The Effect of Hydrolysis Pre-Treatment by Flavourzyme on Meat Quality, Antioxidative Profiles, and Taste-Related Compounds in Samgyetang Breast Supplemented with Black Garlic

BG cloves used in this study were obtained from the Haena Food (20160506929-1, Seoul, Korea). The extraction of the BG was done according to the Kimura et al. (2017). Briefly, peeled BG was added to 10 volumes of deionized water (w/v) and grounded in a food blender (EBR9814W, Electrolux, Stockholm, Sweden) at 13,500×g for 1 min. The solution was placed in a water bath (BW-20G, Biotechnical Services, North Little Rock, AR, USA) at 80°C for 1 h. The obtained BG extract was directly incubated at 4±2°C for 1 h, followed by filtration using Whatman filter paper (No. 1). This solution containing the phenolic extract from BG with the pH value of 4.70 (data not shown) was added to Samgyetang at 5% (v/w) of the total weight.

Conventional Samgyetang was used as the control. It was prepared by placing fat-removed chicken breasts into a retort pouch, added with prepared broth (300 mL) that composed of Astragalus membranaceus root, mulberry branch, Kalopanax septemlobus branch, and licorice, Siberian ginseng with a salt concentration was set at 0.6% (w/w) according to Jeong et al. (2020). Flavourzyme samgyetang (FS) was prepared by hydrolyzing chicken breast prior to retorting with 1% of Flavourzyme (v/w). The hydrolysis was performed as described by Kong et al. (2017) in a water bath at 55°C for 2.5 h. The hydrolyzed chicken samples were added with prepared broth similar to control into a retort pouch. Samgyetang supplemented with BG (NBG group) was prepared as described in our previous study (Barido et al., 2021) wherein, 200 mL of the prepared broth similar to the control was mixed with 100 mL of the prepared BG extract. To prepare hydrolyzed BG Samgyetang (HBG), deionized water was added to the chicken breast at 1:2 (w/v), followed by Flavourzyme at 1% of chicken breast (v/w). All prepared samples were directly subjected to high temperature and pressure of 121°C, 1.5 kg/cm² for 1 h with f₀ 8 using an autoclave (AC-13, Jeio Tech, Daejeon, Korea). Subsequently, the breasts were separated from the broth by filtration using a mesh filter (600 μm) and assigned for further analysis of its quality, antioxidant activity, and taste-related compounds.

The analysis of the proximate composition of the retorted chicken breast samples was done following the procedure by AOAC (2012). The percentage of sample’s moisture was measured on a 1 g sample after oven drying at 105°C for 24 h. Crude protein content was measured following the Kjeltec system procedure (2200 Kjeltec Auto Distillation Unit, Foss, Hillerød, Denmark). Crude fat was determined through the Soxhlet extraction method for 48 h, and the crude ash content was determined after burning in a muffle furnace (LEF-115S, Daihan Labtech, Namyangju, Korea) at 550°C. All analyses were performed in triplicate.

The instrumental color of the cooked breast samples was measured at five different points using a chromameter (CR-400, Konica Minolta Sensing, Osaka, Japan) that was previously calibrated using a white plate (2° observer, Illuminant C: Y=93.6, x=0.3134, y=0.3194). The lightness (CIE L*), redness (CIE a*), and yellowness (CIE b*) were carefully recorded.

The pH value was measured in triplicate on each cooked breast slurry. After mixing 5 g sample with 45 mL of distilled water in a homogenizer (PH91, SMT, Tokyo, Japan), the pH value of the continuously stirred slurry samples was measured using a pH meter probe (Seven Easy pH, Mettler-Toledo GmbH, Schwerzenbach, Switzerland) that previously been calibrated.

The cooked breast samples were made into a 1.5×1.5×1.5 cm³ size and placed under the V blade of the TA-XT2i Plus (Stable Micro Systems, Surrey, UK) texture analyzer. Retorted samples were prepared at the uniform size of 1.5×1.5 cm. The measurement was performed on the interior of each samples parallel with the myofibrils orientation under V blade to avoid high variety of measurement due to the possible hardening that occurs toward the outside cooked edge of the sample. The analyses of shear force value for each samples were done with three replicates.

The determination of the WHC was carried out according to the centrifugation method by Kristensen and Purslow (2001). The unit was expressed as a percentage and was calculated from the ratio of the total moisture content to the remaining water volume after being subjected to boiling in a water bath followed by centrifugation. While the percentage of cooking loss of the cooked breast samples was carefully by calculating the weight before and after cooking [(W₁–W₂) –W₁]. W1 denotes the weight of certain sample before retort processing, while W₂ denotes the weight of certain sample after experience retort processing.

The lipid oxidation rate was measured using 2-TBARS assays to quantify the malondialdehyde (MDA). In brief, cooked breast samples (0.5 g) were prepared in triplicate in a 25 mL TBARS test tube, added with an antioxidant mixture (0.1 mL), 1% TBA in 0.3% NaOH (3 mL), and assigned into a thorough vortex for 30 s. The 17 mL of 2.5% trichloroacetic acid in 36 mM HCl was added, sealed, and heated in a water bath (BW-20G, Biotechnical Services, North Little Rock, AR, USA) at 100°C for 30 min. The tubes were immersed into ice water for 10 min once heating was completed. Each of 5 mL of the aqueous sample was taken into a new 15 mL conical tube, mixed with 3 mL of chloroform, and centrifuged at 2,400×g for 30 min at 4°C (1248R, LaboGene, Lillerød, Denmark). The absorbance was recorded at 532 nm using a UV spectrophotometer (UV-mini 1240 PC, Shimadzu, Kyoto, Japan) and compared against a blank.

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays were performed to measure the antioxidant activity of the cooked breast samples based on the protocol by Islam et al. (2016). The extract solution (100 μL) was placed in 100 μL of methanolic solution containing DPPH radicals (0.2 mM). The mixture was allowed to react for 30 min at 25°C in the dark. The absorbance of each extract solution was measured at 517 nm using a spectrophotometer (UV-mini 1240 PC, Shimadzu). The results were expressed as a scavenging percentage of free radicals.

The concentration of taste-related nucleotides (5’-AMP, 5’-IMP, 5’-GMP, Inosine, and Hypoxanthine) was carried out following the method by Jayasena et al. (2014) with slight modifications via high-performance liquid chromatography (HPLC) (Agilent 1260 series, Agilent Technologies, Santa Clara, CA, USA) that equipped with a 4.6×150 mm C18 column (Agilent Technologies) and a diode array detector. Minced samples (5 g) were mixed with 25 mL 0.7 M perchloric acid and homogenized (Polytron R PT-2500 E, Kinematica, Malters, Switzerland). The homogenate was centrifuged at 2,000×g for 15 min at 0°C, filtered through filter paper (Whatman No. 4). The collected supernatant was adjusted to pH 6.5 with 5 N KOH. The supernatant was placed in a volumetric flask and adjusted to 100 mL with 0.7 M perchloric acid (pH 6.5, adjusted with 5 N KOH). After cooling for 30 min, the mixture was centrifuged at 1,000×g for 10 min (0°C), and the supernatant was filtered using 0.22 μm syringe filter and analyzed by HPLC (Agilent 1260 Infinity, Agilent Technologies). The wavelength was recorded at 254 nm, and the concentrations of 5’-nucleotide are expressed as μg/mg.

The concentration of FAA in cooked breast samples were determined in triplicate according to the method by Rahman et al. (2018) with slight modifications. Briefly, finely ground sample (500 mg) and 6 N HCl (20 mL) were placed into a 25 mL test tube. After being flushed with N₂ gas for 30 s, a sealed tube was subjected to hydrolysis at 110°C for 16 h. Then, 100 μL of the hydrolyzed solution was evaporated under N₂ gas for another 30 s, dissolved with 1 mL of Milli-Q water, vortexed, and the mixtures were filtered through a 0.45 μm PTFE filter. The FAA was then quantified using HPLC (Agilent 1260 series, Agilent Technologies) equipped with a C18 column size 4.6×150 mm, with 5-μm particle size (Agilent Technologies), and a 338 nm detection wavelength at 40°C. Mobile phase A was 40 mM NaH₂PO₄, pH 7.8, and mobile phase B was composed of 45% acetonitrile, 45% methanol, and 10% Milli-Q water.

One-way analysis of variance (ANOVA) using R-version 3.6.1 (The R-foundation for Statistical Computing, Vienna, Austria) was used to analyze the differences in proximate composition, instrumental color, meat quality, antioxidant activity, taste-related nucleotides, and FAA contents among treatments. Each samples in six replications were statistically calculated and the significance values with p-values lower than 0.05 were assigned to Duncan’s multiple range test to obtain superscript.

The proximate composition, including moisture, crude fat, crude protein, and ash percentage, was compared among the treatment groups and is displayed in Table 1. As shown, the highest moisture content was observed in the HBG (73.98%) and Samgyetang that was hydrolyzed with flavorzyme before retorting (FS) group (70.62%), in comparison to the control (66.98%) and NBG (67.71%) groups (p>0.05). These moisture percentages were within the normal range for retorted breast meat from commercial broiler chickens, as per Jeong et al. (2020). Regardless of the BG addition, hydrolysis pre-treatment in this study did significant effect on moisture content. It is possibly attributed to the changes of hydrophilic ability of the meat proteins from enzymolysis that causing more interaction between water molecules and free peptides (Kong et al., 2017). In contrast, the proteins degraded dramatically after hydrolysis unlike in the control and NBG groups (p<0.05). Such macromolecular degradation during hydrolysis may result in altered protein structures, as well as other properties like hydrophilicity of the protein that may increase and consequently retain more water within the inner muscle (Ha et al., 2013; Naveena et al., 2004; Sharma and Vaidya, 2018). However, another outcome from the cleavage of protein macromolecule during hydrolyzation is the formation of shorter chain and lower molecular weight protein called free peptides that easily dissolved into water, causing lower protein content (Dong et al., 2020; Giménez et al., 2009) that also observed in this study. No noticeable differences were observed in crude fat and ash percentages among the treatment groups.

The surface color profile of the cooked breast samples following hydrolyzation were shown in Table 2. The addition of BG extract on Samgyetang was observed to intensify the redness color in our previous study when compared to the conventional one (Barido et al., 2021). This study confirmed the previous finding, wherein the conventional Samgyetang had the lowest redness score among treatments. Moreover, as also seen in this study, the hydrolyzation produced breast samples with the highest redness score among control and BG treatment groups (p<0.05). However, the light color was observed at the lowest score in the hydrolyzed group among treatments. The order of lightness values from the highest to the lowest was control, FS, BG treatment, and hydrolyzed group respectively (p<0.05). The result of this study implied the possible darkening effect from the hydrolyzation process on the color profile of the chicken breast. No statistical differences were observed in terms of the yellow color of the chicken breast following treatments. The basic color of the extract solution may hold the important factor to attribute the red color intensification on the BG-treated Samgyetang, as they could largely alter the surface color through permeation into the muscle environment (Jin et al., 2015). While the darkening effect after hydrolyzation might be due to the dissolution effect of some colored pigments during browning reaction under Maillard reaction, which confirms the study of Gao et al. (2021). These color alterations highly influence consumer preferences toward meat products (Utama et al., 2020).

Table 3 shows the pH values of the cooked breast samples for each treatment. Hydrolysis significantly increased the pH value compared to that in both the control and BG treatments groups (p<0.05). However, this study did not find any further differences in cooking loss percentage between the control and the BG treatment groups (p>0.05). Both BG treatment and hydrolysis were shown to not significantly affect the cooking loss of the Samgyetang. Accordingly, our afore performed studies, cooking loss was tended to be more influenced by the cooking method that the BG extract additions (Barido et al., 2021; Barido et al., 2022). The pH of the breast meat sample in the control, after retorting, was 6.19, slightly lower but within the normal range compared to the previous study on Samgyetang made from commercial broilers that were processed via retort processing (Jeong et al., 2020; Kim et al., 2019b). The increase in pH following hydrolysis with petidases might be attributed to the hydrophilic nature of cleaved protein molecules that influence the water potential or its tendency to pair with free peptides, thus results in increased water retention (Shao et al., 2016), hence supporting the previous report by Ang and Ismail-Fitry (2019). In addition, although biochemical alterations may have a negative correlation with one of the important economic traits such as cooking loss (Barido and Lee, 2021b), hydrolysis did not seem to contribute to it, as the percentage of cooking loss did not differ among treatment groups (p>0.05).

Enzymatic hydrolysis positively affected the textural properties of the cooked breast samples. As seen in Table 3, FS and HBG chicken breast had the lowest shear force value with 1.19 and 1.25 kgf among all treatment groups (p<0.05) with the control and NBG groups having 2.29 and 1.92 kgf, respectively (p>0.05). Enzymatic hydrolysis pre-treatment is one of the methods used to enhance the flavor and texture properties of protein sources. Dong et al. (2020) reasoned the possible improvement in physicochemical quality, including texture, upon hydrolyzation to be a consequence of increased protein solubility and protein degradation, resulting in notable enhancement of short-chain peptides with less effect on organoleptic profiles. Another possible mechanism may be the higher retention of water that allows the heightened juiciness perception toward meat products (Barido and Lee, 2021c). Miller et al. (1995) mentioned that the difference in shear force value of even 0.5 kgf is sufficient to be detectable by the consumer. In accordance, Huffman et al. (1996) also mentioned that a variation of 1 kgf is sufficient for consumers to notice the distinct differences among meat textures. This implied the efficacy of enzymolysis pre-treatment in improving meat texture profiles. Concerning the WHC, the percentage was in the previous reported range by Jeong et al. (2020), with minor variations (p>0.05). Together with cooking loss, WHC is essential economic traits in poultry processing industries due to its high correlation with the yield loss after cooking (Kristensen and Purslow, 2001). In this study, both hydrolyzation and BG extract addition were shown to not detrimentally affect the WHC percentage of the samgyetang samples.

The antioxidant activity of the cooked breast samples was measured using the TBARS assay and DPPH radical scavenging activity. With respect to the lipid oxidation rate, as seen in Fig. 1, both the NBG and HBG groups shared a similar suppression effect on MDA, compared to the control and FS (p<0.05). The lipid oxidation product formation was lowest in the HBG group (0.27 mg MDA/kg), intermediate in the NBG treatment (0.33 mg MDA/kg), and the highest in the FS (0.39 mg MDA/kg) and control group (0.42 mg MDA/kg). As expected, the addition of BG extract to Samgyetang resulted in a remarkable improvement in the antioxidative status of chicken meat, as also observed in our previous report (Barido et al., 2021). This was also in agreement with a previous report by Lee et al. (2018) on pork patties, which implied that BG extracts possessed potent antioxidant compounds. Further consolidation was obtained from the DPPH radical scavenging activity (Fig. 2), which was found to be the highest in the HBG group (50.22%), followed by that in the NBG treatment (46.57%), FS (34.17%) and control (31.20%).

Fig. 1. Lipid oxidation rate measured of Samgyetang breast influenced by enzymatic hydrolysis pre-treatment. ^a,b Mean values with the different superscripts are significantly different among treatments (p<0.05). TBARS, thiobarbituric acid reactive substances; MDA, malondialdehyde; Control, conventional Samgyetang made without addition of black garlic extract and receiving no hydrolyzation; FS, Samgyetang that was hydrolyzed with flavorzyme before retorting; NBG, Samgyetang made with the addition of black garlic extract without hydrolyzation; HBG, enzymatically hydrolyzed black garlic Samgyetang.

Download Original Figure

Fig. 2. DPPH radical scavenging activity of Samgyetang breast influenced by enzymatic hydrolysis pre-treatment. ^a–c Mean values with the different superscripts are significantly different among treatments (p<0.05). DPPH, 2,2-diphenyl-1-picrylhydrazyl; Control, conventional Samgyetang made without addition of black garlic extract and receiving no hydrolyzation; FS, Samgyetang that was hydrolyzed with flavorzyme before retorting; NBG, Samgyetang made with the addition of black garlic extract without hydrolyzation; HBG, enzymatically hydrolyzed black garlic Samgyetang.

Download Original Figure

Based on the DPPH assay obtained by this study, interestingly, the hydrolyzed group tended to exhibit stronger scavenging activity toward free radical when compared to the BG Samgyetang receiving no enzymatic hydrolyzation. However, both hydrolyzed group and BG treatment shared a significantly higher scavenging percentage than that of the control. The proper method used for hydrolyzation is critical for generating end products with improved functionalities. Using Flavourzyme as a the hydrolyzing agent, can allow a broad range of substrate specificities due ability to modulate both endo- and exopeptidase activities, resulting in the enhancement of amino acids with spacious properties (Giménez et al., 2009). The negatively charged amino acids are strong antioxidants that serve to donors the exaggerated electrons thereby scavenging the free radicals (Onuh et al., 2014).

Taste-related nucleotides, like 5’-IMP, 5’-GMP, 5’-AMP, inosine, and hypoxanthine, were analyzed by HPLC, and the results are presented in Table 4. Among the nucleotides recorded in the cooked breast samples, inosine and 5’-IMP were predominantly found across all the treatments. The strong umami nucleotides, 5’-IMP, 5’-GMP were dramatically upregulated following hydrolyzation of BG Samgyetang, with the 5’-IMP concentration being 146.68 μg/mg, 130.72 μg/mg, 123.09 μg/mg, and 127.36 μg/mg for HBG, FS, NBG and control group, respectively. Meanwhile, the concentration observed for 5’-GMP were 15.13 μg/mg, 12.03 μg/mg, 10.62 μg/mg, and 5.78 μg/mg, for HBG, FS, BG, and control group respectively. The results of this study implied the potential flavouring properties from the addition of BG as well as confirmed the efficacy of hydrolysis in enhancing the umami taste properties of meat proteins (Kong et al., 2017).

Two main nucleotide compounds may impart the umami taste in meat: Purine ribonucleotides with a phosphate ester on the 5’-carbon of the ribose section and a hydroxyl group on the 6’-carbon of the purine ring. However, the strength of the umami properties of these nucleotide compounds is highly dependent on their structure. The 5’-GMP, 5’-IMP, and 5’-AMP are known to be strong umami compounds, while the purine nucleotide with 6’-carbon, such as inosine, may also give the umami taste, but is weaker than the ribonucleotide with phosphate ester at the C-5 of the ribose section (Smith and Hong-Sum, 2011). In addition, the purine ribonucleotides phosphorylated at C-2’ or C-3’ of the ribose section can be classified as tasteless or potential umami compounds upon reaction with certain amino acids (Dermiki et al., 2013).

A total of 17 FAAs were recorded in and compared among the groups, in this study. As seen in Table 5, the hydrolysis resulted in the highest total FAA content in HBG group compared with those of the FS, NBG and control groups (p<0.05). Furthermore, the umami-related FAA (Asp and Glu) were remarkably intensified, wherein the concentration of Asp in the HBG group (18.31 μg/mg) was the highest followed by FS (16.35 μg/mg), NBG (16.07 μg/mg) and control group (15.21 μg/mg; p<0.05). Similarly, the Glu concentration in the HBG group (31.08 μg/mg) was higher when compared to FS (26.02 μg/mg), NBG treatment (22.31 μg/mg), and the control group (20.89 μg/mg). Additionally, the HBG and NBG treatment groups shared similar contents of the sweet FAA, alanine (Ala), which was higher than that in the control group (p<0.05). However, the hydrolysis of BG Samgyetang with Flavourzyme may enhance the exposure of hydrophobic amino acids like methionine (Met), possibly imparting a bitter sensation. Apart from its bitterness, Met is a sulfur-containing FAA with antioxidant properties (Smith and Hong-Shum, 2011). The endo- and exopeptidase properties possessed by Flavourzyme may cleave protein molecules both at the amino- and carboxyl-terminal, as well as at the internal cleavage sites (Ang and Ismail-Fitry, 2019; Gao et al., 2021; Kong et al., 2017), resulting in more varied FAAs.