40% KCl; treatment III: 30% NaCl, 70% KCl. Labeled error bars indicate the standard deviation for each treatment. The proteolysis index (PI) was calculated as (NPN/TN) × 100.

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Fig. 5. Peptide nitrogen (PN) content (mg/g muscle dry matter) change during dry-cured bacon processing using three types of salt. Treatment I: control, 100% NaCl; treatment II: 60% NaCl, 40% KCl; treatment III: 30% NaCl, 70% KCl. Labeled error bars indicate the standard deviation for each treatment.

Fig. 6. Amino acid nitrogen (AN) content (mg/g muscle dry matter) change during drycured bacon processing using three types of salt. Treatment I: control, 100% NaCl; treatment II: 60% NaCl, 40% KCl; treatment III: 30% NaCl, 70% KCl. Labeled error bars indicate the standard deviation for each treatment.

maxima at the final stage, indicating the extensive degradation of protein. These results agree with the results obtained by Martín et al. (1998), who found a significant (p b 0.05) increase in PN values during ripening of Iberian ham (Martín et al., 1998). This change of PN values can be due to the behavior of tissue proteases during dry-cured meat products processing. From the existing information in the literature (Prior, 1984; Toldrá & Etherington, 1988; Toldra & Flores, 1998), it shows very likely that the proteolysis phenomenon that take place in most dry-cured meat products during processing are mainly attributed to the action of tissue proteases. Furthermore, other researchers found that the cathepsins can remain active throughout the processing of ham (Toldrá & Etherington, 1988), and the activity of cathepsin D is maximal in comparison with the activity of other endogenous proteolytic enzymes after 6 months of ripening (Sárraga, Gil, & GarcíaRegueiro, 1993). Thus, the activity of cathepsins is of great importance in the degradation of protein during drying-ripening as they release large-sized polypeptides, which are in turn degraded to oligopeptides, and then to free amino acids at the drying-ripening stage (Virgili, Saccani, Gabba, Tanzi, & Soresi Bordini, 2007). Moreover, no significant differences (p ≥ 0.05) were found in the peptide nitrogen (PN) between control and experimental bacons throughout their processing, indicating that the degree of proteolysis was similar in all bacons. This observation indicates that the partial substitution of NaCl by KCl did not affect the activity of cathepsins. These results agree with the results obtained by Armenteros, Aristoy, Barat, and Toldrá (2012), who found that NaCl and KCl exert a similar (p N 0.05) inhibition on cathepsins B, B + L, and H activities in dry-cured ham (Armenteros et al., 2012). Free amino acids are of great importance not only for their contribution to specific taste but also for their involvement in degradation reactions that generate volatile compounds, which provide the flavor in dry curing meat products (Toldra, 1998). The change of amino acid nitrogen (AN) during processing using three types of salt is shown in Fig. 6. Generally, the value of amino acid nitrogen (AN) gradually increased throughout processing in all dry-cured bacons, particularly during the drying-ripening stage. This trend in the change of amino acid nitrogen (AN) was similar to those of other workers who also observed a significant (p b 0.05) increase in amino acid nitrogen (AN) values during ripening of Iberian ham (Córdoba et al., 1994; Martín, Antequera, Ventanas, Benitez-Donoso, & Córdoba, 2001; Martín et al., 1998). Moreover, amino acid nitrogen (AN) contents reached average values of 3.073, 3.2214 and 3.3267 mg/g of muscle dry matter at the end of the ripening stage (15 days) for treatments I, II and III, respectively.

These values were lower than those reported by other authors for hams (Córdoba et al., 1994; Toldrá, Aristoy, & Flores, 2000). Lower quantity of amino acid nitrogen (AN) could be due to the differences in the extent of proteolysis as a result of the shorter ageing period in the present study. No significant (p N 0.05) differences were found in the amino acid nitrogen (AN) values between control and experimental bacons at all sampling ages, which indicated that the extent of proteolysis was similar in all bacons. This can be attributed to that KCl exert a very similar influence to NaCl for aminopeptidase activity (Armenteros, Aristoy, & Toldrá, 2009). Similar results were found in dry-cured loin (Armenteros, Aristoy, Barat, & Toldrá, 2009) and Kefalograviera cheese (Katsiari, Alichanidis, Voutsinas, & Roussis, 2001), in which the proteolysis was not influenced by the type of the salting treatment employed during their processing. Therefore, the result of our present study, that control and experimental bacons had a similar amino acid nitrogen (AN) values could be illustrated on the basis of the results of the above studies.

4. Conclusion The results showed that the substitution of 40% NaCl by KCl did not markedly influence the proteolysis phenomena taking place during processing, as measured by SDS–PAGE, proteolysis index, peptide nitrogen, and amino acid nitrogen. Also, the sensory analysis of the final products indicated it was possible to reduce NaCl by 40% in dry-cured bacon without adverse effects on sensory properties. The substitution of 70% NaCl by KCl would lead to marked bitter taste and softer texture of the dry-cured bacon. Moreover, the replacement up to 40% NaCl by KCl had no significant effect on the moisture content even though the substitution of 70% NaCl by KCl significantly increased the moisture content of dry-cured bacons during processing. These results may be useful as a starting point for the study of NaCl reduction in other dry-cured meat products such as dry-cured ham.

Acknowledgement This work was supported by the National Key Technology R&D Program in the 12th Five-Year Plan of China (2012BAD28B01) and Agro-scientific Research in the Public Interest (201303082-2). The authors acknowledge the staff of the Key Laboratory of Meat Processing and Quality Control, Nanjing Agricultural University.

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References Aliño, M., Grau, R., Baigts, D., & Barat, J. (2009). Influence of sodium replacement on the salting kinetics of pork loin. Journal of Food Engineering, 95(4), 551–557. Aliño, M., Grau, R., Toldrá, F., Blesa, E., Pagán, M. J., & Barat, J. M. (2009). Influence of sodium replacement on physicochemical properties of dry-cured loin. Meat Science, 83(3), 423–430. Aljuraiban, G. S., Chan, Q., Brown, I. J., Griep, L. M.O., Daviglus, M. L., Stamler, J., Van Horn, L., Holmes, E., Elliott, P., & Frost, G. S. (2012). 182 association of number of daily eating episodes with blood pressure and nutrient intake: intermap study. Journal of Hypertension, 30, e54–e55. AOAC (2005). Official Methods of Analysis (18th ed.)Washington, DC: Association of Official Analytical Chemists. Armenteros, M., Aristoy, M., Barat, J., & Toldrá, F. (2009). Biochemical changes in dry-cured loins salted with partial replacements of NaCl by KCl. Food Chemistry, 117(4), 627–633. Armenteros, M., Aristoy, M. -C., Barat, J. M., & Toldrá, F. (2012). Biochemical and sensory changes in dry-cured ham salted with partial replacements of NaCl by other chloride salts. Meat Science, 90(2), 361–367. Armenteros, M., Aristoy, M. -C., & Toldrá, F. (2009). Effect of sodium, potassium, calcium and magnesium chloride salts on porcine muscle proteases. European Food Research and Technology, 229(1), 93–98. Bedia, M., Méndez, L., & Bañón, S. (2011). Evaluation of different starter cultures (Staphylococci plus lactic acid bacteria) in semi-ripened salami stuffed in swine gut. Meat Science, 87(4), 381–386. Careri, M., Mangia, A., Barbieri, G., Bouonl, L., Virgili, R., & Parolari, G. (1993). Sensory property relationships to chemical data of italian-type dry-cured ham. Journal of Food Science, 58(5), 968–972. Córdoba, J., Antequera, T., Ventanas, J., López-Bote, C., García, C., & Asensio, M. (1994). Hydrolysis and loss of extractability of proteins during ripening of Iberian ham. Meat Science, 37(2), 217–227. Doyle, M. E., & Glass, K. A. (2010). Sodium reduction and its effect on food safety, food quality, and human health. Comprehensive Reviews in Food Science and Food Safety, 9(1), 44–56. Gelabert, J., Gou, P., Guerrero, L., & Arnau, J. (2003). Effect of sodium chloride replacement on some characteristics of fermented sausages. Meat Science, 65(2), 833–839. Gimeno, O., Astiasarán, I., & Bello, J. (1998). A mixture of potassium, magnesium, and calcium chlorides as a partial replacement of sodium chloride in dry fermented sausages. Journal of Agricultural and Food Chemistry, 46(10), 4372–4375. Gimeno, O., Astiasarán, I., & Bello, J. (1999). Influence of partial replacement of NaCl with KCl and CaCl2 on texture and color of dry fermented sausages. Journal of Agricultural and Food Chemistry, 47(3), 873–877. Gou, P., Guerrero, L., Gelabert, J., & Arnau, J. (1996). Potassium chloride, potassium lactate and glycine as sodium chloride substitutes in fermented sausages and in dry-cured pork loin. Meat Science, 42(1), 37–48. Huang, M., Huang, F., Ma, H., Xu, X., & Zhou, G. (2012). Preliminary study on the effect of caspase-6 and calpain inhibitors on postmortem proteolysis of myofibrillar proteins in chicken breast muscle. Meat Science, 90(3), 536–542. Ikonić, P., Tasić, T., Petrović, L., Škaljac, S., Jokanović, M., Mandić, A., & Ikonić, B. (2013). Proteolysis and biogenic amines formation during the ripening of Petrovská klobása traditional dry-fermented sausage from Northern Serbia. Food Control, 30(1), 69–75. IOM, Food and Nutrition Board (2004). Dietary reference intakes for water, potassium, sodium, chloride, and sulfate. Washington DC: The National Academies Press. ISO 6658 (2005). Sensory Analysis—Methodology—General Guidance. Geneva: International Organization for Standardization. ISO 8586 (2012). Sensory Analysis—General Guidelines for the Selection, Training and Monitoring of Selected Assessors and Expert Sensory Assessors. Geneva: International Organization for Standarization. ISO 8589 (2007). Sensory Analysis—General Guidance for the Design of Test Rooms. Geneva: International Organization for Standardization. ISO Norm R-1442 (1979). Determination of Moisture. : International Organization for Standardization.

1331

Jin, G., He, L., Zhang, J., Yu, X., Wang, J., & Huang, F. (2012). Effects of temperature and NaCl percentage on lipid oxidation in pork muscle and exploration of the controlling method using response surface methodology (RSM). Food Chemistry, 131(3), 817–825. Jin, G., Zhang, J. H., Yu, X. A., Zhang, Y. P., Lei, Y. X., & Wang, J. M. (2010). Lipolysis and lipid oxidation in bacon during curing and drying-ripening. Food Chemistry, 123(2), 465–471. Katsiari, M., Alichanidis, E., Voutsinas, L., & Roussis, I. (2001). Proteolysis in reduced sodium Kefalograviera cheese made by partial replacement of NaCl with KCl. Food Chemistry, 73(1), 31–43. Ketelaere, A., Demeyer, D., Vandekerckhove, P., & Vervaeke, P. (1974). Stoichiometry of carbohydrate fermentation during dry sausage ripening. Journal of Food Science, 39(2), 297–300. Kilcast, D., & Angus, F. (2007). Reducing Salt in Foods: Practical Strategies. : Woodhead Publishing Ltd. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680–685. Martín, L., Antequera, T., Ventanas, J., Benitez-Donoso, R., & Córdoba, J. (2001). Free amino acids and other non-volatile compounds formed during processing of Iberian ham. Meat Science, 59(4), 363–368. Martín, L., Córdoba, J., Antequera, T., Timón, M., & Ventanas, J. (1998). Effects of salt and temperature on proteolysis during ripening of Iberian ham. Meat Science, 49(2), 145–153. Molina, I., & Toldra, F. (1992). Detection of proteolytic activity in microorganisms isolated from dry-cured ham. Journal of Food Science, 57(6), 1308–1310. Moore, S., & Stein, W. H. (1954). A modified ninhydrin reagent for the photometric determination of amino acids and related compounds. Journal of Biological Chemistry, 211(2), 907–913. Morales, R., Serra, X., Guerrero, L., & Gou, P. (2007). Softness in dry-cured porcine biceps femoris muscles in relation to meat quality characteristics and processing conditions. Meat Science, 77(4), 662–669. Prior, B. (1984). Role of micro-organisms in biltong flavour development. Journal of Applied Microbiology, 56(1), 41–45. Ruiz, J., Garcıa, C., Muriel, E., Andrés, A. I., & Ventanas, J. (2002). Influence of sensory characteristics on the acceptability of dry-cured ham. Meat Science, 61(4), 347–354. Ruusunen, M., & Puolanne, E. (2005). Reducing sodium intake from meat products. Meat Science, 70(3), 531–541. Ruusunen, M., Vainionpää, J., Puolanne, E., Lyly, M., Lähteenmäki, L., Niemistö, M., & Ahvenainen, R. (2003). Physical and sensory properties of low-salt phosphate-free frankfurters composed with various ingredients. Meat Science, 63(1), 9–16. Sárraga, C., Gil, M., & García-Regueiro, J. A. (1993). Comparison of calpain and cathepsin (B, L and D) activities during dry-cured ham processing from heavy and light large white pigs. Journal of the Science of Food and Agriculture, 62(1), 71–75. Toldra, F. (1998). Proteolysis and lipolysis in flavour development of dry-cured meat products. Meat Science, 49, 101–110. Toldrá, F., Aristoy, M., & Flores, M. (2000). Contribution of muscle aminopeptidases to flavor development in dry-cured ham. Food Research International, 33(3), 181–185. Toldrá, F., & Etherington, D. J. (1988). Examination of cathepsins B, D, H and L activities in dry-cured hams. Meat Science, 23(1), 1–7. Toldra, F., & Flores, M. (1998). The role of muscle proteases and lipases in flavor development during the processing of dry-cured ham. Critical Reviews in Food Science, 38(4), 331–352. Virgili, R., Saccani, G., Gabba, L., Tanzi, E., & Soresi Bordini, C. (2007). Changes of free amino acids and biogenic amines during extended ageing of Italian dry-cured ham. LWT–Food Science and Technology, 40(5), 871–878. Zanardi, E., Ghidini, S., Conter, M., & Ianieri, A. (2010). Mineral composition of Italian salami and effect of NaCl partial replacement on compositional, physico-chemical and sensory parameters. Meat Science, 86(3), 742–747. Zhao, G., Tian, W., Liu, Y., Zhou, G., Xu, X., & Li, M. (2008). Proteolysis in biceps femoris during Jinhua ham processing. Meat Science, 79(1), 39–45.