Antioxidant activity of Cinnamomum cassia extract and quality of raw chicken patties added with C. cassia powder and Pleurotus sajor-caju powder as functional ingredients during storage

Preparation of cinnamon extract

Antioxidant activity analysis of cinnamon

Ferrous iron chelating ability

Ferrous iron chelating ability (ICA) was measured using the method described by Le et al [11]. Briefly, 0.5 mL of sample (0.1% to 1.0% in dd-water) was mixed with 0.1 mL of ferrous chloride reagent, 0.5 mL ethylenediamine tetraacetic acid (EDTA), and methanol (0.9 mL). After 5 min, 0.1 mL of ferrozine (5 mM) was added and the sample was held at room temperature for 10 min. The ferrous ICA (%) was determined by measuring the absorbance of the Fe²⁺ ferrozine complex at 562 nm. The EDTA was used as a positive control.

Ferric reducing power

The ferric reducing power (RP) reagent was prepared as described by Huang et al [10]. The reagent was mixed with 2.5 mL of sample (0.1% to 1.0% in dd-water). After 20 min of incubation at 50°C, 2.5 mL of trichloroacetic acid (TCA 10%) was added and the mixture was centrifuged at 1,500 rpm for 10 min. Subsequently, the upper layer (2.5 mL) was taken and added with 2.5 mL of dd-water and 0.5 mL of ferric acid chloride (0.001%). Reducing power was then measured by reading the absorbance at 700 nm.

Total phenolic compounds

Cinnamon sample (0.1 g) was mixed with 10 mL of dd-water. Then 0.1 mL of this mixture was mixed with dd-water (2.8 mL), 2% Na₂CO₃ (2 mL), and 50% Folin-Ciocalteau reagent (0.1 mL) prior for vortexing. After 30 min of incubation at room temperature, the absorbance of the mixture was measured at 750 nm using a spectrophotometer (UV-1601; Shimadzu, Japan). Gallic acid was used as a standard (0 to 200 mg/L). Total phenolic compounds are expressed as GAE/100 g dried powder [12].

Manufacture of raw chicken patties

Fresh chicken breasts were obtained from a wholesale meat market in Gwangju, South Korea. They were ground using a grinder (M-12s; Fujee Plant, Busan, Korea). Ground chicken breast, sodium chloride, and additional ingredients were mixed for 1 min, ground in emulsifier, weighed into approximately 60 g, and formed into individual patties. These patties were placed on a polystyrene plate and held inside a refrigerator at 4°C±1°C during 14 days of refrigerated storage [13]. The following groups were included: control (CTL), 0.1% (w/w) ascorbic acid (REF), 0.1% CP + 0.1% OMP (C1M1), 0.1% CM + 0.2% OMP (C1M2), 0.2% CM + 0.1% OMP (C2M1), and 0.2% CP + 0.2% (OMP) (C2M2) (Table 1).

Table 1

Formulation of raw chicken patties with addition of different levels of cinnamon and oyster mushroom powders

pH and color values

The pH values of raw chicken patties were determined using a pH meter (Mettle-Toledo, Schwarzenbach, Switzerland) for five different locations per sample. Mean values of triplicate samples were calculated to obtain an average pH value. The color of raw chicken patty was determined with a Minolta color reader (Model # CR-10; Minolta, Tokyo, Japan). Results are presented as lightness (L*), redness (a*), and yellowness (b*). Measurement was performed for the surface at six different locations per patty sample. Mean color values of L*, a*, and b* were analyzed to obtain average colorimetric value.

2-Thiobarbituric acid reactive substance

Lipid oxidation of chicken patties during storage was determined by measuring 2-thiobarbituric acid reactive substance (TBARS) [14]. Patty samples were ground, weighed (about 2 g), and mixed with 3 mL of 2.5% thiobarbituric acid (TBA) reagent and 17 mL of 1% TCA in tubes with caps. Tubes were placed in a water bath at 90°C for 30 min. The supernatant of each solution was then mixed with 5 mL chloroform and centrifuged at 2,000×rpm for 5 min (VS-5000 N; Vision Scientific Co. Ltd., Bucheon, Korea). Each supernatant was added with approximately 3 mL of petroleum ether and centrifuged at 2,000×rpm for 10 min. Finally, clear solutions were analyzed with a spectrophotometer (UV-1601; Shimadzu, Japan) at wavelength of 532 nm. TBA value was expressed as mg of malondialdehyde (MDA) per kg meat sample.

Microbial counts

For microbial counts, 10 g of homogenized sample was mixed with 90 mL of sterilized water using a stomacher lab blender. Serial dilutions were then made. After that, about 0.1 mL of each diluted sample was inoculated and spread onto the surface of violet red bile agar and plate count agar medium. Plates were then incubated at 37°C for 24 to 48 hours. All plates were examined visually to count the number of colonies. Microbial colonies were counted and expressed as log 10 colony forming units/g chicken meat.

Volatile basic nitrogen

Volatile basic nitrogen (VBN) values (mg %) of chicken patties were measured using the Conway method [15] with slight modifications. Briefly, approximately 1 g of each patty sample was homogenized with 9 mL of dd-water using a homogenizer (S25N-18G; IKA, Staufen, Germany) for 1 min at 11,000 rpm and filtered with a Whatman No. 1 filter paper. Approximately 1 mL of the filtrate was transferred to a Conway dish to react with 1 mL of saturated potassium carbonate (K₂CO₃) solution at 37°C for 120 min. The incubated solution was then titrated with 0.01 N HCl. VBN value was expressed as mg %.

Statistical analysis

All experiments were carried out in triplicate. Data were analyzed using one-way (experiment 1) or two-way (experiment 2) analysis of variance. In experiment 2, if the interaction between two factors was significant (p<0.05), then data were separated by storage time within a treatment or by treatment within a storage time. If the interaction was not significant (p>0.05), then data were pooled by treatment or storage time. Duncan’s multiple range test was used to determine significant differences at 5% level (p<0.05) using SPSS 21.0 program for Windows.

Experiment 1. Antioxidant activity of cinnamon as affected by different solvent based on DPPH-RSA assay

DPPH assay has been used to estimate antioxidant activities of various food products. DPPH radical, a very stable nitrogen-centered radical, can be used to determine the free radical scavenging ability, which is related to their antioxidant activity. This method is based on the spectrophotometric measurement of DPPH concentration changes resulting from DPPH reaction with an antioxidant [16]. As shown in Table 2, the positive control (AA) had the highest value of DPPH-RSA (p<0.05). Cinnamon powder and CEE 80 (Cinnamon ethanol extract by 80% ethanol) showed higher DPPH-RSA than other treatments (p<0.05). Radical scavenging activities of CP and CEE 80 ranged from 48.1% to 68.8% and from 44.5% to 59.1%, respectively. The DPPH-RSA value was gradually increased with increasing concentration of CP. Mathew and Abraham [17] have reported that the antioxidant activity (RSA) of CP extracted with methanol at 6.25 to 50 μg/mL is increased with increasing concentration. Manchini-Filho et al [18] have reported that phenolic compounds in CP extracts are active components responsible for the antioxidant activity of ethanol-water extracts of CP. In the present study, the antioxidant activity (RSA) of CP was higher with ethanol extraction, particularly with 80% ethanol. Combination of ethanol and water solvent might be suitable for extracting some bioactive compounds with a broad range of polarity. Truong et al [19] have recently found that different extraction solvents with different polarity caused wide variations in the level of bioactive compounds found in the CP extract.

Table 2

Antioxidant activities of cinnamon powder in various concentrations with different solvent

Iron chelating ability

Table 2 shows results of ICA of EDTA, CP, and CP ethanol extract. EDTA as a positive control exhibited the strongest ICA (p<0.05) of 95.8% to 98.6%. The order of ICA in this study was: EDTA>CEE 0>CEE 100>CEE 80>CEE 50>CP (ICA ranges: 95.8% to 98.6%, 21.2% to 83.1%, 15.9% to 53.2%, 19.1% to 39.5%, and 23.5% to 31.9%, respectively). CP alone had the lowest ICA (6.77% to 22.5%). CEE 0 showed higher (p<0.05) ICA than CP and CP ethanol extract. ICA of CP water extract (CEE0) was comparable to that of EDTA. This indicates that the chelating compound in CP might be more soluble in water than in ethanol. These results were similar to a previous study of Cuong and Chin [20]. They reported that water extract of Cudrania tricuspidata (CT) leaves had higher chelating activity than methanol and ethanol extracts. In addition, ICA was increased with increasing CP concentration (0.1% to 1.0%) (p<0.05).

Reducing power

As shown in Table 2, solvent used for extraction affected the RP of CP and CP ethanol extract (p<0.05). However, extracts with different solvent at different concentrations had almost similar values. RP increased with increasing CP concentration (p<0.05). Many studies have indicated that antioxidant activity is related to the development of reductones known to react with certain precursors of peroxide and act as terminators of free radical chain reactions [21]. Ascorbic acid (AA) was used as a positive standard. RP showed the following order: AA>CEE 80>CEE 50>CEE 100>CEE 0>CP. This result indicated that CP ethanol extracts had higher reducing potential than CP and CP water extract. Among different concentrations of ethanol used for extraction, 80% ethanol was the most effective one as CEE 80 showed the highest RP. These results were similar to those of Varalakshmi et al [22]. They reported that RP of aqueous extract of CP was lower than that of methanol or chloroform extract of CP. They also reported that bark extracts functioned as an electron donor which reacted with free radicals to convert them to more stable products, resulting in the termination of radical chain reaction. Different types of solvent can affect the extractability of antioxidants. Water could dissolve alkaloid and glycoside compounds. However, ethanol was effective for extracting sterol, flavonoid, phenolic, and alkaloids [23]. Another study has observed that RP of CP extracted by methanol is the highest, followed by CPs of ethanol and water extracts [24]. Kamleshiya et al [25] reported that methanolic extracts of spices had higher RP than aqueous extracts of spices, with methanolic extracts of Cinnamonum cassia (150 to 200 μg/mL) and Piper nigrum (200 to 250 μg/mL) showing significant activities (>50%).

Total phenolic contents

Total phenolic contents were determined with a spectrometric method according to the Folin-Ciocalteu phenol method. They are expressed as GAE. Phenolics compounds such as flavonoids and phenolic acid possess various biological activities which might be related to their antioxidant activity. As shown in Table 3, total phenolic content in ethanol extract of CP was higher (p<0.05) than that in CP water extract. Total phenolic contents ranged from 11.77 to 19.73 mg GAE/g, showing the following order: CEE 80>CEE 50>CEE 100> CWE>CP. However, total phenolic content showed no significant difference among different ethanol extracts. Dvorackova et al [26] have shown that a binary solvent system is more effective than a single solvent system, depending on their relative polarity. The optimum solvent percentage was about 60% (v/v) ethanol at a ratio of 1:20 with cinnamon sample and the optimal extraction temperature and time were 50°C and 90 min, respectively. Abeyeskera et al [27] have found that cinnamon (Cinnamomum zeylanicum Blume) barks extracted with ethanol have 44.57±0.51 mg GAE/g of total phenolic compound. Cinnamomum zeylanicum (Ceylon cinnamon) leaf and bark extracts had higher antioxidant activities than extracts of other Cinnamomum species such as C. cassia, C. tamala, and C. verum. Another study has also found that the range of total phenolic compounds in extracts of spices including cinnamon (Cinnamomum zeylanicum) was 3.53 to 58.25 mg GAE/g with an average value of 19.9 mg GAE/g. The highest level of total phenolic compounds was observed in galangal, whereas the lowest value was found in white pepper as reported by Lu et al [28]. Six phenolic compounds (catechin, p-coumaric, vannilic acid, caffeic acid, ferulic acid, and protocatechuic acid) have been found in CP extracted with subcritical water [29].

Table 3

Total phenolic compounds (mg GAE/g) of cinnamon powder extracted with different solvents

Ethanol extracts of freeze-dried CP showed higher levels of phenolic compounds than those of oven-dried CP. Therefore, freeze-drying can enhance the extractability of phenolic compounds since ice crystals can form within the sample matrix and rapture the cell structure, which allows cellular components and solvent to come out as reported by Nicoli et al [30]. Furthermore, the loss of phenolic compounds in CP by oven drying at higher temperature (>88°C) was higher than that by freeze drying. However, thermal processing, sometimes, released more phenolic acids from the breakdown of cellular components due to the heat processing, resulting in accumulation of more antioxidants. During the drying processes, oxidative enzymes such as polyphenol oxidase and peroxidase were deactivated, leading to avoid the loss of phenolic compounds as reported by Dewanto et al [31]. Based on the results of the antioxidant activities, data were not consistent. For example, CEE 80 was highest in DPPH, whereas CEE100 was lowest in total plate counts (TPC) in this stduy, which was similar trend to those of RP. Unlike these data, the iron chelating ability was highest in the water extration (CWE) rather than ethanol extraction (CEE), since the CP might be soluble in water rather than ethanol. In TPC, the CEE 80 was better than the CEE 100. The analysis of anitoxidant activities were not similar trend in this study due to the different mechanism of the various antioxidant measurements as reported by Goulas and Manganaris [32].

Experiment 2. Physicochemical and textural properties of chicken patties including pH and color value

Table 4 shows pH values of raw chicken patties with addition of CP and OMP during storage. As shown in Table 2, pH values of raw chicken patties without adding antioxidants (CTL) were higher (p<0.05) than those with addition of CP and OMP. They were also higher than those of REF (0.1% AA). Addition of CP and OMP could decrease pH, regardless of the addition level. However, pH values were found to be similar up to day 3 of storage. They started to increase from 7 days of storage. Then, they became higher toward the end of the storage time. Increased pH values of raw chicken patties with increasing storage time might be partially due to the breakdown of proteins to amino acids or alkali compounds by bacteria during storage. Masniyom et al [33] have reported that pH values of air-stored sea bass are increased during storage. This might be partially due to the production of basic amino acids or amines by bacteria, which are released during protein degradation. These bacteria could use stored glucose. Therefore, products of amino acid decomposition were accumulated, resulting in higher pH values.

Table 4

pH and color values of chicken patties as affected by different levels of cinnamon and oyster mushroom powders

CIE L*, a*, b* color values of raw chicken patties with/without antioxidants are shown in Table 4. CIE color values were affected by the addition of cinnamon and oyster mushroom, especially by the addition of 0.2% CP (C2M1, C2M2). Lightness (L*) values of control chicken patty samples were higher than those of treatments (p<0.05). However, these values remained stable up to 14 days during storage. The addition of 0.2% CP decreased L* value (lightness), but increased a* value (redness) and b* value (yellowness) due to original colors of CP and OMP. These results indicate that addition of CP and OMP alone or in combination might improve color values of chicken patties. Gahruie et al [34] observed natural extracts of cinnamon, shirazi, and rosemary with potential to improve color stabilities of frozen beef burger. These materials were able to control lightness and redness values. A similar study [35] has shown that the addition of spices of Syzygium aromaticum, Cinnamomum cassia, Origanum vulgare, and Brassica nigra to raw chicken meat can result in higher a* values, which indicated by intense red color due to spice carotenoids.

Volatile basic nitrogen value

Byun et al [36] have described that VBN contents in meat are increased due to deamination of amino acids to ammonia during storage. VBN can be used to examine decomposition of fresh meat and poultry products. Volatile compounds such as (CH₃)₃NH (trimethylamine), (CH₃)₂NH (dimethylamine or DMA), and NH₃ (ammonia) are products of microbial degradation. They are known as total volatile basic-nitrogen (TVB-N). Hence, TVB-N level is a potential indicator of fish spoilage [37]. As shown in Table 5, VBN values of raw chicken patties were increased in all treatments with increasing storage period. VBN values reached up to 14.64 mg N/100 g at day 14 of storage. Byun et al [36] reported that total VBN values are 20 and 30 mg N/100 g for beef and pork, respectively. Another study conducted by Balamatsia et al [38] found that chicken meat stored in air have acceptable VBN value of 40 mg N/100 g in a refrigerator. In the present study, chicken patties added with AA (REF) had lower VBN values than those treatments (p<0.05). Although no difference in VBN value was found among treatments (p>0.05), VBN values were still lower than those of CTL (no treatment with powder), indicating that combination of CP and OMP might be effective in retarding protein deterioration. In this study, lower VBN values of meat samples might be partially due to antimicrobial activities of CP and OMP. These results have been suggested by Gupta et al [39] who reported that cinamaldehyde compound on cinnamon bark is highly electro-negative, which is associated with biological processes in electron transfer by reacting with nitrogen-containing components, e.g., proteins and nucleic acids, therefore inhibiting the growth of microorganisms.

Table 5

Volatile basic nitrogen (VBN), total plate counts (TPC), and Enterobacteriaceae (VRB) of chicken patties as affected by different levels of cinnamon and oyster mushroom powders

Microbial counts

As shown in Table 5, both TPC and Enterobacteriaceae were increased rapidly from day 7 throughout the end of storage time. Chicken patties containing C2M1 and C2M2 showed lower TPCs than other treatments, while Enterobacteriaceae counts were lower in all treatments added with natural antioxidant. In this study, addition of CP up to 0.2% (C2) resulted in better antimicrobial activity regardless of OMP addition. These results indicate that the combination of CP and OMP can inhibit the growth of pathogen that partially due to antimicrobial activity of those materials. Hoque et al [40] studied the antimicrobial activity of cinnamon essential oil on ground chicken meat. They found that cinnamon inhibited microbial pathogens such as Pseudomonas, L. monocytogenes, and Salmonella. Iwolakun et al [41] have detected phenolics and terpenoids in oyster mushroom. Those constituents might have potential as antimicrobial agents. In addition, Shan et al [42] reported that total phenolics might be significantly associated with antibacterial activity. Extracts from medicinal herbs which contain high levels of phenolic compounds possess strong antibacterial activities. Thus, medicinal herbs might be potential sources of antioxidants that can help fight against foodborne pathogens.

2-Thiobarbituric acid reactive substances

Since there was an interaction between treatment and storage time, data were separated out by treatment within a storage time or by storage time within a treatment. As shown in Figure 1, TBARS values of all treatments were increased with increasing storage time. However, they were decreased with the addition of CP and OMP. Generally, TBARS values might be correlated with the acceptable level for sensory panelists to consume meat products [43]. CTL had the highest TBARS value and REF showed the lowest TBARS value among treatments, while C2M1 and C2M1 had lower TBARS than 0.1% CP at the initial storage (p<0.05). Interestingly, C2M1 and C2M2 were found the lowest TBARS values (comparable to that of AA/REF). These results indicated that increased level of CP was more effective in inhibiting lipid oxidation than those without CP, regardless of the addition of OMP. These results were consistent with those of El-Alim et al [44] who found that dried spices of cinnamon, peppermint, cloves, nutmeg, marjoram, curry, and caraway could inhibit lipid oxidation of raw minced chicken meat by reducing the TBARS level after 6 months frozen storage to two to three times lower than the control (without any spices). In the present study, adding CP and OMP was effective in inhibiting lipid oxidation and microbial count in ground chicken patties during storage. Arbaayah and Umi [45] have reported that phenolic compounds such as phenolic acids and tannins are major components of the antioxidant system in plants and mushrooms.

Figure 1

Thiobarbituric acid reactive substances(TBARS) values of chicken patties with various levels of cinnamon and oyster mushroom powder during storage. Treatments: CTL = control (without antioxidants); REF = patties mixed with 0.1% ascorbic acid; C1M1 = patties mixed with 0.1% cinnamon powder; 0.1% oyster mushroom powder, C1M2 = patties mixed with 0.1% cinnamon powder; 0.2% oyster mushroom powder, C2M1 = patties mixed with 0.2% cinnamon powder; 0.1% oyster mushroom powder and C2M2 = patties mixed with 0.2% cinnamon powder; 0.2% oyster mushroom powder.