Physicochemical and Functional Characterization of Blue-Shelled Eggs in Korea

The BE used in this study were obtained from farms in Hwasung-si in Korea. All the crossbred blue hens (Araucana × Ogol × White Leghorn) were kept together in brooding and rearing cages after hatching until 14 wk of age. Two hundred pullets were housed indoors at night. However, they spent some time in free range during the day. They were fed a commercial diet, as well as grass and worms, on the range. Among one hundred of laid BE were randomly selected and stored at 23±1°C before the experiment. CE (Hyline Brown commercial eggs) were obtained from the local market in Hwasung-si.

The proximate composition of each egg was determined based on standard Association of Official Analytical Chemists (1995) methods. Moisture content was determined from weight loss after drying for 12 h at 105°C in a drying oven (Vision Scientific Co., Ltd., Korea). Crude fat content was determined using a Soxhlet solvent extraction system. The fat content was calculated from the weight loss after lipid extraction with diethyl ether in the Soxhlet apparatus (Soxtec system HT 1043 extraction unit, Foss tecator, Sweden). Crude protein content was determined by the Kjeldahl method as follows: 0.3 g of the sample in oil paper, one Kjeltec catalyst tablet, and 10 mL H₂SO₄ were added into a Kjeldahl tube and digested for 1 h at 420°C. The product was then analyzed using an automatic Kjeldahl analyzer (Kjeltec auto sampler system 1035 analyzer, Foss tecator, Sweden). Crude ash content was determined from weight loss of sample after heating at furnace (30400, Thermolyne, USA) at 550°C for 24 h.

Egg quality was estimated according to the method reported by Liu et al. (2009). Weight (g), albumen height (mm), yolk color, Haugh units, and shell thickness (mm) were determined using an Egg Multitester model EMT-5200 (Touhoku Rhythm Co., Ltd., Japan). Shell thickness (mm) was determined using a micrometer (Digimatric Outside Micrometer, Mitutoyo Corporation, Japan)

The pH value was estimated according to the method reported by Choe et al. (2011). pH values were determined with a pH meter (Orion 230 A, Thermo Fisher Sci. Inc., USA), after blending 5 g of sample with 20 mL of distilled water for 60 s in a homogenizer (Polytron ® PT-2500 E, Kinematica, Switzerland).

The collagen content was estimated according to the method reported by Kolar (1990). The egg was hydrolyzed in 7 N sulfuric acid at 105°C for 16 h. Then, 1 mL of the hydrolyzed diluted sample was mixed with 0.5 mL of oxidative solution (1.41% chloramine T in collagen buffer solution). The collagen buffer (pH 6.0) was composed of 90 g of sodium acetate trihydrate, 15 g of sodium hydroxide, 30 g of citric acid monohydrate, and 290 mL of 1-propanol per liter. The mixture was incubated in the dark at room temperature for 20 min. The reaction tube was blended and 0.5 mL of reactive color (5 g of 4-methylaminobenzaldehyle with 17.5 mL of 60% sulfuric acid and 32.5 mL of 2-propanol) was added and incubated in a constant-temperature water bath at 60°C for 1 min. After sample incubation, the absorbance of hydroxyproline in the sample was measured at 558 nm using a UV/VIS spectrophotometer (Molecular Device, M2e, USA). A hydroxyproline standard curve was prepared at concentrations of 0.3 μg/mL, 0.6 μg/mL, 1.2 μg/mL, 2.4 μg/mL, and 4.8 μg/mL. Collagen content was calculated using a correction factor of 13.5.

Eggshell color was estimated according to the method reported by Choe et al. (2011). The color measurements were performed using a colorimeter (CR-300 Minolta Co., Japan). The color values (CIE, L*, a*, and b*) of the egg surfaces were measured in triplicate for each egg.

Lipids were extracted according to the method given by Morrison and Smith (1964). Two grams of the freeze-dried extracts of yolk was mixed with 20 μL 0.3% BHA and 20 mL folch solution, which is a mixture of chloroform and methyl alcohol (2:1 vol/vol). After the sample was homogenized, the mixture was filtered using Whatman No.1 filter paper, and the filtered sample was mixed with 0.88% KCl, followed by centrifugation at 1,000×g at 15°C for 10 min. The lower phase was concentrated using nitrogen gas. The sample was blended with 14% boron trifluoride (BF₃) in a constant-temperature water bath at 90°C for 1 h, and then dissolved in hexane. The upper layer was filtered using a 0.22-μm filter. Fatty acid was separated and identified using a gas chromatography (Agilent 6890N, Agilent Technologies, USA) system with a Cp-sil BB capillary column (Agilent CP 7489, 100 cm × 0.25 mm × 0.20 μm). The apparatus was programmed at an initial temperature of 150°C, allowing an increase of 7°C/min until the temperature reaches 200°C, which was maintained for 10 min, followed by an increase of 3°C/min until a final temperature of 250°C was reached, which was maintained for 5 min.

Freeze-dried extracts (2 g each) of BE and CE yolks were mixed with 20 μL 0.3% BHA and 20 mL Folch solution. After incubating overnight, water and chloroform were removed using nitrogen gas. Sample was blended with 14% BF3 in a constant-temperature water bath at 90°C for 1 h and dissolved in ethanol. Then, 0.02 mL sample was mixed with 3 mL enzyme solution kit (Asan Pharmaceutical, Korea) and incubated at 37°C for 5 min. The cholesterol content was measured spectrophotometrically at 500 nm (SpectraMax M2e, Molecular Devices, USA). The concentration was also expressed as milligram per gram yolk weight.

DPPH radical scavenging activity of a sample was estimated according to the method of Blois (1958) with slight modification. In brief, 100 μL of sample solution after extraction was added to 100 μL of methanol (for reference) and to 100 μL of methanolic DPPH solution (0.2 mM). The mixture was vortexed and incubated at room temperature for 30 min. A tube containing 100 μL of distilled water and 100 μL of methanolic DPPH solution (0.2 mM) served as the control. The absorbance of the solution was measured spectrophotometrically at 517 nm. (SpectraMax M2e, Molecular Devices, USA). Trolox was used as a positive control to compare the antioxidation activities of the hydrolysate samples. The percentage of DPPH radical scavenging activity was determined using the following equation:

The α-glucosidase inhibition assay was performed according to a previously described method (Dong et al., 2012) with slight modification. Briefly, 60 μL of the sample solution and 50 μL of 0.1 M phosphate buffer (pH 6.8), containing α-glucosidase solution (0.2 U/mL), was incubated in a 96-well plate at 37°C for 20 min. After pre-incubation, 50 μL of 5 mM p-nitrophenyl-a-D-glucopyranoside solution in 0.1 M phosphate buffer (pH 6.8) was added to each well in the 96-well plate and incubated at 37°C for 30 min. Then, the reaction was stopped by adding 160 μL of 0.2 M Na₂CO₃ into each well, and absorbance readings (O.D) were recorded at 405 nm using a micro-plate reader (Spectra Max M2e, Molecular Devices, USA). The measured values were compared to a control, which contained 60 μL of buffer solution instead of the extract. The α-glucosidase inhibition activity was calculated as follows:

Analyses of variance were conducted using SAS software, and Tukey’s test was used to compare differences among mean values. Mean values and pooled standard errors of the mean (SEM) were reported and the significance was defined at p<0.05.

The proximate compositions of CE and BE are shown in Table 1, divided into three parts: whole egg, egg yolk, and egg white. Moisture, crude protein, crude fat, and crude ash compositions of CE and BE were 75.89% and 74.89%, 11.99% and 12.05%, 10.46% and 10.77%, and 0.78% and 0.69%, respectively. Our findings are consistent with those of U.S. Department of Agriculture (2016) that reported the following proximate compositions of whole egg: 76.15% moisture, 12.56% protein, and 9.51% fat. The whole egg moisture composition of BE was significantly lower than that of CE. The moisture composition is related to the housing management. In this study, the BE layer hens were kept and maintained in the free range condition, however, they were housed indoor at night. Whereas mostly CE hens were housed indoor and controlled environment. In free range housing system hens are living in more variable circumstances and not fully controlled environment. The egg in the outdoor housing system had more contact with the environment, and the moisture composition in the egg loss may due to the water evaporation. Van Den Brand et al. (2004), they reported that indoor housing system had less evaporation. The egg yolk and egg white proximate compositions of CE and BE did not show significant differences.

The average values for all monitored egg quality indicators of CE and BE are presented in Table 2. Internal quality of eggs started to decline soon after they were laid. The egg internal quality was greater when measured soon after laid (fresh) compared when measured after storage (Mohiti-Asli et al., 2008). In this study, most of the egg quality indicators, except weight and shell thickness, showed no significant differences between CE and BE. The weight of CE was significantly higher than that of BE (58.8 g vs. 49.08 g). The BE has a lower moisture content and may this is affected on egg weight. The weight of BE was lower than that of CE may due to the genetic difference of BE. However, the shell of BE was significantly thicker than that of CE (0.32 mm vs. 0.29 mm). The different eggshell thickness might be explained by the amount of dietary Ca and P (Leeson et al., 1993). Although the amount of dietary mineral is not clearly explained in this study, the mineral intake may higher in the BE hens due to free range housing system, because in the range, the hens possibly get an extra mineral intake from grass and worms. The walking activity in the free range housing system also results in more effective mineral metabolism (Van Den Brand et al., 2004). Shell strength and thickness are important parameters for economic consideration, since the thickness of the shell is related to microbial contamination and breakage during processing or storage (Biladeau and Keneer, 2009). Based on the shell thickness, BE may be more resistant to microbial contamination than CE, resulting in longer shelf life. Our findings agree well with those reported by Wang et al. (2009a), who reported the weight, albumen height, yolk color, Haugh unit, and shell thickness of BE (Dongxiang) as 46.42 g, 5.34 mm, 7.98, 76.15, and 0.33 mm, respectively. Moreover, the egg quality can be affected by different housing systems: outdoor and cage housing system (Van Den Brand et al., 2004). In Korea, BE production is not largely prevalent yet, thus necessitating further studies regarding housing, egg laying, and egg collection for blue hens.

pH values were measured on whole egg, egg yolk, and egg white of CE and BE (Table 3). The pH values of CE and BE were not significantly different: 7.56 and 7.59, whole egg; 6.28 and 6.34, egg yolk; and 9.24 and 9.17, egg white, respectively. The pH values of whole eggs in this study are consistent with those reported by Wong et al. (1996), who observed pH values ranging from 7.54 to 7.87. Furthermore, they reported that the pH values can be influenced by eggshell coating, which controls the rate of carbon dioxide transfer through the shell.

Collagen is mostly found in the skin, cartilage, and connective tissues (Khiari et al., 2014). However, eggs also contain collagen; it has been observed in the whole egg, egg yolk, and egg white (Yamauchi et al., 2016). Collagen content in BE and CE in the whole egg was found to be 1.01 mg/mL and 1.10 mg/mL; in the egg yolk, 1.57 mg/mL and 1.66 mg/mL; and in the egg white, 0.63 mg/mL and 0.68 mg/mL, respectively (Table 3). The collagen contents in whole egg and egg yolk of BE were significantly higher than those of CE. The collagen content in BE was higher may due to the higher plant intake (grass) in the free range housing system. In agreement with Yamauchi et al. (2016) study that showed higher collagen content in the egg which treated with the plant processing product diet supplementation. Recently, the demand for the collagen has increased due to their beneficial effects on skin, as collagen is believed to prevent the formation of wrinkles on the skin (Khiari et al., 2014). Our results showed that BE has a higher collagen content than CE, and, thus, BE could have better effect on the skin than CE.

Eggshell color influence the consumer's decision to purchase the egg, making it an important characteristic (Wong et al., 1996). The visual appraisal is the first test that consumers conduct, while making a decision to purchase products. As shown in Table 4, the color values (L*, a*, and b*) were significantly different between BE and CE. The L* value of the shell color of BE was higher than that of CE, possibly due to bright blue color of BE compared to the dull brown color of CE. The a* and b* values were also significantly different. The a* values of CE and BE were 14.20 and −4.75, respectively, indicating that CE was towards red, while BE was towards green. The towards red color in CE which had a brown eggshell color due to protoporphyrin and uroporphyrin content as a red color pigment (Lukanov et al., 2015). The b* values of CE and BE were 30.63 and 10.45, respectively, indicating that CE was towards yellow, while BE was towards blue. It is well known that the pigments in BE are biliverdin IX and biliverdin zinc chelate (Lukanov et al., 2015).

The fatty acid compositions of CE and BE are shown in Table 5. Several fatty acid contents in BE, such as C18: 1n7 cis-vaccenic acid (0.93 vs. 0.20) and C18:3n6 gamma-linoleic acid (0.12 vs. 0.10), were significantly higher than those in CE. The difference of chemical composition including fatty acid on a chicken egg can be affected by many factors such as breeds (Simmons and Somes, 1985), different dietary supplementation (Altuntas and Aydin, 2014), and also housing system (Matt et al., 2009). The C18:3n6 fatty acid was higher in BE due to the higher intake of the plants as a feed supplementation in the free range, agree with those of reported by Altuntas and Aydin (2014) the C18:3n6 fatty acid in the egg was higher in the plant supplementation. Field et al. (2009) reported that consuming vaccenic acid is beneficial for health as it could possibly reduce tumor growth and prevent myocardial infarction (heart attack). Gamma-linoleic acid (GLA) content of BE was significantly higher than that of CE. Fan and Chapkin (1998) demonstrated that dietary GLA reduces the possibility of a cardiovascular disease, as GLA is the fatty acid source of dihomo gamma linoleic acid (DGLA), which becomes prostaglandin of the 1-series (PGE1), known to suppress plaque formation in the blood vessels. They also reported that the GLA could potentially suppress tumor growth and metastasis. Another study reported that the compositions of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) in the egg are 32.2, 48.9, and 19.2%, respectively (Grobas et al., 2001). In this study, the SFA and PUFA contents were higher than those reported in the study of Grobas et al. (2001), while the MUFA content was lower. The concentrations of the different fatty acids in the egg depend on breeds and dietary supplements provided for the chickens, as reported by Millet et al. (2006). The total SFA, PUFA, and MUFA compositions in BE and CE showed no significant differences.

The high cholesterol content is generally an unwanted characteristic of eggs (Millet et al., 2006), as cholesterol is associated with the risk of coronary heart disease (Castelli et al., 1977; Mannien et al.,1992; Naviglio et al., 2012). Krauss et al. (2000) recommend a dietary cholesterol intake for all individuals to be <300 mg/d, and USDA (2016) reported that the cholesterol content of the jumbo egg (63 g edible portion) was 234 mg. Therefore, consuming one egg per day is acceptable. Cholesterol is naturally produced and deposited by hen due to the preparation for the embryo. Many studies have been done to reduce cholesterol content in egg including dietary treatment (Park et al., 2005), storage treatment (Mazalli and Bragagnolo, 2009) and also by mixing the egg yolk with another compound (Valcare et al., 2002). Producing the high cholesterol content will increasing the risk of coronary heart disease, thus the cholesterol content in BE and CE was measured. In agreement with Salma et al. (2007) that genetic selection is one of the efforts for reducing egg cholesterol content.

The cholesterol content in the yolks of BE and CE were measured. The total cholesterol content in the egg yolk of CE and BE are not significantly different (19.72 mg/g and 18.93 mg/g, respectively). This result contradicts the findings of Somes et al. (1977) and Simmons and Somes (1985), who reported that BE (Araucana) has higher cholesterol content than CE. The cholesterol contents of yolks of BE and CE, reported by Somes et al. (1977), were 21.8 mg/g and 20.4 mg/g, respectively. Simmons and Somes (1985) reported that BE has 22.55 mg/g cholesterol content and CE (White Leghorn), 21.56 mg/g. In their study, the cholesterol content of BE was higher than that in our findings. Wang et al. (2015) reported that the cholesterol content of eggs from chickens provided with a different dietary treatment was 15.7 mg/g yolk. Another study, Wang et al. (2009a) reported that the cholesterol content of BE (Dongxiang) was significantly lower when the chickens were raised in an outdoor housing system (8.64 mg/g), as compared to that noted in eggs from chickens raised in a cage housing system (10.32 mg/g). The total cholesterol content was higher in this study, as compared to that reported by other studies (Wang et al., 2009a; Wang et al., 2015). This difference may due to the differences in breed and treatment of the hens, even though they all produced BE. Cunningham (1977) has reported that the differences in total cholesterol content of egg may vary due to multiple factors: differences in breed, experimental conditions, and extraction methods influencing the yolk cholesterol concentration of the egg. The cholesterol contents of CE and BE in this study were not significantly different, possibly because the BE hen, obtained from crossbreeding conventional chicken with Ogol chicken and Araucana, was genetically influenced by the conventional hen.

The DPPH scavenging activity is one of the methods to determine the antioxidation activity. Table 6 shows that the DPPH scavenging activity of BE (40.78%) was significantly higher than that of CE (35.35%). This result is similar to that of Lin et al. (2013), who reported that the DPPH scavenging activity derived from egg white is 44.92%. Another study reported that the DPPH scavenging activity of egg white protein in their study was 39.51% (Liu et al., 2015). DPPH scavenging activity of BE in our study showed a higher value (40.78%), as compared to the reports of Liu et al. (2015). Another study reported that the DPPH radical scavenging activity of the egg white lysozyme hydrolysates by trypsin, papain, and trypsin-papain were 37.2%, 50.4%, and 64.2% respectively (Yazdi et al., 2012). In our study, BE showed a higher DPPH scavenging activity than that of trypsin egg white lysozyme hydrolysate. However, the activity of BE was lower than that of the papain and trypsin-papain hydrolysates from eggs reported by Yazdi et al. (2012). The smaller size of hydrolysates after degradation by enzyme may result in a higher antioxidation activity. Chen et al. (2012) reported that the DPPH scavenging activity of the egg white protein hydrolysates by papain after 3 h of hydrolysis was 73.14%, which was higher than that in our findings.

The α-glucosidase inhibition activity was measured for the whole egg, egg yolk, and egg white. Table 7 shows that in the 90 mg/mL concentration, the α-glucosidase inhibition activity values of BE were significantly higher than those of CE (19.27 vs. 13.95 in the whole egg and 36.06 vs. 32.46 in the egg yolk). No α-glucosidase inhibition activity was detected in the egg white. α-glucosidase is one of the enzymes that hydrolyzes carbohydrates into glucose (Kim et al., 2004b). Preventing absorption of glucose could be beneficial for diabetics. This activity is one of the therapeutic parameters for diabetics (Kim et al., 2004b). One of the antidiabetic agents for commercial use is acarbose, which showed 45.8% of α-glucosidase inhibition activity at a concentration of 1.2 mg/mL (Dong et al., 2012). Acarbose is obtained from the fermentation process of a microorganism Actinoplanes utahensis (Wang et al., 2012). Another study reported that trilobatin, derived from Lithocarpus polystachyus Rehd. at a concentration of 0.8 mg/mL, has an 80% higher α-glucosidase inhibition activity than acarbose at the same concentration (Dong et al., 2012).

Yu et al. (2011) reported that the α-glucosidase inhibition activities of the bioactive peptides (RVPSLM and TPSPR) derived from egg white protein were comparable to that of acarbose. The IC₅₀ values of the peptides RVP-SLM and TPSPR were 23.07 μmol L⁻¹ and 40.02 μmol L⁻¹, respectively, and the IC₅₀ value of acarbose was 60.8 μmol L⁻¹ (Yu et al., 2011). The α-glucosidase inhibition activity from the bioactive peptide derived from BE egg yolk protein is higher than that noted for CE. In this study, BE was shown to have a significantly higher α-glucosidase inhibition activity than CE. Nevertheless, further studies regarding bioactive peptides in BE, which can function as antidiabetics, are necessary.