Investigation of Taste-Related Compounds and Antioxidative Profiles of Retorted Samgyetang Made from Fresh and Dried Cordyceps militaris Mushrooms

A total of 48 carcasses were prepared from commercial broilers (CB; Ross, 4 weeks old) and were purchased 24 h post slaughter from a local slaughterhouse, with an average weight of 550±20 g. Each samgyetang pouch was filled with 450±50 mL of broth that had been previously boiled for 45 min. The boiled broth was prepared with the addition of samgyetang ingredient pouches consisting of 5 g Astragalus membranaceus root, 8.5 g of mulberry branch, 8 g of Kalopanax septemlobus branch, 2 g of licorice, 9 g of Siberian ginseng and 6 g of salt. The broth was also augmented with C. militaris mushrooms under either fresh or dried conditions. Both fresh and dried C. militaris mushroom was purchased from Mushtech (Gangwon, Korea). The fresh mushroom was prepared from 5 months old of C. militaris mushroom, while for the dried one was prepared from 5 months old mushroom subjected to drying process at 80°C for 12 h. The addition concentration was determined based on the broth volume (w/v) ranging from 0% as a control to 1%, 2%, and 3% of C. militaris mushrooms.

To prepare the stuffing, glutinous rice was soaked for 1 h and rinsed prior to use. Garlic, ginseng, and dried jujube were rinsed with cold water. Approximately 35 g of glutinous rice, 8 g of garlic, 5 g of jujube and 7 g of ginseng were placed in rice paper, which was soaked with warm water and wrapped. The wrapped rice, breast and thigh meat were then stuffed into a retort pouch [20 cm (width)×27 cm (length)]. The pouches were then sealed (WB-1150VP; Woobin Tech., Incheon, Korea). Two extra pouches were used to measure the F₀ value during the retorting process. The retort process was performed using a steam-type retort sterilization chamber (Steri-ace, Gyeongsan, Korea). The f₀ value of the retorting process was set to 8.

The sample was ground using a food grinder (HMF-1600 PB, Hanil Electric, Seoul, Korea) at medium speed for 10 s. The proximate composition was determined according to the Association of Official Analytical Chemists method (AOAC, 2002). The percentage of moisture in the sample was determined by drying the samples in an oven at 105°C for 24 h. The crude fat content was determined according to the ether extraction by the Soxhlet system. The nitrogen content was determined using the Kjeltec system (2200 Kjeltec Auto Distillation Unit, Foss, Hilleroed, Sweden), and crude protein was calculated as nitrogen content multiplied by 6.25. Crude ash was determined by burning the samples in the muffle furnace at 550°C for 12 h.

Cooking loss is defined as weight loss after being subjected to the retorting process. Briefly, samples were weighed to obtain an initial weight (W1) before being subjected to retorting. Samples, in triplicate, were then weighed to obtain the weight after manufacturing (W2). The percentage of cooking loss was obtained by calculating weight loss (W1–W2) against W1.

Samples from cooking loss experiments were subsequently used to measure tenderness (toughness) of the meat by performing the Warner-Bratzler Shear Force test using TA-XT2i Plus (Stable Micro Systems, Surrey, UK). The method was performed according to Jeong et al. (2020) with a slight modification, where the sample was made into a 1.5 cm×1 cm size. It was then placed under the V blade and cut with a constant speed through the gap in the instrument table (assay parameters were as follows: pretest speed: 2.0 mm/s; test speed: 1.0 mm/s; posttest speed: 10 mm/s). Each sample was repeated five times.

The measurement of lipid oxidation was performed using 2-thiobarbituric acid reactive substances (TBARS). A sample of 0.5 grams in a 25-mL TBARS test tube was prepared with three repetitions, and 0.1 g of antioxidant mixture (consisting of 54% propylene glycol, 40% Tween 20, 3% butylated hydroxytoluene and 3% butylated hydroxyanisole) was transferred to the tube. Subsequently, 3 mL of 1% thiobarbituric acid in 0.3% NaOH was added to the mixture. Immediately after vortexing, 17 mL of 2.5% trichloroacetic acid in 36 mM HCl was added, and the tube was closed. The sample was subjected to heating in a water bath (BW-20G, Biotechnical Services, North Little Rock, AR, USA) at a temperature of 100°C for 30 min. The tube was then immersed in cold water for another 15 min. A 5 mL aqueous sample was transferred to a new 15 mL centrifuge tube and mixed with 3 mL of chloroform. The mixture was then subjected to centrifugation at 2,400×g for 30 min at 4°C (1248R, Labogene, Lynge, Denmark) to separate it from the pellet. The absorbance was measured at 532 nm by using a UV-spectrophotometer (UV-mini 1240 PC, Shimadzu, Kyoto, Japan) against a blank (distilled water was used to replace the sample). Each sample was repeated three times. Data are expressed in the form of mg malondialdehyde/kg sample.

The determination of bioactive compounds from C. militaris mushrooms, particularly adenosine and cordycepin, was performed according to a method by Wang et al. (2016) with slight modifications. For the extraction of adenosine and cordycepin, 1.00 g of freeze-dried sample was mixed with double-distilled water (100 mL) and extracted for 30 min in an ultrasonic bath at 60 kHz. Then, the supernatant was filtered through a membrane filter (0.45 μm). A high-performance liquid chromatography (HPLC) system (Model 1525, Waters, Milford, MA, USA) furnished with a reverse C18 column (150×4.6 mm) was used for adenosine and cordycepin analysis. The mobile phase, whose flow rate was 0.5 mL min⁻¹ (isocratic elution), was composed of acetonitrile and double-distilled water (20:80, v v⁻¹). The column temperature was 35°C, and a UV–visible detector was applied for detection at a wavelength of 260 nm. HPLC adenosine and cordycepin standards (Sigma-Aldrich, St. Louis, MO, USA) were used for quantitation by the external standard method.

The determination of antioxidant activity was performed according to a method by Islam et al. (2016) with a minor modification. Briefly, a reaction mixture of 1 mL 0.15 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH)-methanol solution, 4 mL methanol, and 2 μL of test samples (10/mL), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), or water (control) was incubated at room temperature for 30 min, and absorbance values were measured at 517 nm using a spectrophotometer (V530, Jasco, Japan). The experiment was repeated three times for each compound. Electron donation ability was defined as the total reduction of absorbance through a spectrophotometer.

The method for analyzing 5’-nucleotide contents (adenosine monophosphate, inosine monophosphate and guanine monophosphate) was modified from the method of Jayasena et al. (2015). The 5’-nucleotide analysis was carried out using HPLC (Shimadzu Nexera X2 HPLC) equipped with an SPD-M20A diode array detector (DAD) at a wavelength of 254 nm. Freeze-dried broth samples (1.00 g) were diluted with distilled water and filtered using a 0.22-μm RC syringe filter (Phenomenex, Torrance, CA, USA). Analytes were separated using a Synergi Hydro-RP column (150×3.0 mm, 4 μm; Phenomenex) with precolumn AQ C18 (4×2.0 mm). The mobile phases were A: 20 mM phosphate buffer (pH 5.9) and B: 100% methanol. The gradient program used was as follows: 0–3 min, 0% B; 3–12 min, 0 to 30% B; 12–13.5 min, 30% B; 13.5–16 min, 30 to 0% B. The total analysis time per sample was 25 min. After every injection, 20% ACN solution and ultrapure H₂O were used to rinse the needle. The flow rate was 0.4 mL/min, the injection volume was 5 μL, and the column oven temperature was 25°C. 5’-Nucleotide concentrations were expressed as mg of compound per 100 g of cooked matter (mg/100 g FM).

Two MSG-related amino acids (L-aspartic acid, L-glutamic acid) were analyzed and identified simultaneously using the HPLC method described in the manufacturer’s technical notes (Shimadzu). A Nexera SIL-30AC autosampler with automated pretreatment functions was used for the derivatization of amino acids into fluorescent substances. The following derivatization reagents were used: OPA and 3-MPA in 0.1 M borate buffer, FMOC in acetonitrile, and acidic phosphate buffer (pH 2.1). After the derivatization procedure, 1 μL of the derivatized standard or sample was injected. The solvents used in the gradient program were A: 20 mM phosphate buffer (pH 6.5) and B: 45/40/15 ACN/MeOH/H₂O. The gradient was as follows: 0–2 min, 11% B; 2–4 min, 11% to 17% B; 4–5.5 min, 17% to 31% B; 5.5–10 min, 31% to 32.5% B; 10–12 min, 32.5% to 46.5% B; 12–15.5 min, 46.5% to 55% B; 15.5–16 min, 55% to 100% B; 16–19.5 min, 100% B; 19.5–20 min, 100% to 11% B. The total analysis time per sample was 25 min. After every injection, 80% MeOH and 20% ACN were used to wash the needle. The flow rate was set to 1.0 mL/min, and the column temperature was maintained at 35°C.

The data results were analyzed by two-way multivariate analysis of variance (MANOVA) using R-version 3.6.1 (The R-foundation for Statistical Computing, Vienna, Austria). The mean value of each group was separated using Duncan’s multiple range test. Differences was considered as significant for p-values lower than 0.05.

Table 1 displays the proximate composition of samgyetang breast and thigh meat after treatment with either fresh or dried C. militaris mushrooms. Different condition of C. militaris mushroom and the level of addition had no significant interaction on overall proximate composition traits (p>0.05). In particular, no significant difference (p>0.05) was observed for the proximate composition of moisture content in breast and thigh meat. These results were in line with the study by Triyannanto et al. (2014), who did not find any difference after applying additional ingredients to samgyetang. The moisture content in this study was still within the range of previously reported studies (Jeong et al., 2020).

The protein, fat and ash percentages were also found to be insignificant for all observed samples (p>0.05). The addition of C. militaris mushrooms at different levels did not significantly contribute to changes in protein, fat and ash percentages in this study. The protein percentage for breast meat ranged from 28.1%–28.9%, while it ranged from 24.1%–25.6% for thigh meat. This value was higher than that found in a previous study by Ali et al. (2007) and still within the range reported in a study by Jeong et al. (2020). However, no effect was observed (p>0.05) by C. militaris mushrooms in this study, which might be due to the small addition percentage.

Cooking loss can be defined as the percentage of cooking yield loss after being subjected to processing stages, and it significantly correlates with economic traits (Barido et al., 2020). Cooking loss is closely correlated with the capability of myofibrillar protein to retain muscle water during processing. Therefore, meat quality traits are important for meat shrinkage prevention. Cooking loss also has a strong relationship with eating satisfaction since muscle water contributes to the juiciness level of meat. Higher cooking loss will lead to less meat product juiciness (Jeong et al., 2020). Regarding cooking loss, we did not observe a statistically significant difference (p>0.05) either for the condition of C. militaris mushroom added and the level of additions, which implies that the addition of C. militaris mushrooms did not significantly affect the cooking yield percentage. The percentage of cooking loss ranged from 24.4% to 26.1%.

Meat tenderness is directly correlated with the acceptability of meat by consumers and leads to consumers’ repurchase intent (Kemp and Wheeler, 2011). Tenderness is determined by complex factors, including muscle water, the degradation of muscle skeleton proteins and protease enzymes that work postmortem (He et al., 2019). Therefore, several attempts were made to improve the tenderness level to increase the eating quality. In this study, C. militaris mushrooms were added at different levels to improve the tenderness of samgyetang meat.

As seen in Table 2, it was found that the tenderness could be improved by the addition of C. militaris mushrooms. The shear force value, which reflects the degree of meat tenderness, seemed to be significantly affected by the addition of C. militaris mushrooms at a level of more than 2%. Both breast and thigh meat with the addition of C. militaris mushrooms at levels of 2% and 3% in fresh and dried conditions contributed to a lower shear force value compared to the control and 1% treatments. However, no significant difference was found (p>0.05) between fresh and dried C. militaris mushroom addition. The increment in tenderness level was seemed only affected by the level of Cordceps militaris mushroom addition as reflected by no significant interaction effect (p>0.05) between different condition and addition level of Cordceps militaris mushroom. The tenderization effect on both breast and thigh meat might be due to the existence of adenosine monophosphate within the C. militaris mushroom, which in turn leads to the increased activation of protease enzymes (Wang et al., 2016). The addition of adenosine monophosphate is often used to improve meat tenderness (Wang et al., 2013).

TBARS measures the degree of lipid deterioration from the sample. It serves to understand the concentration of malondialdehyde, a marker for oxidative stress (McMillin, 2008). Therefore, it can be a useful assay for the determination of a product’s rancidity (Das et al., 2010). Table 3 displays the lipid oxidation rate of the samgyetang meat treated with the addition of C. militaris mushrooms. The malondialdehyde content as an oxidative stress marker was significantly suppressed (p<0.05) by the addition of C. militaris mushrooms at levels of 2% and 3%. However, no significant effect was recorded (p>0.05) from a different condition of C. militaris mushroom added. In addition, insignificant interaction effect between different conditions and addition mushroom level (p>0.05) was observed on malondialdehyde content. C. militaris mushroom extracts could be a strong source of natural antioxidants. The major contributors to most pharmacological functions in C. militaris mushrooms are regulated by the existence of active biological compounds, such as cordycepin, adenosine, adenine, polysaccharide and cordyheptapeptide, as well as D-mannitol (Jayasena et al., 2014). This finding was also supported by Das et al. (2010) who explained that the strong antiradical potency possessed by C. militaris might be the basis of their strong therapeutic efficacy in traditional medicine. However, no effect on the lipid oxidation rate of both breast and thigh meat was found for fresh or dried C. militaris mushroom addition (p>0.05).

DPPH is known as a stable radical at room temperature that accepts electrons or hydrogen radicals to become a stable diamagnetic molecule (Islam et al., 2016). It has been used to determine the antioxidant activity of various neutral products. The effect of antioxidants on DPPH has been thought to be due to their hydrogen donating ability (Adebayo et al., 2012). Antioxidant content within a product is an important variable for consumers because higher antioxidant levels positively correlate with health-promoting functions after consumption. The effect of C. militaris mushroom addition, either fresh or dried, at different concentrations on the antioxidant activity of retorted samgyetang is shown in Table 3. The highest scavenging activities were observed with the addition of dried C. militaris mushrooms at a level of 3% in both breast and thigh samples, with values of 59.96% and 50.16%, respectively. The lowest scavenging activities were recorded for the control sample, with values of 32.92% and 34.92% for thigh and breast meat, respectively. The addition of C. militaris mushrooms at a level of more than 2% significantly improved the antioxidant activities of retorted samgyetang, as noted by a significant difference from the control and 1% addition treatments (p<0.05). C. militaris mushrooms under dried conditions contributed to higher antioxidant activities than those under fresh conditions. Furthermore, this study did not found significant interaction between different condition and addition level of C. militaris mushroom (p>0.05) on DPPH scavenging activity. Drying might lead to important changes in several pharmacological-related compounds (Akinmoladun et al., 2010) through a cell destruction mechanism that inactivates peroxidation enzymes, releases more short bioactive chains and improves their activities (Yu et al., 2009). However, no significant difference was found between the 2% and 3% treatments.

For centuries, C. militaris mushrooms have been widely used as versatile medicines for curing diseases, including liver disease, renal dysfunction, hyperlipidemia, and hyperglycemia (Zhu et al., 2013). Moreover, bioactive compounds, mainly cordycepin, adenosine, adenine, polysaccharide and cordyheptapeptide, as well as D-mannitol, are essential for immunostimulation, a mechanism of improving body defense in elderly people as well as in cancer patients (Katsube et al., 2009). This study investigated the functional effect of samgyetang products after the addition of either fresh or dried C. militaris mushroom, especially the existence of major bioactive compounds, mainly cordycepin and adenosine, after cooking. As displayed in Table 4, the higher concentration of C. militaris mushroom addition significantly improved the contents of bioactive compounds within the broth sample. For the cordycepin content, the highest level was observed in the treatment with the addition of dried C. militaris mushrooms at 3% with 0.66 mg/g db. The dried C. militaris mushroom addition had a higher level of cordycepin than the fresh mushroom addition (p<0.05). However, insignificant interaction between different conditions and an additional level of C. militaris mushroom (p>0.05) was documented, which implies that each treatment variable had a certain effect on samgyetang. High temperature promoted cell wall destruction. This destruction of cells contributed to the exposure of the substrates and the enzymes, while the decrease in moisture content also increased their concentrations. The autocatalytic oxidation triggered by the oxygen contact and decrease in moisture content promoted the increase in cordycepin content (Ke et al., 2011). It also revealed that cordycepin is apparently thermally stable. In addition, a similar trend was found for adenosine, where the highest addition level of C. militaris mushrooms had the highest adenosine content among the samples. However, for the adenosine content, no difference was found between fresh and dried additions in the samgyetang broth sample.

Flavor plays an important role in eating satisfaction and eventually promotes more consumption (Chiang and Yen, 2007). In samgyetang, flavor variations are mainly affected by 5’-nucleotides, free amino acids, soluble sugars and volatile compounds (Jayasena et al., 2015). However, even after being subjected to the processing stage, other essential nutrients, such as carbohydrates, lipids, micronutrients and soluble proteins, are less dissolved into broth (Beluhan and Ranogajec, 2011; Takakura et al., 2014; Zhang et al., 2017). Therefore, this study aimed to enrich the samgyetang flavor through the addition of C. militaris mushroom. Since, aside from its nutrient contents, mushrooms are consumed because of their unique taste and aroma (Mau et al., 1998; Tsai et al., 2008). The effect of C. militaris mushrooms on taste-related compounds in this study is shown in Table 5. Adenosine monophosphate (5’-AMP) was strongly affected by the addition of C. militaris mushrooms with no significant difference between fresh and dried mushroom addition. In this study, the AMP in 2% and 3% was significantly higher compared to that of control and 1% group, while regardless the condition and addition level of C. militaris mushrooms, inosine monophosphate was not significantly affected. It might indicate that high content of AMP within the C. militaris mushroom could contribute to a higher generation of meat AMP. The expected mechanism is that exogenous AMP, which penetrates into the meat muscle was not completely converted into IMP and remain unchanged from a state of AMP residue. AMP is one of the major bioactive compounds within the C. militaris mushroom with a broad range of pharmacological functions (Gamage et al., 2018). Through adenosine monophosphate deaminase (AMPD) pathway, AMP could be converted into ammonia and IMP with the rate of convertion is depend on various factors including heat (Wang et al., 2016). In addition, guanosine monophosphate was not affected regardless the concentration of C. militaris mushroom added (p>0.05). There was no significant interaction effect between different conditions and an additional level of C. militaris mushroom (p>0.05) on taste-related 5’-nucleotide compounds in this study.

L-Aspartic acid and L-glutamic acid were the predominant free amino acids related to MSG-like flavor in this study. As seen in Table 6, compared to the control and 1% addition treatments, L-aspartic acid was significantly affected by the addition of either fresh or dried C. militaris mushrooms at levels of 2% and 3% (p<0.05). In contrast, the highest concentration of L-glutamic acid was observed in the treatment with 3% of either fresh or dried C. militaris mushrooms (p<0.05). The L-glutamic acid level was 9.92 g/100 g dry weight and 9.90 g/100 g dry weight for fresh and dried mushrooms, respectively. However, this study did not found any significant difference between the condition and the addition level of mushroom added (p>0.05) on both L-aspartic acid and L-glutamic acid. Numerous studies have mentioned that mushrooms are rich in umami flavors (Zhang et al., 2013). The umami taste comes from the rich contents of sodium salts, namely, glutamic acids and aspartic acids, also known as umami amino acids (Yang et al., 2001). Studies have also characterized other free amino acids as having a sweet, bitter or neutral taste (Zhang et al., 2013). Therefore, apart from giving the umami taste, mushroom addition into samgyetang could also possibly enrich the overall flavor of samgyetang.