Different Effect of Sodium Chloride Replacement with Calcium Chloride on Proteolytic Enzyme Activities and Quality Characteristics of Spent Hen Samgyetang

Considering the adjustment of SH carcass to the size of the retort pouch, this study only utilized breast meat for chicken soup. It was prepared from SH chicken (Hyline white, 72 weeks old) from 60 birds 24 h postmortem. The breast meat were obtained from commercial slaughterhouse with an average weight of 100±15 g. Left breast samples were randomly allocated into ten different groups consist of control and treatments with different mixtures of salt content, as well as different processing method, particularly boiling and retorting. While the right breast samples were randomly submitted into storage experiments investigated the different responses of proteolytic enzymes after treatment with different salt mixtures measured at day 0, 1, 3, and 5. Each of grouped samples intially subjected to marination with various percentage of salt mixtures that shown in Table 1. The sodium tripolyphosphate (STPP) percentage was adjusted to maximum addition of 0.5% for all treatment groups. Salt mixture solutions were adjusted at 50 mL per 100 g sample. Marination was taken place at a temperature of 4±2°C through immersion for 20 h followed by alleviation of marinade solution from the meat by letting samples to stand for 30 minutes, then wiped by towel tissue.

Broth was previously extracted from chicken soup broth pack containing Astragalus membranaceus root, mulberry branch, Kalopanax septemlobus branch, licorice, Siberian ginseng, and salt that boiled for 45 minutes. Afterward, the broth pack was removed and the solution was filtered using a stainless mesh filter (8 mm). Marinated SH meat samples were brought into different processing stages. In term of boiling, each treatment group consisting six breast meats (6 replications) were boiled together with 1.5 L prepared broth at 100°C for 60 minutes. While for retorting, each retort pouch filled with six marinated breast meat from the same treatment group, stuffed with 1.5 L of prepared broth. The retort process was conducted at a temperature of 121°C with a pressure of 1.5 kgf/cm² for 60 minutes, followed by cooling process for 30 minutes. Cooked samples were subsequently filtered by using stainless mesh filter (600 μm) which subsequently be analyzed for its quality characteristics.

Perfectly ground samples were measured for its moisture, crude protein, crude fat, and ash content according to a method by the Association of Official Analytical Chemists (AOAC, 2002). Each samples were prepared in triplicate.

Cooking loss is a weight loss percentage after subjected to processing stages. It was obtained by calculating the weight loss (Initial weight–weight after process) divided by initial weight.

Ground samples in triplicate was measured for its WHC according to a method by Kristensen and Purslow (2001). It is a centrifugal method where 5 g of the sample was placed in a centrifuge tube equipped with a wire mesh, heated in a water bath at 75°C for 30 min, and centrifugated with (CS-6R Centrifuge; Beckman Instruments, Hialeah, FL, USA) at 980 g for 10 min. WHC is the ratio between total moisture content and remaining water content.

Homogenated samples in triplicate were prepared from 5 g of perfectly ground sample added with 45 mL of distilled water assisted by homogenizer (PH91; SMT, Tokyo, Japan). The pH value was determined by a previously calibrated pH meter probe (Seven Easy pH; Mettler-Toledo GmbH, Schwerzenbach, Switzerland).

The determination of lipid oxidation rate were by using 2-thiobarbituric acid reactive substances (TBARS) according to a method by Sinnhuber and Yu (1977) with a slight modifiction. Analyses were conducted in triplicate and TBARS value was expressed as mg of malondialdehyde (MDA)/kg of meat.

The measurement of shear force value was performed by using a TA-XT2i Plus (Stable Micro Systems, Surrey, UK) on a cooked samples. Cooked samples in rectangular size of 1.5 cm×1.5 cm was placed under the V blade and cut with a constant speed (assay parameters were: pretest speed: 2.0 mm/s; test speed: 1.0 mm/s; posttest speed: 10 mm/s). Each sample was repeated three times.

The determination of myofibrillar fragmentation index was referring to a method by Culler et al. (1978) with slight modifications. Each of the marinated samples was prepared in triplicate. After subjected to isolation with pre-cooled isolating buffer (0.01 mM EDTA, 20 mM K₃PO₄, 100 mM KCl, and 1.0 mM CaCl₂; pH was adjusted to 7.0), the optical density (OD) was measured at 540 nm by using spectrophotometer. The myofibril fragmentation index (MFI) was obtained by multiplying OD with 200.

The collagent content of marinated samples were analyzed based on a method by Jayasena et al. (2013). After subjected to hydrolysis according to a method by Palka and Daun (1999), Collagen conten was calculated by multiplying the hydroxyproline content by a factor of 7.25 and was expressed as mg/g. The insoluble collagent was also expressed as mg/g. All the analyses were repeated three times.

Protein solubility was determined according to procedures of Joo et al. (1999). Sarcoplasmic proteins were extracted from 2-g minced muscle using 20 mL of ice-cold 0.025 M potassium phosphate buffer (pH 7.2). While the total protein (sarcoplasmic+myofibrillar) was extracted from 2-g muscle using by 40 mL of ice-cold 1.1 M potassium iodide in 0.1 M phosphate buffer (pH 7.2). Myofibrillar protein solubility concentrations were obtained by calculate the difference between total and sarcoplasmic protein solubility.

To understand the possible responses of tenderness-related enzymes (calpains, cathepsin-b and caspase-3 enzyme) to treatment with different percentage of sodium chloride replacement, the activity of endogenous enzymes were measured on sixty raw SH breast meat after subjected to marination with various percentage of sodium chloride : calcium chloride at a temperature of 4±2°C through immersion for 20 h, samples were then allocated to storage and determine its change on enzyme activities at day 0, 1, 3, and 5, wherein day 0 was used as reference data. According to a method described by Broadway et al. (2011) with slight modifications, in which a substrate (Suc-LY-AMC) was used for calpains, while ARR-AFC was used for determination of cathepsin-B enzyme activity. To obtain the supernatants, 0.5 g of the homogenated samples in 1.0 mL lysate (100 mmol/L HEPES, 20% glycerol, 0.5 mmol/L EDTA, 5.0 mmol/L DTT, and 0.2% SDS; pH 5.8) were centrifuged at the speed of 12,000×g for 20 min. Regarding the control, the supernatant was replaced with ddH₂O. The absorbance value of the mixture was detected at 380 nm/460 nm (excitation/emission) for calpains and 400 nm/505 nm (excitation/emission) for cathepsin-B. The enzyme activity is shown as the relative absorbance value per min, per mg to the control. In term of Caspase-3 activity was determined according to a method described by He et al. (2019). A total of 1.0 mM Ac-DEVD-pNA (dissolved in Dimethyl sulfoxide (DMSO)) was used as the substrate and incubated with the supernatant at 37°C for 1 h. The absorbance value of the mixture was detected at a wavelength of 405 nm, and caspase-3 enzyme activity is expressed as the relative absorbance value per min, per mg to the control.

The data analyses in this study performing R-version 3.6.1 (The R-foundation for Statistical Computing, Vienna, Austria), CRAN mirror and library USA (CA 1) equipped with Agricolae. two-way multivariate analysis of variance (MANOVA) was used to determine the effect of sodium chloride replacement as well as different manufacturing method on investigated variables. Significant value of the mean for each group was continuesly analyzed using the Duncan’s multiple range test, with a consideration as significant for p-value lower than 0.05.

The effect of sodium chloride replacement with calcium chloride on chicken soup breast meat made from SH chicken is shown in Table 2. Moisture percentage was significantly affected by treatments, with the highest percentage of NaCl replacement, resulting in a significantly higher moisture percentage of the breast meat (p<0.05). However, the replacement of NaCl with CaCl₂ until 75% did not contribute to an increased moisture content (p>0.05). Furthermore, crude protein, crude fat and ash content were not significantly affected by different salt mixture percentage in marinade solution (p>0.05). Besides, regardless of the concentration of NaCl replacement, meat processed in a high temperature and high-pressure condition (retort) significantly lower moisture content (p<0.05) with a less effect on crude protein, crude fat and ash content compared to that processed by boiling (p>0.05). Increased in moisture percentage might be due to the high ionic strength of calcium ion that promotes a more protein-ion bond and consequently assembles a layer-like form on the meat surface to act as a water repulsion inhibitor (Nurmahmudi and Sams, 1997). Coupled with an osmotic-like force with a high affinity to interact and pull water molecules into the meat environment is another hypothesis from the effect of chloride ion on imparting free protein with negative charges (Lee et al., 2012). Following these studies, a replacement of salt type with a stronger ionic strength may lead to an increased of moisture percentage.

Different processing methods significantly affected the shrinkage percentage of SH chicken soup in all treatment samples. As shown in Table 3, breast meat processed by retorting promoted a significantly higher cooking loss compared to that processed by boiling (p<0.001). No significant effect was observed after marination with reduced sodium chloride, indicating a similar calcium chloride attribute to prevent cooking loss as sodium chloride did. In term of WHC, breast meat samples marinated with a solution containing STPP with 100% CaCl₂ resulting in a higher WHC percentage compared to that containing 100% NaCl (p<0.05), meaning a higher portion of CaCl₂ within marinade solution significantly contributed to an increased ability of the muscle to retain water, a factor for the increment of WHC (Barido et al., 2020b). Different cooking methods did not significantly affect WHC, wherein SH meat samples processed through boiling did not differ from that processed by retorting (p>0.05).

A similar trend was also observed for pH value, the value increment was found to be significantly affected by a higher percentage of CaCl₂ within marinade solution (p<0.001). Replacement of NaCl with ≥50% CaCl₂ resulting in a SH breast meat chicken soup with a significantly higher pH value (p<0.001). In addition, along with a significantly lower pH value of chicken soup breast meat processed by retorting compared to that of boiled chicken soup, this study found a significant interaction between treatment and processing method on pH value (p<0.001).

Numerous studies considered WHC to be an important variable that strongly correlated with meat tenderness. It characterizes the moisture retention ability by meat during processing as well as water uptake (Barido et al., 2020a). A higher affinity and ionic strength of CaCl₂ compared to NaCl promotes a greater protein extractability and consequently drive to a more protein-ion bond with negative charges. Although CaCl₂ and NaCl may share similar ion, the tenderization mechanism may differ, wherein calcium chloride could provide faster tenderization via similar conductivity level of ionic strength as sodium chloride, as well as calcium ion-specific effect to activate more proteolytic activities (Koohmaraie et al., 1988). The more assembly of protein-ion filament on meat surface is also consequence by calcium chloride, this condition is therefore assumed to increase the inner meat environment ability to retain water and significantly contribute to an increased WHC (Nurmahmudi and Sams, 1997). Furthermore, this result also confirmed a previous study by Bowker and Zhuang (2015), that categorized breast meat with a higher WHC promoted to a higher pH value, and vice versa.

Table 3 displays the concentration of malondialdehyde content, a highly reactive compound as a marker for lipid oxidation. Different NaCl:CaCl₂ composition within the marinade solution did not significantly contribute to different lipid oxidation rates (p>0.05). However, regardless the percentage of NaCl replacement, samgyteang breast meat processed by retorting significantly resulting in a higher lipid oxidation rate than that of boiled chicken soup (p<0.05). This finding was in accordance with a previous study by Kim et al. (2020), wherein a higher manufacturing condition (temperature and humidity) has a strong interaction with a faster increment of lipid oxidation. TBARS value in this study, however, was still below the threshold in which meat product is characterized as off-flavor and unacceptable by consumer (Nam and Ahn, 2003). These results implied that even complete replacement of NaCl with CaCl₂ salt within the marinade solution did not bring a detrimental effect on meat quality, particularly the formation of lipid oxidation.

The changes in the tenderness level of chicken soup breast meat after treatment with different NaCl:CaCl₂ are shown in Table 4. Although an inconsistent effect on shear force value was observed, the lower shear value tended to result from a higher portion of CaCl₂ within the marinade solution. In addition, higher sodium chloride replacement with calcium chloride promoted a significantly higher fragmentation of myofibrillar protein (p<0.001). In the other hand, the MFI value was significantly affected by both manufacturing method and salt replacement. A higher replacement percentage of NaCl with CaCl₂ significantly increased the MFI value (p<0.001), while accompanied by a higher value in chicken soup breast meat processed through retorting compare to that of by boiling (p<0.001). A decrease in meat tenderness results from a complex interaction between myofibrillar protein thick filament called myosin and actin. Once an animal is sacrificed, biochemical reactions within muscle promote an increased crosslinking between actin and myosin to form actomyosin. The higher formation of actomyosin would generate meat that is characterized as stiff (Broadway et al., 2011). In addition, MFI is another widely used index to determine the occurrence of postmortem tenderization in meat (Volpelli et al., 2005). The degradation of myofibrillar protein is strongly associated with architectural changes in meat muscle, wherein a higher MFI value indicates a higher proteolytic activity. The MFI value in this study significantly affected by CaCl₂ percentage within the marinade solution might be due to the calcium ion effect. The calcium ion was proven to regulate the calpains activity (Nurmahmudi and Sams, 1997), thus a higher concentration of calcium ion leading to a more calpains activity that consequently causes fragmentation of myofibrillar protein and destruction of muscle integrity. This result confirmed a previous study by Li et al. (2017) that mentioned a high concentration of CaCl₂ salt significantly contributed to a higher MFI value of goose breast meat.

Table 4 displays the effect of marination with different percentage of NaCl:CaCl on chicken soup breast meat made from SH chicken. Both total collagen and insoluble collagen content were not differ among samples (p>0.05). Different processing methods also did not significantly modify the collagen content from SH breast meat (p>0.05). Kong et al. (2008) mentioned that along with the muscle integrity, total and insoluble collagen content dominantly dictate the tenderness level of meat. At least two factors influence thermal residual collagen and unextractable collagen; advancing age and different chicken breeds (Nakamura et al., 1975). A study by Jeong et al. (2020) characterize among various chicken breeds used as chicken soup raw material, SH meat was denoted to exert a higher collagen content and, consequently, affect a lower sensory acceptance. While in this study, marination with a diverse percentage of NaCl and CaCl₂ prior to cooking did not generate a SH breast meat with significantly lower collagen content. It was proposed that the calcium-specific effect regulates the intra-cellular calpain activity with substrate limited to tropomodulin protein (Li et al., 2017). Therefore, protein with a large molecular size such as collagen was assumed to not be affected by these events.

Total protein solubility was significantly affected by NaCl:CaCl₂ percentage within marinade solution. A replacement of NaCl with CaCl₂ at ≥50% resulting in a significantly higher total protein solubility among treatments as seen in Table 4, with the highest was observed for complete replacement of NaCl with CaCl₂ (p<0.001)_. Similar trend was also found for myofibrillar protein. A significant effect was found for myofibrillar protein solubility of SH breast meat after marinated with 50:50% (NaCl:CaCl₂), as well as 100% marination with CaCl₂ (p<0.001). Retorting process seemed to impart a significant increase of both total soluble protein and myofibrillar protein solubility of chicken soup breast meat compare to that of boiling groups (p<0.001). The soluble sarcoplasmic protein concentration was not affected by either marination with NaCl replacement or a different processing method (p>0.05). Calcium-specific ion was assumed to regulate calpains’ activity, wherein tropomodulin protein is a potential substrate that consequently promotes protein degradation (Li et al., 2017). An increase in total soluble protein, as well as myofibrillar protein solubility observed by this study, confirmed a previous study by Nurmahmudi and Sams (1997) that besides its calcium specific effect, the ionic strength of CaCl₂ could promote a higher protein extractability and faster tenderization effect compared to NaCl on SH meat.

The activity of cathepsin-B enzyme on SH breast meat after treated with salt mixtures containing different percentage of NaCl and CaCl₂ was shown in Fig. 1. The cathepsin-B enzymes treated with 100% CaCl₂ was observed to had the highest activity among treatments at day 1 (p<0.05). Enzyme activities were shown to be not statistically different in treatment containing 75% and 100% CaCl₂ replacement. In addition, different salt mixtures effect was not differ among treatments at day 3 and day 5 (p>0.05). The upreglation of cathepsin-B enzymes activity treated with complete replacement of NaCl with CaCl₂ were recorded at 8%–33%. It possibly due to the specific effect of Ca²⁺ ion from CaCl₂ that modified the activity of cathepsin-B. As mentioned by Li et al. (2017), CaCl₂ containing Ca²⁺ ion play a significant role for the tropomodulin protein activation (actin filament capping protein). The higher concentration of Ca²⁺ ion within meat environment resulting in a higher depolymerization rate of actin filament through the hydrolization of tropomodulin. These results confirmed previous study by He et al. (2019) who found a significant upregulation of cathepsin-B enzyme by CaCl₂ on duck breast meat.

Fig. 1. Cathepsin-B enzyme activities expressed in (relative absorbance value per min, per mg to control) of spent hen breast meat after treated with different salt solution containing different percentage of NaCl:CaCl₂. 0% (breast meat marinated with 100% NaCl), 25% (breast meat marinated with 75% NaCl:25% CaCl₂), 50% (breast meat marinated with 50% NaCl:50% CaCl₂), 75% (breast meat marinated with 25% NaCl:75% CaCl₂), 100% (breast meat marinated with 100% CaCl₂).

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Similar trends were observed on caspase-3 enzymes, enzyme responsible for the activation of apoptosis. The highest activity of caspase-3 enzymes found by this study as seen in Fig. 2 was recorded on SH breast meat treated with NaCl replacement with CaCl₂ at >75% (p<0.05) at day 1, with a slight effect at day 3 and 5 respectively. Although the caspase-3 enzyme contributions during post-mortem tenderization are still debated and not clearly explained, studies by Nurmahmudi and Sams (1997) revealed that this enzyme could be another important enzymes after calpains and cathepsin families for texture improvement in chicken meat. As well as study by He et al. (2019) who found that CaCl₂ could led to an upregulation of caspase-3 enzyme and eventually promoted to postmortem tenderization.

Fig. 2. Caspase-3 enzyme activities expressed in (relative absorbance value per min, per mg to control) of spent hen breast meat after treated with different salt solution containing different percentage of NaCl:CaCl₂. 0% (breast meat marinated with 100% NaCl), 25% (breast meat marinated with 75% NaCl:25% CaCl₂), 50% (breast meat marinated with 50% NaCl:50% CaCl₂), 75% (breast meat marinated with 25% NaCl:75% CaCl₂), 100% (breast meat marinated with 100% CaCl₂).

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In term of calpains enzyme as shown in Fig. 3, the effect of different percentage of NaCl and CaCl₂ was determined not to differ among treatments throughout storage days, indicated that calpains enzyme activities could independently worked without interference of neither NaCl nor CaCl₂. This results on SH breast meat was consistent with that explained by He et al. (2019), who studied the effect of different enzyme responses after treated with CaCl₂ and MDL-28,170 on duck breast meat, and found no significant effect on the calpains enzyme activities. Moreover, the declining activies of the calpains enzyme throughout storage day was possibly a result of the restricted substrate that could be utilized for the enzyme activation.

Fig. 3. Calpain enzyme activities expressed in (relative absorbance value per min, per mg to control) of spent hen breast meat after treated with different salt solution containing different percentage of NaCl:CaCl₂. 0% (breast meat marinated with 100% NaCl), 25% (breast meat marinated with 75% NaCl:25% CaCl₂), 50% (breast meat marinated with 50% NaCl:50% CaCl₂), 75% (breast meat marinated with 25% NaCl:75% CaCl₂), 100% (breast meat marinated with 100% CaCl₂).

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